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5 DOSIMETRY AND MODE OF ACTION

5.1 Introduction

1 This chapter has two main purposes. The first is to describe the principles that underlie 

2 the dosimetry of O3 and to discuss factors that influence it. The second is to describe the 

3 modes of action leading to the health effects that will be presented in Chapters 6 and 7. 

4 This chapter is not intended to be a comprehensive overview, but rather, it updates the 

5 basic concepts derived from O3 literature presented in previous documents (U.S. EPA, 

6 2006b, 1996a) and introduces the recent relevant literature.

7 In Section 5.2, particular attention is given to dosimetric factors influencing individual 

8 risk of developing effects from O3 exposure. As there have been few O3 dosimetry studies 

9 published since the last AQCD, the reader is referred to previous documents (U.S. EPA, 

10 2006b, 1996a) for more detailed discussion of the past literature. Evaluation of the 

11 progress in the interpretation of past dosimetry studies, as well as studies published since 

12 2005, in the areas of uptake, reactions, and models for O3 dosimetry, is discussed.

13 Section 5.3 highlights findings of studies published since the 2006 O3 AQCD, which 

14 provide insight into the biological pathways by which O3 exerts its actions. Since 

15 common mechanisms lead to health effects from both short- and long-term exposure to 

16 O3, these pathways are discussed in Chapter 5 rather than in later chapters. The related 

17 sections of Chapters 6 and 7 are indicated. Earlier studies that represent the current state 

18 of the science are also discussed. Studies conducted at more environmentally-relevant 

19 concentrations of O3 are of greater interest, since mechanisms responsible for effects at 

20 low O3 concentrations may not be identical to those occurring at high O3 concentrations. 

21 Some studies at higher concentrations are included if they were early demonstrations of 

22 key mechanisms or if they are recent demonstrations of potentially important new 

23 mechanisms. The topics of dosimetry and mode of action are bridged by reactions of O3

24 with components of the extracellular lining fluid (ELF), which play a role in both O3

25 uptake and biological responses (Figure 5-1). 

26 In addition, this chapter discusses interindividual variability in responses, and issues 

27 related to species comparison of doses and responses (Section 5.4 and Section 5.5). These 

28 topics are included in this chapter because they are influenced by both dosimetric and 

29 mechanistic considerations.
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Figure 5-1 Schematic of the ozone exposure and response pathway. Ozone 

transport follows a path from exposure concentration, to inhaled 

dose, to net dose, to the local tissue dose. Chapter 5 discusses the 

concepts of dose and modes of action that result in the health 

effects discussed in Chapters 6 and 7.

5.2 Human and Animal Ozone Dosimetry

5.2.1 Introduction

1 Dosimetry refers to the measurement or estimation of the quantity of or rate at which a 

2 chemical and/or its reaction products are absorbed and retained at target sites. Figure 5-1

3 illustrates the transport of O3 or its reaction products from exposure to dose to the 

4 development of health effects. Ozone exposure has been defined in Section 4.2 and 

5 consists of contact between the human or animal and O3 at a specific concentration for a 

6 specified period of time (i.e., exposure = concentration × time). The amount of O3 present 

7 in a given volume of air for which animals and individuals are exposed is termed 

8 exposure concentration. Ozone exposure will result in some amount (dose) of O3 crossing 

9 an exposure surface to enter a target area. The initial measure of dose after O3 enters the 

10 RT is inhaled dose and is the amount or rate of O3 that crosses the outer RT surface 

before crossing the ELF and is effectively C×t×V̇ 11 E. Ozone may then cross from the gas 

12 phase across the ELF interface where net dose may be measured. Net dose is the amount 

13 or rate of entry of O3 across the gas/ELF interface. In modeling studies, the dose rate is 

14 often expressed as a flux per unit of surface area of a region of respiratory epithelium. 

15 Finally, O3 or its reaction products may reach the tissues and tissue dose of O3 can be 

16 reported. Tissue dose is the amount of O3 or its reaction products absorbed and available 

17 for reacting with tissues and is difficult and rarely measured. In the literature, the 

18 exposure concentration and various measures of dose (i.e., net dose and inhaled dose) are 

19 often used as surrogates for tissue dose. However, ambient or exposure concentrations are 

O3 exposure

Inhaled 

O3 dose

Net O3

dose

Tissue O3 dose and

product formation

Modes of Action Health Effects
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1 not a true measure of dose so understanding the relationship between ambient 

2 concentrations and tissue dose allows for a greater appreciation of the dose-response from 

3 O3 exposure.

Note: Structures are anterior nasal passages, ET1; oral airway and posterior nasal passages, ET2; bronchial airways, BB; 

bronchioles, bb; and alveolar interstitial, AI.

Source: Based on ICRP (1994).

Figure 5-2 Representation of respiratory tract regions in humans.

4 Ozone is a highly reactive, though poorly water soluble, gas at physiological temperature. 

5 The latter feature is believed to be the reason why it is able to penetrate into targets in the 

6 lower respiratory tract (LRT). Figure 5-2 presents the basic structure of the human 

7 respiratory tract (RT). The lung can be divided into three major regions: the extrathoracic 

8 (ET) region or upper respiratory tract (URT, from the nose/mouth to larynx); the 

9 tracheobronchial (TB) tree (from trachea to the terminal bronchioles); and the alveolar or 
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1 pulmonary region (from the respiratory bronchioles to the terminal alveolar sacs). The 

2 latter two regions comprise the LRT. Although the structure varies, the illustrated 

3 anatomic regions are common to all mammalian species with the exception of the 

4 respiratory bronchioles. Respiratory bronchioles, the transition region between ciliated 

5 and fully alveolated airways, are found in humans, dogs, ferrets, cats, and monkeys. 

6 Respiratory bronchioles are absent in rats and mice and abbreviated in hamsters, guinea 

7 pigs, sheep, and pigs. The branching structure of the ciliated bronchi and bronchioles also 

8 differs between species from being a rather symmetric and dichotomous branching 

9 network of airways in humans to a more monopodial branching network in other 

10 mammals.

11 Figure 5-3 illustrates the structure of the LRT with progression from the large airways in 

12 the TB region to the alveolus in the alveolar region. The fact that O3 is so chemically 

13 reactive has suggested to some that its tissue dose at the target sites exists in the form of 

14 oxidation products such as aldehydes and peroxides (see Section 5.2.3). Reaction 

15 products are formed when O3 interacts with components of the ELF such as lipids and 

16 antioxidants. The ELF varies throughout the length of the RT with the bronchial tree 

17 lined with a thin film of mucus and the alveolar region lined with a thinner layer of 

18 surfactant solution (Figure 5-3b). Ozone dose is directly related to the coupled diffusion 

19 and chemical reactions occurring in ELF, a process termed “reactive absorption.” Thus, 

20 the O3 dose depends on both the concentration of O3 as well as the availability of 

21 substrates within the ELF. 

22 Ozone dose is affected by complex interactions between a number of other major factors 

23 including RT morphology, breathing route, frequency, and volume, physicochemical 

24 properties of the gas, physical processes of gas transport, as well as the physical and 

25 chemical properties of the ELF and tissue layers (Figure 5-3c). The role of these 

26 processes varies throughout the length of the RT and as O3 moves from the gas to liquid 

27 compartments of the RT.
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Note: (a) Illustrates basic airway anatomy. Structures are epithelial cells, EP; basement membrane, BM; smooth muscle cells, SM; 

and fibrocartilaginous coat, FC. (b) Illustrates the relative amounts of liquid, tissue, and blood with distal progression. In the bronchi 

there is a thick surface lining over a relatively thick layer of tissues. With distal progress, the lining diminishes allowing increased 

access of compounds crossing the air-liquid interface to the tissues and the blood. (c) Presents the factors acting in the gas and 

liquid phases of O3 transport.

Source: Panel (a) reprinted with permission of McGraw-Hill (Weibel, 1980).

Figure 5-3 Structure of lower airways with progression from the large airways 

to the alveolus.

1 Two types of measurements have been used to arrive at the O3 dose to target sites during 

2 breathing: (1) measurement of removal of O3 from the air stream (termed “uptake”); and 

3 (2) measurement of chemical reactions in tissues or with biomolecules known to be 

4 present in tissues (termed “reactants”). The results of the above measurements have been 

5 incorporated into mathematical models for the purpose of explaining, predicting, and 

c. Transport Factors

Gas Phase

Convection

Diffusion

Dispersion

Liquid Phase

Solubility

Diffusion

Chemical Reaction

Convection

O3

transport
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1 extrapolating O3 dose in different exposure scenarios. Few new studies have investigated 

2 the uptake of O3 in the RT since the last O3 assessment (U.S. EPA, 2006b). The studies 

3 that have been conducted generally agree with the results presented in the past and do not 

4 change the dosimetry conclusions of the last document. 

5.2.2 Ozone Uptake 

5 Past AQCDs provide information on the majority of literature relevant to understanding 

6 the state of the science in O3 dosimetry. Measurements of O3 dose have been inferred 

7 from simultaneous measurements of airflow and O3 concentration at the airway opening 

8 of the nose or mouth (Nodelman and Ultman, 1999; Wiester et al., 1996a) as well as at 

9 internal sampling catheters (Gerrity et al., 1995; Gerrity et al., 1988). One method of 

10 quantifying O3 dose is to measure the amount of O3 removed from the air stream during 

11 breathing (termed “uptake”). The difference in the amount of O3 inhaled and exhaled 

12 relative to the amount of inhaled O3 is termed fractional absorption. Uptake efficiency is 

13 also reported and refers to the fraction of O3 absorbed in a region as a function of the total 

14 amount of O3 entering the given region. Uptake studies have utilized bolus and 

15 continuous O3 breathing techniques as well as modeling to investigate these measures of 

16 uptake and the distribution of O3 uptake between the upper and lower respiratory tract. A 

17 number of the studies that have measured the fractional absorption and uptake efficiency 

18 of O3 in the human RT, URT, and LRT are presented in Table 5-1. For bolus exposure 

19 studies that reported fractional absorption, the total RT uptake efficiency was estimated 

20 as the sum of the products of the experimental bolus absorption and incremental volume 

21 of a bolus into a breath divided by the tidal volume of the breath, or where available, was 

22 taken from Table 1A of Schlesinger et al. (1997).
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Table 5-1 Human respiratory tract uptake efficiency data

Reference Mouth/

Nosea Inspiratory Flow 

(mL/sec)

VT (mL) fB

(bpm)b Uptake Efficiency

URT, 

complete 

breath

URT, 

inspiration

LRT, 

complete 

breath

Total RT, tidal 

breath

Continuous Exposure

Gerrity et al. 

(1988)

OR 509 832 18 0.40 0.91

N 456 754 18 0.36 0.91

OR/N 500 800 18 0.43 0.91

OR/N 350 832 12 0.41 0.93

OR/N 634 778 24 0.38 0.89

Gerrity et al. 

(1994)

OR 1,360 1,650 25 0.37 0.43 0.81

OR 1,360 1,239 35 0.41 0.36 0.78

Gerrity et al. 

(1995)

OR 330 825 12 0.27 0.95 0.91

Wiester et al. 

(1996a)

OR 539 631 16 0.76

N 514 642 16 0.73

Rigas et al. (2000) Face 

mask

480 1,100 27.6 0.86

Santiago et al. 

(2001)

N 50 0.80c

N 250 0.33

Bolus Exposure

Hu et al. (1992) Mouthpiece250 500 0.46 0.88

Kabel et al. (1994) Mouthpiece250 500 0.50 0.88

Mouthpiece250 500 0.53 0.88

N 250 500 0.78 0.94

Hu et al. (1994) Mouthpiece150 500 0.65 0.91

Mouthpiece250 500 0.51 0.87

Mouthpiece500 500 0.26 0.82

Mouthpiece750 500 0.16 0.78

Mouthpiece1,000 500 0.11 0.76

Ultman et al. 

(1994)

Mouthpiece250 500d 15 0.30

Mouthpiece250 500 15 0.47
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5.2.2.1 Gas Transport Principles

1 The three-dimensional transport of O3 in the lumen of an airway is governed by diffusion 

2 and bulk flow or convection. When modeled as a one-dimensional process in which the 

3 radial profiles of axial velocity and O3 concentration profiles are flat, O3 transport along 

4 an airway lumen occurs by convection, axial diffusion and a coupled diffusion-reaction 

5 process called dispersion. Simultaneously, O3 diffuses into the ELF where it undergoes 

6 radial diffusion and chemical reaction (Figure 5-3c) (Miller, 1995). The relative 

7 importance of these transport mechanisms varies among RT regions for a given level of 

8 ventilation. In the URT and major bronchi, bulk airflow tends to be the predominant 

9 mechanism for axial transport in the airway lumen. However, in the alveolar region of the 

10 lung, diffusion is the major gas transport mechanism.

11 Gas transport in the TB region occurs by a combination of bulk flow and mixing 

12 (Ultman, 1985). Mixing can occur by diffusion processes associated with the molecular 

13 nature of the gas and by convection, which depends on local velocity patterns. The 

14 complexity of the airway structure and surface affects the bulk airflow patterns so that not 

15 all nasal and lung surfaces receive the same O3 exposure or dose (Miller and Kimbell,

16 1995). For example, it has been reported that the larger surface-to-volume ratio 

17 associated with the smaller airways in women enhances local O3 uptake and reduces the 

18 distal penetration volume of O3 into the RT of women relative to men (Ultman et al., 

19 2004). Also, it was reported that changes in cross-sectional area available for gas 

20 diffusion are related to overall O3 retention (Reeser et al., 2005; Ultman et al., 2004).

Reference Mouth/

Nosea Inspiratory 

Flow 

(mL/sec)

VT (mL) fB

(bpm)b Uptake Efficiency

URT, 

complete 

breath

URT, 

inspiration

LRT, 

complete 

breath

Total RT, tidal 

breath

Bush et al. (1996) Mouthpiece250 500 0.51 0.89

Nodelman and 

Ultman (1999)

Nasal 

Cannula

150 500 18 0.90 0.92

Nasal 

Cannula

1,000 500 120 0.50 0.84

Mouthpiece150 500 18 0.77 0.91

Mouthpiece1,000 500 120 0.25 0.75

Ultman et al. 

(2004)

OR 490 450d 32.7 0.87

OR 517 574 27 0.91

a

OR = oral exposure during spontaneous breathing; N = nasal exposure during spontaneous breathing; OR/N = pooled data from oral 

and nasal exposure; mouthpiece = exposure by mouthpiece. b

fB is either measured or is computed from flows and VT. c FURT from Santiago et al. (2001) represents nasal absorption (Fnose). d

VT is computed from flow and fB.
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1 The principal influence on mixing in the TB region comes from the axial velocity profile 

2 and diffusion. When air flows through an airway, O3 located near the tube center moves 

3 faster than O3 near the tube wall where frictional forces retard the flow. This 

4 non-uniformity in the radial profile of velocity gives rise to an axial spreading or 

5 dispersion of the O3 that operates in parallel with bulk flow and axial diffusion (a process 

6 caused by the ever-present Brownian motion of individual O3 molecules). The shape of 

7 the velocity profile is affected by the flow direction through bifurcating airway branches 

8 (Schroter and Sudlow, 1969). The velocity profile is nearly parabolic during inhalation 

9 but quite flat during exhalation. Thus, there tends to be greater axial dispersion during 

10 inhalation than during exhalation. Dispersion also depends on the nature of the flow, that 

11 is, whether it is laminar (i.e., streamlined) or turbulent (i.e., possessing random velocity 

12 fluctuations). Because turbulent flow flattens velocity profiles, it may actually diminish 

13 dispersion. In humans, turbulent flow persists only a few generations into the RT. The 

14 persistence of turbulence into the RT also varies by species and flow rates. For example, 

15 airflow is nonturbulent in the rat nose at any physiologic flow rate but may be highly 

16 turbulent in the human nose during exercise (Miller, 1995). Diffusive forces and 

17 resistance vary along the RT. Diffusive resistance increases with distal penetration into 

18 the RT with a study reporting that the gas boundary layer contributes 53% of the overall 

19 diffusive resistance in the URT, 78% in the proximal LRT, and 87% in the distal LRT 

20 (Hu et al., 1994). 

21 Conversely, the principal mechanism of gas mixing in the lung periphery is molecular 

22 diffusion (Engel, 1985). While moving into more distal areas of the RT, the 

23 cross-sectional area of the airways rapidly increases and linear velocities decrease, 

24 leading to a greater role for molecular diffusion of gases. Gas molecules close to the 

25 alveolar-capillary membrane have almost zero convective velocity with respect to the 

26 membrane, and this creates a substantial boundary layer resistance to O3 transfer across 

27 the gas-eLF interface. 

5.2.2.2 Target Sites for Ozone Dose

28 A primary uptake site of O3 delivery to the lung epithelium is believed to be the 

29 centriacinar region (CAR). The CAR refers to the zone at the junction of the TB airways 

30 and the gas exchange region. This area is also termed the proximal alveolar region (PAR) 

31 and is defined as the first generation distal to the terminal bronchioles. Contained within 

32 the CAR, the respiratory bronchioles were confirmed as the site receiving the greatest O3

dose (18 33 O mass/lung weight) in resting O3 exposed rhesus monkeys, when not considering 

34 the nose (Plopper et al., 1998). Furthermore, the greatest cellular injury occurred in the 

35 vicinity of the respiratory bronchioles and was dependent on the delivered O3 dose to 
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these tissues (see also Section 5.4.1). However, 18 1 O label was detected to a lesser extent 

2 in other regions of the TB airway tree, showing that O3 is delivered to these 

3 compartments as well, although in a smaller dose. These studies agree with earlier model 

4 predictions showing that the tissue O3 dose (O3 flux to liquid-tissue interface) was low in 

5 the trachea, increased to a maximum in the terminal bronchioles and the CAR, and then 

6 rapidly decreased in the alveolar region (Miller et al., 1985). It was also predicted that the 

7 net O3 dose (total absorption, O3 flux to air-liquid interface) gradually decreased with 

8 distal progression from the trachea to the end of the TB region and then rapidly decreased 

9 in the alveolar region. Despite the exclusion of the URT and appreciable O3 reactions 

10 with ELF constituents after the 16th generation, the results from the model agree with 

11 experimental results showing that the greatest O3 tissue dose was received in the CAR 

12 (Miller et al., 1985). 

13 Inhomogeneity in the RT structure may affect the dose delivered to this target site. 

14 Models have predicted that the farther the PAR is from the trachea, the less the O3 tissue 

15 dose to the region. Ultman and Anjilvel (1990) and Overton and Graham (1989)

16 predicted approximately a 50 to 300% greater PAR dose for the shortest path relative to 

17 the longest path in humans and rats, respectively. In addition, Mercer et al. (1991) found 

18 that both path distance and ventilatory unit size affected dose. The variation of O3 dose 

19 among anatomically equivalent ventilatory units was predicted to vary as much as 6-fold, 

20 as a function of path length from the trachea. This could have implications in regional 

21 damage to the LRT, such that even though the average LRT dose may be at a level where 

22 health effects would not be predicted, local regions of the RT may receive considerably 

23 higher than average doses and therefore be at greater risk of effects.

5.2.2.3 Upper Respiratory Tract Ozone Removal and Dose

24 Total O3 uptake in the entire RT in rats and guinea pigs ranges from 40-54% efficient 

25 (Hatch et al., 1989; Wiester et al., 1988; Wiester et al., 1987), while in humans at rest it 

26 ranges from 80-95% efficient (Hu et al., 1992). The URT provides a defense against O3

27 entering the lungs by removing half of the O3 that will be absorbed from the airstream. In 

28 both animals and humans, about 50% of the O3 that was absorbed in the RT was removed 

29 in the head (nose, mouth, and pharynx), about 7% in the larynx/trachea, and about 43% in 

30 the lungs (Hu et al., 1992; Hatch et al., 1989; Miller et al., 1979). However, experimental 

31 studies in dogs have reported 75-100% uptake in the URT (Yokoyama and Frank, 1972; 

32 Vaughan et al., 1969). The fraction of O3 taken up was inversely related to flow rate and 

33 to inlet O3 concentration (Yokoyama and Frank, 1972; Vaughan et al., 1969). The 

34 limiting factors in nasal O3 uptake were simultaneous diffusion and chemical reaction of 

35 O3 in the nasal ELF layer (Santiago et al., 2001). The ELF layer in the nose is thicker 
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1 than in the rest of the RT, and mathematical estimates predicted that O3 penetrates less 

2 than the thickness of the ELF layer; reaction products are likely the agents damaging the 

3 nasal tissue and not O3 itself. It was hypothesized that the nasal non-linear kinetics of O3

4 uptake fraction result from the depleting substrates in the nasal ELF becoming the 

5 limiting factor of the reaction (Santiago et al., 2001). 

6 Uptake efficiencies have been measured for various segments of the URT (Table 5-1). 

7 Gerrity et al. (1995) reported unidirectional uptake efficiencies of O3 inhaled from a 

8 mouthpiece; of 17.6% from the mouth to vocal cords, 9.5% from the vocal cords to the 

9 upper trachea (totaling 27.1%), 8.4% from the upper trachea to the main bifurcation 

10 carina (totaling 35.5%), and essentially zero between the carina and the bronchus 

11 intermedius (totaling 32.5%). These values are lower than those calculated by Hu et al. 

12 (1992) that reported uptake efficiencies of 21, 36, 44, and 46% during a complete breath 

13 in which an O3 bolus penetrated between the mouth and the vocal cords, the upper 

14 trachea, the main bifurcation carina, and the bronchus intermedius, respectively. The 

15 lower efficiencies seen in Gerrity et al. (1995) may have resulted because these 

16 investigators measurements were based on inhalation alone or was caused by O3

17 scrubbing by the mouthpiece. 

18 Past studies investigating nasal uptake of O3 have shown that the nose partially protects 

19 the LRT from damage from inspired O3 (Santiago et al., 2001; Gerrity et al., 1988). 

20 Sawyer et al. (2007) further investigated nasal uptake of O3 in healthy adults during 

21 exercise. Fractional O3 uptake, acoustic rhinometry (AR), and nasal NO measurements 

22 were taken on ten adults (8 women, 2 men) exposed to 200 ppb O3 before and after 

23 moderate exercise at two flow rates (10 and 20 L/min). The percent nasal uptake of O3

24 was ~50% greater at 10 L/min compared to 20 L/min both pre- and post-exercise. 

25 However, the inhaled O3 dose delivered to the LRT (i.e., flow rate × exposure 

26 concentration × (1 - nasal absorbed fraction)) was 1.6-fold greater at the higher flow than 

27 at the lower flow (2.5 compared to 0.9 ppm∙L/min). These results are similar to those 

28 published earlier that found air pollutant retention increased with increasing airflow by 

29 more than what would be predicted by just the increased partial pressure difference of the 

30 gas (Aharonson et al., 1974). Prior exercise did not affect O3 uptake at either flow rate, 

31 but did significantly increase nasal volume (Vn) and AR measurements of nasal 

32 cross-sectional area (minimum cross-sectional area (MCA) that corresponds to the nasal 

33 valve, CSA2 that corresponds to the anterior edge of the nasal turbinates, and CSA3 that 

34 corresponds to the posterior edge of the nasal turbinates) (p ≤ 0.05) (Sawyer et al., 2007). 

35 Conversely, exercise decreased nasal resistance (Rn) (p <0.01) and NO production 

36 (nonsignificant, p >0.05). The change in Vn and CSA2:MCA ratio was correlated with 

37 the percent change in nasal uptake, however the overall effect was small and sensitive to 

38 elimination of outliers and gender segregation. 
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1 Overall, the majority of studies suggest that the URT removes about half of the O3 that 

2 will be absorbed by reactions in the nasal ELF. The exact uptake efficiency will change 

3 due to variations in flow rate and inhaled concentration. 

5.2.2.4 Lower Respiratory Tract Ozone Uptake and Dose

4 Approximately 43% of the O3 absorption occurs in the LRT of both humans and animals. 

5 Models predicted that the net O3 dose decreases distally from the trachea toward the end 

6 of the TB region and then rapidly decreases in the alveolar region (Miller et al., 1985). 

7 Further, these models predicted low tissue O3 dose in the trachea and large bronchi. 

8 Uptake efficiency depends on a number of variables, including O3 exposure 

9 concentration, exposure time, and breathing pattern. For breaths of similar waveforms, 

10 respiratory patterns are uniquely described by breathing frequency (fB) and tidal volume 

(VT); by minute ventilation (V̇

E = fB x VT) and fB; or by V̇ 11 E and VT. Simulations from the 

12 Overton et al. (1996) single-path anatomical respiratory tract model, where the upper and 

13 lower respiratory tracts were modeled but uptake by the URT was not considered, 

14 predicted that fractional uptake and PAR O3 dose increased with VT when fB was held 

15 constant. Likewise, experimental studies found that O3 uptake was positively correlated 

16 with changes in VT (Ultman et al., 2004; Gerrity et al., 1988). Also, O3 exposure led to a 

17 reflex mediated increase in fB and reduction in VT, hypothesized to be protective by 

decreasing the dose delivered to the lung at a particular V̇ 18 E (Gerrity et al., 1994). Nasal O3

19 uptake efficiency was inversely proportional to flow rate (Santiago et al., 2001), so that 

an increase in V̇

E will increase O3 delivery to the lower airways. At a fixed V̇ 20 E, increasing 

21 VT (corresponding to decreasing fB) drove O3 deeper into the lungs and increased total 

22 respiratory uptake efficiency (Figure 5-4) (Ultman et al., 2004; Wiester et al., 1996a; 

23 Gerrity et al., 1988). Modeling predicted a decrease in fractional uptake with increased fB 

24 when VT was held constant, but an increase in PAR dose with increased fB (Overton et 

25 al., 1996). Similarly, increased fB (80 - 160 bpm) and shallow breathing in rats decreased 

midlevel tracheal 18O content and an increased 18 26 O content in the mainstem bronchi 

27 (Alfaro et al., 2004). This dependence may be a result of frequency-induced alterations in 

28 contact time that affects the first-order absorption rate for O3 (Postlethwait et al., 1994). 

Also, an association of O3 uptake efficiency was found with V̇ 29 E and exposure time. 

30 Increasing flow leads to deeper penetration of O3 into the lung, such that a smaller 

31 fraction of O3 is absorbed in the URT and uptake shifts to the TB airways and respiratory 

32 airspaces (Nodelman and Ultman, 1999; Hu et al., 1994; Ultman et al., 1994). Hu et al. 

33 (1994) and Ultman et al. (1994) found that O3 absorption increased with volumetric 

34 penetration (Vp) of a bolus of O3 into the RT. Ozone uptake efficiency and Vp were not 
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1 affected by bolus O3 concentration (Kabel et al., 1994; Hu et al., 1992), indicating that 

2 under these experimental conditions O3 uptake was a linear absorption process, where the 

3 diffusion and chemical reaction rates of O3 were proportional to the O3 concentration. 

4 The absorption relationship would not be linear once interfacial mass transfer is 

5 saturated. As mentioned above, a weak negative relationship between O3 concentration 

6 and uptake efficiency was reported for the nasal cavities by Santiago et al. (2001). Rigas 

7 et al. (2000) also found a weak but significant negative dependence of O3 concentration 

8 on RT uptake efficiency in exercising individuals. This study also found that exposure 

9 time had a small but significant influence on uptake efficiency; however, this negative 

10 dependence may be an artifact of progressive depletion of reactive substrates from the 

11 ELF.

Note: Subjects breathed 250 ppb O3 oronasally via a breathing mask. The uptake efficiency was well correlated with breathing 

frequency (r = -0.723, p <0.001) and tidal volume (not illustrated; r = 0.490, p <0.001).

Source: Reprinted with permission of Health Effects Institute (Ultman et al., 2004).

Figure 5-4 Total ozone uptake efficiency as a function of breathing frequency 

at a constant minute ventilation of 30 L/min.

12 Past studies have shown that O3-induced epithelial damage to the lung occurs with a 

13 reproducible pattern of severity between daughter branches of individual bifurcations that 

14 is dependent on the O3 concentration-time profile of the inhaled gas. A 3-D 

15 computational fluid dynamics model was created to investigate the O3 transport in a 
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1 single airway bifurcation (Taylor et al., 2007). The model consisted of one parent branch 

2 and two symmetrical daughter branches with a branching angle of 90° and a sharp carinal 

3 ridge. Various flow scenarios were simulated using Reynolds numbers (Re) ranging from 

4 100 to 500. The Re that corresponds to a certain airway generation is dependent upon 

both lung size and V̇ 5 E, such that the range in Re from 100-500 would encompass 

6 generations 1-5, 3-7, and 6-10 for an adult during quiet breathing, light exertion, and 

7 heavy exercise, respectively, whereas the same Re range corresponds to generations 0-4, 

8 1-6, and 4-8 for a 4-year-old child. This model predicted velocity distributions that were 

9 consistent with earlier work of Schroter and Sudlow (1969), and also reported O3

10 concentration and wall uptake distributions. The model predicted that during inspiration, 

11 the velocity and O3 concentration distribution were axisymmetric throughout the parent 

12 branch, but skewed towards the inner wall within the daughter branches. During 

13 expiration, the model predicted that the velocity and O3 concentration distribution was 

14 slightly skewed towards the outer walls of the daughter branches. Hot spots of wall flux 

15 existed at the carina during inspiration and expiration with Re >100. Additional hot spots 

16 were found during expiration on the parent branch wall downstream of the branching 

17 region. 

18 Overall O3 inhalation uptake in humans is over 80% efficient, but the exact efficiency 

19 that determines how much O3 is available at longitudinally distributed compartments in 

20 the lung is sensitive to changes in VT, fB, and to a minor extent, exposure time.

5.2.2.5 Mode of Breathing

21 Ozone uptake and distribution is sensitive to the mode of breathing. Variability in TB 

22 airways volume had a weaker influence on O3 absorption during nasal breathing 

23 compared to oral breathing. This could be a result of O3 scrubbing in the nasal 

24 passageways that are bypassed by oral breathing. Studies by Ultman and colleagues using 

25 bolus inhalation demonstrated that O3 uptake fraction was greater during nasal breathing 

26 than during oral breathing at each Vp (e.g., 0.90 during nasal breathing and 0.80 during 

27 oral breathing at 150 mL/sec and 0.45 during nasal breathing and 0.25 during oral 

28 breathing at 1,000 mL/sec) (Nodelman and Ultman, 1999; Kabel et al., 1994; Ultman et 

29 al., 1994). Therefore, oral breathing results in deeper penetration of O3 into the RT with a 

30 higher absorbed fraction in the TB and alveolar airways (Nodelman and Ultman, 1999). 

31 Similar results were obtained from O3 uptake studies in dogs (Yokoyama and Frank, 

32 1972). Earlier human studies suggested that oral or oronasal breathing results in a higher 

33 O3 uptake efficiency than nasal breathing (Wiester et al., 1996a; Gerrity et al., 1988). 

34 Overall, the mode of breathing may have a seemingly small effect on the RT uptake 
Draft – Do Not Cite or Quote 5-15 June 2012

1 efficiency; however, it does play an important role in the distribution of O3 deposited in 

2 the distal airways.

5.2.2.6 Interindividual Variability in Dose

3 Similarly exposed individuals vary in the amount of actual dose delivered to the LRT 

4 (Santiago et al., 2001; Rigas et al., 2000; Bush et al., 1996). Interindividual variability 

5 accounted for between 10-50% of the absolute variability in O3 uptake measurements 

(Santiago et al., 2001; Rigas et al., 2000). When concentration, time, and V̇ 6 E were held 

7 constant, fractional absorption ranged from 0.80 to 0.91 (Rigas et al., 2000). It has been 

8 hypothesized that interindividual variation in O3 induced responses such as FEV1 is the 

9 result of interindividual variation in net dose or regional O3 uptake among exposed 

10 individuals. 

11 Recent studies have reiterated the importance of intersubject variation in O3 uptake. The 

12 intersubject variability in nasal O3 uptake determined by Sawyer et al. (2007) ranged 

13 from 26.8 to 65.4% (pre- and post-exercise). A second study investigating the use of the 

14 CO2 expirogram to quantify pulmonary responses to O3 found that intersubject variability 

15 accounted for 50% of the overall variance in the study (Taylor et al., 2006). 

16 Variability in net or tissue dose may be attributed to differences in the pulmonary 

17 physiology, anatomy, and biochemistry. Since the URT and TB airways remove the 

18 majority of inhaled O3 before it reaches the gas exchange region, the volume and surface 

19 area of these airways will influence O3 uptake. Models predicted that fractional O3 uptake 

20 and PAR dose (flux of O3 to the PAR surfaces divided by exposure concentration) 

21 increase with decreasing TB volume and decreasing TB region expansion. On the 

22 contrary, alveolar expansion had minimal effect on uptake efficiency as relatively little 

23 O3 reaches the peripheral lung (Bush et al., 2001; Overton et al., 1996). Ozone uptake 

24 was virtually complete by the time O3 reaches the alveolar spaces of the lung 

25 (Postlethwait et al., 1994). Experimental studies have found that differences in TB 

26 volumes may account for 75% of the variation in absorption between subjects (Ultman et 

27 al., 2004). In support of this concept, regression analysis showed that O3 absorption was 

28 positively correlated with anatomical dead space (VD) and TB volume (i.e., VD minus 

29 VURT), but not total lung capacity (TLC), forced vital capacity (FVC), or functional 

30 residual capacity (FRC) (Ultman et al., 2004; Bush et al., 1996; Hu et al., 1994; 

31 Postlethwait et al., 1994). Variability in VD was correlated more with the variability in the 

32 TB volume than the URT volume. Similarly, uptake was correlated with changes in 

33 individual bronchial cross-sectional area, indicating that changes in cross-sectional area 

34 available for gas diffusion are related to overall O3 retention (Reeser et al., 2005; Ultman 
Draft – Do Not Cite or Quote 5-16 June 2012

1 et al., 2004). When coupled, these results suggest that the larger surface-to-volume ratio 

2 associated with the smaller airways in women enhances local O3 uptake, thereby reducing 

3 the distal penetration volume of O3 into the female respiratory system. When absorption 

4 data were normalized to Vp/VD, variability attributed to gender differences were not 

5 distinguishable (Bush et al., 1996). These studies provide support to the RT anatomy, 

6 especially the TB volume and surface area, playing a key role in variability of O3 uptake 

7 between individuals. 

8 In addition, variability between individuals is influenced by age. Overton and Graham 

9 (1989) predicted that the total mass of O3 absorbed per minute (in units of: μg/min per 

[μg/m3 10 of ambient O3]) increased with age from birth to adulthood. This model predicted 

11 that during quiet breathing the LRT distribution of absorbed O3 and the CAR O3 tissue 

12 dose were not sensitive to age. However, during heavy exercise or work O3 uptake was 

13 dependent on age. A physiologically based pharmacokinetic model simulating O3 uptake 

14 predicted that regional extraction of O3 was relatively insensitive to age, but extraction 

15 per unit surface area was 2-fold to 8-fold higher in infants compared to adults, due to the 

16 fact that children under age 5 have much a much smaller airway surface area in the 

17 extrathoracic (nasal) and alveolar regions (Sarangapani et al., 2003). Additionally, 

18 children tend to have a greater oral breathing contribution than adults at rest and during 

19 exercise (Bennett et al., 2008; Becquemin et al., 1999; James et al., 1997). Normalized to 

20 lung surface area, the dose rate to the lower airways of children compared to adults is 

21 increased further because children breathe at higher minute ventilations relative to their 

22 lung volumes.

23 Smoking history, with its known increase in mucus production, was not found to affect 

24 the fractional uptake of a bolus of O3 in apparently healthy smokers with limited smoking 

25 history (Bates et al., 2009). Despite similar internal O3 dose distribution, the smokers 

26 exhibited greater pulmonary responses to O3 bolus exposures, measured as FEV1

27 decrements and increases in the normalized slope of the alveolar plateau (SN). This was 

28 contrary to previous studies conducted in smokers with a greater smoking history that 

29 found decreased O3 induced decrements in FEV1 in smokers during continuous O3

30 exposure (Frampton et al., 1997a; Emmons and Foster, 1991). 

5.2.2.7 Physical Activity

31 Exercise increases the overall exposure of the lung to inhaled contaminants due, in most 

32 part, to the increased intake of air. Thus, human studies have used exercise, at a variety of 

33 activity levels, to enhance the effects of O3 (Table 5-2). Further explanation of the effects 

34 of physical activity on ventilation can be found in Chapters 4 and 6. Table 4-5 presents 
Draft – Do Not Cite or Quote 5-17 June 2012

1 the mean ventilation rates at different activity levels for different age groups. Table 6-1

2 provides activity levels as detailed in specific human exposure studies.

Table 5-2 General adult human inhalation rates by activity levels.

3 As exercise increases from a light to moderate level, VT increases. This increase in VT is 

4 achieved by encroaching upon both the inspiratory and expiratory reserve volumes of the 

5 lung (Dempsey et al., 1990). After VT reaches about 50% of the vital capacity, generally 

6 during heavy exercise, further increases in ventilation are achieved by increasing fB. 

7 Ventilatory demands of very heavy exercise require airway flow rates that often exceed 

8 10 times resting levels and VT that approach 5 times resting levels (Dempsey et al., 

9 2008).

10 This increase in VT and flow associated with exercise in humans shifts the net O3 dose 

11 further into the periphery of the RT causing a disproportionate increase in distal lung 

12 tissue dose. In addition to increasing the bulk transport of O3 into the lung, exercise also 

13 leads to a switch from nasal to oronasal breathing. Higher ventilatory demand 

14 necessitates a lower-resistance path through the mouth. Modeling heavy exercise by 

15 increasing ventilatory parameters from normal respiration levels predicted a 10-fold 

16 increase in total mass uptake of O3 (Miller et al., 1985). This model also predicted that as 

17 exercise and ventilatory demand increased, the maximum tissue dose, the O3 reaching the 

18 tissues, moved distally into the RT (Figure 5-5). By increasing flow to what is common 

19 in moderate or heavy exercise (respiratory flow = 45-60 L/min compared to 15 L/min), 

20 the URT absorbed a smaller fraction of the O3 (~0.50 at low flow rate to 0.10 at high flow 

21 rate); however, the trachea and more distal TB airways received higher doses during 

22 higher flow rates than at lower flow rates (0.65 absorbed in the lower TB airways, and 

23 0.25 absorbed in the alveolar zone with high flow compared to 0.5 in the TB with almost 

24 no O3 reaching the alveolar zone at low flow) (Hu et al., 1994). The same shift in the O3

25 dose distribution more distally in the lung occurred in other studies mimicking the effects 

26 of exercise (Nodelman and Ultman, 1999). Also, LRT uptake efficiency was sensitive to 

27 age only under exercise conditions (Overton and Graham, 1989). The total mass of O3

Activity Level Inhalation Rate

Light 2 to 3 × resting V̇

E

a

Moderate 4 to 6 × resting V̇

E

Heavy 7 to 8 × resting V̇

E

Very Heavy >9 × resting V̇

E

a

Resting V̇

E approximates 8 L/min

Source: U.S. EPA (1986).
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absorbed per minute (μg/min per [μg/m3 1 of ambient O3]) was predicted to increase with 

2 age during heavy work or exercise. A recent study by Sawyer et al. (2007) approximated 

3 that doubling minute ventilation led to only a 1.6-fold higher delivered dose rate of O3 to 

4 the lung (delivered dose was calculated as: flow rate × [O3 ppm] × (100-percent nasal O3

5 uptake)). Past models have predicted the increase in uptake during exercise is distributed 

6 unevenly in the RT compartments and regions. Tissue and mucus layer dose in the TB 

7 region increased ~1.4-fold during heavy exercise compared to resting conditions, whereas 

8 the alveolar region surfactant and tissue uptake increased by factors of 5.2 and 13.6, 

9 respectively (Miller et al., 1985). 

Note: Curve 1: VT = 500 mL; fB = 15 breaths/min. Curve 2: VT = 1,000 mL; fB = 15 breaths/min. Curve 3: VT = 1,750 mL; fB = 20.3 

breaths/min. Curve 4: VT = 2,250 mL; fB = 30 breaths/min. TB = tracheobronchial region; P = pulmonary region.

Source: Reprinted with permission of Elsevier (Miller et al., 1985).

Figure 5-5 Modeled effect of exercise on tissue dose of the LRT.
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5.2.2.8 Summary

1 In summary, O3 uptake is affected by complex interactions between a number of factors 

2 including RT morphology, breathing route, frequency, and volume, physicochemical 

3 properties of the gas, physical processes of gas transport, as well as the physical and 

4 chemical properties of the ELF and tissue layers. The role of these processes varies 

5 throughout the length of the RT and as O3 moves from the gas into liquid compartments 

6 of the RT. The primary uptake site of O3 delivery to the lung epithelium is believed to be

7 the CAR, however inhomogeneity in the RT structure may affect the dose delivered to 

8 this target site with larger path lengths leading to smaller locally delivered doses. This 

9 could have implications in regional damage to the LRT, such that even though the

10 average LRT dose may be at a level where health effects would not be predicted , local 

11 regions of the RT may receive considerably higher than average doses and therefore be at 

12 greater risk of effects. Recent studies have provided evidence for hot spots of O3 flux 

13 around bifurcations in airways. Experimental studies and models have suggested that the 

14 net O3 dose gradually decreases distally from the trachea toward the end of the TB region 

15 and then rapidly decreases in the alveolar region. However, the tissue O3 dose is low in 

16 the trachea, increases to a maximum in the terminal bronchioles and the CAR, and then 

17 rapidly decreases distally into the alveolar region. 

18 O3 uptake efficiency is sensitive to a number of factors. Fractional absorption will 

decrease with increased flow and increase proportional to VT, so that at a fixed V̇ 19 E, 

20 increasing VT (or decreasing fB) drives O3 deeper into the lungs and increases total 

21 respiratory uptake efficiency. Individual total airway O3 uptake efficiency is also 

sensitive to large changes in O3 concentration, exposure time, and V̇ 22 E. Major sources of 

variability in absorption of O3 include O3 concentration, exposure time, fB, V̇ 23 E, and VT, 

24 but the interindividual variation is the greatest source of variability uptake efficiency. The 

25 majority of this interindividual variability is due to differences in TB volume and surface 

26 area.

27 An increase in VT and fB are both associated with increased physical activity. These 

28 changes and a switch to oronasal breathing during exercise results in deeper penetration 

29 of O3 into the lung with a higher absorbed fraction in the ET, TB, and alveolar airways. 

30 For these reasons, increased physical activity acts to move the maximum tissue dose of 

31 O3 distally into the RT and into the alveolar region. 
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5.2.3 Ozone Reactions and Reaction Products

1 Ozone dose is affected by the chemical reactions or the products of these reactions that 

2 result from O3 exposure. The process by which O3 moves from the airway lumen and into 

3 the ELF is related to the coupled diffusion and chemical reactions occurring in ELF is 

4 called “reactive absorption”. Ozone is chemically reactive with a wide spectrum of 

5 biomolecules and numerous studies have evaluated the loss of specific molecules such as 

6 GSH and the appearance of plausible products such as nonanal. Both in vitro and in vivo 

7 studies contribute to the understanding of O3 reactions and reaction products.

8 Ozone may interact with many of the components in the ELF including phospholipids, 

9 neutral lipids like cholesterol, free fatty acids, proteins, and low molecular weight 

10 antioxidants as has been demonstrated in in vitro studies (Perez-Gil, 2008; Uppu et al., 

11 1995). It was estimated that 88% of the O3 that does not come in contact with 

12 antioxidants will react with unsaturated fatty acids in the ELF including those contained 

13 within phospholipids or neutral lipids (Uppu et al., 1995). Ozone reacts with the double 

14 bond of unsaturated fatty acids to form stable and less reactive ozonide, aldehyde, and 

15 hydroperoxide reaction products via chemical reactions such as the Criegee ozonolysis 

16 mechanism (Figure 5-6) (Pryor et al., 1991). Lipid ozonation products, such as the 

17 aldehydes hexanal, heptanal, and nonanal, have been recovered after O3 exposure in 

18 human BAL fluid (BALF), rat BALF, isolated rat lung, and in vitro systems (Frampton et 

19 al., 1999; Postlethwait et al., 1998; Pryor et al., 1996). Adducts of the aldehyde 

20 4-hydroxynonenal were found in human alveolar macrophages after O3 exposure in vivo

21 (Hamilton et al., 1998). Polyunsaturated fatty acid (PUFA) reactions are limited by the 

22 availability of O3 since lipids are so abundant in the ELF. Yields of O3-induced aldehydes 

23 were increased by the decrease in other substrates such as ascorbic acid (AH2) 

24 (Postlethwait et al., 1998). Free radicals are also generated during O3-mediated oxidation 

25 reactions with PUFA (Pryor, 1994). These reactions are reduced by the presence of the 

26 lipid-soluble free radical scavenger α-tocopherol (α-TOH) (Pryor, 1994; Fujita et al., 

27 1987; Pryor, 1976). PUFA reactions may not generate sufficient bioactive materials to 

28 account for acute cell injury, however only modest amounts of products may be 

29 necessary to induce cytotoxicity (Postlethwait and Ultman, 2001; Postlethwait et al., 

30 1998). 
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Note: Not all secondary reaction products are shown.

Source: U.S. EPA (2006b).

Figure 5-6 Schematic overview of ozone interaction with PUFA in ELF and 

lung cells.

1 Cholesterol is the most abundant neutral lipid in human ELF. Reaction of cholesterol 

2 with O3 results in biologically active cholesterol products such as the oxysterols, 

3 β-epoxide and 6-oxo-3,5-diol (Murphy and Johnson, 2008; Pulfer et al., 2005; Pulfer and 

4 Murphy, 2004). Product yields depend on ozonolysis conditions, however cholesterol 

5 ozonolysis products form in similar abundance to phospholipid-derived ozonolysis 

6 products in rat ELF (Pulfer and Murphy, 2004). 

7 The ELF also contains proteins derived from blood plasma as well as proteins secreted by 

8 surface epithelial cells. Ozone reactions with proteins have been studied by their in vitro 

9 reactions as well as reactions of their constituent amino acids (the most reactive of which 

10 are cysteine, histidine, methionine, tyrosine, and tryptophan). Ozone preferentially reacts 

11 with biomolecules in the following order: thiosulfate >ascorbate >cysteine ≈ methionine 

12 >glutathione (Kanofsky and Sima, 1995). Rate constants for the reaction of amino acids 

13 with O3 vary between studies due to differing reaction conditions and assumptions; 

14 however aliphatic amino acids were consistently very slow to react with O3 (e.g., alanine: 

15 25-100 moles/L/sec) (Kanofsky and Sima, 1995; Ignatenko and Cherenkevich, 1985; 

16 Pryor et al., 1984; Hoigné and Bader, 1983). Uppu et al. (1995) predicted that 12% of 

17 inhaled O3 that does not react with antioxidants will react with proteins in the ELF.

18 Reactions of O3 with low molecular weight antioxidants have been extensively studied. 

19 The consumption of antioxidants such as uric acid (UA), ascorbate (AH2), and reduced 

20 glutathione (GSH) by O3 was linear with time and positively correlated with initial 

21 substrate concentration and O3 concentration (Mudway and Kelly, 1998; Mudway et al., 

22 1996). Endogenous antioxidants are present in relatively high concentrations in the ELF 

23 of the human airways (obtained as BALF) and display high (but not equal) intrinsic 
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1 reactivities toward O3. In individual and in limited composite mixtures, UA was the most 

2 reactive antioxidant tested, followed by AH2 (Mudway and Kelly, 1998). GSH was 

3 consistently less reactive than UA or AH2 (Mudway and Kelly, 1998; Mudway et al., 

4 1996; Kanofsky and Sima, 1995). To quantify these reactions, Kermani et al. (2006)

5 evaluated the interfacial exposure of aqueous solutions of UA, AH2, and GSH 

6 (50-200 μM) with O3 (1-5 ppm). Similar to the results of Mudway and Kelly (1998), this 

7 study found the hierarchy in reactivity between O3 and these antioxidants to be 

8 UA≥ AH2>>GSH. UA and AH2 shared a 1:1 stoichiometry with O3, whereas 2.5 moles of 

9 GSH were consumed per mole of O3. Using these stoichiometries, reaction rate constants 

were derived (5.8×104 M-1 sec-1, 5.5×104 M-1 sec-1, and 57.5 M-0.75 sec-1 [20.9 M-1 sec-1 10 ] for 

11 the reaction of O3 with UA, AH2, and GSH, respectively). Other studies report reactive 

12 rate constants that are two to three orders of magnitude larger, however these studies used 

13 higher concentrations of O3 and antioxidants under less physiologically relevant 

14 experimental conditions (Kanofsky and Sima, 1995; Giamalva et al., 1985; Pryor et al., 

15 1984). However, O3 acts through competition kinetics so the effective concentration of 

16 the reactants present in the ELF will determine the reactions that occur in vivo. For 

17 example, the pKa of GSH is about 8.7 so that at physiological pH very little is in the 

reactive form of thiolate (GS- 18 ). On the other hand, ascorbic acid has a pKa of about 4.2 so 

it exists almost entirely as ascorbate (AH- 19 ) in the ELF. Thus, the effective concentration 

20 of GSH that is available to react with O3 will be much lower than that of ascorbate in 

21 ELF. 

22 A series of studies used new techniques to investigate the reaction products resulting 

23 from initial air-liquid interface interactions of O3 with ELF components 

24 (e.g., antioxidants and proteins) in ~1 millisecond (Enami et al., 2009a, b, c, 2008a, b). 

25 Solutions of aqueous UA, AH2, GSH, α-TOH, and protein cysteines (CyS) were sprayed 

26 as microdroplets in O3/N2 mixtures at atmospheric pressure and analyzed by electrospray 

27 mass spectrometry. These recent studies demonstrated different reactivity toward AH2, 

28 UA, and GSH by O3 when the large surface to volume ratio of microdroplets promote an 

29 interfacial reaction compared to previous studies using bulk liquid phase bioreactors. This 

30 artificial system does not recapitulate the lung surface so caution must be taken in 

31 translating the results of these studies to in vivo conditions.

32 As was seen in previous studies (Kermani et al., 2006; Kanofsky and Sima, 1995), the 

33 hierarchy of reactivity of these ELF components with O3 was determined to be AH2 ≈ UA 

34 > CyS >GSH. There was some variance between the reaction rates and product formation 

35 of UA, AH2, and GSH with O3 as investigated by Enami et al. versus O3 reacting with 

36 bulk liquid phase bioreactors as described previously. UA was more reactive than AH2

37 toward O3 in previous studies, but in reactions with O3 with microdroplets, these 

38 antioxidants had equivalent reactivity (Enami et al., 2008b). As O3 is a kinetically slow 
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1 one-electron acceptor but very reactive O-atom donor, products of the interaction of O3

2 with UA, AH2, GSH, CyS, and α-TOH result from addition of n O-atoms (n = 1-4). These 

products included epoxides (e.g., U-O-), peroxides (e.g., U-O2

- 3 ), and ozonides 

(e.g., U-O3

-

). For instance, GSH was oxidized to sulfonates (GSO3

-

/GSO3

2- 4 ), not 

5 glutathione disulfide (GSSG) by O3 (Enami et al., 2009b). However, it is possible that 

6 other oxidative species are oxidizing GSH in vivo, since sulfonates are not detected in O3

7 exposed ELF whereas GSSG is. This is also supported by the fact that O3 is much less 

8 reactive with GSH than other antioxidants, such that <3% of O3 will be scavenged by 

9 GSH when in equimolar amounts with AH2 (Enami et al., 2009b). 

10 This series of studies also demonstrated that ozonolysis product yields and formation 

11 were affected by pH. Acidified conditions (pH ≈ 3-4), such as those that may result from 

12 acidic particulate exposure or pathological conditions like asthma (pH ≈ 6), decreased the 

13 scavenging ability of UA and GSH for O3; such that at low pH, the scavenging of O3

14 must be taken over by other antioxidants, such as AH2 (Enami et al., 2009b, 2008b). 

15 Also, under acidic conditions (pH ≈ 5), the ozonolysis products of AH2 shifted from the 

16 innocuous dehydroascorbic acid to the more persistent products, AH2 ozonide and 

17 threonic acid (Enami et al., 2008a). It is possible that the acidification of the ELF by 

18 acidic copollutant exposure will increase the toxicity of O3 by preventing some 

19 antioxidant reactions and shifting the reaction products to more persistent compounds.

20 Since ELF exists as a complex mixture, it is important to look at O3 reactivity in substrate 

21 mixtures. Individual antioxidant consumption rates decreased as the substrate mixture 

22 complexity increased (e.g., antioxidant mixtures and albumin addition) (Mudway and 

23 Kelly, 1998). However, O3 reactions with AH2 predominated over the reaction with 

24 lipids, when exposed to substrate solution mixtures (Postlethwait et al., 1998). It was 

25 suggested that O3 may react with other substrates once AH2 concentrations within the 

26 reaction plane fall sufficiently. Additionally, once AH2 was consumed, the absorption 

27 efficiency diminished, allowing inhaled O3 to be distributed to more distal airways 

28 (Postlethwait et al., 1998). Multiple studies have concluded O3 is more reactive with AH2

29 and UA than with the weakly reacting GSH (or cysteine or methionine) or with amino 

30 acid residues and protein thiols (Kanofsky and Sima, 1995; Cross et al., 1992). 

31 In a red blood cell (RBC) based system, AH2 augmented the in vitro uptake of O3 by 

32 6-fold, as computed by the mass balance across the exposure chamber (Ballinger et al., 

33 2005). However, estimated in vitro O3 uptake was not proportional to the production of 

34 O3-derived aldehydes from exposing O3 to RBC membranes (Ballinger et al., 2005). In 

35 addition, O3 induced cell membrane oxidation that required interactions with AH2 and 

36 GSH, but not UA or the vitamin E analog Trolox. Further, aqueous phase reactions 

37 between O3 and bovine serum albumin did not result in membrane oxidation (Ballinger et 
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1 al., 2005). The presence of UA or bovine serum albumin protected against lipid and 

2 protein oxidation resulting from the reaction of O3 and AH2 (Ballinger et al., 2005). This 

3 study provided evidence that antioxidants may paradoxically facilitate O3-mediated 

4 damage. This apparent contradiction should be viewed in terms of the 

5 concentration-dependent role of the ELF antioxidants. Reactions between O3 and 

6 antioxidant species exhibited a biphasic concentration response, with oxidation of protein 

7 and lipid occurring at lower, but not higher, concentrations of antioxidant. In this way, 

8 endogenous reactants led to the formation of secondary oxidation products that were 

9 injurious and also led to quenching reactions that were protective. Moreover, the 

10 formation of secondary oxidation products mediated by some antioxidants was opposed 

11 by quenching reactions involving other antioxidants. 

12 Alterations in ELF composition can result in alterations in O3 uptake. Bolus O3 uptake in 

13 human subjects can be decreased by previous continuous O3 exposure (120-360 ppb), 

14 possibly due to depletion of compounds able to react with O3 (Rigas et al., 1997; Asplund 

15 et al., 1996). Conversely, O3 (360 ppb) bolus uptake was increased with prior NO2

16 (360-720 ppb) or SO2 (360 ppb) exposure (Rigas et al., 1997). It was hypothesized that 

17 this increased fractional absorption of O3 could be due to increased production of reactive 

18 substrates in the ELF due to oxidant-induced airway inflammation. 

19 Besides AH2, GSH and UA, the ELF contains numerous antioxidant substances that 

20 appear to be an important cellular defense against O3 including α-TOH, albumin, 

21 ceruloplasmin, lactoferrin, mucins, and transferrin (Mudway et al., 2006; Freed et al., 

22 1999). The level and type of antioxidant present in ELF varies between species, regions 

23 of the RT, and can be altered by O3 exposure. Mechanisms underlying the regional 

24 variability are not well-understood. It is thought that both plasma ultrafiltrate and locally 

25 secreted substances contribute to the antioxidant content of the ELF (Mudway et al., 

26 2006; Freed et al., 1999). In the case of UA, the major source appears to be the plasma 

27 (Peden et al., 1995). Repletion of UA in nasal lavage fluid was demonstrated during 

28 sequential nasal lavage in human subjects (Mudway et al., 1999a). When these subjects, 

29 exercising at a moderate level, were exposed to 200 ppb O3 for 2 hours , nasal lavage 

30 fluid UA was significantly decreased while plasma UA levels were significantly 

31 increased (Mudway et al., 1999a). The finding that UA, but not AH2 or GSH, was 

32 depleted in nasal lavage fluid indicated that UA was the predominant antioxidant with 

33 respect to O3 reactivity in the nasal cavity (Mudway et al., 1999a). However, in human 

34 BALF samples, the mean consumption of AH2 was greater than UA (Mudway et al., 

35 1996). In addition, concentrations of UA were increased by cholinergic stimulation of the 

36 airways in human subjects, which suggested that increased mucosal gland secretions were 

37 an important source (Peden et al., 1993). Using the O3-specific antioxidant capacity assay 

38 on human nasal lavage samples, Rutkowski et al. (2011) concluded that about 30% of the 
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1 antioxidant capacity of the nasal liquid lining layer was attributed to UA activity. 

2 Additionally, more than 50% of the subject-to-subject differences in antioxidant capacity 

3 were driven by differences in UA concentration. However, day-to-day within-subject 

4 variations in measured antioxidant capacity were not related to the corresponding 

5 variations in UA concentration in the nasal lavage fluid. Efforts to identify the 

6 predominant antioxidant(s) in other RT regions besides the nasal cavity have failed to 

7 yield definitive results. 

8 Regulation of AH2, GSH and α-TOH concentrations within the ELF is less clear than that 

9 of UA (Mudway et al., 2006). In a sequential nasal lavage study in humans, wash-out of 

10 AH2 and GSH occurred, indicating the absence of rapidly acting repletion mechanisms 

11 (Mudway et al., 1999a). Other studies demonstrated increases in BALF GSH and 

12 decreases in BALF and plasma AH2 levels several hours following O3 exposure (200 ppb 

13 for 2 h, while exercising at a moderate level) (Mudway et al., 2001; Blomberg et al., 

14 1999; Mudway et al., 1999b). Other investigators have demonstrated cellular uptake of 

15 oxidized AH2 by several cell types leading to intracellular reduction and export of 

16 reduced AH2 (Welch et al., 1995). Studies with rats exposed to 0.4-1.1 ppm O3 for 

17 1-6 hours have shown consumption of AH2 that correlates with O3 exposure (Gunnison 

18 and Hatch, 1999; Gunnison et al., 1996; Vincent et al., 1996b).

19 A body of evidence suggests that reaction of O3 within the ELF limits its diffusive 

20 transport through the ELF; direct contact of O3 with the apical membranes of the 

21 underlying epithelial cells therefore might be negligible (Ballinger et al., 2005; Connor et 

22 al., 2004; Postlethwait and Ultman, 2001; Pryor, 1992). This conclusion is based on 

23 computational analyses and in vitro studies. Direct confirmation using in vivo studies is 

24 lacking. Nevertheless, when predicting exposure-related outcomes across species and 

25 anatomic sites, whether O3 directly contacts the apical membranes of the epithelial cells 

26 is an important consideration, given that the extracellular surface milieu of the RT 

27 appreciably varies in terms of the types and concentrations of the substrates present and 

28 the thickness of the ELF. 

29 For O3 or its reaction products to gain access to the underlying cellular compartments, O3

30 must diffuse at the air-liquid interface of the airway surface and travel through the ELF 

31 layer. In vitro experiments have shown that O3 disappearance from the gas phase depends 

32 on the characteristics of the ELF substrates (Postlethwait et al., 1998; Hu et al., 1994). 

33 The ELF is comprised of the airway surface lining that includes the periciliary sol layer 

34 and overlying mucus gel layer, and the alveolar surface lining that includes the subphase 

35 of liquid and vesicular surfactant and the continuous surfactant monolayer (Bastacky et 

36 al., 1995). There is a progressive decrease in ELF thickness and increase in interfacial 

37 surface with progression from the TB region to the alveolus (Mercer et al., 1992). The 
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1 progressive thinning of the ELF while moving further down the RT decreases the radial 

2 distance O3 or its reaction products must travel to reach the cells lining the RT. 

3 Taking into account the high reactivity and low water solubility of O3, calculations 

4 suggest that O3 will not penetrate ELF layers greater than 0.1 μm without being 

5 transformed to other more long-lived reactive species, thus initiating a reaction cascade 

6 (Pryor, 1992). These calculations utilize the Einstein-Smoluchowski equation (Equation 

7 5-1) that combines Fick’s second law of diffusion and a stochastic view of motion to 

8 compare the half-life of O3 in the ELF layer to the time it takes, t, for O3 to travel a 

distance, d, with a diffusion coefficient of D (~2×10-5 cm2 9 /sec). 

t = d2/2D

Equation 5-1

The transit time through an ELF layer of 10-5 cm was estimated to be 2.5×10-6 10 seconds. 

11 The half-life of O3 can be approximated by dividing the pseudo-first order rate constant, 

k1, into ln 2. Pryor (1992) assumed the reaction rate constant 109 12 L/mol/sec for O3 with 

13 GSH and the concentration of GSH equaled 1 mM in the ELF. Using these values and 

neglecting reactions of O3 with other ELF species, the half-life of O3 would be 7×10-7 14

15 seconds. Under these assumptions of GSH concentration and ELF thickness, the half-life 

16 of O3 is about one third of the time necessary for O3 to diffuse through the ELF layer.

17 Further, assuming that 0.5 ppm O3 enters the trachea and the intrapulmonary gas-phase 

18 concentration is reduced only 5 fold during transport to the terminal bronchioles, by using 

19 a Henry’s law constant and assuming equilibrium, the ELF O3 concentration could be 

calculated to be <1.4 × 10-9 20 M or approximately 0.0014 μM. Further, assuming that 

21 ascorbate = 100 μM, GSH = 300 μM, and uric acid = 250 μM, while ignoring unsaturated 

22 lipids and reactive proteins, the most facile reactants would equate to an approximately 

23 500,000-fold excess over O3. If one then assumes a lumped reaction rate constant of 

107 M-1 sec-1 24 , any O3 in solution would be consumed by reaction almost instantaneously, 

25 thereby constraining its diffusion as an unreacted species to within <0.1 μm, which is less 

26 than the thickness values estimated for distal airway ELF. If unsaturated lipids 

(~ 106 M-1 sec-1 27 ) and proteins (for which the rate constant will vary depending on low 

28 pKa thiolates and other amino acid-reactive sites) are included, the penetration depth is 

29 further reduced. 

30 Similarly, model calculations of the nasal cavity based on diffusion equations and 

31 reaction rates of O3 with model substrates predict an O3 penetration distance (0.5 μm) 

32 less than the thickness of the nasal lining layer (10 μm) (Santiago et al., 2001). 

33 A computational fluid dynamics model was able to predict experimentally measured 
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1 O3 uptake when nasal mucus layer thickness was considered (Cohen-Hubal et al., 1996), 

2 reaffirming the importance of the resistance imparted by the ELF layer in dose and lesion 

3 patterns in the nasal passage. 

4 Despite calculations and in vitro studies suggesting that reactions of O3 with underlying 

5 epithelial cells may be negligible, there is some evidence that suggests direct interaction 

6 of O3 with epithelial cells is possible. While moving distally in the lung, the ELF 

7 thickness decreases and becomes ultrathin in the alveolar region, possibly allowing for 

8 direct interaction of O3 with the underlying epithelial cells. One definitive study

9 conducted in excised rat lung measured alveolar lining layer thickness over relatively flat 

10 portions of the alveolar wall to be 0.14 μm, to be 0.89 μm at the alveolar wall junctions, 

11 and 0.09 μm over the protruding features (Bastacky et al., 1995). The area-weighted 

12 average thickness of the alveolar lining fluid was found to be about 0.2 μm and the 

13 alveolar lining layer was continuous over the entire alveolar surface measured. The 

14 surface appeared smooth, and no epithelial surface features or macrophage features 

15 protruded above the air-liquid interface. It was noted that measurements of alveolar lining 

16 layer thickness were made in lungs prepared in a state of roughly 80% of total lung 

17 capacity, and as a result, the values reported would be approaching the lowest values 

18 possible during the respiratory cycle. However, 4% of the surface area in the alveolar 

19 compartment was covered by alveolar lining fluid layer of less than 20 nm (Bastacky et 

20 al., 1995), suggesting the possibility that unreacted O3 could penetrate to the cell layer in 

21 this region. Further it remains a possibility that airways macrophages may protrude into 

22 the gas phase, allowing for direct contact between O3 and airways epithelial cells.

23 Still, direct reaction of O3 with alveolar epithelial cells or macrophages may be limited by 

24 the presence of dipalmitoyl phosphatidylcholine (DPPC), the major component of 

25 surfactant, which has been shown in vitro to inhibit uptake of O3 into an aqueous 

26 compartment containing ascorbate, glutathione, and uric acid (Connor et al., 2004). 

27 Further, the amount of O3 available to the alveolar compartment may be limited by 

uptake of O3 in nasal and TB compartments (Figure 5-5). In fact, the amount of 18 28 O 

29 reaction product was lower in the alveolar tissues than in TB tissues of rhesus monkeys 

immediately following a 2 hour exposure to 18 30 O-labeled O3 (0.4 and 1 ppm) (Plopper et 

31 al., 1998). These considerations illustrate the difficulty in determining whether O3 reacts 

32 directly with cells in the alveolar compartment. 

33 In some cases, however, with regard to the initiating mechanisms of cellular 

34 perturbations, the precise reactive species that encounters the epithelia might or might not 

35 have specificity to O3 per se or to its secondary oxidants. Many of the measureable 

36 products formed as a consequence of O3 exposure have limited specificity to O3, such as 

37 4-hydroxynonenal that is formed by autoxidation, an event that can be initiated by O3 but 
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1 also by a multitude of other oxidants. Although some classes of lipid oxidation products 

2 (e.g., specific aldehydes, cholesterol products) are specific to O3, measurement in either 

3 BALF or in tissue does not necessarily provide insight on the compartment in which they 

4 were formed (i.e., the ELF, cell membrane, intracellular space) because the ELF is a 

5 dynamic compartment and, once formed, hydrophobic species can partition. Oxidation of 

6 membrane components might produce similar cellular outcomes regardless of the 

7 initiating oxidant. Lipid ozonides, which could be generated either within the ELF or 

8 from ozonation of cell membrane unsaturated lipids, could bind to receptors, activate 

9 signaling cascades, and act in other ways, making differences between pure extracellular 

10 reaction and direct membrane reaction indistinguishable. Thus, in some cases 

11 documenting whether O3 per se reacts directly with cellular constituents might be 

12 essential (despite the challenges of in vivo demonstrations), while in other cases precisely 

13 where O3 reacts might be of less concern with regard to characterizing mechanisms of 

14 health outcomes.

15 Thus, components of the ELF are major targets for O3 and the resulting secondary 

16 oxidation products key mediators of toxicity in the airways (the role of reaction products 

17 in O3-induced toxicity is discussed in Section 5.3). The reaction cascade resulting from 

18 the interaction of O3 with ELF substrates can then carry the oxidative burden deeper into 

19 cells lining the RT to elicit the health effects observed.

5.2.3.1 Summary

20 The ELF is a complex mixture of lipids, proteins, and antioxidants that serve as the first 

21 barrier and target for inhaled O3 (Figure 5-7). The thickness of the lining fluid and mucus 

22 layer is an important determinant of the dose of O3 to the tissues. The antioxidant 

23 substances present in the ELF appear in most cases to limit interaction of O3 with 

24 underlying tissues and to prevent penetration of O3 deeper into the lung. The formation of 

25 secondary oxidation products is likely related to the concentration of antioxidants present 

26 and the quenching ability of the lining fluid. Mechanisms are present to replenish the 

27 antioxidant substrate pools as well as to remove secondary reaction products from tissue 

28 interactions. Important differences exist in the reaction rates for O3 and these ELF 

29 biomolecules and the reactivity of the resulting products. Overall, studies suggest that UA 

30 and AH2 are more reactive with O3 than GSH, proteins, or lipids. In addition to 

31 contributing to the driving force for O3 uptake, formation of secondary oxidation 

32 products may lead to increased cellular injury and cell signaling (discussed in 

33 Section 5.3). Studies indicate that the antioxidants might be participating in reactions 

34 where the resulting secondary oxidation products might penetrate into the tissue layer and 

35 lead to perturbations.
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Note: Contents of this figure not discussed in Section 5.2 will be discussed in Section 5.3. Clara cell secretory protein, CCSP; 

Surfactant Protein-A, SP-A; Platelet activating factor, PAF. Ozone will react with components of the ELF to produce reaction 

products that may lead to cellular injury and cell signaling as discussed in Section 5.3.

Figure 5-7 Details of the ozone interaction with the airway ELF to form 

secondary oxidation products.

5.3 Possible Pathways/Modes of Action

5.3.1 Introduction

1 Mode of action refers to a sequence of key events and processes that result in a given 

2 toxic effect (U.S. EPA, 2005). Elucidation of mechanisms provides a more detailed 

3 understanding of these key events and processes (U.S. EPA, 2005). Moreover, toxicity 

4 pathways describe the processes by which perturbation of normal biological processes 

5 produce changes sufficient to lead to cell injury and subsequent events such as adverse 

Oxidized proteins

Aldehydes

Ozonized Cholesterol Species

Lipid Peroxides

Eicosanoids and PAF

Hyaluronan Fragments

Ozonized Radical

Reactive Oxygen Species

Liquid Layer Antioxidants

Uric Acid, Ascorbate, 

Glutathione, α-Tocopherol

Cellular Macromolecules

Plasma membrane proteins 

and phospholipids

Free fatty acids and 

carbohydrates 

Mechanisms for Antioxidant 

Repletion

• Secretion by epithelial cells

• Transport from plasma 

• Reduction of oxidized ascorbate

Mechanisms for Reaction 

Product Removal 

• Quenching reactions by ELF 

antioxidants and proteins

• Non-enzymatic reactions with 

cellular antioxidants 

• Metabolism by cellular GST/NQO1

• Receptor-mediated uptake by 

macrophages

Ozone

ELF Macromolecules

Surfactant components 

e.g. proteins, phospholipids/

cholesterol, CCSP, 

Albumin, Hyaluronan, SP-A

Secondary Oxidation Products

Mucus Layer

Mucins

Phospholipids

Cellular injury

Cellular signaling
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1 health effects (U.S. EPA, 2009f). The purpose of this section of Chapter 5 is to describe 

2 the key events and toxicity pathways that contribute to health effects resulting from short3 term and long-term exposures to O3. The extensive research carried out over several 

4 decades in humans and in laboratory animals has yielded numerous studies on 

5 mechanisms by which O3 exerts its effects. This section will discuss some of the 

6 representative studies with particular emphasis on studies published since the 2006 O3 

7 AQCD and on studies in humans that inform biological mechanisms underlying 

8 responses to O3.

9 It is well-appreciated that secondary oxidation products, which are formed as a result of 

10 O3 exposure, initiate numerous responses at the cellular, tissue and whole organ level of 

11 the respiratory system. These responses include the activation of neural reflexes, 

12 initiation of inflammation, alteration of epithelial barrier function, sensitization of 

13 bronchial smooth muscle, modification of innate/adaptive immunity and airways 

14 remodeling, as will be discussed below. These have the potential to result in effects on 

15 other organ systems such as the cardiovascular, central nervous, hepatic and reproductive 

16 systems or result in developmental effects. It has been proposed that lipid ozonides and 

17 other secondary oxidation products, which are bioactive and cytotoxic in the respiratory 

18 system, are responsible for systemic effects. However it is not known whether they gain 

19 access to the vascular space (Chuang et al., 2009). Recent studies in animal models show 

20 that inhalation of O3 results in systemic oxidative stress. The following subsections 

21 describe the current understanding of potential pathways and modes of action responsible 

22 for the pulmonary and extrapulmonary effects of O3 exposure.

5.3.2 Activation of Neural Reflexes

23 Acute O3 exposure results in reversible effects on lung function parameters through 

24 activation of neural reflexes. The involvement of bronchial C-fibers, a type of nociceptive 

25 sensory nerve, has been demonstrated in dogs exposed through an endotracheal tube to 

26 2-3 ppm O3 for 20-70 minutes (Coleridge et al., 1993; Schelegle et al., 1993). This vagal 

27 afferent pathway was found to be responsible for O3-mediated rapid shallow breathing 

28 and other changes in respiratory mechanics in O3-exposed dogs (Schelegle et al., 1993). 

29 Ozone also triggers neural reflexes that stimulate the autonomic nervous system and alter 

30 electrophysiologic responses of the heart. For example, bradycardia, altered HRV and 

31 arrhythmia have been demonstrated in rodents exposed for several hours to 0.1-0.6 ppm 

32 O3 (Hamade and Tankersley, 2009; Watkinson et al., 2001; Arito et al., 1990). Another 

33 effect is hypothermia, which in rodents occurred subsequent to the activation of neural 

34 reflexes involving the parasympathetic nervous system (Watkinson et al., 2001). Vagal 

35 afferent pathways originating in the RT may also be responsible for O3-mediated 
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1 activation of nucleus tractus solitarius neurons that resulted in neuronal activation in 

2 stress-responsive regions of the central nervous system (CNS) (rats, 0.5-2.0 ppm O3 for 

3 1.5-120 hours) (Gackière et al., 2011).

4 Recent studies in animals provide new information regarding the effects of O3 on reflex 

5 responses mediated by bronchopulmonary C-fibers. In ex vivo mouse lungs, O3 exposure 

6 (30 μM solubilized) selectively activated a subset of C-fiber receptors that are TRPA1 

7 ion channels (Taylor-Clark and Undem, 2010). TRPA1 ion channels are members of the 

8 TRP family of ion channels, which are known to mediate the responses of sensory 

9 neurons to inflammatory mediators (Caceres et al., 2009). In addition to TRPA1 ion 

10 channels possibly playing a key role in O3-induced decrements in pulmonary function, 

11 they may mediate allergic asthma (Caceres et al., 2009). Activation of TRPA1 ion 

12 channels following O3 exposure is likely initiated by secondary oxidation products such 

13 as aldehydes and prostaglandins (Taylor-Clark and Undem, 2010) through covalent 

14 modification of cysteine and lysine residues (Trevisani et al., 2007). Ozonation of 

15 unsaturated fatty acids in the ELF was found to result in the generation of aldehydes 

16 (Frampton et al., 1999) such as 4-hydroxynonenal and 4-oxononenal (Taylor-Clark et al., 

17 2008; Trevisani et al., 2007). 4-oxononenal is a stronger electrophile than 

18 4-hydroxynonenal and exhibits greater potency towards the TRPA1 channels (Taylor19 Clark et al., 2008; Trevisani et al., 2007). In addition, PGE2 is known to sensitize TRPA1 

20 channels (Bang et al., 2007). 

21 In humans exercising at a moderate level, the response to O3 (500 ppb for 2 h) was 

22 characterized by substernal discomfort, especially on deep inspiration, accompanied by 

23 involuntary truncation of inspiration (Hazucha et al., 1989). This latter response led to 

24 decreased inspiratory capacity and to decreased forced vital capacity (FVC) and forced 

25 expiratory volume in one second (FEV1), as measured by spirometry. These changes, 

26 which occurred during O3 exposure, were accompanied by decreased VT and increased 

27 respiratory frequency in human subjects. Spirometric changes in FEV1 and FVC were not 

28 due to changes in respiratory muscle strength (Hazucha et al., 1989). In addition, 

29 parasympathetic involvement in the O3-mediated decreases in lung volume was minimal 

30 (Mudway and Kelly, 2000), since changes in FVC or symptoms were not modified by 

31 treatment with bronchodilators such as atropine in human subjects exposed to 400 ppb O3 

32 for 2 hours while exercising at a heavy level (Beckett et al., 1985). However, the loss of 

33 vital capacity was reversible with intravenous administration of the rapid-acting opioid 

34 agonist, sufentanyl, in human subjects exercising at a moderate level and exposed to 

35 420 ppb O3 for 2 hours, which indicated the involvement of opioid receptor-containing 

36 nerve fibers and/or more central neurons (Passannante et al., 1998). The effects of 

37 sufentanyl may be attributed to blocking C-fiber stimulation by O3 since activation of 

38 opioid receptors downregulated C-fiber function (Belvisi et al., 1992). Thus, nociceptive 
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1 sensory nerves, presumably bronchial C-fibers, are responsible for O3-mediated 

2 responses in humans (Passannante et al., 1998). This vagal afferent pathway is 

3 responsible for pain-related symptoms and inhibition of maximal inspiration in humans 

4 (Hazucha et al., 1989).

5 There is some evidence that eicosanoids (see Section 5.3.3) play a role in the neural 

6 reflex since cyclooxygenase inhibition with indomethacin (Alexis et al., 2000; Schelegle 

7 et al., 1987) or ibuprofen, which also blocks some lipoxygenase activity (Hazucha et al., 

8 1996), before exposure to O3 significantly blunted the spirometric responses. These 

9 studies involved exposures of 1-2 hours to 350-400 ppb O3 in human subjects exercising 

10 at light, moderate and heavy levels. In the latter study, ibuprofen treatment resulted in 

11 measurable decreases in BALF levels of PGE2 and TXB2 at 1-hour postexposure 

12 (Hazucha et al., 1996). Although an earlier study demonstrated that PGE2 stimulated 

13 bronchial C-fibers (Coleridge et al., 1993; Coleridge et al., 1976) and suggested that 

14 PGE2 mediated O3-induced decreases in pulmonary function, no correlation was observed 

15 between the degree of ibuprofen-induced inhibition of BALF PGE2 levels and blunting of 

16 the spirometric response to O3 (Hazucha et al., 1996). These results point to the 

17 involvement of a lipoxygenase product. Further, as noted above, PGE2 may play a role in 

18 the neural reflex by sensitizing TRPA1 channels. A recent study in human subjects 

19 exercising at a moderate to high level and exposed for 1 hour to 350 ppb O3 also provided 

20 evidence that arachidonic acid metabolites, as well as oxidative stress, contribute to 

21 human responsiveness to O3 (Alfaro et al., 2007). 

22 In addition to the spirometric changes, mild airways obstruction occurred in human 

23 subjects exercising at a moderate level during O3 exposure (500 ppb for 2 hours) 

24 (Hazucha et al., 1989). This pulmonary function decrement is generally measured as 

25 specific airway resistance (sRaw) which is the product of airway resistance and thoracic 

26 gas volume. In several studies involving human subjects exercising at a moderate to 

27 heavy level and exposed for 1-4 hours to 200-300 ppb O3, changes in sRaw correlated 

28 with changes in inflammatory and injury endpoints measured 18-hours postexposure, but 

29 did not follow the same time course or change to the same degree as spirometric changes 

30 (i.e., FEV1, FVC) measured during exposure (Balmes et al., 1996; Aris et al., 1993; 

31 Schelegle et al., 1991). In addition, a small but persistent increase in airway resistance 

32 associated with narrowing of small peripheral airways (measured as changes in 

33 isovolumetric FEF25-75) was demonstrated in O3-exposed human subjects (350 ppb for 

34 130 minutes, moderate exercise level) (Weinmann et al., 1995c; Weinmann et al., 1995b). 

35 A similar study (400 ppb O3 for 2 hours in human subjects exercising at a heavy level) 

36 found decreases in FEF25-75 concomitant with increases in residual volume, which is 

37 suggestive of small airways dysfunction (Kreit et al., 1989). In separate studies, a 

38 statistically significant increase in residual volume (500 ppb for 2 hours) (Hazucha et al., 
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1 1989) and a statistically significant decrease in FEF25-75 (160 ppb for 7.6 hours) 

2 (Horstman et al., 1995) were observed following O3 exposure in human subjects 

3 exercising at moderate and light levels, respectively, providing further support for an 

4 O3-induced effect on small airways.

5 Mechanisms underlying this rapid increase in airway resistance following O3 exposure 

6 are incompletely understood. Pretreatment with atropine decreased baseline sRaw and 

7 prevented O3-induced increases in sRaw in human subjects exercising at a heavy level

8 (400 ppb for 0.5 hours) (Beckett et al., 1985), indicating the involvement of muscarinic 

9 cholinergic receptors of the parasympathetic nervous system. Interestingly, atropine 

10 pretreatment partially blocked the decrease in FEV1, but had no effect on the decrease in 

11 FVC, breathing rate, tidal volume or respiratory symptoms (Beckett et al., 1985). Using a 

12 β-adrenergic agonist, it was shown that smooth muscle contraction, not increased airway 

13 mucus secretion, was responsible for O3-induced increases in airway resistance (Beckett 

14 et al., 1985). Thus, pulmonary function decrements measured as FEV1 may reflect both 

15 restrictive (such as decreased inspiratory capacity) and obstructive (such as 

16 bronchoconstriction) type changes in airway responses. This is consistent with findings of 

17 McDonnell et al. (1983) who observed a relatively strong correlation between sRaw and 

18 FEV1 (r = -0.31, p = 0.001) and a far weaker correlation between sRaw and FVC 

19 (r = -0.16, p = 0.10) in human subjects exercising at a heavy level and exposed for 

20 2.5 hours to 120-400 ppb O3.

21 Furthermore, tachykinins may contribute to O3-mediated increases in airway resistance. 

22 In addition to stimulating CNS reflexes, bronchopulmonary C-fibers mediate local axon 

23 responses by releasing neuropeptides such as substance P (SP), neurokinin (NK) A and 

24 calcitonin gene-related peptide (CGRP). Tachykinins bind to NK receptors resulting in 

25 responses such as bronchoconstriction. Recent studies in animals demonstrated that NK-1 

26 receptor blockade had no effect on O3-stimulated physiologic responses such as VT and fB

27 in rats over the 8 hour exposure to 1 ppm O3 (Oslund et al., 2008). However, SP and NK 

28 receptors contributed to vagally-mediated bronchoconstriction in guinea pigs 3 days after 

29 a single 4-hour exposure to 2 ppm O3 (Verhein et al., 2011). In one human study in which 

30 bronchial biopsies were performed and studied by immunohistochemistry, SP was 

31 substantially diminished in submucosal sensory nerves 6 hours following O3 exposure 

32 (200 ppb for 2 hours, light exercise) (Krishna et al., 1997). A statistically significant 

33 correlation was observed between loss of SP immunoreactivity from neurons in the 

34 bronchial mucosa and changes in FEV1 measured 1-hour postexposure (Krishna et al., 

35 1997). Another study found that SP was increased in lavage fluid of human subjects 

36 immediately after O3 challenge (250 ppb for 1 hour, heavy exercise) (Hazbun et al., 

37 1993). These results provide evidence that the increased airway resistance observed 
Draft – Do Not Cite or Quote 5-34 June 2012

1 following O3 exposure is due to vagally-mediated responses and possibly by local axon 

2 reflex responses through bronchopulmonary C-fiber-mediated release of SP. 

3 A role for antioxidant defenses in modulating neural reflexes has been proposed given the 

4 delay in onset of O3-induced pulmonary function responses that has been noted in 

5 numerous studies. Recently, this delay was characterized in terms of changes in fB

6 (Schelegle et al., 2007). In humans exposed for 1-2 hours to 120-350 ppb O3 while 

7 exercising at a high level, no change in fB was observed until a certain cumulative inhaled 

8 dose of O3 had been reached. Subsequently, the magnitude of the change in fB was 

9 correlated with the inhaled dose rate (Schelegle et al., 2007). These investigators 

10 proposed that initial reactions of O3 with ELF resulted in a time-dependent depletion of 

11 ELF antioxidants, and that activation of neural reflexes occurred only after the 

12 antioxidant defenses were overwhelmed (Schelegle et al., 2007). 

5.3.3 Initiation of inflammation

13 As described previously (Section 5.2.3), O3 mainly reacts with components of the ELF 

14 and cellular membranes resulting in the generation of secondary oxidation products. 

15 Higher concentrations of these products may directly injure RT epithelium. Subsequent 

16 airways remodeling may also occur (Section 5.3.7) (Mudway and Kelly, 2000). Lower 

17 concentrations of secondary oxidation products may initiate cellular responses including 

18 cytokine generation, adhesion molecule expression, and modification of tight junctions 

19 leading to inflammation and increased permeability across airway epithelium 

20 (Section 5.3.4) (Dahl et al., 2007; Mudway and Kelly, 2000). 

21 An important hallmark of acute O3 exposure in humans and animals is neutrophilic 

22 airways inflammation. Although neutrophil influx into nasal airways has been 

23 demonstrated in human subjects (400 ppb O3 2 hours, heavy exercise) (Graham and 

24 Koren, 1990), most studies of neutrophil influx have focused on the lower airways 

25 (Hazucha et al., 1996; Aris et al., 1993). The time course of this response in the lower 

26 airways and its resolution appears to be slower than that of the decrements in pulmonary 

27 function in exercising human subjects (Hazucha et al., 1996). In several studies, airways 

28 neutrophilia was observed by 1-3 hours, peaked by 6 hours and was returning to baseline 

29 levels at 18-24 hours in human subjects exercising at a heavy level and exposed for 

30 1-2 hours to 300-400 ppb O3 (Schelegle et al., 1991; Koren et al., 1989; Seltzer et al., 

31 1986). Neutrophils are thought to be injurious and a study in guinea pigs demonstrated 

32 that the influx and persistence of neutrophils in airways following O3 exposure correlated 

33 with the temporal profile of epithelial injury (0.26-1 ppm O3, 72 hours) (Hu et al., 1982). 

34 However, neutrophils have also been shown to contribute to repair of O3-injured 
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1 epithelium in rats exposed for 8 hours to 1 ppm O3, possibly by removing necrotic 

2 epithelial cells (Mudway and Kelly, 2000; Vesely et al., 1999). Nonetheless, the degree 

3 of airways inflammation due to O3 is thought to have more important long-term 

4 consequences than the more quickly resolving changes in pulmonary function since 

5 airways inflammation is often accompanied by tissue injury (Balmes et al., 1996). 

6 Ozone exposure results in alterations in other airways inflammatory cells besides 

7 neutrophils, including lymphocytes, macrophages, monocytes and mast cells. Influx of 

8 some of these cells accounts for the later (i.e., 18-20 hours) phase of inflammation 

9 following O3 exposure. Numbers of lymphocytes and total cells in BALF were decreased 

10 early after O3 exposure in human subjects exercising at a light to moderate level and 

11 exposed for 2 hours to 200 ppb O3, which preceded the neutrophil influx (Mudway and 

12 Kelly, 2000; Blomberg et al., 1999; Krishna et al., 1997). The decrease in total cells was 

13 thought to reflect decreases in macrophages, although it was not clear whether the cells 

14 were necrotic or whether membrane adhesive properties were altered making them more 

15 difficult to obtain by lavage (Mudway and Kelly, 2000; Blomberg et al., 1999; Mudway 

16 et al., 1999b; Frampton et al., 1997b; Pearson and Bhalla, 1997). A recent study in human 

17 subjects exercising at a moderate level and exposed for 6.6 hours to 80 ppb O3 

18 demonstrated an increase in numbers of sputum monocytes and dendritic-like cells with 

19 increased expression of innate immune surface proteins and antigen presentation markers 

20 (Peden, 2011; Alexis et al., 2010) (see Section 6.2.3.1). An increase in submucosal mast 

21 cells was observed 1.5 hours after a 2 hour-exposure to 200 ppb O3 (Blomberg et al., 

22 1999) and an increase in BAL mast cell number was observed 18 hours after a 4-hour 

23 exposure to 220 ppb O3 exposure in human subjects exercising at a moderate level 

24 (Frampton et al., 1997b). Mast cells may play an important role in mediating neutrophil 

25 influx since they are an important source of several pro-inflammatory cytokines and since 

26 their influx preceded that of neutrophils in human subjects exercising at a moderate level 

27 and exposed for 2 hours to 200 ppb O3 (Stenfors et al., 2002; Blomberg et al., 1999). 

28 Further, a study using mast cell-deficient mice demonstrated decreased neutrophilic 

29 inflammation in response to O3 (1.75 ppm, 3 hours) compared with wild type mice 

30 (Kleeberger et al., 1993). Influx of these inflammatory cell types in the lung is indicative 

31 of O3-mediated activation of innate immunity as will be discussed in Section 5.3.6. 

32 Much is known about the cellular and molecular signals involved in inflammatory 

33 responses to O3 exposure (U.S. EPA, 2006b). Eicosanoids are one class of secondary 

34 oxidation products that may be formed rapidly following O3 exposure and that may 

35 mediate inflammation. Eicosanoids are metabolites of arachidonic acid—a 20-carbon 

36 PUFA—that are released from membrane phospholipids by phospholipase A2-mediated 

37 catalysis. Activation of phospholipase A2 occurs by several cell signaling pathways and 

38 may be triggered by O3-mediated lipid peroxidation of cellular membranes (Rashba-Step 
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1 et al., 1997). Additionally, cellular phospholipases A2, C and D may be activated by lipid 

2 ozonation products (Kafoury et al., 1998). While the conversion of arachidonic acid to 

3 prostaglandins, leukotrienes and other eicosanoid products is generally catalyzed by 

4 cyclooxygenases and lipoxygenases, non-enzymatic reactions also occur during oxidative 

5 stress leading to the generation of a wide variety of eicosanoids and reactive oxygen 

6 species. Further, the release of arachidonic acid from phospholipids is accompanied by 

7 the formation of lysophospholipids that are precursors for platelet activating factors 

8 (PAFs). Thus, formation of eicosanoids, reactive oxygen species and PAFs accompanies 

9 O3-mediated lipid peroxidation. 

10 In addition, secondary reaction products may stimulate macrophages to produce 

11 cytokines such as IL-1, IL-6 and TNF-α that in turn activate IL-8 production by epithelial 

12 cells. Although IL-8 has been proposed to play a role in neutrophil chemotaxis, 

13 measurements of IL-8 in BALF from humans exposed to O3 found increases that were 

14 too late to account for this effect (Mudway and Kelly, 2000). The time-course profiles of 

15 PGE2 and IL-6 responses suggest that they may play a role in neutrophil chemotaxis in 

16 humans (Mudway and Kelly, 2000). However, pretreatment with ibuprofen attenuated 

17 O3-induced increases in BALF PGE2 levels, but had no effect on neutrophilia in human 

18 subjects exercising at a heavy level and exposed for 2 hour to 400 ppb O3 (Hazucha et al., 

19 1996). 

20 One set of studies in humans focused on the earliest phase of airways inflammation 

21 (1-2 hours following exposure). Human subjects, exercising at a moderate level, were 

22 exposed to 200 ppb O3 for 2 hours and bronchial biopsy tissues were obtained 1.5 and 

23 6 hours after exposure (Bosson et al., 2009; Bosson et al., 2003; Stenfors et al., 2002; 

24 Blomberg et al., 1999). Results demonstrated upregulation of vascular endothelial 

25 adhesion molecules P-selectin and ICAM-1 at both 1.5 and 6 hours (Stenfors et al., 2002; 

26 Blomberg et al., 1999). Submucosal mast cell numbers were increased at 1.5 hours in the 

27 biopsy samples without an accompanying increase in neutrophil number (Blomberg et al., 

28 1999). Pronounced neutrophil infiltration was observed at 6 hours in the bronchial 

29 mucosa (Stenfors et al., 2002). Surprisingly, suppression of the NF-κB and AP-1 

30 pathways at 1.5 hours and a lack of increased IL-8 at 1.5 or 6 hours in bronchial 

31 epithelium were observed (Bosson et al., 2009). The authors suggested that vascular 

32 endothelial adhesion molecules, rather than redox sensitive transcription factors, play key 

33 roles in early neutrophil recruitment in response to O3. 

34 Increases in markers of inflammation occurred to a comparable degree in human subjects 

35 with mild (least sensitive) and more remarkable (more sensitive) spirometric responses to 

36 O3 (200 ppb, 4 hours, moderate exercise) (Balmes et al., 1996). Two other studies 

37 (200 ppb for 4 hours with moderate exercise and 300 ppb for 1 hour with heavy exercise) 
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1 found that acute spirometric changes were not positively correlated with cellular and 

2 biochemical indicators of inflammation (Aris et al., 1993; Schelegle et al., 1991). 

3 However inflammation was correlated with changes in sRaw (Balmes et al., 1996). In 

4 another study, pretreatment with ibuprofen had no effect on neutrophilia although it 

5 blunted the spirometric response in human subjects exercising at heavy level and exposed 

6 for 2 hours to 400 ppb O3 (Hazucha et al., 1996). Taken together, results from these 

7 studies indicate different mechanisms underlying the spirometric and inflammatory 

8 responses to O3. 

9 A common mechanism underlying both inflammation and impaired pulmonary function 

10 was suggested by Krishna et al. (1997). This study, conducted in human subjects 

11 exercising at a light level and exposed to 200 ppb O3 for 2 hours, demonstrated a 

12 correlation between loss of SP immunoreactivity from neurons in the bronchial mucosa 

13 and numbers of neutrophils and epithelial cells (shed epithelial cells are an index of 

14 injury) in the BALF 6-hours postexposure. Furthermore, the loss of SP immunoreactivity 

15 was correlated with the observed changes in FEV1. Another study found that SP was 

16 increased in lavage fluid of exercising human subjects immediately after O3 challenge 

17 (250 ppb, 1 hour, heavy exercise) (Hazbun et al., 1993). SP is a neuropeptide released by 

18 sensory nerves which mediates neurogenic edema and bronchoconstriction (Krishna et 

19 al., 1997). Collectively, these findings suggest that O3-mediated stimulation of sensory 

20 nerves that leads to activation of central and local axon reflexes is a common effector 

21 pathway leading to impaired pulmonary function and inflammation. 

22 Studies in animal models have confirmed many of these findings and provided evidence 

23 for additional mechanisms involved in O3-induced inflammation. A study in mice (2 ppm 

24 O3, 3 hours) demonstrated that PAF may be important in neutrophil chemotaxis 

25 (Longphre et al., 1999), while ICAM-1 and macrophage inflammatory protein-2 (MIP-2), 

26 the rodent IL-8 homologue, have been implicated in a rat model (1 ppm O3, 3 hours) 

27 (Bhalla and Gupta, 2000). Key roles for CXCR2, a receptor for keratinocyte-derived 

28 chemokine (KC) and MIP-2, and for IL-6 in O3-mediated neutrophil influx were 

29 demonstrated in mice (1 ppm O3, 3 hours) (Johnston et al., 2005a; Johnston et al., 2005b). 

30 Activation of JNK and p38 pathways and cathepsin-S were also found to be important in 

31 this response (3 ppm O3, 3 hours) (Williams et al., 2009a; Williams et al., 2008a; 

32 Williams et al., 2007a). Matrix metalloproteinase-9 (MMP-9) appeared to confer 

33 protection against O3-induced airways inflammation and injury in mice (0.3 ppm O3, 

34 6-72 hours) (Yoon et al., 2007). Interleukin-10 (IL-10) also appeared to be protective 

35 since IL-10 deficient mice responded to O3 exposure (0.3 ppm, 24-72 hours) with 

36 enhanced numbers of BAL neutrophils, enhanced NF-κB activation and MIP-2 levels 

37 compared with IL-10 sufficient mice (Backus et al., 2010).
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1 In addition, lung epithelial cells may release ATP in response to O3 exposure (Ahmad et 

2 al., 2005). ATP and its metabolites (catalyzed by ecto-enzymes) can bind to cellular 

3 purinergic receptors resulting in activation of cell signaling pathways (Picher et al., 

4 2004). One such metabolite, adenine, is capable of undergoing oxidation leading to the 

5 formation of UA which, if present in high concentrations, could activate inflammasomes 

6 and result in caspase 1 activation and the maturation and secretion of IL-1β and IL-18 

7 (Dostert et al., 2008). A recent study in human subjects exercising at a moderate level and 

8 exposed for 2 hours to 400 ppb O3 demonstrated a correlation between ATP metabolites 

9 and inflammatory markers (Esther et al., 2011), which provides some support for this 

10 mechanism. 

11 Several recent studies have focused on the role of Toll-like receptor (TLR) and its related 

12 adaptor protein MyD88 in mediating O3-induced neutrophilia. Hollingsworth et al. (2004)

13 demonstrated airways neutrophilia that was TLR4-independent following acute (2 ppm, 

14 3 hours) and subchronic (0.3 ppm, 72 hours) O3 exposure in a mouse model. However, 

15 Williams et al. (2007b) found that MyD88 was important in mediating O3-induced 

16 neutrophilia in mice (3 ppm, 3 hours), with TLR4 and TLR2 contributing to the speed of 

17 the response. Moreover, MyD88, TLR2 and TLR4 contributed to inflammatory gene 

18 expression in this model and O3 upregulated MyD88, TLR4 and TLR4 gene expression 

19 (Williams et al., 2007a). Neutrophilic inflammation was also found to be partially 

20 dependent on MyD88 in mice exposed to 1 ppm O3 for 3 hours (Li et al., 2011). 

21 Hyaluronan was found to mediate a later phase (24 hours) of O3-induced inflammation in 

22 mice (Garantziotis et al., 2010; Garantziotis et al., 2009). Hyaluronan is an extracellular 

23 matrix component that is normally found in the ELF as a large polymer. Exposure to 

24 2 ppm O3 for 3 hours resulted in elevated levels of soluble low molecular weight 

25 hyaluronan in the BALF 24-hours postexposure (Garantziotis et al., 2010; Garantziotis et 

26 al., 2009). Similar results were found in response to 3 hour exposure to 1 ppm O3 (Li et 

27 al., 2011). Ozone may have caused the depolymerization of hyaluronan to soluble 

28 fragments that are known to be endogenous ligands of the CD44 receptor and TLR4 in 

29 the macrophage (Jiang et al., 2005). Binding of hyaluronan fragments to the CD44 

30 receptor activates hyaluronan clearance, while binding to TLR4 results in signaling 

31 through MyD88 to produce chemokines that stimulate the influx of inflammatory cells 

32 (Jiang et al., 2005). Activation of NF-κB occurred in both airway epithelia and alveolar 

33 macrophages 24-hours postexposure to O3. Increases in BALF pro-inflammatory factors 

34 KC, IL-1β, MCP-1, TNF-α and IL-6 observed 24 hours following O3 exposure were 

35 found to be partially dependent on TLR4 (Garantziotis et al., 2010) while increases in 

36 BAL inflammatory cells, which consisted mainly of macrophages, were dependent on 

37 CD44 (Garantziotis et al., 2009). BAL inflammatory cells number and injury markers 
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1 following O3 exposure were similar in wild-type and TLR4-deficient animals 

2 (Garantziotis et al., 2010). 

3 Since exposure to O3 leads to airways inflammation characterized by neutrophilia, and 

4 since neutrophil-derived oxidants often consume ELF antioxidants, concentrations of 

5 ELF antioxidants have been examined during airways neutrophilia (Long et al., 2001; 

6 Gunnison and Hatch, 1999; Mudway et al., 1999b). In human subjects exercising at a 

7 moderate level and exposed to 200 ppb O3 for 2 hours, UA, GSH and α-TOH levels 

8 remained unchanged in BALF 6-hours postexposure while AH2 was decreased 

9 significantly in both BALF and plasma (Mudway et al., 1999b). A second study 

10 involving the same protocol reported a loss of AH2 from bronchial wash fluid and BALF, 

11 representing proximal and distal airway ELF respectively, as well as an increase in 

12 oxidized GSH in both compartments (Mudway et al., 2001). No change was observed in 

13 ELF UA levels in response to O3 (Mudway et al., 2001). Further, O3 exposure (0.8 ppm, 

14 4 hours) in female rats resulted in a 50% decrease in BALF AH2 immediately 

15 postexposure (Gunnison and Hatch, 1999). These studies suggested a role for AH2 and 

16 GSH in protecting against the oxidative stress associated with inflammation.

17 The relationship between inflammation, antioxidant status and O3 dose has also been 

investigated. The degree of inflammation in rats has been correlated with 18 18 O-labeled O3

19 dose markers in the lower lung. In female rats exposed to 0.8 ppm O3 for 4 hours, BAL 

neutrophil number and 18 20 O reaction product were directly proportional (Gunnison and 

21 Hatch, 1999). Kari et al. (1997) observed that a 3-week caloric restriction (75%) in rats 

22 abrogated the toxicity of O3 (2 ppm, 2 hours), measured as BALF increases in protein, 

23 fibronectin and neutrophils, that was seen in normally fed rats. Accompanying this 

resistance to O3 toxicity was a reduction (30%) in the accumulation of 18 24 O reaction 

25 product in the lungs. These investigations also demonstrated an inverse relationship 

26 between AH2 levels and O3 dose and provided evidence for AH2 playing a protective 

27 role following O3 exposure in these studies. Pregnant and lactating rats had lower AH2 

content in BALF and exhibited a greater increase in accumulation of 18 28 O reaction 

29 products compared with pre-pregnant rats in response to O3 exposure (Gunnison and 

30 Hatch, 1999). In the calorie restricted model, a 30% higher basal BALF AH2 

31 concentration and a rapid accumulation of AH2 into the lungs to levels 60% above 

32 normal occurred as result of O3 exposure (Kari et al., 1997). However, this relationship 

33 between AH2 levels and O3 dose did not hold up in every study. Aging rats (9 and 

34 24 months old) had 49% and 64% lower AH2 in lung tissue compared with month-old 

rats but the aging-induced AH2 loss did not increase the accumulation of 18 35 O reaction 

36 products following O3 exposure (0.4-0.8 ppm, 2-6 hours) (Vincent et al., 1996b). 
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1 A few studies have examined the dose- or concentration-responsiveness of airways 

2 neutrophilia in O3-exposed humans (Holz et al., 1999; Devlin et al., 1991). No 

3 concentration-responsiveness was observed in healthy human subjects exposed for 1 hour 

4 to 125-250 ppb O3 while exercising at a light level and a statistically significant increase 

5 in sputum neutrophilia was observed only at the higher concentration (Holz et al., 1999). 

6 However, concentration-dependent and statistically significant increases in BAL 

7 neutrophils and the inflammatory mediator IL-6 were reported following exposure to 80 

8 and 100 ppb O3 for 6.6 hours in human subjects exercising at a moderate level (Devlin et 

9 al., 1991). Additional evidence is provided by a meta-analysis of the O3

10 dose-inflammatory response in controlled human exposure studies involving exposure to 

11 80-600 ppb O3 for 60-396 minutes and exercise levels ranging from light to heavy 

12 (Mudway and Kelly, 2004b). Results demonstrated a linear relationship between inhaled 

13 O3 dose (determined as the product of concentration, ventilation and time) and BAL 

14 neutrophils at 0-6 hours and 18-24 hours following O3 exposure (Mudway and Kelly, 

15 2004b). 

5.3.4 Alteration of Epithelial Barrier Function

16 Following O3 exposure, injury and inflammation can lead to altered airway barrier 

17 function. Histologic analysis has demonstrated damage to tight junctions between 

18 epithelial cells, suggesting an increase in epithelial permeability. In addition, the presence 

19 of shed epithelial cells in the BALF and increased epithelial permeability, which is 

20 measured as the flux of small solutes, have been observed and are indicative of epithelial 

21 injury. This could potentially lead to the loss of ELF solutes that could diffuse down their 

22 concentration gradient from the lung to the blood. Increases in vascular permeability, as 

23 measured by BALF protein and albumin, have also been demonstrated (Costa et al., 

24 1985; Hu et al., 1982). 

25 An early study in sheep measured changes in airway permeability as the flux of inhaled 

26 radiolabeled histamine into the plasma (Abraham et al., 1984). Exposure of sheep to 

27 0.5 ppm O3 for 2 hours via an endotracheal tube resulted in an increased rate of histamine 

28 appearance in the plasma at 1 day postexposure. Subsequently, numerous studies have 

measured epithelial permeability as the flux of the small solute 99m 29 Tc-DTPA that was 

30 introduced into the air spaces in different regions of the RT. Increased pulmonary 

epithelial permeability, measured as the clearance of 99m 31 Tc-DTPA from lung to blood, 

32 was demonstrated in humans 1-2 hours following a 2-hour exposure to 400 ppb O3 while 

33 exercising at a heavy level (Kehrl et al., 1987). Another study in human subjects found 

34 increased epithelial permeability 19-hours postexposure to 240 ppb O3 for 130 minutes 

35 while exercising at moderate level (Foster and Stetkiewicz, 1996). Increased bronchial 
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1 permeability was also observed in dogs 1-day postexposure (0.4 ppm O3 by endotracheal 

2 tube for 6 hours) and did not resolve for several days (Foster and Freed, 1999). 

3 A role for tachykinins in mediating airway epithelial injury and decreased barrier 

4 function has been suggested. Nishiyama et al. (1998) demonstrated that capsaicin, which 

5 depletes nerve fibers of substance P, blocked the O3-induced increase in permeability of 

6 guinea pig tracheal mucosa (0.5-3 ppm O3, 0.5 hours). Pretreatment with propranolol or 

7 atropine failed to inhibit this response, suggesting that adrenergic and cholinergic 

8 pathways were not involved. In another study, tachykinins working through NK-1 and 

9 CGRP receptors were found to contribute to airway epithelial injury in O3-exposed rats 

10 (1 ppm, 8 hours) (Oslund et al., 2009, 2008).

11 Kleeberger et al. (2000) evaluated genetic susceptibility to O3-induced altered barrier 

12 function in recombinant inbred strains of mice. Lung hyperpermeability, measured as 

13 BALF protein, was evaluated 72 hours after exposure to 0.3 ppm O3 and found to be 

14 associated with a functioning Tlr4 gene. This study concluded that Tlr4 was a strong 

15 candidate gene for susceptibility to hyperpermeability in response to O3 (Kleeberger et 

16 al., 2000). A subsequent study by these same investigators found that Tlr4 modulated 

17 mRNA levels of the Nos2 genes and suggested that the protein product of Nos2, iNOS, 

18 plays an important role in O2-induced lung hyperpermeability (0.3 ppm, 72 hours) 

19 (Kleeberger et al., 2001). More recently, HSP70 was identified as part of the TLR4 

20 signaling pathway (0.3 ppm, 6-72 hours) (Bauer et al., 2011). 

21 Antioxidants have been shown to confer resistance to O3-induced injury. In a recent 

22 study, lung hyperpermeability in response to O3 (0.3 ppm, 48 hours) was unexpectedly 

23 reduced in mice deficient in the glutamate-cysteine ligase modifier subunit gene 

24 compared with sufficient mice (Johansson et al., 2010). Since the lungs of these mice 

25 exhibited 70% glutathione depletion, protection against O3-induced injury was 

26 unexpected (Johansson et al., 2010). However it was found that several other antioxidant 

27 defenses, including metallothionein, were upregulated in response to O3 to a greater 

28 degree in the glutathione-deficient mice compared with sufficient mice (Johansson et al., 

29 2010). The authors suggested that resistance to O3-induced lung injury was due to 

30 compensatory augmentation of antioxidant defenses (Johansson et al., 2010). Antioxidant 

31 effects have also been attributed to Clara cell secretory protein (CCSP) and surfactant 

32 protein A (SP-A). CCSP was found to modulate the susceptibility of airway epithelium to 

33 injury in mice exposed to O3 (0.2 or 1 ppm for 8 hours) by an unknown mechanism 

34 (Plopper et al., 2006). SP-A appeared to confer protection against O3-induced airways 

35 inflammation and injury in mice (2 ppm, 3 hours) (Haque et al., 2007).

36 Increased epithelial permeability has been proposed to play a role in allergic sensitization 

37 (Matsumura, 1970), in activation of neural reflexes and in stimulation of smooth muscle 
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1 receptors (Dimeo et al., 1981). Abraham et al. (1984) reported a correlation between 

2 airway permeability and airways hyperresponsiveness (AHR) in O3-exposed sheep. 

3 However a recent study in human subjects exposed to 220 ppb O3 for 135 minutes while 

4 exercising at a light to moderate level did not find a relationship between O3-induced 

5 changes in airway permeability and AHR (Que et al., 2011). 

5.3.5 Sensitization of Bronchial Smooth Muscle

6 Bronchial reactivity is generally determined in terms of a response to a challenge agent. 

7 Non-specific bronchial reactivity in humans is assessed by measuring the effect of 

8 inhaling increasing concentrations of a bronchoconstrictive drug on lung mechanics 

9 (sRaw or FEV1). Methacholine is most commonly employed but histamine and other 

10 agents are also used. Specific bronchial reactivity is assessed by measuring effects in 

11 response to an inhaled allergen in individuals (or animals) already sensitized to that 

12 allergen. An increase in sRaw in response to non-specific or specific challenge agents 

13 indicates AHR.

14 In addition to causing mild airways obstruction as discussed above, acute O3 exposure 

15 results in reversible increases in bronchial reactivity by mechanisms that are not well 

16 understood. In one study, bronchial reactivity of healthy subjects was significantly 

17 increased 19-hours postexposure to O3 (120-240 ppb O3 for 2 hours with moderate 

18 exercise) (Foster et al., 2000). These effects may be more considerable in human subjects 

19 with already compromised airways (Section 5.4.2.2). 

20 Ozone may sensitize bronchial smooth muscle to stimulation through an exposure-related 

21 effect on smooth muscle or through effects on the sensory nerves in the epithelium or on 

22 the motor nerves innervating the smooth muscle (O'Byrne et al., 1984; O'Byrne et al., 

23 1983; Holtzman et al., 1979). It is also recognized that increased bronchial reactivity can 

24 be both a rapidly occurring and a persistent response to O3 (Foster and Freed, 1999). 

25 Tachykinins and secondary oxidation products of O3 have been proposed as mediators of 

26 the early response and inflammation-derived products have been proposed as mediators 

27 of the later response (Foster and Freed, 1999). Furthermore, bronchial reactivity may be 

28 increased as a result of O3-mediated generation of ROS. 

29 Ozone-induced increases in epithelial permeability, which could improve access of 

30 agonist to smooth muscle receptors, may be one mechanism of sensitization through a 

31 direct effect on bronchial smooth muscle (Holtzman et al., 1979). As noted above, a 

32 correlation between airway permeability and AHR has been reported in O3-exposed sheep 

33 (Abraham et al., 1984) but not in O3-exposed human subjects (Que et al., 2011). 
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1 Neurally-mediated sensitization has been demonstrated. In human subjects exposed for 

2 2 hours to 600 ppb O3 while exercising at a light level, pretreatment with atropine 

3 inhibited O3-induced AHR, suggesting the involvement of cholinergic postganglionic 

4 pathways (Holtzman et al., 1979). Animal studies have demonstrated that O3-induced 

5 AHR involved vagally-mediated responses (rabbits, 0.2 ppm O3, 72 hours) (Freed et al., 

6 1996) and local axon reflex responses through bronchopulmonary C-fiber-mediated 

7 release of SP (guinea pigs, 0.8 ppm O3, 2 hours) (Joad et al., 1996). Further, pretreatment 

8 with capsaicin to deplete nerve fibers of SP blocked O3-mediated AHR (guinea pigs, 

9 1-2 ppm O3, 2-2.25 hours) (Tepper et al., 1993). Other investigators demonstrated that SP 

10 released from airway nociceptive neurons in ferrets contributed to O3-induced AHR 

11 (2 ppm O3, 3 hours) (Wu et al., 2008c; Wu et al., 2003). 

12 Some evidence suggests the involvement of arachidonic acid metabolites and neutrophils 

13 in mediating O3-induced AHR (Seltzer et al., 1986; Fabbri et al., 1985). Increased BAL 

14 neutrophils and cyclooxygenase products were found in one study demonstrating AHR in 

15 human subjects exercising at a heavy level immediately postexposure to 600 ppb O3 for 

16 2 hours (Seltzer et al., 1986). Another study found that ibuprofen pretreatment had no 

17 effect on AHR in human subjects exercising at a heavy level following exposure to 

18 400 ppb O3 for 2 hours, although spirometric responses were blunted (Hazucha et al., 

19 1996). This study measured arachidonic acid metabolites and provided evidence that that 

20 the arachidonic acid metabolites whose generation was blocked by ibuprofen, 

21 (i.e., prostaglandins, thromboxanes and some leukotrienes) did not play a role in AHR. 

22 Experiments in dogs exposed for 2 hours to 2.1 ppm O3 demonstrated a close correlation 

23 between O3-induced AHR and airways neutrophilic inflammation measured in tissue 

24 biopsies (Holtzman et al., 1983). Furthermore, the increased AHR observed in dogs 

25 following O3 exposure (3 ppm, 2 hours) was inhibited by neutrophil depletion (O'Byrne 

26 et al., 1983) and by pre-treatment with inhibitors of arachidonic acid metabolism. In one 

27 of these studies, indomethacin pre-treatment did not prevent airways neutrophilia in 

28 response to O3 (3 ppm, 2 hours) providing evidence that the subset of arachidonic acid 

29 metabolites whose generation was inhibitable by the cyclooxygenase inhibitor 

30 indomethacin (i.e., prostaglandins and thromboxanes) was not responsible for neutrophil 

31 influx (O'Byrne et al., 1984). It should be noted that these studies did not measure 

32 whether the degree to which the inhibitor blocked arachidonic acid metabolism and thus 

33 their results should be interpreted with caution.Taken together, these findings suggest that 

34 arachidonic acid metabolites may be involved in the AHR response following O3

35 exposure in dogs. Studies probing the role of neutrophils in mediating the AHR response 

36 have provided inconsistent results (Al-Hegelan et al., 2011). 

37 Evidence for cytokine and chemokine involvement in the AHR response to O3 has been 

38 described. Some studies have suggested a role for TNF-α (mice, 0.5 and 2 ppm O3, 
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1 3 hours) (Cho et al., 2001; Shore et al., 2001) and IL-1 (mice and ferrets, 2 ppm O3, 

2 3 hours) (Wu et al., 2008c; Park et al., 2004). The latter study found that SP expression in 

3 airway neurons was upregulated by IL-1 that was released in response to O3. Other 

4 studies in mice have demonstrated a key role for CXCR2, the chemokine receptor for the 

5 neutrophil chemokines KC and MIP-2, but not for IL-6 in O3-mediated AHR (1 ppm O3, 

6 3 hours) (Johnston et al., 2005a; Johnston et al., 2005b). In contrast, CXCR2 and IL-6 

7 were both required for neutrophil influx in this model (Johnston et al., 2005a; Johnston et 

8 al., 2005b), as discussed above. Williams et al. (2008b) demonstrated that the Th2 

9 cytokine IL-13 contributed to AHR, as well as to airways neutrophilia, in mice (3 ppm 

10 O3, 3 hours).

11 Other studies have focused on the role of TLR4. Hollingsworth et al. (2004) measured 

12 AHR, as well as airways neutrophilia, in mice 6 and 24 hours following acute (2 ppm O3

13 for 3 hours) and subchronic (0.3 ppm for 3 days) exposure to O3. TLR4 is a key 

14 component of the innate immune system and is responsible for the immediate 

15 inflammatory response seen following challenge with endotoxin and other pathogen16 associated substances. In this study, a functioning TLR4 was required for the full AHR 

17 response following O3 exposure but not for airways neutrophilia (Hollingsworth et al., 

18 2004). These findings are complemented by an earlier study demonstrating that O3 effects 

19 on lung hyperpermeability required a functioning TLR4 (mice, 0.3 ppm O3, 72 hours) 

20 (Kleeberger et al., 2000). Williams et al. (2007b) found that TLR2, TLR4 and the TLR 

21 adaptor protein MyD88 contributed to AHR in mice (3 ppm O3, 3 hours). Ozone was also 

22 found to upregulate MyD88, TLR4 and TLR4 gene expression in this model (Williams et 

23 al., 2007b). Furthermore, a recent study reported O3-induced AHR that required TLR4 

24 and MyD88 in mice exposed to 1 ppm O3 for 3 hours (Li et al., 2011). 

25 A newly recognized mechanistic basis for O3-induced AHR is provided by studies 

26 focusing on the role of hyaluronan following O3 exposure in mice (Garantziotis et al., 

27 2010; Garantziotis et al., 2009). Hyaluronan is an extracellular matrix component that is 

28 normally found in the ELF as a large polymer. Briefly, TLR4 and CD44 were found to 

29 mediate AHR in response to O3 and hyaluronan. Exposure to 2 ppm O3 for 3 hours 

30 resulted in enhanced AHR and elevated levels of soluble low molecular weight 

31 hyaluronan in the BALF 24-hours postexposure (Garantziotis et al., 2010; Garantziotis et 

32 al., 2009). Ozone may have caused the depolymerization of hyaluronan to soluble 

33 fragments that are known to be endogenous ligands of the CD44 receptor and TLR4 in 

34 the macrophage (Jiang et al., 2005). In the two recent studies, O3-induced AHR was 

35 attenuated in CD44 and TLR4-deficient mice (Garantziotis et al., 2010; Garantziotis et 

36 al., 2009). Hyaluronan fragment-mediated stimulation of AHR was found to require 

37 functioning CD44 receptor and TLR4 (Garantziotis et al., 2010; Garantziotis et al., 2009). 

38 In contrast, high-molecular-weight hyaluronan blocked AHR in response to O3
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1 (Garantziotis et al., 2009). In another study high-molecular-weight hyaluronan enhanced 

2 repair of epithelial injury (Jiang et al., 2005). These studies provide a link between innate 

3 immunity and the development of AHR following O3 exposure, and indicate a role for 

4 TLR4 in increasing airways responsiveness. While TLR4-dependent responses usually 

5 involve activation of NF-κB and the upregulation of proinflammatory factors, the precise 

6 mechanisms leading to AHR are unknown (Al-Hegelan et al., 2011). 

7 In guinea pigs, AHR was found to be mediated by different pathways at 1- and 3-days 

8 postexposure to a single exposure of O3 (2 ppm for 4 hours) (Verhein et al., 2011; Yost et 

9 al., 2005). At 1 day, AHR was due to activation of airway parasympathetic nerves rather 

10 than to an exposure-related effect on smooth muscle (Yost et al., 2005). This effect 

11 occurred as a result of O3-stimulated release of major basic protein from eosinophils 

12 (Yost et al., 2005). Major basic protein is known to block inhibitory M2 muscarinic 

13 receptors that normally dampen acetylcholine release from parasympathetic nerves (Yost 

14 et al., 2005). The resulting increase in acetylcholine release caused an increase in smooth 

15 muscle contraction following O3 exposure (Yost et al., 2005). Eosinophils played a 

16 different role 3-days postexposure to O3 in guinea pigs (Yost et al., 2005). Ozone17 mediated influx of eosinophils into lung airways resulted in a different population of cells 

18 present 3-days postexposure compared to those present at 1 day (Yost et al., 2005). At 

19 this time point, eosinophil-derived major basic protein increased smooth muscle 

20 responsiveness to acetylcholine which also contributed to AHR (Yost et al., 2005). 

21 However, the major effect of eosinophils was to protect against vagal hyperreactivity 

22 (Yost et al., 2005). The authors suggested that these beneficial effects were due to the 

23 production of nerve growth factor (Yost et al., 2005). Further work by these investigators 

24 demonstrated a key role for IL-1β in mediating AHR 3-days postexposure to O3 (Verhein 

25 et al., 2011). In this study, IL-1β increased nerve growth factor and SP that acted through 

26 the NK1 receptor to cause vagally-mediated bronchoconstriction (Verhein et al., 2011). 

27 The mechanism by which SP caused acetylcholine release from parasympathetic nerves 

28 following O3 exposure was not determined (Verhein et al., 2011). Taken together, the 

29 above study results indicate that mechanisms involved in O3-mediated AHR can vary 

30 over time postexposure and that eosinophils and SP can play a role. Results of this animal 

31 model may provide some insight into allergic airways disease in humans that is 

32 characterized by eosinophilia (Section 5.4.2.2).

5.3.6 Modification of Innate/Adaptive Immune System Responses

33 Host defense depends on effective barrier function and on innate immunity and adaptive 

34 immunity (Al-Hegelan et al., 2011). The effects of O3 on barrier function in the airways 

35 was discussed above (Section 5.3.4). This section focuses on the mechanisms by which 
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1 O3 impacts innate and adaptive immunity. Both tissue damage and foreign pathogens are 

2 triggers for the activation of the innate immune system. This results in the influx of 

3 inflammatory cells such as neutrophils, mast cells, basophils, eosinophils, monocytes and 

4 dendritic cells and the generation of cytokines such as TNF-α, IL-1, IL-6, KC and IL-17. 

5 Further, innate immunity encompasses the actions of complement and collections, and 

6 the phagocytic functions of macrophages, neutrophils and dendritic cells. Airway 

7 epithelium also contributes to innate immune responses. Innate immunity is highly 

8 dependent on cell signaling networks involving TLR4. Adaptive immunity provides 

9 immunologic memory through the actions of B and T-cells. Important links between the 

10 two systems are provided by dendritic cells and antigen presentation. Recent studies 

11 demonstrate that exposure to O3 modifies cells and processes which are required for 

12 innate immunity, contributes to innate-adaptive immune system interaction and primes 

13 pulmonary immune responses to endotoxin. 

14 Ozone exposure of human subjects resulted in recruitment of activated innate immune 

15 cells to the airways. Healthy individuals were exposed to 80 ppb O3 for 6.6 hours while 

16 exercising at a moderate level and airways inflammation was characterized in induced 

17 sputum 18-hours postexposure (Alexis et al., 2010). Previous studies demonstrated that 

18 induced sputum contains liquid and cellular constituents of the ELF from central 

19 conducting airways (Alexis et al., 2001b) and also identified these airways as a site of 

20 preferential O3 absorption during exercise (Hu et al., 1994). Ozone exposure resulted in 

21 increased numbers of neutrophils, airway monocytes and dendritic-like cells in sputum 

22 (Alexis et al., 2010). In addition, increased expression of cell surface markers 

23 characteristic of innate immunity and antigen presentation (i.e., CD-14 and HLA-DR) 

24 was demonstrated on airway monocytes (Alexis et al., 2010). Enhanced antigen 

25 presentation contributes to exaggerated T-cell responses and promotes Th2 inflammation 

26 and an allergic phenotype (Lay et al., 2007). Upregulation of pro-inflammatory cytokines 

27 was also demonstrated in sputum of O3-exposed subjects (Alexis et al., 2010). One of 

28 these cytokines, IL-12p70, correlated with numbers of dendritic-like cells in the sputum, 

29 and is an indicator of dendritic cell activation (Alexis et al., 2010). These authors have 

30 previously reported that exposure of human subjects exercising at a light to moderate 

31 level to 400 ppb O3 for 2 hours resulted in activation of monocytes and macrophages 

32 (Lay et al., 2007), which could play a role in exacerbating existing asthma by activating 

33 allergen-specific memory T-cells. The current study confirms these findings and extends 

34 them by suggesting a potential mechanism whereby O3-activated dendritic cells could 

35 stimulate naïve T-cells to promote the development of asthma (Alexis et al., 2010). A 

36 companion study by these same investigators (described in detail in Section 5.4.2.1) 

37 provides evidence of dendritic cell activation, measured as increased expression of HLA38 DR, in a subset of the human subjects (GSTM1 null) exposed to 400 ppb O3 for 2 hours 

39 while exercising at a light to moderate level (Alexis et al., 2009). Since dendritic cells are 
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1 a link between innate and adaptive immunity, these studies provide evidence for an 

2 O3-mediated interaction between the innate and adaptive immune systems. 

3 Another recent study linked O3-mediated activation of the innate immune system to the 

4 development of non-specific AHR in a mouse model (Pichavant et al., 2008). Repeated 

5 exposure to 1 ppm O3 for 3 hours (3 days over a 5 day period) induced non-specific AHR 

6 measured 24 hours following the last exposure (Pichavant et al., 2008). This response 

7 was found to require NKT-cells, which are effector lymphocytes of innate immunity, as 

8 well as IL-17 and airways neutrophilia (Pichavant et al., 2008). Since glycolipids such as 

9 galactosyl ceramide are ligands for the invariant CD1 receptor on NKT-cells and serve as 

10 endogenous activators of NKT-cells, a role for O3-oxidized lipids in activating NKT-cells 

11 was proposed (Pichavant et al., 2008). The authors contrasted this innate immunity 

12 pathway with that of allergen-provoked specific AHR which involves adaptive immunity, 

13 the cytokines IL-4, IL -13, IL-17, and airways eosinophilia (Pichavant et al., 2008). 

14 Interestingly, NKT-cells were required for both the specific AHR provoked by allergen 

15 and the non-specific AHR provoked by O3 (Pichavant et al., 2008). Different cytokine 

16 profiles of the NKT-cells from allergen and O3-exposed mice were proposed to account 

17 for the different pathways (Pichavant et al., 2008). More recently, NKT-cells have been 

18 found to function in both innate and adaptive immunity (Vivier et al., 2011).

19 An interaction between allergen and O3 in the induction of nonspecific AHR was shown 

20 in another animal study (Larsen et al., 2010). Mice were sensitized with the aerosolized 

21 allergen OVA on 10 consecutive days followed by exposure to O3 (0.1-0.5 ppm for 

22 3 hours) (Larsen et al., 2010). While allergen sensitization alone did not alter airways 

23 responsiveness to a nonspecific challenge, O3 exposure of sensitized mice resulted in 

24 nonspecific AHR at 6- and 24-hours postexposure (Larsen et al., 2010). The effects of O3

25 on AHR were independent of airways eosinophilia and neutrophilia (Larsen et al., 2010). 

26 However, OVA pretreatment led to goblet cell metaplasia which was enhanced by O3

27 exposure (Larsen et al., 2010). It should be noted that OVA sensitization using only 

28 aerosolized antigen in this study is less common than the usual procedure for OVA 

29 sensitization achieved by one or more initial systemic injections of OVA and adjuvant 

30 followed by repeated inhalation exposure to OVA. This study also points to an interaction 

31 between innate and adaptive immune systems in the development of the AHR response. 

32 Furthermore, O3 was found to act as an adjuvant for allergic sensitization (Hollingsworth 

33 et al., 2010). Oropharyngeal aspiration of OVA on day 0 and day 6 failed to lead to 

34 allergic sensitization unless mice were first exposed to 1 ppm O3 for 2 hours 

35 (Hollingsworth et al., 2010). The O3-mediated response involved Th2 (IL-4, IL-5 and 

36 IL-9) and Th17 cytokines (IL-17) and was dependent on a functioning TLR4 

37 (Hollingsworth et al., 2010). Ozone exposure also activated OVA-bearing dendritic cells 
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1 in the thoracic lymph nodes, as measured by the presence of the CD86 surface marker, 

2 which suggests naïve T-cell stimulation and the involvement of Th2 pathways 

3 (Hollingsworth et al., 2010). Thus the adjuvant effects of O3 may be due to activation of 

4 both innate and adaptive immunity. 

5 Priming of the innate immune system by O3 was reported by Hollingsworth et al. (2007). 

6 In this study, exposure of mice to 2 ppm O3 for 3 hours led to nonspecific AHR at 24-

7 and 48-hours postexposure, an effect which subsided by 72 hours (Hollingsworth et al., 

8 2007). However, in mice treated with aerosolized endotoxin immediately following O3

9 exposure, AHR was greatly enhanced at 48-and 72-hours postexposure (Hollingsworth et 

10 al., 2007). In addition, O3 pre-exposure was found to reduce the number of inflammatory 

11 cells in the BALF, to increase cytokine production and total protein in the BALF and to 

12 increase systemic IL-6 following exposure to endotoxin (Hollingsworth et al., 2007). 

13 Furthermore, O3 stimulated the apoptosis of alveolar macrophages 24-hours 

14 postexposure, an effect which was greatly enhanced by endotoxin treatment. Apoptosis of 

15 circulating blood monocytes was also observed in response to the combined exposures 

16 (Hollingsworth et al., 2007). Ozone pre-exposure enhanced the response of lung 

17 macrophages to endotoxin (Hollingsworth et al., 2007). Taken together, these findings 

18 demonstrated that O3 exposure increased innate immune responsiveness to endotoxin. 

19 The authors attributed these effects to the increased surface expression of TLR4 and 

20 increased signaling in macrophages observed in the study (Hollingsworth et al., 2007). It 

21 was proposed that the resulting decrease in airway inflammatory cells could account for 

22 O3-mediated decreased clearance of bacterial pathogens observed in numerous animal 

23 models (Hollingsworth et al., 2007). 

24 More recently, these authors demonstrated that hyaluronan contributed to the O3-primed 

25 response to endotoxin (Li et al., 2010). In this study, exposure of mice to 1 ppm O3 for 

26 3 hours resulted in enhanced responses to endotoxin, which was mimicked by 

27 intratracheal instillation of hyaluronan fragments (Li et al., 2010). Hyaluronan, like O3, 

28 was also found to induce TLR4 receptor peripheralization in the macrophage membrane 

29 (Li et al., 2010; Hollingsworth et al., 2007), an effect which is associated with enhanced 

30 responses to endotoxin. This study and previous ones by the same investigators showed 

31 elevation of BALF hyaluronan in response to O3 exposure (Garantziotis et al., 2010; Li et 

32 al., 2010; Garantziotis et al., 2009), providing evidence that the effects of O3 on innate 

33 immunity are at least in part mediated by hyaluronan fragments. The authors note that 

34 excessive TLR4 signaling can lead to lung injury and suggest that O3 may be responsible 

35 for an exaggerated innate immune response which may underlie lung injury and 

36 decreased host defense (Li et al., 2010). 
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1 Activation or upregulation of the immune system has not been reported in all studies. 

2 Impaired antigen-specific immunity was demonstrated following subacute O3 exposure 

3 (0.6 ppm, 10 h/day for 15 days) in mice (Feng et al., 2006). Specifically, O3 exposure 

4 altered the lymphocyte subset and cytokine profile and impacted thymocyte early 

5 development leading to immune dysfunction. Further, recent studies demonstrated SP-A 

6 oxidation in mice exposed for 3-6 hours to 2 ppm O3. SP-A is an important innate 

7 immune protein which plays a number of roles in host defense including acting as 

8 opsonin for the recognition of some pathogens (Haque et al., 2009). These investigations 

9 found that O3-mediated carbonylation of purified SP-A was associated with impaired 

10 macrophage phagocytosis in vitro (Mikerov et al., 2008c). In addition, O3 exposure 

11 (2 ppm for 3 hours) in mice was found to increase susceptibility to pneumonia infection 

12 in mice through an impairment of SP-A dependent phagocytosis (Mikerov et al., 2008b; 

13 Mikerov et al., 2008a). Furthermore, early life exposure to O3 in infant monkeys followed 

14 by a recovery period led to hyporesponsiveness to endotoxin (Maniar-Hew et al., 2011), 

15 as discussed below and in Section 5.4.2.4 and Section 7.2.3.1. 

16 Taken together, results of recent studies provide evidence that O3 alters host 

17 immunologic response and leads to immune system dysfunction through its effects on 

18 innate and adaptive immunity.

5.3.7 Airways Remodeling

19 The nasal airways, conducting airways and distal airways (i.e., respiratory bronchioles or 

20 CAR depending on the species) have all been identified as sites of O3-mediated injury 

21 and inflammation (Mudway and Kelly, 2000). At all levels of the RT, loss of sensitive 

22 epithelial cells, degranulation of secretory cells, proliferation of resistant epithelial cells 

23 and neutrophilic influx have been observed as a result of O3 exposure (Mudway and 

24 Kelly, 2000; Cho et al., 1999). An important study (Plopper et al., 1998) conducted in 

25 adult rhesus monkeys (0.4 and 1.0 ppm O3 for 2 hours at rest) found that 1 ppm O3

26 resulted in the greatest epithelial injury in the respiratory bronchioles immediately 

27 postexposure although injury was observed at all of the RT sites studied except for the 

28 lung parenchyma. Exposure to 0.4 ppm O3 resulted in epithelial injury only in the 

29 respiratory bronchioles. Initial cellular injury correlated with site-specific O3 dose since 

the respiratory bronchioles received the greatest O3 dose (18 30 O mass/lung weight) and 

31 sustained the greatest initial cellular injury. The respiratory bronchioles were also the site 

32 of statistically significant GSH reduction. In addition, a study in isolated perfused rat 

33 lungs found greater injury in conducting airways downstream of bifurcations where local 

34 doses of O3 were higher (Postlethwait et al., 2000).
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1 In addition to the degree of initial injury, the degree of airways inflammation due to O3

2 may have important long-term consequences since airways inflammation may lead to 

3 tissue injury (Balmes et al., 1996). Persistent inflammation and injury, observed in animal 

4 models of chronic and intermittent exposure to O3, are associated with airways 

5 remodeling, including mucous cell metaplasia of nasal transitional epithelium (Harkema 

6 et al., 1999; Hotchkiss et al., 1991) and bronchiolar metaplasia of alveolar ducts 

7 (Mudway and Kelly, 2000). Fibrotic changes such as deposition of collagen in the 

8 airways and sustained lung function decrements especially in small airways have also 

9 been demonstrated as a response to chronic O3 exposure (Mudway and Kelly, 2000; 

10 Chang et al., 1992). These effects, described in detail in Section 7.2.3.1, have been 

11 demonstrated in rats exposed to levels of O3 as low as 0.25 ppm. Mechanisms responsible 

12 for the resolution of inflammation and the repair of injury remain to be clarified and there

13 is only a limited understanding of the biological processes underlying long-term 

14 morphological changes. However, a recent study in mice demonstrated a key role for the 

15 TGF-β signaling pathway in the deposition of collagen in the airways wall following 

16 chronic intermittent exposure to 0.5 ppm O3 (Katre et al., 2011). Studies in infant 

17 monkeys have also documented effects of chronic intermittent exposure to 0.5 ppm O3 on 

18 the developing lung and immune system. Extensive discussion of this topic is found in 

19 Section 5.4.2.4 (Lifestage) and in Section 7.2.3.1. 

20 It should be noted that repeated exposure to O3 results in attenuation of some O3-induced 

21 responses, including those associated with the activation of neural reflexes 

22 (e.g., decrements in pulmonary function), as discussed in Section 5.3.2. However, 

23 numerous studies demonstrate that some markers of injury and inflammation remain 

24 increased during multi-day exposures to O3. Mechanisms responsible for attenuation, or 

25 the lack thereof, are incompletely understood. 

5.3.8 Systemic Inflammation and Oxidative/Nitrosative Stress

26 Extrapulmonary effects of O3 have been noted for decades (U.S. EPA, 2006b). It has 

27 been proposed that lipid oxidation products resulting from reaction of O3 with lipids in 

28 the ELF are responsible for systemic effects, however it is not known whether they gain 

29 access to the vascular space (Chuang et al., 2009). Alternatively, extrapulmonary release 

30 of diffusible mediators may initiate or propagate inflammatory responses in the vascular 

31 or systemic compartments (Cole and Freeman, 2009). A role for O3 in modulating 

32 endothelin, a potent vasoconstrictor, has also been proposed. Studies in rats found that 

33 exposure to 0.4 and 0.8 ppm O3 induced endothelin system genes in the lung and 

34 increased circulating levels of endothelin (Thomson et al., 2006; Thomson et al., 2005). 

35 Systemic oxidative stress may be suggested by studies in humans which reported 
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1 associations between O3 exposure and levels of plasma 8-isoprostanes and the presence 

2 of peripheral blood lymphocyte micronuclei (Chen et al., 2007a; Chen et al., 2006a). 

3 However, plasma isoprostanes are not a direct measure of systemic oxidative stress since 

4 they are stable and can be generated in any compartment before diffusion into the 

5 vascular space. Evidence of O3-mediated systemic oxidative stress is better provided by 

6 new animal studies described below. 

7 Ozone-induced perturbations of the cardiovascular system were recently investigated in 

8 young mice and monkeys (Chuang et al., 2009) and in rats (Kodavanti et al., 2011; 

9 Perepu et al., 2010) (see Section 6.3.3 and Section 7.3.1.2). These are the first studies to 

10 suggest that systemic oxidative stress and inflammation play a mechanistic role in 

11 O3-induced effects on the systemic vascular and heart. Exposure to 0.5 ppm O3 for 5 days 

12 resulted in oxidative/nitrosative stress, vascular dysfunction and mitochondrial DNA 

13 damage in the aorta (Chuang et al., 2009). Chronic exposure to 0.8 ppm O3 resulted in an 

14 enhancement of inflammation and lipid peroxidation in the heart following an ischemia15 reperfusion challenge (Perepu et al., 2010). In addition, chronic intermittent exposure to 

16 0.4 ppm O3 increased aortic levels of mRNA for biomarkers of oxidative stress, 

17 thrombosis, vasoconstriction and proteolysis and aortic lectin-like oxidized-low density 

18 lipoprotein receptor-1(LOX-1) mRNA and protein levels (Kodavanti et al., 2011). The 

19 latter study suggests a role for circulating oxidized lipids in mediating the effects of O3. 

20 Systemic inflammation and oxidative/nitrosative stress may similarly affect other organ 

21 systems as well as the plasma compartment. Circulating cytokines have the potential to 

22 enter the brain through diffusion and active transport and to contribute to 

23 neuroinflammation, neurotoxicity, cerebrovascular damage and a break-down of the 

24 blood brain barrier (Block and Calderón-Garcidueñas, 2009) (see Section 6.4 and 

25 Section 7.5). They can also activate neuronal afferents (Block and Calderón-Garcidueñas, 

26 2009). Vagal afferent pathways originating in the RT may also be responsible for 

27 O3-mediated activation of nucleus tractus solitarius neurons which resulted in neuronal

28 activation in stress-responsive regions of the CNS in rats (0.5 or 2 ppm O3 for 

29 1.5-120 hours) (Gackière et al., 2011). Recent studies have demonstrated O3-induced 

30 brain lipid peroxidation, cytokine production in the brain and upregulated expression of 

VEGF in rats (0.5 ppm O3 31 , 3 hours or 0.25-0.5 ppm O3, 4 h/day, 15-60 days) (Guevara32 Guzmán et al., 2009; Araneda et al., 2008; Pereyra-Muñoz et al., 2006). Further, 

33 O3-induced oxidative stress resulted in increased plasma lipid peroxides (0.25 ppm, 

34 4h/day, 15-60 days) (Santiago-López et al., 2010), which was correlated with damage to 

35 specific brain regions (Pereyra-Muñoz et al., 2006). 

36 Oxidative stress is one mechanism by which testicular and sperm function may be 

37 disrupted (see Section 7.4.1). Studies in Leydig cells in vitro have demonstrated that 
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1 steroidogenesis is blocked by oxidative stress (Diemer et al., 2003). It has been proposed 

2 that lipid peroxidation of sperm plasma membrane may lead to impaired sperm mobility 

3 and decreased sperm quality (Agarwal et al., 2003). Further, it has been proposed that 

4 oxidative stress may damage DNA in the sperm nucleus and lead to apoptosis and a 

5 decline in sperm counts (Agarwal et al., 2003). One study reported an association 

6 between O3 exposure and semen quality and suggested oxidative stress as an underlying 

7 mechanism (Sokol et al., 2006). Additional evidence is required to substantiate this link. 

8 A role for plasma antioxidants in modulating O3-induced respiratory effects was 

9 suggested by a recent study (Aibo et al., 2010). In this study, pretreatment of rats with a

10 high dose of acetaminophen resulted in increased levels of plasma cytokines and the 

11 influx of inflammatory cells into the lung following O3 exposure (0.25-0.5 ppm, 6 hours) 

12 (Aibo et al., 2010). These effects were not observed in response to O3 alone. 

13 Furthermore, acetaminophen-induced liver injury was exacerbated by O3 exposure. A 

14 greater increase in hepatic neutrophil accumulation and greater alteration in gene 

15 expression profiles was observed in mice exposed to O3 and acetaminophen compared 

16 with either exposure alone (Aibo et al., 2010). Although not measured in this study, 

17 glutathione depletion in the liver is known to occur in acetaminophen toxicity. Since liver 

18 glutathione is the source of plasma glutathione, acetaminophen treatment may have 

19 lowered plasma glutathione levels and altered the redox balance in the vascular 

20 compartment. These findings indicate interdependence between RT, plasma and liver 

21 responses to O3, possibly related to glutathione status. 

5.3.9 Impaired Alveolar-Arterial Oxygen Transfer 

22 O3 may impair alveolar-arterial oxygen transfer and reduce the supply of arterial oxygen 

23 to the myocardium. This may have a greater impact in individuals with compromised 

24 cardiopulmonary systems. Gong et al. (1998) provided evidence of a small decrease in 

25 arterial oxygen saturation in human subjects exposed for 3 hours to 300 ppb O3 while 

26 exercising at a light to moderate level. In addition, Delaunois et al. (1998) demonstrated 

27 pulmonary vasoconstriction in O3-exposed rabbits (0.4 ppm, 4 hours). Although of 

28 interest, the contribution of this pathway to O3-induced cardiovascular effects remains 

29 uncertain. 

5.3.10 Summary

30 This section summarizes the modes of action and toxicity pathways resulting from O3

31 inhalation (Figure 5-8). These pathways provide a mechanistic basis for the health effects 
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1 which are described in detail in Chapters 6 and 7. Three distinct short-term responses 

2 have been well-characterized in humans challenged with O3: decreased pulmonary 

3 function, airways inflammation, and increased bronchial reactivity. In addition, O3

4 exposure exacerbates, and possibly also causes, asthma and allergic airways disease in 

5 humans. Animal studies have demonstrated airways remodeling and fibrotic changes in 

6 response to chronic and intermittent O3 exposures and a wide range of other responses. 

7 While the RT is the primary target tissue, cardiovascular and other organ effects occur 

8 following short- and long-term exposures of animals to O3. 

Figure 5-8 The modes of action/possible pathways underlying the health 

effects resulting from inhalation exposure to ozone.

9 The initial key event in the toxicity pathway of O3 is the formation of secondary oxidation 

10 products in the RT. This mainly involves direct reactions with components of the ELF. 

11 The resulting secondary oxidation products transmit signals to the epithelium, 

12 nociceptive sensory nerve fibers and, if present, dendritic cells, mast cells and 

13 eosinophils. Thus, the effects of O3 are mediated by components of ELF and by the 

Mode of Action/Possible Pathways

Formation of secondary oxidation products

Ozone + Respiratory Tract 

Systemic inflammation and 

oxidative/nitrosative stress

Activation 

of neural 

reflexes

Airways 

remodeling

Sensitization 

of bronchial 

smooth muscle

Alteration 

of epithelial 

barrier 

function

Initiation of 

inflammation 

Modification

of innate and 

adaptive

immunity

Extrapulmonary Effects

Decrements in pulmonary function

Pulmonary inflammation/oxidative stress

Increases in airways permeability

Airways hyperresponsiveness

Exacerbation/induction of asthma

Decreased host defenses

Epithelial metaplasia and fibrotic changes

Altered lung development 
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1 multiple cell types found in the RT. Further, oxidative stress is an implicit part of this 

2 initial key event. 

3 Another key event in the toxicity pathway of O3 is the activation of neural reflexes which 

4 lead to decrements in pulmonary function (see Section 6.2.1). Evidence is accumulating 

5 that secondary oxidation products are responsible for this effect. Eicosanoids have been 

6 implicated in humans, while both eicosanoids and aldehydes are effective in animal 

7 models. Different receptors on bronchial C-fibers have been shown to mediate separate 

8 effects of O3 on pulmonary function. Nociceptive sensory nerves are involved in the 

9 involuntary truncation of inspiration which results in decreases in FVC, FEV1, tidal 

10 volume and pain upon deep inspiration. Opioids block these responses while atropine has 

11 only a minimal effect. New evidence in an animal model suggests that TRPA1 receptors 

12 on bronchial C-fibers mediate this pathway. Ozone exposure also results in activation of 

13 vagal sensory nerves and a mild increase in airway obstruction measured as increased 

14 sRaw. Atropine and β-adrenergic agonists greatly inhibit this response in humans 

15 indicating that the airways obstruction is due to bronchoconstriction. Other studies in 

16 humans implicated SP release from bronchial C-fibers resulting in airway narrowing due 

17 to either neurogenic edema or bronchoconstriction. New evidence in an animal model 

18 suggests that the SP-NK receptor pathway caused bronchoconstriction following O3

19 exposure. Activation of neural reflexes also results in extrapulmonary effects such as 

20 bradycardia. 

21 Initiation of inflammation is also a key event in the toxicity pathway of O3. Secondary 

22 oxidation products, as well as chemokines and cytokines elaborated by airway epithelial 

23 cells and macrophages, have been implicated in the initiation of inflammation. Vascular 

24 endothelial adhesion molecules may also play a role. Work from several laboratories

25 using human subjects and animal models suggest that O3 triggers the release of 

26 tachykinins such as SP from airway sensory nerves which could contribute to 

27 downstream effects including inflammation (see Section 6.2.3 and Section 7.2.4). 

28 Airways neutrophilia has been demonstrated in BALF, mucosal biopsy and induced 

29 sputum samples. Influx of mast cells, monocytes and macrophages also occur. 

30 Inflammation further contributes to O3-mediated oxidative stress. Recent investigations 

31 show that O3 exposure leads to the generation of hyaluronan fragments from high 

32 molecular weight polymers of hyaluronan normally found in the ELF in mice. 

33 Hyaluronan activates TLR4 and CD44-dependent signaling pathways in macrophages, 

34 and results in an increased number of macrophages in the BALF. Activation of these 

35 pathways occurs later than the acute neutrophilic response suggesting that they may 

36 contribute to longer-term effects of O3. The mechanisms involved in clearing 

37 O3-provoked inflammation remain to be clarified. It should be noted that inflammation, 
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1 as measured by airways neutrophilia, is not correlated with decrements in pulmonary 

2 function as measured by spirometry. 

3 A fourth key event in the toxicity pathway of O3 is alteration of epithelial barrier 

4 function. Increased permeability occurs as a result of damage to tight junctions between 

5 epithelial cells subsequent to O3-induced injury and inflammation. It may play a role in 

6 allergic sensitization and in AHR (see Section 6.2.2, Section 6.2.6, and Section 7.2.5). 

7 Tachykinins mediate this response while antioxidants may confer protection. Genetic 

8 susceptibility has been associated with a functioning Tlr4 and Nos2 genes. 

9 A fifth key event in the toxicity pathway of O3 is the sensitization of bronchial smooth 

10 muscle. Increased bronchial reactivity can be both a rapidly occurring and a persistent 

11 response. The mechanisms responsible for early and later AHR are not well-understood 

12 (see Section 6.2.2). One proposed mechanism of sensitization, O3-induced increases in 

13 epithelial permeability, would improve access of agonist to smooth muscle receptors. The 

14 evidence for this mechanism is not consistent. Another proposed mechanism, for which 

15 there is greater evidence, is neurally-mediated sensitization. In humans exposed to O3, 

16 atropine blocked the early AHR response indicating the involvement of cholinergic 

17 postganglionic pathways. Animal studies demonstrated that O3-induced AHR involved 

18 vagally-mediated responses and local axon reflex responses through bronchopulmonary 

19 C-fiber-mediated release of SP. Later phases of increased bronchial reactivity may 

20 involve the induction of IL-1β which in turn upregulates SP production. In guinea pigs, 

21 eosinophil-derived major basic protein contributed to the stimulation of cholinergic 

22 postganglionic pathways. A novel role for hyaluronan in mediating the later phase effects 

23 O3-induced AHR has recently been demonstrated. Hyaluronan fragments stimulated AHR 

24 in a TLR4- and CD44 receptor-dependent manner. Tachykinins and secondary oxidation 

25 products of O3 have been proposed as mediators of the early response and inflammation26 derived products have been proposed as mediators of the later response. Inhibition of 

27 arachidonic acid metabolism was ineffective in blocking O3-induced AHR in humans 

28 while in animal models mixed results were found. Other cytokines and chemokines have 

29 been implicated in the AHR response to O3 in animal models. 

30 A sixth key event in the toxicity pathway of O3 is the modification of innate/adaptive 

31 immunity. While the majority of evidence for this key event comes from animal studies, 

32 there are several studies suggesting that this pathway may also be relevant in humans. 

33 Ozone exposure of human subjects resulted in recruitment of activated innate immune 

34 cells to the airways. This included macrophages and monocytes with increased 

35 expression of cell surface markers characteristic of innate immunity and antigen 

36 presentation, the latter of which could contribute to exaggerated T-cell responses and the 

37 promotion of an allergic phenotype. Evidence of dendritic cell activation was observed in 
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1 GSTM1 null human subjects exposed to O3, suggesting O3-mediated interaction between 

2 the innate and adaptive immune systems. Animal studies further linked O3-mediated 

3 activation of the innate immune system to the development of nonspecific AHR, 

4 demonstrated an interaction between allergen and O3 in the induction of nonspecific 

5 AHR, and found that O3 acted as an adjuvant for allergic sensitization through the 

6 activation of both innate and adaptive immunity. Priming of the innate immune system by 

7 O3 was reported in mice. This resulted in an exaggerated response to endotoxin which 

8 included enhanced TLR4 signaling in macrophages. Ozone-mediated impairment of the 

9 function of SP-A, an innate immune protein, has also been demonstrated. Taken together 

10 these studies provide evidence that O3 can alter host immunologic response and lead to 

11 immune system dysfunction. These mechanisms may underlie the exacerbation and 

12 induction of asthma (see Section 6.2.6 and Section 7.2.1), as well as decreases in host 

13 defense (see Section 6.2.5 and Section 7.2.6). 

14 Another key event in the toxicity pathway of O3 is airways remodeling. Persistent 

15 inflammation and injury, which are observed in animal models of chronic and 

16 intermittent exposure to O3, are associated with morphologic changes such as mucous 

17 cell metaplasia of nasal epithelium, bronchiolar metaplasia of alveolar ducts and fibrotic 

18 changes in small airways (see Section 7.2.3). Mechanisms responsible for these responses 

19 are not well-understood. However a recent study in mice demonstrated a key role for the 

20 TGF-β signaling pathway in the deposition of collagen in the airway wall following 

21 chronic intermittent exposure to O3. Chronic intermittent exposure to O3 has also been 

22 shown to result in effects on the developing lung and immune system. 

23 Systemic inflammation and vascular oxidative/nitrosative stress are also key events in the 

24 toxicity pathway of O3. Extrapulmonary effects of O3 occur in numerous organ systems, 

25 including the cardiovascular, central nervous, reproductive and hepatic systems (see 

26 Section 6.3 to Section 6.5 and Section 7.3 to Section 7.5). It has been proposed that lipid 

27 oxidation products resulting from reaction of O3 with lipids and/or cellular membranes in 

28 the ELF are responsible for systemic responses, however it is not known whether they 

29 gain access to the vascular space. Alternatively, release of diffusible mediators from the 

30 lung into the circulation may initiate or propagate inflammatory responses in the vascular 

31 or in systemic compartments. 

5.4 Interindividual Variability in Response

32 Responses to O3 exposure are variable within the population (Mudway and Kelly, 2000). 

33 Some studies have shown a large range of pulmonary function responses to O3 among 

34 healthy young adults (i.e., 4 hours to 200 ppb O3 or for 1.5 hours to 420 ppb O3 while 
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1 exercising at a moderate level) (Hazucha et al., 2003; Balmes et al., 1996). Since 

2 individual responses were relatively consistent across time in these studies, it was thought 

3 that responsiveness reflected an intrinsic characteristic of the subject (Mudway and Kelly, 

4 2000). Other studies have shown that age and body mass index may influence 

5 responsiveness to O3. In human subjects exercising moderately and exposed to 420 ppb 

6 O3 for 1.5 hours, older adults were generally not responsive to O3 (Hazucha et al., 2003), 

7 while obese young women appeared to be more responsive than lean young women 

8 (Bennett et al., 2007). In another study, the observed lack of spirometric responsiveness 

9 in one group of human subjects was not attributable to the presence of endogenous 

10 endorphins, which could vary between individuals and which could potentially block C11 fiber stimulation by O3 (420 ppb, 2 hours, moderate exercise (Passannante et al., 1998). 

12 Other responses to O3 have also been characterized by a large degree of interindividual 

13 variability. For example, interindividual variability in the neutrophilic response has been 

14 noted in human subjects (Holz et al., 1999; Devlin et al., 1991; Schelegle et al., 1991). 

15 One study demonstrated a 3-fold difference in airways neutrophilia, measured as percent 

16 of total cells in proximal BALF, among human subjects exposed to 300 ppb O3 for 1 hour 

17 while exercising at a heavy level (Schelegle et al., 1991). Another study reported a 

18 20-fold difference in BAL neutrophils following exposure to 80-100 ppb O3 for 6.6 hours 

19 in human subjects exercising at a moderate level (Devlin et al., 1991). In contrast, 

20 reproducibility of intraindividual responses to 1-hour exposure to 250 ppb O3 in human 

21 subjects exercising at a light level, measured as sputum neutrophilia, was demonstrated 

22 by Holz et al. (1999). While the basis for the observed interindividual variability in 

23 responsiveness to O3 is not clear, both dosimetric and mechanistic factors are likely to 

24 contribute and will be discussed below. 

5.4.1 Dosimetric Considerations

25 Two studies have investigated the correlation of O3 uptake with the pulmonary function 

26 responses to O3 exposure (Reeser et al., 2005; Gerrity et al., 1994). These studies found 

27 that the large subject-to-subject variability in %ΔFEV1 response to O3 does not appear to 

28 have a dosimetric explanation. Reeser et al. (2005) found no significant relationship 

29 between %ΔFEV1 and fractional absorption of O3 using the bolus method. Contrary to 

30 previous findings, the percent change in dead space volume of the respiratory tract 

31 (%ΔVD) did not correlate with O3 uptake, possibly due to the contraction of dead space 

32 caused by airway closure. Gerrity et al. (1994) found that intersubject variability in FEV1

33 and airway resistance was not related to differences in the O3 dose delivered to the lower 

34 airways, whereas minute ventilation was predictive of FEV1 decrement. No study has yet 

35 demonstrated that subjects show a consistent pattern of O3 retention when re-exposed 
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1 over weeks of time, as has been shown to be the case for the FEV1 response, or that 

2 within-subject variation in FEV1 response is related to fluctuations in O3 uptake. 

3 However, these studies did not control for the differences in conducting airway volume 

4 between individuals. By controlling for conducting airway volume, it may be possible to 

5 estimate how much of the intersubject variation in FEV1 response at a given O3 exposure 

6 is due to actual inter-individual variability in dose. 

5.4.2 Mechanistic Considerations

7 This section considers mechanistic factors that may contribute to variability in responses 

8 between individuals. It has been proposed that some of the variability may be genetically 

9 determined (Yang et al., 2005a). Besides gene-environment interactions, other factors 

10 such as pre-existing diseases and conditions, nutritional status, lifestage, attenuation, and 

11 co-exposures may also contribute to inter-individual variability in responses to O3 and are 

12 discussed below. 

5.4.2.1 Gene-environment Interactions 

13 The pronounced interindividual variation in responses to O3 infers that genetic 

14 background may play an important role in responsiveness to O3 (Cho and Kleeberger, 

15 2007; Kleeberger et al., 1997) (see also Section 8.4). Strains of mice which are prone or 

16 resistant to O3-induced effects have been used to systematically identify candidate 

17 susceptibility genes. Using these recombinant inbred strains of mice and exposures to 

18 0.3 ppm O3 for up to 72 hours, genome wide linkage analyses (also known as positional 

19 cloning) demonstrated quantitative trait loci for O3-induced lung inflammation and 

20 hyperpermeability on chromosome 17 (Kleeberger et al., 1997) and chromosome 4 

21 (Kleeberger et al., 2000), respectively. More specifically, these studies found that Tnf, 

22 whose protein product is the inflammatory cytokine TNF-α, and Tlr4, whose protein 

23 product is TLR4, were candidate susceptibility genes (Kleeberger et al., 2000; Kleeberger 

24 et al., 1997). Other studies, which used targeted deletion, identified genes encoding iNOS 

25 and heat shock proteins as TLR4 effector genes (Bauer et al., 2011; Kleeberger et al., 

26 2001) and found that IL-10 protects against O3-induced pulmonary inflammation (Backus 

27 et al., 2010). Investigations in inbred mouse strains found that differences in expression 

28 of certain proteins, such as CCSP (1.8 ppm O3 for 3 hours) (Broeckaert et al., 2003) and 

29 MARCO (0.3 ppm O3 for up to 48 hours) (Dahl et al., 2007), were responsible for 

30 phenotypic characteristics, such as epithelial permeability and scavenging of oxidized 

31 lipids, respectively, which confer sensitivity to O3. 
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1 Genetic polymorphisms have received increasing attention as modulators of O3-mediated 

2 effects. Functionally relevant polymorphisms in candidate susceptibility genes have been 

3 studied at the individual and population level in humans, and also in animal models. 

4 Genes whose protein products are involved in antioxidant defense/oxidative stress and 

5 xenobiotic metabolism, such as glutathione-S-transferase M1 (GSTM1) and 

6 NADPH:quinone oxidoreductase 1 (NQO1), have also been a major focuses of these 

7 efforts. This is because oxidative stress resulting from O3 exposure is thought to 

8 contribute to the pathogenesis of asthma, and because xenobiotic metabolism detoxifies 

9 secondary oxidation products formed by O3 which contribute to oxidative stress (Islam et 

10 al., 2008). TNF-α is of interest since it is linked to a candidate O3 susceptibility gene and 

11 since it plays a key role in initiating airways inflammation (Li et al., 2006d). 

12 Polymorphisms of genes coding for GSTM1, NQO1 and TNF-α have been associated 

13 with altered risk of O3-mediated effects (Li et al., 2006d; Yang et al., 2005a; Romieu et 

14 al., 2004b; Corradi et al., 2002; Bergamaschi et al., 2001). Additional studies have 

15 focused on functional variants in other genes involved in antioxidant defense such as 

16 catalase (CAT), myeloperoxidase, heme oxygenase (HMOX-1) and manganese

17 superoxide dismutase (MnSOD) (Wenten et al., 2009; Islam et al., 2008). These studies 

18 are discussed below. 

19 GSTM1 is a phase II antioxidant enzyme which is transcriptionally regulated by 

20 NF-e2-related factor 2-antioxidant response element (Nrf2-ARE) pathway. A large 

21 proportion (40-50%) of the general public (across ethnic populations) has the 

22 GSTM1-null genotype, which has been linked to an increased risk of health effects due to 

23 exposure to air pollutants (London, 2007). A role for GSTs in metabolizing electrophiles 

24 such as 4-hydroxynonenal, which is a secondary oxidation product resulting from O3

25 exposure, has been demonstrated (Awasthi et al., 2004). A recent study found that the 

26 GSTM1 genotype modulated the time course of the neutrophilic inflammatory response 

27 following acute O3 exposure (400 ppb for 2 hours with light to moderate exercise) in 

28 healthy adults (Alexis et al., 2009). In GSTM1-null and -sufficient subjects, O3-induced 

29 sputum neutrophilia was similar at 4 hours. However, neutrophilia resolved by 24 hours 

30 in sufficient subjects but not in GSTM1-null subjects. In contrast, no differences in 

31 24 hour sputum neutrophilia were observed between GSTM1-null and -sufficient human 

32 subjects exposed to 60 ppb O3 for 2 hours with moderate exercise (Kim et al., 2011). It is 

33 not known whether the effect seen at the higher exposure level (Alexis et al., 2009) was 

34 due to the persistence of pro-inflammatory stimuli, impaired production of 

35 downregulators or impaired neutrophil apoptosis and clearance. However, a subsequent 

36 in vitro study by these same investigators found that GSTM1 deficiency in airway 

37 epithelial cells enhanced IL-8 production in response to 0.4 ppm O3 for 4 hours (Wu et 

38 al., 2011). Furthermore, NF-κB activation was required for O3-induced IL-8 production 

39 (Wu et al., 2011). Since IL-8 is a potent neutrophil activator and chemotaxin, this study 
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1 provides additional evidence for the role of GSTM1 as a modulator of inflammatory 

2 responses due to O3 exposure. 

3 In addition, O3 exposure increased the expression of the surface marker CD14 in airway 

4 neutrophils of GSTM1-null subjects to a greater extent than in sufficient subjects (Alexis 

5 et al., 2009). Furthermore, differences in airway macrophages were noted between the 

6 GSTM1-sufficient and -null subjects. Numbers of airway macrophages were decreased at 

7 4 and 24 hours following O3 exposure in GSTM1-sufficient subjects (Alexis et al., 2009). 

8 Airway macrophages in GSTM1-null subjects were greater in number and found to have 

9 greater oxidative burst and phagocytic capability than those of sufficient subjects. Airway 

10 macrophages and dendritic cells from GSTM1-null subjects exposed to O3 expressed 

11 higher levels of the surface marker HLA-DR, suggesting activation of the innate immune 

12 system (Alexis et al., 2009). These differences in inflammatory responses between the 

13 GSTM1-null and -sufficient subjects may provide biological plausibility for the 

14 differences in O3-mediated effects reported in controlled human exposure studies 

15 (Corradi et al., 2002; Bergamaschi et al., 2001). It should also be noted that GSTM1 

16 genotype did not affect the acute pulmonary function (i.e., spirometric) response to O3

17 which provides additional evidence for separate mechanisms underlying the effects of O3

18 on pulmonary function and inflammation in adults (Alexis et al., 2009). However, 

19 GSTM1-null asthmatic children were previously found to be more at risk of O3-induced 

20 effects on pulmonary function than GSTM1-sufficient asthmatic children (Romieu et al., 

21 2004b).

22 Another enzyme involved in the metabolism of secondary oxidation products is NQO1. 

23 NQO1 catalyzes the 2-electron reduction by NADPH of quinones to hydroquinones. 

24 Depending on the substrate, it is capable of both protective detoxification reactions and 

25 redox cycling reactions resulting in the generation of reactive oxygen species. A recent 

26 study using NQO1-null mice demonstrated that NQO1 contributes to O3-induced 

27 oxidative stress, AHR and inflammation following a 3-hour exposure to 1 ppm O3

28 (Voynow et al., 2009). These experimental results may provide biological plausibility for 

29 the increased biomarkers of oxidative stress and increased pulmonary function 

30 decrements observed in O3-exposed individuals bearing both the wild-type NQO1 gene 

31 and the null GSTM1 gene (Corradi et al., 2002; Bergamaschi et al., 2001). 

32 Besides enzymatic metabolism, other mechanisms participate in the removal of 

33 secondary oxidation products formed as a result of O3 inhalation. One involves 

34 scavenging of oxidized lipids via the macrophage receptor with collagenous structure 

35 (MARCO) expressed on the cell surface of alveolar macrophages. A recent study 

36 demonstrated increased gene expression of MARCO in the lungs of an O3-resistant C3H 

37 mouse strain (HeJ) but not in an O3-sensitive, genetically similar strain (OuJ) (Dahl et al., 
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1 2007). Upregulation of MARCO occurred in mice exposed to 0.3 ppm O3 for 

2 24-48 hours; inhalation exposure for 6 hours at this concentration was insufficient for this 

3 response. Animals lacking the MARCO receptor exhibited greater inflammation and 

4 injury, as measured by BAL neutrophils, protein and isoprostanes, following exposure to 

5 0.3 ppm O3 (Dahl et al., 2007). MARCO also protected against the inflammatory effects 

6 of oxidized surfactant lipids (Dahl et al., 2007). Scavenging of oxidized lipids may limit 

7 O3-induced injury since ozonized cholesterol species formed in the ELF (mice, 0.5-3 ppm 

8 O3, 3 hours) (Pulfer et al., 2005; Pulfer and Murphy, 2004) stimulated apoptosis and 

9 cytotoxicity in vitro (Gao et al., 2009b; Sathishkumar et al., 2009; Sathishkumar et al., 

10 2007b; Sathishkumar et al., 2007a).

11 Two studies reported relationships between TNF promoter variants and O3-induced 

12 effects in humans. In one study, O3-induced change in lung function was significantly 

13 lower in adult subjects with TNF promoter variants -308A/A and -308G/A compared with 

14 adult subjects with the variant -308G/G (Yang et al., 2005a). This response was 

15 modulated by a specific polymorphism of LTA (Yang et al., 2005a), a previously 

16 identified candidate susceptibility gene whose protein product is lymphotoxin-α 

17 (Kleeberger et al., 1997). In the second study, an association between the TNF promoter 

18 variant -308G/G and decreased risk of asthma and lifetime wheezing in children was 

19 found (Li et al., 2006d). The protective effect on wheezing was modulated by ambient O3

20 levels and by GSTM1 and GSTP1 polymorphisms. The authors suggested that the 

21 TNF-308 G/G genotype may have a protective role in the development of childhood 

22 asthma (Li et al., 2006d).

23 Similarly, a promoter variant of the gene HMOX-1, consisting of a smaller number of 

24 (GT)n repeats, was associated with a reduced risk for new-onset asthma in non-Hispanic 

25 white children (Islam et al., 2008). The number of (GT)n repeats in this promoter has 

26 been shown to be inversely related to the inducibility of HMOX-1. A modulatory effect of 

27 O3 was demonstrated since the beneficial effects of this polymorphism were seen only in 

28 children living in low O3 communities (Islam et al., 2008). This study also identified an 

29 association between a polymorphism of the CAT gene and increased risk of new-onset 

30 asthma in Hispanic children; however no modulation by O3 was seen (Islam et al., 2008). 

31 No association was observed in this study between a MnSOD polymorphism and asthma 

32 (Islam et al., 2008).

33 Studies to date indicate that some variability in individual responsiveness to O3 may be 

34 accounted for by functional genetic polymorphisms. Further, the effects of 

35 gene-environment interactions may be different in children and adults.
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5.4.2.2 Pre-existing Diseases and Conditions

1 Pre-existing diseases and conditions can alter the response to O3 exposure. For example, 

2 responsiveness to O3, as measured by spirometry, is decreased in smokers and individuals 

3 with COPD (U.S. EPA, 2006b). Asthma and allergic diseases are of major importance in 

4 this discussion. In individuals with asthma, there is increased responsiveness to 

5 bronchoconstrictor challenge. This results from a combination of structural and 

6 physiological factors including increased airway inner-wall thickness, smooth muscle 

7 responsiveness and mucus secretion. Although inflammation is likely to contribute, its 

8 relationship to AHR is not clear (U.S. EPA, 2006b). However, some asthmatics have 

9 higher baseline levels of neutrophils, lymphocytes, eosinophils and mast cells in 

10 bronchial washes and bronchial biopsy tissue (Stenfors et al., 2002). It has been proposed 

11 that enhanced sensitivity to O3 is conferred by the presence of greater numbers of resident 

12 airway inflammatory cells in disease states such as asthma (Mudway and Kelly, 2000).

13 In order to determine whether asthmatics exhibit greater responses to O3, several earlier 

14 studies compared pulmonary function in asthmatic and non-asthmatic subjects following 

15 O3 exposure. Some also probed mechanisms which could account for enhanced 

16 sensitivity. While the majority focused on measurements of FEV1 and FVC and found no 

17 differences between the two groups following exposures of 2-4 hours to 125-250 ppb O3 

18 or to a 30-minute exposure to 120-180 ppb O3 by mouthpiece in human subjects 

19 exercising at a light to moderate level (Stenfors et al., 2002; Mudway et al., 2001; Holz et 

20 al., 1999; Scannell et al., 1996; Koenig et al., 1987; Linn et al., 1978), there were notable 

21 exceptions. In one study, greater airways obstruction in asthmatics compared with non22 asthmatic subjects was observed immediately following a 2-hour exposure to 400 ppb O3

23 while exercising at a heavy level (Kreit et al., 1989). These changes were measured as 

24 statistically significant greater decreases in FEV1 and in FEF25-75 (but not in FVC) in the 

25 absence of a bronchoconstrictor challenge (Kreit et al., 1989). These results suggest that 

26 this group of asthmatics responded to O3-exposure with a greater degree of vagally27 mediated bronchoconstriction compared with the non-asthmatics. A second study 

28 demonstrated a statistically significant greater decrease in FEV1 and in FEV1/FVC (but 

29 not in FVC) in asthmatics compared with non-asthmatics exposed to 160 ppb O3 for 

30 7.6 hours with light exercise (Horstman et al., 1995). These responses were accompanied 

31 by wheezing and inhaler use in the asthmatics (Horstman et al., 1995). Aerosol bolus 

32 dispersion measurements demonstrated a statistically significant greater change in 

33 asthmatics compared with non-asthmatics, which was suggestive of O3-induced small 

34 airway dysfunction (Horstman et al., 1995). Furthermore, a statistically significant 

35 correlation was observed between the degree of baseline airway status and the FEV1

36 response to O3 in the asthmatic subjects (Horstman et al., 1995). A third study found 

37 similar decreases in FVC and FEV1 in both asthmatics and non-asthmatics exposed to 
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1 400 ppb O3 for 2 hours with light exercise (Alexis et al., 2000). However, a statistically 

2 significant decrease in FEF75, a measure of small airway function, was observed in 

3 asthmatics but not in non-asthmatics (Alexis et al., 2000). Taken together, these latter 

4 studies indicate that while the magnitude of restrictive type spirometric decline was 

5 similar in asthmatics and non-asthmatics, that obstructive type changes 

6 (i.e., bronchoconstriction) were greater in asthmatics. Further, asthmatics exhibited 

7 greater sensitivity to O3 in terms of small airways function. 

8 Since asthma exacerbations occur in response to allergens and/or other triggers, some 

9 studies have focused on O3-induced changes in AHR following a bronchoconstrictor 

10 challenge. No difference in sensitivity to methacholine bronchoprovocation was observed 

11 between asthmatics and non-asthmatics exposed to 400 ppb O3 for 2 hours while 

12 exercising at a heavy level (Kreit et al., 1989). However, increased bronchial reactivity to 

13 inhaled allergens was demonstrated in mild allergic asthmatics exposed to 160 ppb for 

14 7.6 hours, 250 ppb for 3 hours and 120 ppb for 1 hour while exercising at a light level or 

15 at rest (Kehrl et al., 1999; Jorres et al., 1996; Molfino et al., 1991) and in allergen16 sensitized guinea pigs following O3 exposure (1 ppm, 1 hour) (Sun et al., 1997). Similar, 

17 but modest, responses were reported for individuals with allergic rhinitis (Jorres et al., 

18 1996). Further, the contractile response of isolated airways from human donor lung 

19 tissue, which were sensitized and challenged with allergen, was increased by 

20 pre-exposure to 1 ppm O3 for 20 (Roux et al., 1999). These studies provide support for 

21 O3-mediated enhancement of responses to allergens in allergic subjects. 

22 In terms of airways neutrophilia, larger responses were observed in asthmatics compared 

23 to non-asthmatics subjects, who were exercising at a light to moderate level and exposed 

24 to O3, in some (Balmes et al., 1997; Scannell et al., 1996; Basha et al., 1994) but not all 

25 (Mudway et al., 2001) of the earlier studies. While each of these studies involved 

26 exposure of exercising human subjects to 200 ppb O3, the duration of exposure was 

27 longer (i.e., 4-6 hours) in the former studies than in the latter study (2 hours). Further, 

28 statistically significantly increases in myeloperoxidase levels (an indicator of neutrophil 

29 activation) in bronchial washes was observed in mild asthmatics compared with non30 asthmatics, despite no difference in O3-stimulated neutrophil influx between the 2 groups 

31 following exposure to 200 ppb O3 for 2 hours with moderate exercise (Stenfors et al., 

32 2002). A more recent study found that atopic asthmatic subjects exhibited an enhanced 

33 inflammatory response to O3 (400 ppb, 4 hours, with light to moderate exercise) 

34 (Hernandez et al., 2010). This response was characterized by greater numbers of 

35 neutrophils, higher levels of IL-6, IL-8 and IL-1β and greater macrophage cell-surface 

36 expression of TLR4 and IgE receptors in induced sputum compared with healthy 

37 subjects. This study also reported a greater increase in hyaluronan in atopic subjects and 

38 atopic asthmatics compared with healthy subjects following O3 exposure. Animal studies 
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1 have previously reported that hyaluronic acid activates TLR4 signaling and results in 

2 AHR (see Section 5.3.5). Furthermore, levels of IL-10, a potent anti-inflammatory 

3 cytokine, were greatly reduced in atopic asthmatics compared to healthy subjects. These 

4 results provide evidence that innate immune and adaptive responses are different in 

5 asthmatics and healthy subjects exposed to O3. 

6 Eosinophils may be an important modulator of responses to O3 in asthma and allergic 

7 airways disease. Eosinophils and associated proteins are thought to affect muscarinic 

8 cholinergic receptors which are involved in vagally-mediated bronchoconstriction 

9 (Mudway and Kelly, 2000). Studies described in Section 5.3.5 which demonstrated a key 

10 role of eosinophils in O3-mediated AHR may be relevant to human allergic airways 

11 disease which is characterized by airways eosinophilia (Yost et al., 2005). Furthermore, 

12 O3 exposure sometimes results in airways eosinophilia in allergic subjects or animal 

13 models. For example, eosinophilia of the nasal and other airways was observed in 

14 individuals with pre-existing allergic disease following O3 inhalation (160 ppb, 7.6 hours 

15 with light exercise and 270 ppb, 2 hours with moderate exercise) (Vagaggini et al., 2002; 

16 Peden et al., 1997). Further, O3 exposure (0.5 ppm, 8 hours/day for 1-3 days) increased 

17 allergic responses, such as eosinophilia and augmented intraepithelial mucosubstances, in 

18 the nasal airways of ovalbumin (OVA)-sensitized rats (Wagner et al., 2002). In contrast, 

19 Stenfors et al. (2002) found no stimulation of eosinophil influx measured in bronchial 

20 washes and BALF of mild asthmatics following exposure to a lower concentration 

21 (200 ppb, 2 hours, with moderate exercise) of O3. 

22 The role of mast cells in O3-mediated asthma exacerbations has been investigated. Mast 

23 cells are thought to play a key role in O3-induced airways inflammation, since airways 

24 neutrophilia was decreased in mast cell-deficient mice exposed to O3 (Kleeberger et al., 

25 1993). However, another study found that mast cells were not involved in the 

26 development of increased bronchial reactivity in O3-exposed mice (Noviski et al., 1999). 

27 Nonetheless, mast cells release a wide variety of important inflammatory mediators 

28 which may lead to asthma exacerbations (Stenfors et al., 2002). A large increase in mast 

29 cell number in bronchial submucosa was observed in non-asthmatics and a significant 

30 decrease in mast cell number in bronchial epithelium was observed in mild asthmatics 

31 6 hours following exposure to 200 ppb O3 for 2 hours during mild exercise (Stenfors et 

32 al., 2002). While these results point to an O3-mediated flux in bronchial mast cell 

33 populations which differed between the non-asthmatics and mild asthmatics, 

34 interpretation of these findings is difficult. Furthermore, mast cell number did not change 

35 in airway lavages in either group in response to O3 (Stenfors et al., 2002)

36 Cytokine profiles in the airways have been investigated as an indicator of O3 sensitivity. 

37 Differences in epithelial cytokine expression were observed in bronchial biopsy samples 
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1 in non-asthmatic and asthmatic subjects both at baseline and 6-hours postexposure to 

2 200 ppb O3 for 2 hours with moderate exercise (Bosson et al., 2003). The asthmatic 

3 subjects had a higher baseline expression of IL-4 and IL-5 compared to non-asthmatics. 

4 In addition, expression of IL-5, IL-8, GM-CSF, and ENA-78 in asthmatics was increased 

5 significantly following O3 exposure compared to non-asthmatics (Bosson et al., 2003). 

6 Some of these (IL-4, IL-5 and GM-CSF) are Th2-related cytokines or neutrophil 

7 chemoattractants, and play a role in IgE production, airways eosinophilia and suppression 

8 of Th1-cytokine production (Bosson et al., 2003). These findings suggest a link between 

9 adaptive immunity and enhanced responses of asthmatics to O3. 

10 A further consideration is the compromised status of ELF antioxidants in disease states 

11 such as asthma (Mudway and Kelly, 2000). This could possibly be due to ongoing 

12 inflammation which causes antioxidant depletion or to abnormal antioxidant transport or 

13 synthesis (Mudway and Kelly, 2000). For example, basal levels of AH2 were 

14 significantly lower and basal levels of oxidized GSH and UA were significantly higher in 

15 bronchial wash fluid and BALF of mild asthmatics compared with healthy control 

16 subjects (Mudway et al., 2001). Differences in ELF antioxidant content have also been 

17 noted between species. These observations have led to the suggestion that the amount and 

18 composition of ELF antioxidants, the capacity to replenish antioxidants in the ELF or the 

19 balance between beneficial and injurious interactions between antioxidants and O3 may 

20 contribute to O3 sensitivity, which varies between individuals and species (Mudway et 

21 al., 2006; Mudway and Kelly, 2000; Mudway et al., 1999a). The complexity of these 

22 interactions was demonstrated by a study in which a 2-hour exposure to 200 ppb O3, 

23 while exercising at a moderate level, resulted in similar increases in airway neutrophils 

24 and decreases in pulmonary function in both mild asthmatics and healthy controls, 

25 despite differences in ELF antioxidant concentrations prior to O3 exposure (Mudway et 

26 al., 2001). Further, the O3-induced increase in oxidized GSH and decrease in AH2 

27 observed in ELF of healthy controls was not observed in mild asthmatics (Mudway et al., 

28 2001). While the authors concluded that basal AH2 and oxidized GSH concentrations 

29 were not predictive of responsiveness to O3, they also suggested that the increased basal 

30 UA concentrations in the mild asthmatics may have played a protective role (Mudway et 

31 al., 2001). Thus compensatory mechanisms resulting in enhanced total antioxidant 

32 capacity may play a role in modulating responses to O3. 

33 Collectively these older and more recent studies provide insight into mechanisms which 

34 may contribute to enhanced responses of asthmatic and atopic individuals following O3 

35 exposure. Greater airways inflammation and/or greater bronchial reactivity have been 

36 demonstrated in asthmatics compared to non-asthmatics. This pre-existing inflammation 

37 and altered baseline bronchial reactivity may contribute to the enhanced 

38 bronchoconstriction seen in asthmatics exposed to O3. Furthermore, O3-induced 
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1 inflammation may contribute to O3-mediated AHR. An enhanced neutrophilic response to 

2 O3 has been demonstrated in some asthmatics. A recent study in humans provided 

3 evidence for differences in innate immune responses related to TLR4 signaling between 

4 asthmatics and healthy subjects. Animal studies have demonstrated a role for eosinophil5 derived proteins in mediating the effects of O3. Since airways eosinophilia occurs in both 

6 allergic humans and allergic animal models, this pathway may underlie the exacerbation 

7 of allergic asthma by O3. In addition, differences have been noted in epithelial cytokine 

8 expression in bronchial biopsy samples of healthy and asthmatic subjects. A Th2 

9 phenotype, indicative of adaptive immune system activation and enhanced allergic 

10 responses, was observed before O3 exposure and was increased by O3 exposure in 

11 asthmatics. These findings support links between innate and adaptive immunity and 

12 sensitivity to O3-mediated effects in asthmatics and allergic airways disease. 

13 In addition to asthma and allergic diseases, obesity may alter responses to O3. While O3 is 

14 a trigger for asthma, obesity is a known risk factor for asthma (Shore, 2007). The 

15 relationship between obesity and asthma is not well understood but recent investigations 

16 have focused on alterations in endocrine function of adipose tissue in obesity. It is 

17 thought that the increases in serum levels of factors produced by adipocytes 

18 (i.e., adipokines), such as cytokines, chemokines, soluble cytokine receptors and energy 

19 regulating hormones, may contribute to the relationship between obesity and asthma. 

20 Some of these same mechanisms may be relevant to insulin resistant states such as 

21 metabolic syndrome. 

22 In a re analysis of the data of Hazucha et al. (2003), increasing body mass index in 

23 young women was associated with increased O3 responsiveness, as measured by 

24 spirometry following a 1.5-hour exposure to 420 ppb O3 while exercising at a moderate 

25 level (Bennett et al., 2007). In several mouse models of obesity, airways were found to be 

26 innately more hyperresponsive and responded more vigorously to acute O3 exposure than 

27 lean controls (Shore, 2007). Pulmonary inflammatory and injury in response to O3 were 

28 also enhanced (Shore, 2007). It was postulated that oxidative stress resulting from 

29 obesity-related hyperglycemia could account for these effects (Shore, 2007). However, 

30 responses to O3 in the different mouse models are somewhat variable and depend on 

31 whether exposures are acute or subacute. For example, diet-induced obesity augmented 

32 inflammation and injury, as measured by BALF markers, and enhanced AHR in mice 

33 exposed acutely to O3 (2 ppm, 3 hours) (Johnston et al., 2008). In contrast, the 

34 inflammatory response following sub-acute exposure to O3 was dampened by obesity in a 

35 different mouse model (0.3 ppm, 72 hours) (Shore et al., 2009). It is not known whether 

36 differences in responsiveness to O3 are due to differences in lung development in 

37 genetically-modified animals which result in smaller lungs and thus to differences in 

38 inhaled dose because of the altered body mass to lung size ratio. 
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5.4.2.3 Nutritional Status

1 A further consideration is the compromised status of ELF antioxidants in nutritional 

2 deficiencies. Thus, many investigations have focused on antioxidant deficiency and 

3 supplementation as modulators of O3-mediated effects. One study in mice found that 

4 vitamin A deficiency enhanced lung injury induced by exposure to 0.3 ppm O3 for 

5 72 hours (Paquette et al., 1996). Ascorbate deficiency was shown to increase the effects 

6 of acute (0.5-1 ppm for 4 hours), but not subacute (0.2-0.8 ppm for 7 days), O3 exposure 

7 in guinea pigs (Kodavanti et al., 1995; Slade et al., 1989). Supplementation with AH2 

8 and α-TOH was protective in healthy adults who were on an AH2-deficient diet and 

9 exposed to 400 ppb O3 for 2 hours while exercising at a moderate level (Samet et al., 

10 2001). In this study, the protective effect consisted of a smaller reduction in FEV1

11 following O3 exposure (Samet et al., 2001). However the inflammatory response (influx 

12 of neutrophils and levels of IL-6) measured in BALF 1 hour after O3 exposure was not 

13 different between supplemented and non-supplemented subjects (Samet et al., 2001). 

14 Other investigators found that AH2 and α-TOH supplementation failed to ameliorate the 

15 pulmonary function decrements or airways neutrophilia observed in humans exposed to 

16 200 ppb O3 for 2 hours while exercising at a moderate level (Mudway et al., 2006). It was 

17 suggested that supplementation may be ineffective in the absence of antioxidant 

18 deficiency (Mudway et al., 2006).

19 In asthmatic adults, these same dietary antioxidants reduced O3-induced bronchial 

20 hyperresponsiveness (120 ppb, 45 min, light exercise) (Trenga et al., 2001). Furthermore, 

21 supplementation with AH2 and α-tocopherol protected against pulmonary function 

22 decrements and nasal inflammatory responses which were associated with high levels of 

23 ambient O3 in asthmatic children living in Mexico City (Sienra-Monge et al., 2004; 

24 Romieu et al., 2002). Similarly, supplementation with ascorbate, α-tocopherol and 

25 β-carotene improved pulmonary function in Mexico City street workers (Romieu et al., 

26 1998b). 

27 Protective effects of supplementation with α-tocopherol alone have not been observed in 

28 humans experimentally exposed to O3 (Mudway and Kelly, 2000). Alpha-TOH 

29 supplementation also failed to protect against O3-induced effects in animal models of 

30 allergic rhinosinusitis and lower airways allergic inflammation (rats, 1 ppm O3 for 

31 2 days) (Wagner et al., 2007). However, protection in these same animal models was 

32 reported using γ-TOH supplementation (Wagner et al., 2009; Wagner et al., 2007). 

33 Whether or not this effect was due to enhanced antioxidant status or to activated signaling 

34 pathways is not known. Other investigators found that α-TOH deficiency led to an 

35 increase in liver lipid peroxidation (rats, 0.3 ppm 3 hours/day for 7 months) (Sato et al., 

36 1980) and a drop in liver α-TOH levels following O3 exposure (mice, 0.5 ppm, 
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1 6 hours/day for 3 days) (Vasu et al., 2010). A recent study used α-TOH transfer protein 

2 null mice as a model of α-TOH deficiency and demonstrated an altered adaptive response 

3 of the lung genome to O3 exposure (Vasu et al., 2010). Taken together, these studies 

4 provide evidence that the tocopherol system modulates O3-induced responses. 

5.4.2.4 Lifestage

5 Responses to O3 are modulated by factors associated with lifestage. On one end of the 

6 lifestage spectrum is aging. The spirometric response to O3 appears to be lost in humans 

7 as they age, as was demonstrated in two studies involving exposures of human subjects 

8 exercising at levels ranging from light to heavy to 420-450 ppb O3 for 1.5-2 hours 

9 (Hazucha et al., 2003; Drechsler-Parks, 1995). In mice, physiological responses to O3

10 (600 ppb, 2 hours) were diminished with age (Hamade et al., 2010). Mechanisms 

11 accounting for this effect have not been well-studied but could include altered number 

12 and sensitivity of receptors, altered signaling pathways involved in neural reflexes or 

13 compromised status of ELF antioxidants. 

14 On the other side of the lifestage spectrum is pre/postnatal development. Critical 

15 windows of development during the pre/postnatal period are associated with an enhanced 

16 sensitivity to environmental toxicants. Adverse birth outcomes and developmental 

17 disorders may occur as a result (Section 7.4). 

18 Adverse birth outcomes may result from stressors which impact transplacental oxygen 

19 and nutrient transport by a variety of mechanisms including oxidative stress, placental 

20 inflammation and placental vascular dysfunction (Kannan et al., 2006). These 

21 mechanisms may be linked since oxidative/nitrosative stress is reported to cause vascular 

22 dysfunction in the placenta (Myatt et al., 2000). As described earlier in this chapter and in 

23 Section 7.4, systemic inflammation and oxidative/nitrosative stress and modification of 

24 innate and adaptive immunity are key events underlying the health effects of O3 and as 

25 such they may contribute to adverse birth outcomes. An animal toxicology study showing 

26 that exposure to 2 ppm O3 led to anorexia (Kavlock et al., 1979) (see Section 7.4.2) in 

27 exposed rat dams provide an additional mechanism by which O3 exposure could lead to 

28 diminished transplacental nutrient transport. Disturbances of the pituitary-adrenocortico29 placental system (Ritz et al., 2000) may also impact normal intrauterine growth and 

30 development. Further, restricted fetal growth may result from pro-inflammatory 

31 cytokines which limit trophoblast invasion during the early stages of pregnancy (Hansen 

32 et al., 2008). Direct effects on maternal health, such as risk of infection, and on fetal 

33 health, such as DNA damage, have also been proposed as mechanisms underlying 

34 adverse birth outcomes (Ritz et al., 2000). In addition to restricted fetal growth, preterm 
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1 birth may contribute to adverse birth outcomes. Preterm birth may result from the 

2 development of premature contractions and/or premature rupture of membranes as well 

3 as from disrupted implantation and placentation which results in suboptimal placental 

4 function (Darrow et al., 2009; Ritz et al., 2000). Genetic mutations are thought to be an 

5 important cause of placental abnormalities in the first trimester, while vascular alterations 

6 may be the main cause of placental abnormalities in later trimesters (Jalaludin et al., 

7 2007). Ozone-mediated systemic inflammation and oxidative stress/nitrosative stress may 

8 possibly be related to these effects although there is no firm evidence. 

9 Enhanced sensitivity to environmental toxicants during critical windows of development 

10 may also result in developmental disorders. For example, normal migration and 

11 differentiation of neural crest cells are important for heart development and are 

12 particularly sensitive to toxic insults (Ritz et al., 2002). Further, immune dysregulation 

13 and related pathologies are known to be associated with pre/postnatal environmental 

14 exposures (Dietert et al., 2010). Ozone exposure is associated with developmental effects 

15 in several organ systems. These include the lung and immune system (see below) and 

16 neurobehavioral changes which could reflect the effect of O3 on CNS plasticity or the 

17 hypothalamic-pituitary axis (Auten and Foster, 2011) (see Section 7.4.9). 

18 The majority of developmental effects due to O3 have been described for the respiratory 

19 system (see Section 7.2.3 and 7.4.8). Since its growth and development take place during 

20 both the prenatal and early postnatal periods, both prenatal and postnatal exposures to O3

21 have been studied. Maternal exposure to 0.4-1.2 ppm O3 during gestation resulted in 

22 developmental health effects in the RT of mice (Sharkhuu et al., 2011). Recent studies 

23 involving postnatal exposure to O3 have focused on differences between developing and 

24 adult animals in antioxidant defenses, respiratory physiology and sensitivity to cellular 

25 injury, and on mechanisms, such as lung structural changes, antigen sensitization, 

26 interaction with nitric oxide signaling, altered airway afferent innervation and loss of 

27 alveolar repair capacity, by which early O3 exposure could lead to asthma pathogenesis or 

28 exacerbations in later life (Auten and Foster, 2011).

29 An interesting set of studies conducted over the last 10 years in the infant rhesus monkey 

30 has identified numerous O3-mediated perturbations in the developing lung and immune 

31 system (Plopper et al., 2007). These investigations were prompted by the dramatic rise in 

32 the incidence of childhood asthma and focused on the possible interaction of O3 and 

33 allergens in promoting remodeling of the epithelial-mesenchymal trophic unit during 

34 postnatal development of the tracheobronchial airway wall. In humans, airways growth 

35 during the 8-12 year period of postnatal development is not well understood. Rhesus 

36 monkeys were used in these studies because the branching pattern and distribution of 

37 airways in this model are more similar to humans than those of rodents are to humans. In 
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1 addition, a model of allergic airways disease, which exhibits the main features of human 

2 asthma, had already been established in the adult rhesus monkey. Studies in infant 

3 monkeys were designed to determine whether repeated exposure to O3 altered postnatal 

4 growth and development, and if so, whether such effects were reversible. In addition, 

5 exposure to O3 was evaluated for its potential to increase the development of allergic 

6 airways disease. Exposures were to cyclic episodic O3 over 5 months which involved 5 

7 biweekly cycles of alternating filtered air and O3 - 9 consecutive days of filtered air and 5 

8 consecutive days of 0.5 ppm O3, 8 h/day – and to house dust mite allergen (HDMA) for 

9 2 hours per day for 3 days on the last 3 days of O3 exposure followed by 11 days of 

10 filtered air.

11 Key findings were numerous. First, baseline airway resistance and AHR in the infant 

12 monkeys were dramatically increased by combined exposure to both HDMA and O3

13 (Joad et al., 2006; Schelegle et al., 2003). Secondly, O3 exposure led to a large increase in 

14 BAL eosinophils (Schelegle et al., 2003) while HDMA exposure led to a large increase of 

15 eosinophils in airways tissue (Joad et al., 2006; Schelegle et al., 2003). Thirdly, the 

16 growth pattern of distal airways was changed to a large extent by exposure to O3 alone 

17 and in combination with HDMA. More specifically, longer and narrower airways resulted 

18 and the number of conducting airway generations between the trachea and the gas 

19 exchange area was decreased (Fanucchi et al., 2006). This latter effect was not 

20 ameliorated by a recovery period of 6 months. Fourthly, exposure to both HDMA and O3

21 altered the abundance and distribution of CD25+ lymphocytes in the airways (Miller et 

22 al., 2009). Lastly, several effects were seen at the level of the epithelial mesenchymal 

23 trophic unit in response to O3. These include altered organization of the airways 

24 epithelium (Schelegle et al., 2003), increased abundance of mucous goblet cells 

25 (Schelegle et al., 2003), disruption of the basement membrane zone (Evans et al., 2003), 

26 reduced innervation (Larson et al., 2004), increased neuroendocrine-like cells (Joad et al., 

27 2006), and altered orientation and abundance of smooth muscle bundles (Plopper et al., 

28 2007; Tran et al., 2004). Six months of recovery in filtered air led to reversal of some but 

29 not all of these effects (Kajekar et al., 2007; Plopper et al., 2007; Evans et al., 2004). The 

30 authors concluded that cyclic challenge of infant rhesus monkeys to allergen and O3

31 during the postnatal period compromised airway growth and development and resulted in 

32 changes which favor allergic airways responses and persistent effects on the immune 

33 system (Plopper et al., 2007). A more recent study in this same model reported that early 

34 life exposure to O3 resulted in decreased total peripheral blood leukocyte numbers and 

35 increased blood eosinophils as well as persistent effects on pulmonary and systemic 

36 innate immunity in the infant rhesus monkey model (Maniar-Hew et al., 2011). 

37 Furthermore, the effect of cyclic episodic O3 exposure on nasal airways was studied in 

38 the infant rhesus monkey model. The three-dimensional detail of the nasal passages was 
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1 analyzed for developing predictive dosimetry models and exposure-dose-response 

2 relationships (Carey et al., 2007). The authors reported that the relative amounts of the 

3 five epithelial cell types in the nasal airways of monkeys remained consistent between 

4 infancy and adulthood [comparing to (Gross, 1987; Gross, 1982)]. Cyclic episodic O3

5 exposure (as described in the previous paragraphs) resulted in 50-80% decreases in 

6 epithelial thickness and epithelial cell volume of the ciliated respiratory and transitional 

7 epithelium, confirming that these cell types in the nasal cavity were the most sensitive to 

8 O3 exposure. The character and location of nasal lesions resulting from O3 exposure were 

9 similar in the infant monkeys and adult monkeys similarly exposed. However, the nasal 

10 epithelium of infant monkeys did not undergo nasal airway epithelial remodeling or 

11 adaptation which occurs in adult animals following O3-mediated injury and which may 

12 protect against subsequent O3 challenge. The authors suggested that infant monkeys may 

13 be prone to developing persistent necrotizing rhinitis following episodic longer-term 

14 exposures. 

5.4.2.5 Attenuation of Responses

15 Repeated daily exposure to O3 often results in a reduction in the degree of a response, 

16 i.e., an attenuation of response. This phenomenon may reflect compensatory mechanisms 

17 and is not necessarily beneficial. Furthermore, there is variability among the different 

18 O3-related endpoints in terms of response attenuation, as will be described below. As a 

19 result, attenuation of some responses occurs concomitantly with the exacerbation of 

20 others.

21 In responsive individuals, a striking degree of attenuation of the FEV1 response occurred 

22 following repeated daily exposures to O3. Generally, the young O3 responder was no 

23 longer responsive on the fourth or fifth day of consecutive daily O3 exposure 

24 (200-500 ppb O3 for 2-4 hours with light to heavy levels of exercise) and required days to 

25 weeks of non-exposure in order for the subject to regain O3 responsiveness (Christian et 

26 al., 1998; Devlin et al., 1997; Linn et al., 1982b; Horvath et al., 1981; Hackney et al., 

27 1977b). This phenomena has been reported for both lung function and symptoms such as 

28 upper airway irritation, nonproductive cough, substernal discomfort and pain upon deep 

29 inspiration (Linn et al., 1982b; Horvath et al., 1981; Hackney et al., 1977b). Repeated 

30 daily exposures also led to an attenuation of the sRaw response in moderately exercising 

31 human subjects exposed for 4 hours to 200 ppb O3 (Christian et al., 1998) and to a 

32 dampened AHR response compared with a single day exposure in light exercising human 

33 subjects exposed for 2 hours to 400 ppb O3 (Dimeo et al., 1981). However, one group 

34 reported persistent small airway dysfunction despite attenuation of the FEV1 response on 
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1 the third day of consecutive O3 exposure (250 ppb, 2 hours, with moderate exercise) 

2 (Frank et al., 2001). 

3 Studies in rodents also indicated an attenuation of the physiologic response measured by 

4 breathing patterns and tidal volume following five consecutive days of exposure to 

5 0.35-1 ppm O3 for 2.25 hours (Tepper et al., 1989). Attenuation of O3-induced 

6 bradycardic responses, which also result from activation of neural reflexes, has been 

7 reported in rodents (0.5-0.6 ppm O3, 2-6 h/day, 3-5 days (Hamade and Tankersley, 2009; 

8 Watkinson et al., 2001). 

9 Multi-day exposure to O3 has been found to decrease some markers of inflammation 

10 compared with a single day exposure (Christian et al., 1998; Devlin et al., 1997). For 

11 example, in human subjects exposed for 4 hours to 200 ppb O3 during moderate exercise, 

12 decreased numbers of BAL neutrophils and decreased levels of BALF fibronectin and 

13 IL-6 were observed after 4 days of consecutive exposure compared with responses after 

14 1 day (Christian et al., 1998). Results indicated an attenuation of the inflammatory 

15 response in both proximal airways and distal lung. However markers of injury, such as 

16 lactate dehydrogenase (LDH) and protein in the BALF, were not attenuated in this study 

17 (Christian et al., 1998). Other investigators found that repeated O3 exposure (200 ppb O3

18 for 4 hours on 4 consecutive days with light exercise) resulted in increased numbers of 

19 neutrophils in bronchial mucosal biopsies despite decreased BAL neutrophilia (Jorres et 

20 al., 2000). Other markers of inflammation, including BALF protein and IL-6 remained 

21 elevated following the multi-day exposure (Jorres et al., 2000).

22 In rats, the increases in BALF levels of proteins, fibronectin, IL-6 and inflammatory cells 

23 observed after one day of exposure to 0.4 ppm O3 for 12 hours were no longer observed 

24 after 5 consecutive days of exposure (Van Bree et al., 2002). A separate study in rats 

25 exposed to 0.35-1 ppm O3 for 2.25 hours for 5 consecutive days demonstrated a lack of 

26 attenuation of the increase in BALF protein, persistence of macrophages in the 

27 centriacinar region and histological evidence of progressive tissue injury (Tepper et al., 

28 1989). Findings that injury, measured by BALF markers or by histopathology, persist in 

29 the absence of BAL neutrophila or pulmonary function decrements suggested that 

30 repeated exposure to O3 may have serious long-term consequences such as airway 

31 remodeling. In particular, the small airways were identified as a site where cumulative 

32 injury may occur (Frank et al., 2001). 

33 Some studies examined the recovery of responses which were attenuated by repeated O3

34 exposure. In a study of humans undergoing heavy exercise who were exposed for 2 hours 

35 to 400 ppb O3 for five consecutive days (Devlin et al., 1997), recovery of the 

36 inflammatory responses which were diminished by repeated exposure required 

37 10-20 days following the exposure (Devlin et al., 1997). In an animal study conducted in 
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1 parallel (Van Bree et al., 2002), full susceptibility to O3 challenge following exposure to 

2 O3 for five consecutive days required 15-20 days recovery. 

3 Several mechanisms have been postulated to explain the attenuation of some responses 

4 observed in human subjects and animal models following repeated exposure to O3. First, 

5 the upregulation of antioxidant defenses (or conversely, a decrease in critical O3-reactive 

6 substrates) may protect against O3-mediated effects. Increases in antioxidant content of 

7 the BALF have been demonstrated in rats exposed to 0.25 and 0.5 ppm O3 for 

8 several hours on consecutive days (Devlin et al., 1997; Wiester et al., 1996b; Tepper et 

9 al., 1989). Second, IL-6 was demonstrated to be an important mediator of attenuation in 

10 rats exposed to 0.5 ppm for 4 hours on two consecutive days (Mckinney et al., 1998). 

11 Third, a protective role for increases in mucus producing cells and mucus concentrations 

12 in the airways has been proposed (Devlin et al., 1997). Fourth, epithelial hyperplasia or 

13 metaplasia may decrease further effects due to subsequent O3 challenge (Carey et al., 

14 2007; Harkema et al., 1987a; Harkema et al., 1987b). These morphologic changes have 

15 been observed in nasal and lower airways in monkeys exposed chronically to

16 0.15-0.5 ppm O3 and reflect a persistent change in epithelial architecture which may lead 

17 to other long-term sequelae. Although there is some evidence to support these 

18 possibilities, there is no consensus on mechanisms underlying response attenuation. 

19 Recent studies demonstrating that O3 activates TRP receptors suggest that modulation of 

20 TRP receptor number or sensitivity by repeated O3 exposures may also contribute to the 

21 attenuation of responses.

22 In summary, the attenuation of pulmonary function responses by repeated exposure to O3

23 has been linked to exacerbation of O3-mediated injury. Enhanced exposure to O3 due to a 

24 dampening of the O3-mediated truncation of inspiration may be one factor which 

25 contributes to this relationship. 

5.4.2.6 Co-exposures with Particulate Matter

26 Numerous studies have investigated the effects of co-exposure to O3 and PM because of 

27 the prevalence of these pollutants in ambient air. Results are highly variable and depend 

28 on whether exposures are simultaneous or sequential, the type of PM employed and the 

29 endpoint examined. Additive and interactive effects have been demonstrated. For 

30 example, simultaneous exposure to O3 (120 ppb for 2 hours at rest) and concentrated 

31 ambient particles (CAPs) in human subjects resulted in a diminished systemic IL-6 

32 response compared with exposure to CAPs alone (Urch et al., 2010). However, exposure 

33 to O3 alone did not alter blood IL-6 levels (Urch et al., 2010). The authors provided 

34 evidence that O3 mediated a switch to shallow breathing which may have accounted for 
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1 the observed antagonism (Urch et al., 2010). Further, simultaneous exposure to O3

2 (114 ppb for 2 hours at rest) and CAPs but not exposure to either alone, resulted in 

3 increased diastolic blood pressure in human subjects (Fakhri et al., 2009). Mechanisms 

4 underlying this potentiation of response were not explored. In some strains of mice, 

5 pre-exposure to O3 (0.5 ppm for 2 hours) modulated the effects of carbon black PM on 

6 heart rate, HRV and breathing patterns (Hamade and Tankersley, 2009). Another recent 

7 study in mice demonstrated that treatment with carbon nanotubes followed 12 hours later 

8 by O3 exposure (0.5 ppm for 3 hours) resulted in a dampening of some of the pulmonary 

9 effects of carbon nanotubes measured as markers of inflammation and injury in the 

10 BALF (Han et al., 2008). Further, Harkema and Wagner (2005) found that epithelial and 

11 inflammatory responses in the airways of rats were enhanced by co-exposure to O3

12 (0.5 ppm for 3 days) and LPS (used as a model of biogenic PM) or to O3 (1 ppm for 

13 2 days) and OVA (used as a model of an aeroallergen). Lastly, a recent study 

14 demonstrated that maternal exposure to particulate matter (PM) resulted in augmented 

15 lung inflammation, airway epithelial mucous metaplasia and AHR in young mice 

16 exposed chronically and intermittently to 1 ppm O3 (Auten et al., 2009). 

17 In summary, many of the demonstrated responses to co-exposure were more than 

18 additive. These findings are hard to interpret but demonstrate the complexity of responses 

19 following combined exposure to PM and O3. 

5.4.2.7 Summary

20 Collectively, these earlier and more recent studies provide some evidence for 

21 mechanisms that may underlie the variability in responsiveness seen among individuals 

22 (Figure 5-9). Certain functional genetic polymorphisms, pre-existing conditions and 

23 diseases, nutritional status, lifestage and co-exposures contribute to altered risk of 

24 O3-induced effects. Attenuation of responses may also be important, but it is 

25 incompletely understood, both in terms of the pathways involved and the resulting 

26 consequences. 
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Figure 5-9 Some factors, illustrated in yellow, that likely contribute to the 

interindividual variability in responses resulting from inhalation of 

ozone.

5.5 Species Homology and Interspecies Sensitivity

1 The previous O3 AQCDs discussed the suitability of animal models for comparison with 

2 human O3 exposure and concluded that the acute and chronic functional responses of 

3 laboratory animals to O3 appear qualitatively homologous to human responses. Thus, 

4 animal studies can provide important data in determining cause-effect relationships 

5 between exposure and health outcome that would be impossible to collect in human 

6 studies. Furthermore, animal studies add to a better understanding of the full range of 

7 potential O3-mediated effects. 

8 Still, care must be taken when comparing quantitative dose-response relationships in 

9 animal models to humans due to obvious interspecies differences. This section will 

10 qualitatively describe basic concepts in species homology concerning both dose and 

11 response to O3 exposure. Overall, there have been few new publications examining 

12 interspecies differences in dosimetry and response to O3 since the last AQCD. These 

13 studies do not overtly change the conclusions discussed in the previous document. 

Ozone + Respiratory Tract 

Formation of secondary oxidation products

Systemic inflammation and 

oxidative/nitrosative stress

Activation 

of neural 

reflexes

Airways 

remodeling

Sensitization 

of bronchial 

smooth muscle

Alteration 

of epithelial 

barrier 

function

Initiation of 

inflammation 

Modification

of innate and 

adaptive

immunity

Extrapulmonary Effects

Respiratory System Effects

Dosimetric factors

Nutritional status

Lifestage

Attenuation factors

Co-exposures

Gene-environment interactions

Pre-existing diseases/conditions

COPD/smoking status

Asthma/allergic airways disease

Obesity/metabolic syndrome

Obesity/

Metabolic Stress

Lifestage

Attenuation 

factors
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5.5.1 Interspecies Dosimetry

1 As discussed in Section 5.2.1, O3 uptake depends on complex interactions between RT 

2 morphology, breathing route, rate, and depth, physicochemical properties of the gas, 

3 physical processes of gas transport, as well as the physical and chemical properties of the 

4 ELF and tissue layers. Understanding differences in these variables between humans and 

5 experimental animals is important to interpreting delivered doses in animal and human 

6 toxicology studies. 

7 Physiological and anatomical differences exist between experimental species. The 

8 structure of the URT is vastly different between rodents and humans but scales according 

9 to body mass. The difference in the cross-sectional shape and size of the nasal passages 

10 affects bulk airflow patterns, particularly the shape of major airflow streams. The nasal 

11 epithelium is lined by squamous, respiratory, or olfactory cells, depending on location. 

12 The differences in airflow patterns in the URT mean that not all nasal surfaces and cell 

13 types receive the same exposure to inhaled O3 leading to differences in local absorption 

14 and potential for site-specific tissue damage. The morphology of the LRT also varies 

15 within and among species. Rats and mice do not possess respiratory bronchioles; 

16 however, these structures are present in humans, dogs, ferrets, cats, and monkeys. 

17 Respiratory bronchioles are abbreviated in hamsters, guinea pigs, sheep, and pigs. The 

18 branching structure of the ciliated bronchi and bronchioles also differs between species 

19 from being a rather symmetric and dichotomous branching network of airways in humans 

20 and primates to a more monopodial branching network in other mammals. In addition, 

21 rodents have fewer terminal bronchioles due to a smaller lung size compared to humans 

22 or canines (McBride, 1992). The cellular composition in the pulmonary region is similar 

23 across mammalian species; at least 95% of the alveolar epithelial tissue is composed of 

24 Type I cells. However, considerable differences exist between species in the number and 

25 type of cells in the TB airways. Differences also exist in breathing route and rate. 

26 Primates are oronasal breathers, while rodents are obligate nasal breathers. Past studies of 

27 the effect of body size on resting oxygen consumption also suggest that rodents inhale 

28 more volume of air per lung mass than primates. These distinctions as well as differences 

29 in nasal structure between primates and rodents affect the amount of O3 uptake. 

30 As O3 absorption and reactivity relies on ELF antioxidant substances (see Section 5.2.3), 

31 variability in antioxidant concentrations and metabolism between species may affect dose 

32 and O3-induced health outcomes. The thickness of the ELF in the TB airways varies 

33 among species. Mercer et al. (1992) found that the human ELF thickness in bronchi and 

34 bronchioles was 6.9 and 1.8 μm, respectively, compared to 2.6 and 1.9 μm for the same 

35 locations in the rat. Guinea pigs and mice have a lower basal activity of GSH transferase 

36 and GSH peroxidase, and lower α-TOH levels in the lung compared to rats (Ichinose et 
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1 al., 1988; Sagai et al., 1987). Nasal lavage fluid analysis shows that humans have a higher 

2 proportion of their nasal antioxidants as UA and low levels of AH2 whereas mice, rats, or 

3 guinea pigs have high levels of AH2 and undetectable levels of UA. GSH is not detected 

4 in the nasal lavage of most of these species, but is present in monkey nasal lavage. 

5 Guinea pigs and rats have a higher antioxidant to protein ratio in nasal lavage and BALF 

6 than humans (Hatch, 1992). The BALF profile differs from the nasal lavage. Humans 

7 have a higher proportion of GSH and less AH2 making up their BALF content compared 

8 to the guinea pigs and rats (Slade et al., 1993; Hatch, 1992). Similar to the nose, rats have 

9 the highest antioxidant to protein mass ratio found in BALF (Slade et al., 1993). 

10 Antioxidant defenses also vary with age (Servais et al., 2005) and exposure history (Duan 

11 et al., 1996). Duan et al. (1996); Duan et al. (1993) reported that differences in 

12 antioxidant levels between species and lung regions did not appear to be the primary 

13 factor in O3 induced tissue injury. However, a close correlation between site-specific O3

14 dose, the degree of epithelial injury, and reduced glutathione depletion was observed in 

15 monkeys (Plopper et al., 1998).

16 Even with these differences humans and animals are similar in the pattern of regional O3

17 dose distribution. As discussed for humans in Section 5.2.2, O3 flux to the air-liquid 

18 interface of the ELF slowly decreases distally in the TB region and then rapidly decreases 

19 distally in the alveolar region (Miller et al., 1985). Modeled tissue dose in the human RT, 

20 representing O3 flux to the liquid-tissue interface, is very low in the trachea, increases to 

21 a maximum in the CAR, and then rapidly decreases distally in the alveolar region 

22 (Figure 5-10). Similar patterns of O3 tissue dose profiles normalized to inhaled O3

23 concentration were predicted for rat, guinea pig, and rabbit (Miller et al., 1988; Overton 

24 et al., 1987) (Figure 5-10a). Overton et al. (1987) modeled rat and guinea pig O3 dose 

25 distribution and found that after comparing two different morphometrically based 

26 anatomical models for each species, considerable difference in predicted percent RT and 

27 alveolar region uptakes were observed. This was due to the variability between the two 

28 anatomical models in airway path distance to the first alveolated duct. As a result, the 

29 overall dose profile was similar between species however the O3 uptake efficiency varied 

30 due to RT size and path length (Section 5.2.2). A similar pattern of O3 dose distribution 

was measured in monkeys exposed to 0.4 and 1.0 ppm 18 31 O3 (Plopper et al., 1998)

(Figure 5-10b). Less 18 32 O was measured in the trachea, proximal bronchus, and distal 

33 bronchus than was observed in the respiratory bronchioles. Again indicating the highest 

34 concentration of O3 tissue dose is localized to the CAR, which are the respiratory 

bronchioles in nonhuman primates. In addition, the lowest 18 35 O detected in the RT was in 

36 the parenchyma (i.e., alveolar region), mimicking the rapid decrease in tissue O3 dose 

37 predicted by models for the alveolar regions of humans and other animals. 
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a.

Note: Panel (a) presents the predicted tissue dose of O3 (as μg of O3 per cm2 of segment surface area per min, standardized to a 

tracheal O3 value of 1 μg/m3

) for various regions of the rabbit, guinea pig, rat, and human RT. TB = tracheobronchial region, 

A = alveolar region. Panel (b) presents a comparison of excess 18O in the five regions of the TB airways of rhesus monkeys exposed 

to O3 for 2h. *p <0.05 comparing the same O3 concentration across regions. **p <0.05 comparing different O3 concentrations in the 

same region.

Source: Panel (a) U.S. EPA (1996a) (b) Plopper et al. (1998)

Figure 5-10 Humans and animals are similar in the regional pattern of ozone

tissue dose distribution.

0.4 ppm O3

1.0 ppm O3

Oxygen-18-Content (μg/g dry weight)

0

10

20

30

40

50

TRACHEA PROXIMAL

BRONCHUS

DISTAL

BRONCHUS

RESPIRATORY

BRONCHIOLE

PARENCHYMA

a.

b.

A
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1 Humans and animal models are similar in the pattern of regional O3 dose, but absolute 

2 values differ. Hatch et al. (1994) reported that exercising humans exposed to oxygen-18 

3 labeled O3 (400 ppb) accumulated 4-5 times higher concentrations of O3 reaction product 

4 in BAL cells, surfactant and protein fractions compared to resting rats similarly exposed 

(400 ppb) (Figure 5-11). The use of 18 5 O was specifically employed in an attempt to 

6 accurately measure O3 dose to BALF fractions and lung tissue and was normalized to the 

7 dried mass of lavaged constituents. It was necessary to expose resting rats to 2 ppm O3 to 

achieve the same BALF accumulation of 18 8 O reaction product that was observed in 

humans exposed to 400 ppb with intermittent heavy exercise (V̇ 9 E ~60 L/min). The 

concentration of 18 10 O reaction product in BALF paralleled the accumulation of BALF 

11 protein and cellular effects of the O3 exposure observed such that these responses to 

12 2.0 ppm O3 were similar to those of the 400 ppb O3 in exercising humans. This suggests 

13 that animal data obtained in resting conditions would underestimate the reaction of O3

14 with cells in the RT and presumably the resultant risk of effect for humans. However 

these results should be interpreted with caution given an important limitation in the 18 15 O 

16 labeling technique when used for interspecies comparisons. The reaction between O3 and 

some reactants such as ascorbate produce 18 17 O-labeled products that are lost during sample 

18 processing. When levels of ascorbate or other such reactants vary between species, this 

lost portion of the total 18 19 O-reaction products will also vary, leading to uncertainty in 

20 interspecies comparisons. 
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Note: The excess 18O in each fraction is expressed relative to the dry weight of that fraction. Fractions assayed include cells, high 

speed pellet (HSP), high speed supernatant (HSS), and lavaged lung homogenates.

Source: Hatch et al. (1994)

Figure 5-11 Oxygen-18 incorporation into different fractions of BALF from 

humans and rats exposed to 0.4 and 2.0 ppm 18O3.

1 Recently, a quantitative comparison of O3 transport in the airways of rats, dogs, and 

2 humans was conducted using a three-compartment airways model, based on upper and 

3 lower airway casts and mathematical calculation for alveolar parameters (Tsujino et al., 

4 2005). This one-dimensional gas transport model examined how interspecies anatomical 

5 and physiological differences affect intra-airway O3 concentrations and the amount of gas 

6 absorbed. The morphological model consisted of cylindrical tubes with constant volume 

7 and no airway branching patterns. Peak, real-time, and mean O3 concentrations were 

8 higher in the upper and lower airways of humans compared to rats and dogs, but lowest 

9 in the alveoli of humans. The amount of O3 absorbed was lowest in humans when 

10 normalized by body weight. The intra-airway concentration decreased distally in all 

11 species. Sensitivity analysis demonstrated that VT, fB, and upper and lower airways 
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1 surface area had a statistically significant impact on model results. The model is limited 

2 in that it did not account for chemical reactions in the ELF or consider gas diffusion as a 

3 driving force for O3 transport. Also, the model was run at a respiratory rate of 16/min 

4 simulating a resting individual, however exercise may cause a further deviation from 

5 animal models as was seen in Hatch et al. (1994).

6 Overall, animal models exhibit qualitatively similar patterns of O3 net and tissue dose 

7 distribution with the largest tissue dose delivered to the CAR. However, due to 

8 anatomical and biochemical RT differences the absolute values of O3 dose delivered 

9 differs. Past results suggest that animal data obtained in resting conditions would 

10 underestimate the O3 reactions with cells in the BALF and presumably the resultant risk 

11 of effect for humans, especially for humans during exercise.

5.5.2 Interspecies Homology of Response

12 Biological response to O3 exposure broadly shows commonalities in many species. 

13 Among rodents, non-human primates, and humans, for example, ample data suggest that 

14 O3 induces oxidative stress, cell injury, upregulation of cytokines/chemokines, 

15 inflammation, alterations in lung function, and disruption of normal lung growth and 

16 development (See Chapters 6 and 7). 

17 The effects related to early life exposures can differ appreciably across species due to the 

18 maturation stage of the lung and immune systems at birth. Evidence from non-human 

19 primate studies shows that early life O3 exposure disrupts lung development producing 

20 physiologic perturbations that are similar to those observed in children exposed to urban 

21 air pollution (Fanucchi et al., 2006; Joad et al., 2006). Studies of O3 effects on lung 

22 surface chemistry also show some degree of homology. Lipid oxidation products specific 

23 to O3 reactions with unsaturated fatty acids have been reported, for example, in lavage 

24 fluids from both rodents and humans (Frampton et al., 1999; Pryor et al., 1996). In 

25 humans, the extent to which systemic effects occur is less well studied; plasma indices of 

26 lipid oxidation such as isoprostanes unfortunately do not pinpoint the compartment(s) 

27 where oxidative stress has transpired. That oxidative stress occurs systemically in both 

28 rodents and non-human primates (Chuang et al., 2009), nevertheless, suggests that it 

29 likely also occurs in humans. 

30 Despite the overall similarities in responses to O3 among species, studies have reported 

31 variability in the responsiveness to O3 between and within species, as well as between 

32 endpoints. Rodents appear to have a slightly higher tachypneic response to O3 and are 

33 less sensitive to changes in pulmonary function responses than humans (U.S. EPA, 

34 1996a). However, rats experience attenuation of pulmonary function and tachypneic 
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1 ventilatory responses, similar to humans (Wiester et al., 1996b). Hatch et al. (1986)

2 reported that guinea pigs were the most responsive to O3-induced inflammatory cell and 

3 protein influx. Rabbits were the least responsive and rats, hamsters, and mice were 

4 intermediate responders. Further analysis of this study by Miller et al. (1988) found that 

5 the protein levels in BALF from guinea pigs increased more rapidly with predicted 

6 pulmonary tissue dose than in rats and rabbits. Alveolar macrophages isolated from 

7 guinea pigs and humans mounted similar qualitative and quantitative cytokine responses 

8 to in vitro O3 (0.1-1.0 ppm for 60 minutes) exposure (Arsalane et al., 1995). 

9 Also, because of their higher body surface to volume ratio, rodents can rapidly lower 

10 body temperature during exposure leading to lowered O3 dose and toxicity (Watkinson et 

11 al., 2003; Iwasaki et al., 1998; Slade et al., 1997). In addition to lowering the O3 dose to 

12 the lungs, this hypothermic response may cause: (1) lower metabolic rate, (2) altered 

13 enzyme kinetics, and (3) altered membrane function. The thermoregulatory mechanisms 

14 also may affect disruption of heart rate that may lead to: (1) decreased cardiac output, (2) 

15 lowered blood pressure, and (3) decreased tissue perfusion (Watkinson et al., 2003). 

16 These responses have not been observed in humans except at very high exposures, thus 

17 further complicating extrapolation of effects from animals to humans.

18 The degree to which O3 induces injury and inflammation responses appears to be variable 

19 between species. However, the majority of those studies did not normalize the response 

20 to the dose received to account for species differences in O3 absorption. For example, 

21 Dormans et al. (1999) found that rats, mice, and guinea pigs all exhibited O3-induced (0.2 

22 - 0.4 ppm for 3-56 days) inflammation; however, guinea pigs were the most responsive 

23 with respect to alveolar macrophage elicitation and pulmonary cell density in the 

24 centriacinar region. Mice were the most responsive in terms of bronchiolar epithelial 

25 hypertrophy and biochemical changes (e.g., LDH, glutathione reductase, glucose26 6-phosphate dehydrogenase activity), and had the slowest recovery from O3 exposure. All 

27 species displayed increased collagen in the ductal septa and large lamellar bodies in Type 

28 II pneumocytes at the longest exposure and highest concentration; whereas this response 

29 occurred in the rat and guinea pig at lower O3 levels (0.2 ppm) as well. Overall, the 

30 authors rated mice as most responsive, followed by guinea pigs, then rats (Dormans et al., 

31 1999). Rats were also less responsive in terms of epithelial necrosis and inflammatory 

32 responses as a result of O3 exposure (1.0 ppm for 8 hours) compared with monkeys and 

33 ferrets, which manifested a similar response (Sterner-Kock et al., 2000). Results of this 

34 study should be interpreted with caution since no dose metric was used to normalize the 

35 total inhaled dose or local organ dose between species. 

36 To further understand the genetic basis for age-dependent differential response to O3, 

37 adult (15 week old) and neonatal (15-16 day old) mice from 8 genetically diverse strains 
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1 were examined for O3-induced (0.8 ppm for 5 hours) pulmonary injury and lung 

2 inflammation (Vancza et al., 2009). Ozone exposure increased polymorphonuclear 

3 leukocytes (PMN) influx in all strains of neonatal mice tested, but significantly greater 

4 PMNs occurred in neonatal compared to adult mice for only some sensitive strains, 

5 suggesting a genetic background effect. This strain difference was not due to differences 

in delivered dose of O3 to the lung, evidenced by 18 6 O lung enrichment. The sensitivity of 

7 strains for O3-induced increases in BALF protein and PMNs was different for different 

8 strains of mice suggesting that genetic factors contributed to heightened responses. 

Interestingly, adult mice accumulated more than twice the levels of 18 9 O reaction product 

10 of O3 than corresponding strain neonates. Thus, it appeared that the infant mice showed a 

11 2-fold- to 3-fold higher response than the adults when expressed relative to the 

12 accumulated O3 reaction product in their lungs. The apparent decrease in delivered O3

13 dose in neonates could be a result of a more rapid loss of body temperature in infant 

14 rodents incident to maternal separation and chamber air flow.

15 In animal studies, inhaled O3 concentration and exposure history rarely reflect actual 

16 human environmental exposures. Generally, very high exposure concentrations are used 

17 to induce murine AHR, which in some human subjects is observed at far more relevant 

18 concentrations. This calls into question whether the differences in airway reactivity are 

19 simply a function of differential nasopharyngeal scrubbing or whether the complexities 

20 encompassing a variety of contributory biological pathways show species divergence. 

21 Furthermore, in non-human primates exposed during early life, eosinophil trafficking 

22 occurs, which has not been observed in rodents (unless sensitized) (Maniar-Hew et al., 

23 2011). This response has been shown to be persistent when O3 challenges are 

24 administered after a recovery period of ≥9 months during which no exposure transpired. 

25 Quantitative extrapolation is challenging due to a number of uncertainties. Unfortunately, 

26 many input parameters needed to conduct quantitative extrapolations across species have 

27 not been obtained or currently remain undefined. It is not clear whether characterization 

28 of the ELF provides the information needed to compute a profile of reaction products or 

29 whether environmentally relevant exposure has altered the physicochemical interactions 

30 that occur within the RT surface compartment (e.g., O3 diffusion through regions where 

31 the ELF is thin). That systemic effects have been documented in both rodents and non32 human primates leads to the question of whether reaction products, 

33 cytokines/chemokines, or both enter the nasopharyngeal or bronchial circulation, both of 

34 which show species-dependent differences (Chuang et al., 2009; Cole and Freeman, 

35 2009).

36 In addition, the response to O3 insult across species and more recent health effects such as 

37 immune system development are uncertain. Non-human primate studies have shown 
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1 hypo-responsiveness to endotoxin challenge as a consequence of exposure; whether this 

2 occurs in rodents and humans is largely unknown (Maniar-Hew et al., 2011). In addition, 

3 structural changes (e.g., airways remodeling, fibrogenesis) might differ appreciably 

4 across species. Moreover, whether the upper airways differentially contribute to either 

5 distal lung or systemic impacts has not been explored. 

6 Some outcomes (e.g., inflammation) support the conclusion of homologous responses 

7 across species. However, factors such as age, exposure history, diet, endogenous 

8 substrate generation and homeostatic regulation, the cellular machinery that regulates 

9 inflammatory cell trafficking, responses to other environmental challenges, and the 

10 precise chemical species (whether ELF or cell membrane-derived) that account for 

11 exposure-related initiation of pathophysiologic sequelae might differ across species, but 

12 the extent of species-specific contributing factors remains unknown. Consequently, some 

13 level of uncertainty cannot be dismissed. Nonetheless, if experimental animals show 

14 pathophysiological consequences of exposure, assuming that qualitatively similar human 

15 health impacts could occur is not unreasonable.

5.5.3 Summary

16 In summary, biological response to O3 exposure broadly shows commonalities in many 

17 species and thus supports the use of animal studies in determining mechanistic and cause18 effect relationships and as supporting evidence that similar effects could occur in humans 

19 if O3 exposure is sufficient. However, there is uncertainty regarding the similarity of 

20 response to ozone across species for some recently described endpoints. Differences exist 

21 between species in a number of factors that influence O3 dosimetry and responses, such 

22 as RT anatomy, breathing patterns, and ELF antioxidant concentrations and chemical 

23 species. While humans and animals are similar in the pattern of regional O3 dose 

24 distribution, these differences will likely result in differences in the absolute values of 

25 O3 dose delivered throughout the RT. These considerations limit quantitative comparison 

26 between species.

5.6 Chapter Summary

27 Ozone is a highly reactive gas and a powerful oxidant with a short half-life. Both O3

28 uptake and responses are dependent upon the formation of secondary reaction products in 

29 the ELF; however more complex interactions occur. Uptake in humans at rest is 80-95% 

30 efficient and it is influenced by a number of factors including RT morphology, breathing 

31 route, frequency, and volume, physicochemical properties of the gas, physical processes 
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1 of gas transport, as well as the physical and chemical properties of the ELF and tissue 

2 layers. In fact, even though the average LRT dose may be at a level where health effects 

3 would not be predicted, local regions of the RT may receive considerably higher than 

4 average doses due to RT inhomogeneity and differences in the pathlengths, and therefore 

5 be at greater risk of effects. The primary uptake site of O3 delivery to the lung epithelium 

6 is believed to be the CAR, however changes in a number of factors (e.g., physical 

7 activity) can alter the distribution of O3 uptake in the RT. Ozone uptake is chemical 

8 reaction-dependent and the substances present in the ELF appear in most cases to limit 

9 interaction of O3 with underlying tissues and to prevent penetration of O3 distally into the 

10 RT. Still, reactions of O3 with soluble ELF components or possibly plasma membranes 

11 result in distinct products, some of which are highly reactive and can injure and/or 

12 transmit signals to RT-cells.

13 Thus, in addition to contributing to the driving force for O3 uptake, formation of 

14 secondary oxidation products initiates pathways that provide the mechanistic basis for 

15 health effects that are described in detail in Chapters 6 and 7 and that involve the RT as 

16 well as extrapulmonary systems. These pathways include activation of neural reflexes, 

17 initiation of inflammation, alteration of epithelial barrier function, sensitization of 

18 bronchial smooth muscle, modification of innate and adaptive immunity, airways 

19 remodeling, and systemic inflammation and oxidative/nitrosative stress. With the 

20 exception of airways remodeling, these pathways have been demonstrated in both 

21 animals and human subjects in response to the inhalation of O3. 

22 Both dosimetric and mechanistic factors contribute to the understanding of 

23 interindividual variability in responses to O3. This variability is influenced by differences 

24 in RT volume and surface area, certain genetic polymorphisms, pre-existing conditions 

25 and disease, nutritional status, lifestages, attenuation, and co-exposures. Some of these 

26 factors also underlie differences in species homology and sensitivity. Qualitatively, 

27 animal models exhibit similar patterns of O3 net and tissue dose distribution with the 

28 largest tissue dose of O3 delivered to the CAR. However, due to anatomical and 

29 biochemical RT differences, the absolute value of delivered O3 dose differs, with animal 

30 data obtained in resting conditions underestimating the dose to the RT and presumably 

31 the resultant risk of effect for humans, especially humans during exercise. Even though 

32 interspecies differences limit quantitative comparison between species, many short-term

33 responses of laboratory animals to O3 appear qualitatively homologous to those of the 

34 human. Furthermore, animal studies add to a better understanding of the full range of 

35 potential O3-mediated effects. Given the commonalities in many responses across 

36 species, animal studies that observe O3-induced effects may be used as supporting 

37 evidence that similar effects could occur in humans or in determining mechanistic and 

38 cause-effect relationships if O3 exposure is sufficient.
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