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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 which underlie 

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

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

4 7. 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 

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

10 EPA, 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 relevant 

17 sections of Chapters 6 and 7 are indicated. Older studies which 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 The topics of dosimetry and mode of action are bridged by reactions of O3 with 

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

23 and biological responses (Figure 5-1). 

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

25 related to species comparison of doses and responses (Sections 5.4 and 5.5). These topics 

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

27 mechanistic considerations.
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Figure 5-1 Schematic of the O3 exposure and response pathway. O3

concentrations can be reported as the exposure concentration, 

inhaled dose, the net dose, or the local tissue dose. The net dose 

refers to the total absorption of O3 and is the sum of all the tissue 

compartmental doses. 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. Dose refers 

3 to the amount of O3 crossing an exposure surface to enter a target area. In the literature, 

4 surrogates of dose of reactive gases, such as O3, can range in refinement from their 

5 concentration in the ambient exposure atmosphere to the “effective” dose of the chemical 

6 or its reaction products that actively participate in toxic reactions (Dahl, 1990). However, 

7 ambient concentrations are not a true measure of dose. Ideally, the units for the 

8 expression of the dose of O3 might range from the quantity of gas inhaled as the product 

9 of gas concentration × minute ventilation × time (units of ppm × L × h), to the quantity of 

10 gas retained by the whole body, to the concentration of gas molecules that have been 

11 absorbed or reacted with the tissue (moles/g tissue weight). In modeling studies, the dose 

12 rate is often expressed as a flux per unit of surface area of a region of respiratory 

13 epithelium.

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

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

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

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

O3 exposure

Inhaled 

O3 dose

Net O3

dose

Tissue O3 dose and

product formation

Modes of Action Health Effects
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1 (ET) region or upper respiratory tract (URT, from the nose/mouth to larynx); the 

2 tracheobronchial (TB) tree (from trachea to the terminal bronchioles); and the alveolar or 

3 pulmonary region (from the respiratory bronchioles to the terminal alveolar sacs). The 

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

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

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

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

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

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

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

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

12 mammals.

Source: Based on ICRP (1994)

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

are anterior nasal passages, ET1; oral airway and posterior nasal 

passages, ET2; bronchial airways, BB; bronchioles, bb; and 

alveolar interstitial, AI.
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Source: Panel (a) reprinted with permission from McGraw-Hill (Weibel, 1980)

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

to the alveolus. (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.

c. Transport Factors

Gas Phase

Convection

Diffusion

Dispersion

Liquid Phase

Solubility

Diffusion

Chemical Reaction

Convection

O3

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

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

3 reactive has suggested to some that its effective dose at the target sites exists in the form 

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

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

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

7 with a thin film of mucus and the alveolar region lined with a thinner layer of surfactant. 

8 Ozone toxicity is observed to some extent in the nasal cavity, however further toxicity 

9 exists in the LRT where the thinness of the ELF layer allows O3 to react directly with 

10 cells protruding from the ELF (Figure 5-3b). Ozone uptake relates directly to these ELF 

11 substrate reactions and is termed “reactive absorption.” Thus, the uptake of O3 is related 

12 to both the concentration of O3 as well as the availability of substrates within the ELF. 

13 Chemical reactions are not the only processes controlling the uptake of O3 from the 

14 airstream into compartments of the RT (Figure 5-3c). Ozone uptake is affected by 

15 complex interactions between a number of major factors including RT morphology, 

16 breathing route, frequency, and volume, physicochemical properties of the gas, physical 

17 processes of gas transport, as well as the physical and chemical properties of the ELF and 

18 tissue layers. The role of these processes varies throughout the length of the RT and as O3

19 moves from the gas to liquid compartments of the RT.

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

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

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

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

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

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

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

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

28 change the dosimetry conclusions of the last document. 

5.2.2 Ozone Uptake 

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

30 the state of the science in O3 dosimetry. One method of quantifying O3 dosimetry is to 

31 measure the amount of O3 removed from the air stream during breathing (termed 

32 “uptake”). The O3 in the breath that is removed during the breathing period is termed 

33 “uptake efficiency” or fractional absorption. Uptake studies have utilized bolus and 

34 continuous O3 breathing techniques as well as modeling to investigate uptake efficiency 
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1 and distribution of O3 uptake between the upper and lower respiratory tract. A number of 

2 the studies that have measured the fractional uptake of O3 in the human RT (FRT), URT 

3 (FURT), and LRT (FLRT) are presented in Table 5-1. 

4 Table 5-1 Human respiratory tract uptake efficiency data

5.2.2.1 Gas Transport Principles

5 Transport of O3 in the gas phase is governed by bulk flow or convection, effective axial 

6 dispersion, and loss to airway walls (Figure 5-3c) (Miller, 1995). The relative importance 

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

8 ventilation in any species. For example, bulk airflow is the predominant mechanism for 

9 gas transport in the URT and bronchi, while diffusion is the major transport mechanism in 

10 the alveolar region of the lung. 

Reference Mouth/Nosea Inspiratory Flow 

(mL/s) VT (mL) fB (bpm)b FRT FURT FLRT

CONTINUOUS EXPOSURE

Gerrity et al. (1988) M 509 832 18 0.40 0.91

N 456 754 18 0.36 0.91

M/N 350 832 12 0.41 0.93

M/N 634 778 24 0.38 0.89

Gerrity et al. (1994)

c M 1,360 1,650 25 0.81 0.37 0.43

M 1,360 1,239 35 0.78 0.41 0.36

Gerrity et al. (1995) Mouthpiece 330 825 12 0.91 0.27 0.95

Wiester et al. (1996c) M 539 631 16 0.76

N 514 642 16 0.73

Santiago et al. (2001) N 50 0.80d

N 250 0.33

Rigas et al. (2000) Face mask 480 1,100 27.6 0.86

BOLUS EXPOSURE

Hu et al. (1992) Mouthpiece 250 0.96 0.46

Ultman et al. (1994) Mouthpiece 250 500e 15 0.30

Mouthpiece 250 500 15 0.47

Ultman et al. (2004) M 490 450e 32.7 0.87

M 517 574 27 0.91

Nodelman and Ultman (1999) Nasal Cannula 150 500 18 0.90

Nasal Cannula 1,000 500 120 0.45

Mouthpiece 150 500 18 0.80 0.95

Mouthpiece 1,000 500 120 0.25 0.90

a

M = mouth exposure during spontaneous breathing; N = nasal exposure during spontaneous breathing; M/N = pooled data from mouth and 

nasal exposure; mouthpiece = exposure by mouthpiece; FRT = total RT uptake; FURT = upper RT uptake; FLRT = lower RT uptake.

b

fB is either measured or is computed from flows and VT.

c

Total RT uptake reported by Gerrity et al. (1988) and Gerrity et al. (1994) did not include the contribution from URT uptake efficiency during 

expiration. The data include an expiratory URT contribution, assuming it equals inspiratory URT uptake efficiency.

d

FURT from Santiago et al. (2001) represents nasal absorption (Fnose).

e

VT is computed from flow and fB.
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1 Gas transport in the TB region occurs by a combination of bulk flow and mixing 

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

3 nature of the gas or by dispersion processes which depend on local velocity patterns. The 

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

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

6 1995). The principal influence on mixing in the TB region comes from the axial velocity 

7 profile and diffusion. When passing through a bifurcation a velocity profile is altered; the 

8 inspiratory profile is sharper than the more flattened expiratory profile (Schroter and 

9 Sudlow, 1969). The longitudinal dispersion of inspired air depends on the flow regime, 

10 laminar flow (i.e., streamlined) or turbulent flow (i.e., possessing random velocity 

11 fluctuations), and is influenced by Taylor dispersion forces. In humans, turbulent flow 

12 regime persists only a few generations into the RT. Turbulence generation also varies by 

13 species and flow rates. For example, airflow is nonturbulent in the rat nose at any 

14 physiologic flow rate but may be highly turbulent in the human nose during exercise 

15 (Miller, 1995).

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

17 diffusion (Engel, 1985). While moving into more distal areas of the RT, the cross18 sectional area of the airways rapidly increases and linear velocities decrease, leading to a 

19 greater role of molecular diffusion of gases. Gas molecules close to the alveolocapillary 

20 membrane have almost zero convective velocity with respect to the membrane. Overall, 

21 the diffusion of O3 into the ELF where chemical reactions occur drives alveolar gas 

22 uptake. 

5.2.2.2 Target Sites for Ozone Dose

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

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

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

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

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

dose (18 28 O mass/lung weight) in resting O3 exposed rhesus monkeys, when not 

29 considering the nose (Plopper et al., 1998). Furthermore, the greatest cellular injury 

30 occurred in the vicinity of the respiratory bronchioles and was dependent on the delivered 

O3 dose to these tissues (see also Section 5.4.1). However, 18 31 O label was detected to a 

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

33 compartments as well, although in a smaller dose. Earlier models predicted that the net 

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

35 progression from the trachea to the end of the TB region and then rapidly decreased in the 
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1 alveolar region (Miller et al., 1985). However, the tissue O3 dose (O3 flux to liquid-tissue 

2 interface) was low in the trachea, increased to a maximum in the terminal bronchioles 

3 and the CAR, and then rapidly decreased in the alveolar region. Despite the exclusion of 

4 the URT and O3 reactions with ELF constituents after the 16th generation, the model 

5 predicted experimental results showing that the CAR received the greatest O3 tissue dose 

6 (Miller et al., 1985). 

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

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

9 dose to the region. Ultman and Anjilvel (1990) and Overton et al. (1989) predicted 

10 approximately a 50 to 300% greater PAR dose for the shortest path relative to the longest 

11 path in humans and rats, respectively. In addition, Mercer et al. (1991) found that both 

12 path distance and ventilatory unit size affected dose. The variation of O3 dose among 

13 anatomically equivalent ventilatory units was predicted to vary as much as six-fold, as a 

14 function of path length from the trachea. This could have implications in regional damage 

15 to the LRT, such that even though the average LRT dose may be at a level that would be 

16 considered insignificant, local regions of the RT may receive significantly higher than 

17 average doses and therefore be at greater risk of effects.

5.2.2.3 Upper Respiratory Tract Ozone Removal and Dose

18 The URT provides a defense against O3 entering the lungs by removing half of the 

19 inhaled O3 from the airstream. In both animals and humans, about 50% of the absorbed 

20 O3 was removed in the head (nose, mouth, and pharynx), about 7% in the larynx/trachea, 

21 and about 43% in the lungs (Hu et al., 1992; Hatch et al., 1989; Miller et al., 1979). The 

22 fraction of O3 taken up was inversely related to flow rate and weakly related to inlet O3

23 concentration (Yokoyama and Frank, 1972). The limiting factors in nasal O3 uptake were 

24 simultaneous diffusion and chemical reaction of O3 in the nasal ELF layer (Santiago et 

25 al., 2001). The ELF layer in the nose is thicker than in the rest of the RT, and 

26 mathematical estimates predicted that O3 penetrates less than the thickness of the ELF 

27 layer; reaction products are likely the agents damaging the nasal tissue and not O3 itself. 

28 It was hypothesized that the nasal nonlinear reaction kinetics could result from the 

29 depleting substrates in the nasal ELF becoming the limiting factor of the reaction 

30 (Santiago et al., 2001). 

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

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

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

34 upper trachea (totaling 27.1%), 8.4% from the upper trachea to the main bifurcation 
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1 carina (totaling 35.5%), and essentially zero between the carina and the bronchus 

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

3 (1992) that reported uptake efficiencies of 21, 36, 44, and 46% between the mouth and 

4 the vocal cords, the upper trachea, the main bifurcation carina, and the bronchus 

5 intermedius, respectively. The lower efficiencies seen in Gerrity et al. (1995) may have 

6 resulted from the mouthpiece scrubbing O3 from the breath during inhalation. 

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

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

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

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

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

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

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

14 However, the inhaled O3 delivered dose to the LRT (i.e., flow rate × [O3 ppm] × nasal O3

15 penetration) was 1.6-fold greater at the higher flow than at the lower flow (2.5 compared 

16 to 0.9 ppm·L/min). Prior exercise did not affect O3 uptake at either flow rate, but did 

17 significantly increase nasal volume (Vn) and AR measurements of nasal cross-sectional 

18 area (minimum cross-sectional area (MCA) which corresponds to the nasal valve, CSA2 

19 which corresponds to the anterior edge of the nasal turbinates, and CSA3 which 

20 corresponds to the posterior edge of the nasal turbinates) (p ≤ 0.05). Conversely, exercise 

21 decreased nasal resistance (Rn) (p < 0.01) and NO production (nonsignificant, p > 0.05). 

22 The change in Vn and CSA2:MCA ratio was correlated with the percent change in nasal 

23 uptake, however the overall effect was small and sensitive to elimination of outliers and 

24 gender segregation. 

25 Overall, the URT removes half of the inhaled O3 by reactions in the nasal ELF. The exact 

26 uptake efficiency will change due to variations in flow rate and inhaled concentration. 

5.2.2.4 Lower Respiratory Tract Ozone Uptake and Dose

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

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

29 ranges from 80-95% efficient (Hu et al., 1992). Approximately 43% of inhaled O3 is 

30 absorbed in the LRT of both humans and animals. Models predicted that the net O3 dose 

31 decreases distally from the trachea toward the end of the TB region and then rapidly 

32 decreases in the alveolar region (Miller et al., 1985). However, these models predicted 

33 low tissue O3 dose in the trachea and large bronchi. As injury has been seen in these 

34 areas, net dose may be a better predictor of local toxic tissue dose. 
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1 Uptake efficiency depends on a number of variables, including O3 exposure 

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

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

4 (VT); by minute volume (MV = fB x VT) and fB; or by MV and VT. Simulations from the 

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

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

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

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

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

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

11 decreasing the dose delivered to the lung at a particular MV (Gerrity et al., 1994). Nasal 

12 O3 uptake was inversely proportional to flow rate (Santiago et al., 2001), so that an 

13 increase in MV will increase O3 delivery to the lower airways. At a fixed MV, increasing 

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

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

16 Gerrity et al., 1988). Modeling also predicted a decrease in fractional uptake with 

17 increased fB when VT was held constant, but an increase in PAR dose with increased fB

18 (Overton et al., 1996). Similarly, increased fB (80 - 160 bpm) and shallow breathing in 

rats decreased midlevel tracheal 18O content and an increased 18 19 O content in the mainstem 

20 bronchi (Alfaro et al., 2004). This dependence may be a result of frequency-induced 

21 alterations in contact time that affects the first-order absorption rate for O3 (Postlethwait 

22 et al., 1994). Also, an association of O3 uptake efficiency was found with MV and 

23 exposure time. 

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

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

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

27 (1994) and Ultman et al. (1994) found that O3 absorption increased with volumetric 

28 penetration (Vp) of a bolus of O3 into the RT (Figure 5-5). Ozone uptake efficiency and 

29 Vp were not affected by bolus O3 concentration (Kabel et al., 1994; Hu et al., 1992), 

30 indicating that O3 uptake is a linear absorption process, where the diffusion and chemical 

31 reaction rates of O3 are proportional to the O3 concentration. This relationship was also 

32 true for nasal cavity uptake (Santiago et al., 2001). Rigas et al. (2000) found a weak but 

33 significant negative dependence of O3 concentration on uptake efficiency in exercising 

34 individuals; however, only due to large changes in O3 concentration suggesting that O3

35 uptake is likely still essentially linear with respect to O3 concentration. This study also 

36 found that exposure time had a small but significant influence on uptake efficiency; 

37 however, this negative dependence may be an artifact of progressive depletion of reactive 

38 substrates from the ELF.
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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. Subjects breathed 

0.25 ppm 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).

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

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

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

4 computational fluid dynamics model was created to investigate the dose-response 

5 relationship leading to the distribution of damage in a single airway bifurcation (Taylor et 

6 al., 2007). The model consisted of one parent branch and two symmetrical daughter 

7 branches with a branching angle of 90° and a sharp carinal ridge. Various flow scenarios 

8 were simulated using Reynolds numbers (Re) ranging from 100 to 500. The Re that 

9 corresponds to a certain airway generation is dependent upon both lung size and MV, 

10 such that the range in Re from 100-500 would encompass generations 1-5, 3-7, and 6-10 

11 for an adult during quiet breathing, light exertion, and heavy exercise, respectively, 

12 whereas the same Re range corresponds to generations 0-4, 1-6, and 4-8 for a 4-year-old 

13 child. Consistent with early physical models of Schroter and Sudlow (1969), the model 

14 predicted that during inspiration, the velocity and O3 concentration distribution were 

15 axisymmetric throughout the parent branch, but skewed towards the inner wall within the 

16 daughter branches. During expiration, the model predicted that the velocity and O3

17 concentration distribution was slightly skewed towards the outer walls of the daughter 

18 branches. Hot spots of wall flux existed at the carina during inspiration and expiration 
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1 with Re >100. Additional hot spots were found during expiration on the parent branch 

2 wall downstream of the branching region. 

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

Figure 5-5 Ozone uptake fraction as a function of volumetric penetration (Vp) 

in a representative subject. Each point represents the O3 uptake of 

a bolus inspired through a mouthpiece by the subject. The 

volumes, VUA and VD, are the volume of the upper airways and 

anatomical dead space, respectively, and Vp50% is the Vp at which 

50% of the inspired bolus was absorbed. In 47 healthy subjects, 

Ultman et al. (2004) found that Vp50% was well correlated with VD

and better correlated with the volume of the conducting airways, 

i.e., VD minus VUA.

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

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

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

6 Decreased fB at a fixed penetration volume will shift the O3 uptake from the upper 

7 airways to the central airways and respiratory airspaces.

5.2.2.5 Mode of Breathing

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

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

10 compared to oral breathing. This could be a result of O3 scrubbing in the nasal 
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1 passageways that are bypassed by oral breathing. Studies by Ultman and colleagues using 

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

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

4 oral breathing at 150 mL/s and 0.45 during nasal breathing and 0.25 during oral breathing 

5 at 1,000 mL/s) (Nodelman and Ultman, 1999; Kabel et al., 1994; Ultman et al., 1994). 

6 Therefore, oral breathing results in deeper penetration of O3 into the RT with a higher 

7 absorbed fraction in the URT, TB, and alveolar airways (Nodelman and Ultman, 1999). 

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

9 1972). Earlier human studies suggesedt that oral or oronasal breathing results in a higher 

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

11 however the difference observed between inspired O3 taken up during oral versus nasal 

12 breathing may not be biologically significant. These human studies measured total RT 

13 absorption after continuous O3 exposure using a pharyngeal sampling tube, which may 

14 decrease sensitivity and lead to measurement errors. Overall, the mode of breathing may 

15 have little effect of the RT uptake efficiency, but does play an important role in the 

16 distribution of O3 deposited in the distal airways.

5.2.2.6 Interindividual Variability in Dose

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

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

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

20 (Santiago et al., 2001; Rigas et al., 2000). When concentration, time, and MV were held 

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

22 hypothesized that interindividual variation in O3 induced response such as FEV1 is the 

23 result of interindividual variation in delivered dose or regional O3 uptake among exposed 

24 individuals. 

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

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

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

28 CO2 expirogram to quantify pulmonary responses to O3 found that intersubject 

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

30 Variability in local dose may be attributed to differences in the pulmonary physiology, 

31 anatomy, and biochemistry. Since the TB airways remove the majority of inhaled O3

32 before it reaches the gas exchange region, the volume and surface area of the upper 

33 airways will influence O3 uptake. Models predicted that fractional O3 uptake and PAR 

34 dose (flux of O3 to the PAR surfaces divided by exposure concentration) increase with 
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1 decreasing TB volume and decreasing TB region expansion. On the contrary, alveolar 

2 expansion had minimal effect on uptake efficiency as relatively little O3 reaches the 

3 peripheral lung (Bush et al., 2001; Overton et al., 1996). Ozone uptake was virtually 

4 complete by the time O3 reaches the alveolar spaces of the lung (Postlethwait et al., 

5 1994). Experimental studies have found that differences in TB volumes may account for 

6 75% of the variation in absorption between subjects (Ultman et al., 2004). In support of 

7 this concept, regression analysis showed that O3 absorption was positively correlated 

8 with anatomical dead space (VD) and TB volume (i.e., VD minus VURT), but not total lung 

9 capacity (TLC), forced vital capacity (FVC), or functional residual capacity (FRC) 

10 (Ultman et al., 2004; Bush et al., 1996; Hu et al., 1994; Postlethwait et al., 1994). 

11 Variability in VD was correlated more with the variability in the TB volume than the URT 

12 volume. Similarly, uptake was correlated with changes in individual bronchial cross13 sectional area, indicating that changes in cross-sectional area available for gas diffusion 

14 are related to overall O3 retention (Reeser et al., 2005; Ultman et al., 2004). These studies 

15 provide support to the pulmonary physiology, especially the TB volume and surface area, 

16 playing a key role in variability of O3 uptake between individuals.

17 When absorption data were normalized to Vp/VD, variability attributed to gender 

18 differences were not distinguishable (Bush et al., 1996). However, variability due to age 

19 has been predicted. Overton and Graham (1989) predicted that the total quantity of O3

20 absorbed per minute increased with age from birth to adulthood. This model predicted 

21 that the LRT distribution of absorbed O3 and the CAR O3 tissue dose were not sensitive 

22 to age during quiet breathing. However, during heavy exercise or work O3 uptake was 

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

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

25 per unit surface area was two- to eightfold higher in infants compared to adults, due to 

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

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

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

29 significantly affect the fractional uptake of a bolus dose of O3 in apparently healthy 

30 smokers with limited smoking history (Bates et al., 2009). Despite similar internal O3

31 dose distribution, the smokers exhibited greater pulmonary responses to O3 bolus 

32 exposures, measured as FEV1 decrements and increases in the normalized slope of the 

33 alveolar plateau (SN). This was contrary to previous studies conducted in smokers with a 

34 greater smoking history that found decreased O3 induced decrements in FEV1 in smokers 

35 during continuous O3 exposure (Frampton et al., 1997b; Emmons and Foster, 1991). 
Draft – Do Not Cite or Quote 5-15 September 2011

5.2.2.7 Physical Activity

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

2 part, to the increased intake of air. As exercise increases from a low to moderate level, VT

3 increases. This increase in VT is achieved by encroaching upon both the inspiratory and 

4 expiratory reserve volumes of the lung (Dempsey et al., 1990). After VT reaches about 

5 50% of the vital capacity, generally during heavy exercise, further increases in ventilation 

6 are achieved by increasing fB. Ventilatory demands of heavy exercise require airway flow 

7 rates that often exceed 10 times resting levels and VT that approach 5 times resting levels 

8 (Dempsey et al., 2008).

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

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

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

12 to a switch from nasal to oronasal breathing. Higher ventilatory demand necessitates a 

13 lower-resistance path through the mouth. Modeling heavy exercise by increasing 

14 ventilatory parameters from normal respiration levels predicted a 10-fold increase in total 

15 mass uptake of O3 (Miller et al., 1985). This model also predicted that as exercise and 

16 ventilatory demand increased the maximum tissue dose moved distally into the RT 

17 (Figure 5-6). By increasing flow to what is common in moderate exercise (respiratory 

18 flow = 750 -1,000 mL/s compared to 250 mL/s at rest), the URT absorbed a smaller 

19 fraction of the O3 (~0.50 at rest to 0.10 at exercise); however, the trachea and more distal 

20 TB airways received higher doses during exercise than rest (0.65 absorbed in the lower 

21 TB airways, and 0.25 absorbed in the alveolar zone with exercise compared to 0.5 in the 

22 TB with almost no O3 reaching the alveolar zone at rest) (Hu et al., 1994). The same shift 

23 in the O3 dose distribution more distally in the lung occurred in other studies mimicking 

24 the effects of exercise (Nodelman and Ultman, 1999). Also, LRT uptake efficiency was 

25 sensitive to age only under exercise conditions (Overton and Graham, 1989). The total 

26 quantity of O3 absorbed per minute was predicted to increase with age during heavy work 

27 or exercise. A recent study by Sawyer et al. (2007) showed that doubling minute 

28 ventilation led to only a 1.6-fold higher delivered dose rate of O3 to the lung. Past models 

29 have predicted the increase in uptake during exercise is distributed unevenly in the RT 

30 compartments and regions. Tissue and mucus layer dose in the TB region increased ~1.4-

31 fold during heavy exercise compared to resting conditions, whereas the alveolar region 

32 surfactant and tissue uptake increased by factors of 5.2 and 13.6, respectively (Miller et 

33 al., 1985). 
Draft – Do Not Cite or Quote 5-16 September 2011

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

Figure 5-6 Modeled effect of exercise on tissue dose of the LRT. 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.

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. Recent 

9 studies have provided evidence for hot spots of O3 flux around bifurcations in airways. 

10 Experimental studies and models have suggested that the net O3 dose gradually decreases 

11 distally from the trachea toward the end of the TB region and then rapidly decreases in 

12 the alveolar region. However, the tissue O3 dose is low in the trachea, increases to a 
Draft – Do Not Cite or Quote 5-17 September 2011

1 maximum in the terminal bronchioles and the CAR, and then rapidly decreases distally 

2 into the alveolar region. 

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

4 decrease with increased flow and increase proportional to VT, so that at a fixed MV, 

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

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

7 sensitive to large changes in O3 concentration, exposure time, and MV. Major sources of 

8 variability in absorption of O3 include O3 concentration, exposure time, fB, MV, and VT, 

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

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

11 area.

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

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

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

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

16 O3 distally into the RT and into the alveolar region. 

5.2.3 Ozone Reactions and Reaction Products

17 Ozone dose can be examined by the chemical reactions or the products of these reactions 

18 that result from O3 exposure. Since O3 is chemically reactive with a wide spectrum of 

19 biomolecules, it is not feasible to delineate its many reaction products. Measurements of 

20 reaction product formation have included either the loss of a specific molecule and 

21 appearance of plausible products, or the addition of O3-derived oxygen to biomolecules 

22 through the use of oxygen-18 labeling. In vitro exposure of ELF showed that O3

23 disappearance from the gas phase depends on the characteristics of the ELF substrates 

24 (Postlethwait et al., 1998; Hu et al., 1994). 

25 For O3 to gain access to the underlying cellular compartments, O3 must dissolve at the 

26 air-liquid interface of the airway surface and travel through the ELF layer. The ELF is 

27 comprised of the airway surface lining that includes the periciliary sol layer and

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

29 liquid and vesicular surfactant and the continuous surfactant monolayer (Bastacky et al., 

30 1995). There is a progressive decrease in ELF thickness and increase in interfacial 

31 surface with progression from the large airways to the alveolus (Mercer et al., 1992). 

32 Some cells, such as macrophages, may protrude into the gas phase, allowing for direct 

33 contact between O3 and cell membranes. The progressive thinning of the ELF while 

34 moving further down the RT decreases the radial distance O3 must travel to reach the 
Draft – Do Not Cite or Quote 5-18 September 2011

1 cellular tissue layer. A computational fluid dynamics model was able to predict 

2 experimentally measured O3 uptake, but only with nasal mucus layer thickness 

3 considered (Cohen-Hubal et al., 1996), reaffirming the importance of the resistance 

4 imparted by the ELF layer in dose and lesion patterns in the nasal passage. 

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

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

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

8 (Pryor, 1992). However, the surfactant layer in the pulmonary region becomes ultrathin, 

9 possibly allowing for direct interaction of O3 with the underlying epithelial cells. One 

10 study measured pulmonary liquid lining thickness over relatively flat portions of the 

11 alveolar wall to be 0.14 μm, to be 0.89 μm at the alveolar wall junctions, and 0.09 μm 

12 over the protruding features (Bastacky et al., 1995). Still, the ELF should be considered 

13 an important target for O3 and the resulting secondary oxidation products should be 

14 considered key mediators of toxicity in the airways (role of reaction products in O3

15 induced toxicity is discussed in Section 5.3). Model calculations of the nasal cavity based 

16 on diffusion equations and reaction rates of O3 with model substrates predict an O3

17 penetration distance (0.5 μm) less than the thickness of the mucus layer (10 μm) 

18 (Santiago et al., 2001). Experimental support for this concept comes from several studies 

19 which measured the total oxygen-addition product of O3 reactions in the airways through 

20 the use of oxygen-18 labeled O3. High concentrations of O3 reaction products were found 

21 in the bronchoalveolar lavage (BAL) mucus and surfactant providing evidence that O3

22 reacts at the air-liquid interface. Thus, O3 may cause injury by direct reaction with 

23 constituents of the lining layer, with cells protruding from it and in some cases with cells 

24 underlying the lining fluid. The reaction cascade resulting from the interaction of O3 with 

25 ELF substrates acts to carry the oxidative burden deeper into the tissues. 

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

27 neutral lipids, free fatty acids, proteins, and low molecular weight antioxidants (Perez28 Gil, 2008; Uppu et al., 1995). It was estimated that 88% of the O3 that does not come in 

29 contact with antioxidants will react with unsaturated fatty acids in the ELF including 

30 those contained within phospholipids or neutral lipids (Uppu et al., 1995). Ozone reacts 

31 with the double bond of lipids such as unsaturated fatty acids, a large component of ELF, 

32 to form stable and less reactive ozonide, aldehyde, and hydroperoxide reaction products 

33 via chemical reactions such as the Criegee ozonolysis mechanism (Figure 5-7) (Pryor et 

34 al., 1991). Lipid ozonation products, such as the aldehydes hexanal, heptanal, and 

35 nonanal, have been recovered after O3 exposure in human BAL fluid (BALF), rat BALF, 

36 isolated rat lung, and in vitro systems (Frampton et al., 1999; Postlethwait et al., 1998; 

37 Pryor et al., 1996). Nonanal has been suggested as a relatively specific biomarker for O3

38 exposure since the monounsaturated fatty acid parent compound, oleic acid, does not 
Draft – Do Not Cite or Quote 5-19 September 2011

1 undergo autoxidation (Pryor et al., 1996). Adducts of the aldehyde 4-hydroxynonenal 

2 were found in human alveolar macrophages after O3 exposure (Hamilton et al., 1998). 

3 Polyunsaturated fatty acid (PUFA) reactions are limited by the availability of O3 since 

4 lipids are so abundant in the ELF. Yields of O3-induced aldehydes were increased by the 

5 decrease in other substrates such as ascorbic acid (AH2) (Postlethwait et al., 1998). Free 

6 radicals are also generated during O3-mediated oxidation reactions with PUFA (Pryor, 

7 1994). These reactions are reduced by the presence of the lipid-soluble free radical 

8 scavenger α-tocopherol (α-TOH) (Pryor, 1994; Fujita et al., 1987; Pryor, 1976). PUFA 

9 reactions may not generate sufficient bioactive materials to account for acute cell injury, 

10 however only modest amounts of products may be necessary to induce cytotoxicity 

11 (Postlethwait and Ultman, 2001; Postlethwait et al., 1998). 

Source: U.S. EPA (2006b)

Figure 5-7 Schematic overview of ozone interaction with PUFA in ELF and 

lung cells. It should be noted that not all secondary reaction 

products are shown.

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

13 O3 results in biologically active cholesterol products such as the oxysterols, β-epoxide 

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

15 2004). Product yields will depend on ozonolysis conditions, however cholesterol 

16 ozonolysis products were formed in similar abundance to phospholipid-derived 

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

18 The ELF also contains proteins present in blood plasma as well as proteins secreted by 

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

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

21 are cysteine, histidine, methionine, tyrosine, and tryptophan). Ozone has been shown to 
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1 preferentially react with biomolecules in the following order: thiosulfate > ascorbate > 

2 cysteine ≈ methionine > glutathione (Kanofsky and Sima, 1995). Rate constants for the 

3 reaction of amino acids with O3 vary between investigations due to differing reaction 

4 conditions and assumptions; however aliphatic amino acids consistently were very slow 

5 to react with O3 (e.g., alanine: 25-100 moles/L/sec) (Kanofsky and Sima, 1995; 

6 Ignatenko and Cherenkevich, 1985; Pryor et al., 1984; Hoigné and Bader, 1983). Uppu et 

7 al. (1995) predicted that 12% of inhaled O3 that does not react with antioxidants will 

8 react with proteins in the ELF, whereas 88% will react with PUFAs. 

9 Reactions of ozone with low molecular weight antioxidants have been extensively 

10 studied. The consumption of antioxidants such as uric acid (UA), ascorbate (AH2), and 

11 reduced glutathione (GSH) by O3 was linear with time and positively correlated with 

12 initial substrate concentration and chamber O3 concentration (Mudway and Kelly, 1998; 

13 Mudway et al., 1996). Endogenous antioxidants are present in relatively high 

14 concentrations in the ELF of the human TB airways and display high intrinsic reactivities 

15 toward O3, but do not possess equal O3 reactivity. In individual and in limited composite 

16 mixtures, UA was the most reactive antioxidant tested, followed by AH2 (Mudway and 

17 Kelly, 1998). GSH was consistently less reactive than UA or AH2 (Mudway and Kelly, 

18 1998; Mudway et al., 1996; Kanofsky and Sima, 1995). To quantify these reactions, 

19 Kermani et al. (2006) recently evaluated the interfacial exposure of aqueous solutions of 

20 UA, AH2, and GSH (50-200 μM) with O3 (1-5 ppm). Similar to the results of Mudway 

21 and Kelly (1998), this study found the hierarchy in reactivity between O3 and these 

22 antioxidants to be UA≥AH2>>GSH. UA and AH2 shared a 1:1 stoichiometry with O3, 

23 whereas 2.5 moles of 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- 24

0.75/sec [20.9 M-1 sec-1 25 ] for the reaction of O3 with UA, AH2, and GSH, respectively). 

26 These values are similar to those derived from data presented in Mudway and Kelly 

27 (1998). Other studies reported reactive rate constants that are two to three orders of 

28 magnitude larger, however these studies used higher concentrations of O3 and 

29 antioxidants under less physiologically relevant experimental conditions (Kanofsky and 

30 Sima, 1995; Giamalva et al., 1985; Pryor et al., 1984). 

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

32 from initial air-liquid interface interactions of O3 with ELF components (e.g., 

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

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

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

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

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

38 interfacial reaction compared to previous studies using bulk liquid phase bioreactors, thus 
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1 supporting the relevance of reactions between gas phase O3 and antioxidants found in the 

2 ELF.

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

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

5 UA > CyS > GSH. There was some variance between the reaction rates and product 

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

7 reacting with bulk liquid phase bioreactors as described previously. UA was more 

8 reactive than AH2 toward O3 in previous studies, but in reactions with O3 with 

9 microdroplets, these antioxidants had equivalent reactivity (Enami et al., 2008b). As O3 is 

10 a kinetically slow one-electron acceptor but very reactive O-atom donor, products of the 

11 interaction of O3 with UA, AH2, GSH, CyS, and α-TOH result from addition of n Oatoms (n = 1-4). These products included epoxides (e.g., U-O-

), peroxides (e.g. U-O2

- 12 ), 

and ozonides (e.g., U-O3

-

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

-

/GSO3

2- 13

14 ), not glutathione disulfide (GSSG) by O3 (Enami et al., 2009b). However, it is possible 

15 that other oxidative species are oxidizing GSH in vivo, since sulfonates are not detected 

16 in O3 exposed ELF whereas GSSG is. This is also supported by the fact that O3 is much 

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

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

19 Ozonolysis product yields and formation were affected by pH. Acidified conditions (pH ≈ 

20 3-4), such as those that may result from acidic particulate exposure or pathological 

21 conditions like asthma (pH ≈ 6), decreased the scavenging ability of UA and GSH for O3; 

22 such that at low pH, the scavenging of O3 must be taken over by other antioxidants, such 

23 as AH2 (Enami et al., 2009b, 2008b). Also, under acidic conditions (pH ≈ 5), the 

24 ozonolysis products of AH2 shifted from the innocuous dehydroascorbic acid to the more 

25 persistent products, AH2 ozonide and threonic acid (Enami et al., 2008a). It is possible 

26 that the acidification of the ELF by acidic copollutant exposure will increase the toxicity 

27 of O3 by preventing some antioxidant reactions and shifting the reaction products to more 

28 persistent compounds.

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

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

31 2005). However, estimated in vitro O3 uptake was not proportional to the production of 

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

addition, O3 33 induced cell membrane oxidation which required interactions with AH2 and 

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

35 between O3 and bovine serum albumin did not result in membrane oxidation (Ballinger et 

36 al., 2005). The presence of UA or bovine serum albumin protected against lipid and 

37 protein oxidation resulting from the reaction of O3 and AH2 (Ballinger et al., 2005). This 
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1 study provided evidence that antioxidants may paradoxically facilitate O3-mediated 

2 damage. This apparent contradiction should be viewed in terms of the concentration3 dependent role of the ELF antioxidants. Reactions between O3 and antioxidant species 

4 exhibited a biphasic concentration response, with oxidation of protein and lipid occurring 

5 at lower, but not higher, concentrations of antioxidant. In this way, endogenous reactants 

6 led to the formation of secondary oxidation products which were injurious and also led to 

7 quenching reactions which were protective. Moreover, the formation of secondary 

8 oxidation products mediated by some antioxidants was opposed by quenching reactions 

9 involving other antioxidants. 

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

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

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

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

14 NO2 (360-720 ppb) or SO2 (360 ppb) exposure (Rigas et al., 1997). It was hypothesized 

15 that this increased fractional absorption of O3 could be due to increased production of 

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

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

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

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

20 1999). The level and type of antioxidant present in ELF varies between species, regions 

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

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

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

24 2006; Freed et al., 1999). In the case of UA, the major source appears to be the plasma 

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

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

27 were exposed to 200 ppb O3 for 2 hours while exercising, nasal lavage fluid UA was 

28 significantly decreased while plasma UA levels were significantly increased (Mudway et 

29 al., 1999a). The finding that UA, but not AH2 or GSH, was depleted in nasal lavage fluid 

30 indicated that UA was the predominant antioxidant with respect to O3 reactivity in the 

31 nasal cavity (Mudway et al., 1999a). In addition, concentrations of UA were increased by 

32 cholinergic stimulation of the airways in exercising human subjects exposed to 400 ppb 

33 O3 for 2 hours, which suggested that increased mucosal gland secretions were an 

34 important source (Peden et al., 1995). Using the O3-specific antioxidant capacity assay on 

35 human nasal lavage samples, Rutkowski et al. (2011) concluded that about 30% of the 

36 antioxidant capacity of the nasal liquid lining layer was attributed to UA activity. This 

37 assay predicted that more than 50% of the subject-to-subject differences in antioxidant 

38 capacity were driven by differences in UA concentration. However, day-to-day within-
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1 subject variations in measured antioxidant capacity were not related to the corresponding 

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

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

4 yield definitive results. However, in human BALF samples, the mean consumption of 

5 AH2 was greater than UA (Mudway et al., 1996). 

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

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

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

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

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

11 for 2 h, while exercising) (Mudway et al., 2001; Blomberg et al., 1999; Mudway et al., 

12 1999b). Furthermore, high levels of dehydroascorbate, the oxidized form of AH2, have 

13 been reported in human ELF (Mudway et al., 2006). Other investigators have 

14 demonstrated cellular uptake of oxidized AH2 by several cell types leading to 

15 intracellular reduction and export of reduced AH2 (Welch et al., 1995). Studies with rats 

16 exposed to 0.4-1.1 ppm O3 for 1-6 hours have shown consumption of AH2 that correlates 

17 with O3 exposure (Gunnison and Hatch, 1999; Gunnison et al., 1996; Vincent et al., 

18 1996a).

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

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

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

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

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

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

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

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

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

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

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

30 In addition to reactions with components of the ELF, O3 may react with plasma 

31 membranes of cells which reside in the RT. Eicosanoids are an important class of 

32 secondary oxidation products which may be formed rapidly by this mechanism. 

33 Eicosanoids are metabolites of arachidonic acid, a 20-carbon PUFA, which is released 

34 from membrane phospholipids by phospholipase A2-mediated catalysis. Activation of 

35 phospholipase A2 occurs by several cell signaling pathways and may be triggered by O3-

36 mediated lipid peroxidation of cellular membranes (Rashba-Step et al., 1997). 

37 Additionally, cellular phospholipases A2, C and D may be activated by lipid ozonation 
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1 products (Kafoury et al., 1998). While the conversion of arachidonic acid to 

2 prostaglandins, leukotrienes and other eicosanoid products is generally catalyzed by 

3 cyclooxygenases and lipoxygenases, non-enzymatic reactions also occur during oxidative 

4 stress leading to the generation of a wide variety of eicosanoids and reactive oxygen 

5 species. Further, the release of arachidonic acid from phospholipids is accompanied by 

6 the formation of lysophospholipids which are precursors for platelet activating factors 

7 (PAFs). Thus, formation of eicosanoids, reactive oxygen species and PAFs accompanies 

8 O3-mediated lipid peroxidation. 

5.2.3.1 Summary

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

10 barrier and target for inhaled O3 (Figure 5-8). The thickness of the lining fluid and mucus 

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

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

Figure 5-8 Details of the O3 interaction with the airway ELF to form secondary 

oxidation products. 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. 

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

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

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

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

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

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

7 and AH2 are more reactive with O3 than GSH, proteins, or lipids. In addition to contri8 buting to the driving force for O3 uptake, formation of secondary oxidation products may 

9 lead to increased cellular injury and cell signaling (discussed in Section 5.3). Studies 

10 indicate that the antioxidants might be participating in reactions where the resulting 

11 secondary oxidation products might penetrate into the tissue layer and cause injury.
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5.3 Possible Pathways/Modes of Action

5.3.1 Introduction

1 Mode of action refers to a sequence of key events and processes which 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 

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

7 the key events and toxicity pathways which contribute to health effects resulting from 

8 short-term and long-term exposures to O3. The extensive research carried out over 

9 several decades in humans and in laboratory animals has yielded numerous studies on 

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

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

12 AQCD and on studies in humans which inform biological mechanisms underlying 

13 responses to O3.

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

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

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

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

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

19 remodeling, as will be discussed below. Exposure to O3 also may result in effects on 

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

21 systems. It is unlikely that lipid ozonides and other secondary oxidation products, which 

22 are bioactive and cytotoxic in the respiratory system, gain access to the vascular space 

23 (Chuang et al., 2009). However the inhalation of O3 may result in systemic oxidative 

24 stress. The following subsections describe the current understanding of potential 

25 pathways and modes of action responsible for the pulmonary and extrapulmonary effects 

26 of O3 exposure.

5.3.2 Activation of Neural Reflexes 

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

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

29 sensory nerve, has been demonstrated in dogs exposed through an endotracheal tube to 2-

30 3 ppm O3 for 20-70 minutes (Coleridge et al., 1993; Schelegle et al., 1993). This vagal 
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1 afferent pathway was found to be responsible for O3-mediated rapid shallow breathing 

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

3 Ozone also triggers neural reflexes which stimulate the autonomic nervous system and 

4 alter electrophysiologic responses of the heart. For example, bradycardia, altered HRV 

5 and arrhythmia have been demonstrated in rodents exposed to 0.1-0.6 ppm O3 (Hamade 

6 and Tankersley, 2009; Watkinson et al., 2001; Arito et al., 1990). Another effect is 

7 hypothermia, which in rodents occurred subsequent to the activation of neural reflexes 

8 involoving the parasympathetic nervous system (Watkinson et al., 2001). Vagal afferent 

9 pathways originating in the respiratory tract may also be responsible for O3-mediated 

10 activation of nucleus tractus solitarius neurons which resulted in neuronal activation in 

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

12 1.5-120 hours) (Gackière et al., 2011).

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

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

15 selectively activated a subset of C-fiber receptors which are TRPA1 ion channels (Taylor16 Clark and Undem, 2010). TRPA1 ion channels are members of the TRP family of ion 

17 channels, which are known to mediate the responses of sensory neurons to inflammatory 

18 mediators (Caceres et al., 2009). In addition to TRPA1 ion channels possibly playing a 

19 key role in O3-induced decrements in pulmonary function, they may mediate allergic 

20 asthma (Caceres et al., 2009). Activation of TRPA1 ion channels following O3 exposure 

21 is likely initiated by secondary oxidation products such as aldehydes and prostaglandins 

22 (Taylor-Clark and Undem, 2010) through covalent modification of cysteine and lysine 

23 residues (Trevisani et al., 2007). Ozonation of unsaturated fatty acids in the ELF was 

24 found to result in the generation of aldehydes (Frampton et al., 1999) such as 

25 4-hydroxynonenal and 4-oxononenal (Taylor-Clark et al., 2008; Trevisani et al., 2007). 4-

26 oxononenal is a stronger electrophile than 4-hydroxynonenal and exhibits greater potency 

27 towards the TRPA1 channels (Taylor-Clark et al., 2008). (Trevisani et al., 2007). In 

28 addition, PGE2 is known to sensitize TRPA1 channels (Bang et al., 2007). 

29 In exercising humans, the response to O3 (500 ppb for 2 h) was characterized by 

30 substernal discomfort, especially on deep inspiration, accompanied by involuntary 

31 truncation of inspiration (Hazucha et al., 1989). This led to decreased inspiratory capacity 

32 and to decreased forced vital capacity (FVC) and forced expiratory volume in one second 

33 (FEV1), as measured by spirometry. These changes, which occurred during O3 exposure, 

34 were accompanied by decreased VT and increased respiratory frequency in human 

35 subjects. Spirometric changes in FEV1 and FVC were not due to changes in respiratory

36 muscle strength (Hazucha et al., 1989). In addition, parasympathetic involvement in the 

37 O3-mediated decreases in lung volume was minimal (Mudway and Kelly, 2000), since 

38 changes in FVC or symptoms were not modified by treatment with bronchodilators such 
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1 as atropine in exercising human subjects exposed to 400 ppb O3 for 0.5 hour (Beckett et 

2 al., 1985). However, the loss of vital capacity was reversible with intravenous 

3 administration of the rapid-acting opioid agonist, sufentanyl, in exercising human 

4 subjects exposed to 420 ppb O3 for 2 hours, which indicated the involvement of opioid 

5 receptor-containing nerve fibers and/or more central neurons (Passannante et al., 1998). 

6 The effects of sufentanyl may be attributed to blocking C-fiber stimulation by O3 since 

7 activation of opioid receptors downregulated C-fiber function (Belvisi et al., 1992). Thus, 

8 nociceptive sensory nerves, presumably bronchial C-fibers, are responsible for O3-

9 mediated responses in humans (Passannante et al., 1998). This vagal afferent pathway is 

10 responsible for pain-related symptoms and inhibition of maximal inspiration in humans 

11 (Hazucha et al., 1989).

12 There is some evidence that eicosanoids (see Section 5.3.3) play a role in the neural 

13 reflex since cyclooxygenase inhibition with indomethacin (Alexis et al., 2000; Schelegle 

14 et al., 1987) or ibuprofen, which also blocks some lipoxygenase activity (Hazucha et al., 

15 1996), before exposure to O3 significantly blunted the spirometric responses. These 

studies involved exposures of 1-2 hours to 350-400 ppb O3 16 in exercising human subjects. 

17 In the latter study, ibuprofen treatment resulted in measurable decreases in BALF levels 

18 of PGE2 and TXB2 at 1-hour postexposure (Hazucha et al., 1996). Although an earlier 

19 study demonstrated that PGE2 stimulated bronchial C-fibers (Coleridge et al., 1993; 

20 Coleridge et al., 1976) and suggested that PGE2 mediated O3-induced decreases in 

21 pulmonary function, no correlation was observed between the degree of ibuprofen22 induced inhibition of BALF PGE2 levels and blunting of the spirometric response to O3

23 (Hazucha et al., 1996). These results point to the involvement of a lipoxygenase product. 

24 Further, as noted above, PGE2 may play a role in the neural reflex by sensitizing TRPA1 

25 channels. A recent study in exercising human subjects exposed for 1 hour to 350 ppb O3

26 also provided evidence that arachidonic acid metabolites, as well as oxidative stress, 

27 contribute to human responsiveness to O3 (Alfaro et al., 2007). 

28 In addition to the spirometric changes, mild airways obstruction occurred in exercising 

29 humans during O3 exposure (500 ppb for 2 hours) (Hazucha et al., 1989). This pulmonary 

30 function decrement is generally measured as specific airway resistance (sRaw) which is 

31 the product of airway resistance and thoracic gas volume. In several studies involving 

32 exercising human subjects exposed for 1-4 hours to 200-300 ppb O3, changes in sRaw 

33 correlated with changes in inflammatory and injury endpoints measured 18-hours 

34 postexposure, but did not follow the same time course or change to the same degree as 

35 spirometric changes (i.e. FEV1, FVC) measured during exposure (Balmes et al., 1996; 

36 Aris et al., 1993; Schelegle et al., 1991). In addition, a small but persistent increase in 

37 airway resistance associated with narrowing of small peripheral airways (measured as 

38 changes in isovolumetric FEF25-75) was demonstrated in O3-exposed human subjects (350 
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1 ppb for 130 minutes with exercise) (Weinmann et al., 1995a; Weinmann et al., 1995b). A 

2 similar study (400 ppb O3 for 2 hours in exercising human subjects) found decreases in 

3 FEF25-75 concomitant with increases in residual volume, which is suggestive of small 

4 airways dysfunction (Kreit et al., 1989). In separate studies, a statistically significant 

5 increase in residual volume (500 ppb for 2 hours) (Hazucha et al., 1989) and a 

6 statistically significant decrease in FEF25-75 (160 ppb for 7.6 hours) (Horstman et al., 

7 1995) were observed following O3 exposure in exercising human subjects, providing 

8 further support for an O3-induced effect on small airways.

9 Mechanisms underlying this rapid increase in airway resistance following O3 exposure 

10 are incompletely understood. Pretreatment with atropine decreased baseline sRaw and 

11 prevented O3-induced increases in sRaw in exercising human subjects (400 ppb for 0.5 

12 hours) (Beckett et al., 1985), indicating the involvement of muscarinic cholinergic 

13 receptors of the parasympathetic nervous system. Interestingly, atropine pretreatment 

14 partially blocked the decrease in FEV1, but had no effect on the decrease in FVC, 

15 breathing rate, tidal volume or respiratory symptoms (Beckett et al., 1985). Using a β16 adrenergic agonist, it was shown that smooth muscle contraction, not increased airway 

17 mucus secretion, was responsible for O3-induced increases in airway resistance (Beckett 

18 et al., 1985). Thus, pulmonary function decrements measured as FEV1 may reflect both 

19 restrictive (such as decreased inspiratory capacity) and obstructive (such as 

20 bronchoconstriction) type changes in airway responses. This is consistent with 

21 McDonnell et al. (1983) who observed a relatively strong correlation between sRaw and 

22 FEV1 (r=-0.31, p=0.001) and a far weaker correlation between sRaw and FVC (r=-0.16, 

23 p=0.10) in exercising human subjects exposed for 2.5 hours to 120-400 ppb O3.

24 Furthermore, tachykinins may contribute to O3-mediated increases in airway resistance. 

25 In addition to stimulating CNS reflexes, bronchopulmonary C-fibers mediate local axon 

26 responses by releasing neuropeptides such as substance P (SP), neurokinin (NK) A and 

27 calcitonin gene-related peptide (CGRP). Tachykinins bind to NK receptors resulting in 

28 responses such as bronchoconstriction. Recent studies in animals demonstrated that NK-1 

29 receptor blockade had no effect on O3-stimulated physiologic responses such as VT and 

30 fB in rats over the 8 hour exposure to 1 ppm O3 (Oslund et al., 2008). However, SP and 

31 NK receptors contributed to vagally-mediated bronchoconstriction in guinea pigs 3 days 

32 after a single 4-hour exposure to 2 ppm O3 (Verhein et al., 2011). In one human study in 

33 which bronchial biopsies were performed and studied by immunohistochemistry, SP was 

34 substantially diminished in submucosal sensory nerves 6 hours following O3 exposure 

35 (200 ppb for 2 hours with exercise) (Krishna et al., 1997). A statistically significant 

36 correlation was observed between loss of SP immunoreactivity from neurons in the 

37 bronchial mucosa and changes in FEV1 measured 1-hour postexposure (Krishna et al., 

38 1997). Another study found that SP was increased in lavage fluid of human subjects 
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1 immediately after O3 challenge (250 ppb for 1 hour with exercise) (Hazbun et al., 1993). 

2 These results provide evidence that the increased airway resistance observed following 

3 O3 exposure is due to vagally-mediated responses and possibly by local axon reflex 

4 responses through bronchopulmonary C-fiber-mediated release of SP. 

5.3.3 Initiation of inflammation

5 As described previously (5.2.3), O3 reacts with components of the ELF and cellular 

6 membranes resulting in the generation of secondary oxidation products. Higher 

7 concentrations of these products may directly injure respiratory tract epithelium. Lower 

8 concentrations may initiate cellular responses including cytokine generation, adhesion 

9 molecule expression and modification of tight junctions leading to inflammation and 

10 increased permeability across airway epithelium (Section 5.3.4) (Dahl et al., 2007; 

11 Mudway and Kelly, 2000). Subsequent airways remodeling may also occur (Section 

12 5.3.7) (Mudway and Kelly, 2000). 

13 An important hallmark of acute O3 exposure in humans and animals is neutrophilic 

14 airways inflammation. Although neutrophil influx into nasal airways has been 

15 demonstrated in exercising human subjects (400 ppb O3, 2 hours) (Graham and Koren, 

16 1990), most studies of neutrophil influx have focused on the lower airways (Hazucha et 

17 al., 1996; Aris et al., 1993). The time course of this response in the lower airways and its 

18 resolution was slower than that of the decrements in pulmonary function in exercising 

19 human subjects exposed for 2 hours to 500 ppb O3 (Hazucha et al., 1996). In several 

20 studies, airways neutrophilia was observable within 1-2 hours, peaked at 4-6 hours and 

21 was returning to baseline levels at 24 hours following exposure of 1-2 hours to 300-400 

22 ppb O3 in exercising humans (Devlin et al., 1991; Schelegle et al., 1991). Since the influx 

23 and persistence of neutrophils in airways following O3 exposure correlated with the 

24 temporal profile of epithelial injury (guinea pigs, 0.26-1 ppm O3, 72 hours) (Hu et al., 

25 1982), neutrophils were probably injurious. However, neutrophils have also been shown 

26 to contribute to repair of O3-injured epithelium in rats exposed for 8 hours to 1 ppm O3,

27 possibly by removing necrotic epithelial cells (Mudway and Kelly, 2000; Vesely et al., 

28 1999). Nonetheless, the degree of airways inflammation due to O3 is thought to have 

29 more important long-term consequences than the more quickly resolving changes in 

30 pulmonary function since airways inflammation is often accompanied by tissue injury 

31 (Balmes et al., 1996). 

32 Ozone exposure results in alterations in other airways inflammatory cells besides 

33 neutrophils, including lymphocytes, macrophages, monocytes and mast cells. Influx of 

34 some of these cells accounts for the later (i.e. 18-20 hours) phase of inflammation 
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1 following O3 exposure. Numbers of lymphocytes and total cells in BALF were decreased 

2 early after O3 exposure in exercising humans exposed for 2 hours to 200 ppb O3, which 

3 preceded the neutrophil influx (Mudway and Kelly, 2000; Blomberg et al., 1999; Krishna 

4 et al., 1997). The decrease in total cells was thought to reflect decreases in macrophages, 

5 although it was not clear whether the cells were necrotic or whether membrane adhesive 

6 properties were altered making them more difficult to obtain by lavage (Mudway and 

7 Kelly, 2000; Blomberg et al., 1999; Mudway et al., 1999b; Frampton et al., 1997a; 

8 Pearson and Bhalla, 1997). A recent study in exercising human subjects exposed for 6.6 

9 hours to 80 ppb O3 demonstrated an increase in numbers of sputum monocytes and 

10 dendritic-like cells with increased expression of innate immune surface proteins and 

11 antigen presentation markers (Peden, 2011; Alexis et al., 2010) (see Section 6.2.3.1). An 

12 increase in submucosal mast cells was observed 1.5 hours after a 2 hour-exposure to 200 

13 ppb O3 (Blomberg et al., 1999) and an increase in BAL mast cell number was observed 

14 18 hours after a 4-hour exposure to 220 ppb O3 exposure in exercising human subjects 

15 (Frampton et al., 1997a). Mast cells may play an important role in mediating neutrophil 

16 influx since they are an important source of several pro-inflammatory cytokines and since 

17 their influx preceded that of neutrophils in exercising human subjects exposed for 2 hours 

18 to 200 ppb O3 (Stenfors et al., 2002; Blomberg et al., 1999). Further, a study using mast 

19 cell-deficient mice demonstrated decreased neutrophilic inflammation in response to O3

20 (1.75 ppm, 3 hours) compared with wild type mice (Kleeberger et al., 1993). Influx of 

21 these inflammatory cell types in the lung is indicative of O3-mediated activation of innate 

22 immunity as will be discussed in Section 5.3.6. 

23 Much is known about the cellular and molecular signals involved in inflammatory 

24 responses to O3 exposure (U.S. EPA, 2006b). Eicosanoids are one class of secondary 

25 oxidation products which may be formed rapidly following O3 exposure and which may 

26 mediate inflammation. In addition, secondary reaction products may stimulate 

27 macrophages to produce cytokines such as IL-1, IL-6 and TNF-α which in turn activate 

28 IL-8 production by epithelial cells. Although IL-8 has been proposed to play a role in 

29 neutrophil chemotaxis, measurements of IL-8 in BALF from humans exposed to O3

30 found increases that were too late to account for this effect (Mudway and Kelly, 2000). 

31 The time-course profiles of PGE2 and IL-6 responses suggest that they may play a role in 

32 neutrophil chemotaxis in humans (Mudway and Kelly, 2000). However, pretreatment 

33 with ibuprofen attenuated O3-induced increases in BALF PGE2 levels, but had no effect 

34 on neutrophilia in exercising human subjects exposed for 2 hour to 400 ppb O3 (Hazucha 

35 et al., 1996). 

36 One set of studies in humans focused on the earliest phase of airways inflammation (1-2 

37 hours following exposure). Exercising subjects were exposed to 200 ppb O3 for 2 hours 

38 and bronchial biopsy tissues were obtained 1.5 and 6 hours after exposure (Bosson et al., 
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1 2009; Bosson et al., 2003; Stenfors et al., 2002; Blomberg et al., 1999). Results 

2 demonstrated upregulation of vascular endothelial adhesion molecules P-selectin and 

3 ICAM-1 at both 1.5 and 6 hours (Stenfors et al., 2002; Blomberg et al., 1999). 

4 Submucosal mast cell numbers were increased at 1.5 hours in the biopsy samples without 

5 an accompanying increase in neutrophil number (Blomberg et al., 1999). Pronounced 

6 neutrophil infiltration was observed at 6 hours in the bronchial mucosa (Stenfors et al., 

7 2002). Surprisingly, suppression of the NF-κB and AP-1 pathways at 1.5 hours and a lack 

8 of increased IL-8 at 1.5 or 6 hours in bronchial epithelium was observed (Bosson et al., 

9 2009). The authors suggested that vascular endothelial adhesion molecules, rather than 

10 redox sensitive transcription factors, play key roles in early neutrophil recruitment in 

11 response to O3. 

12 Increases in markers of inflammation occurred to a comparable degree in exercising 

13 human subjects with mild (least sensitive) and more remarkable (more sensitive) 

14 spirometric responses to O3 (200 ppb, 4 hours) (Balmes et al., 1996). Two other studies 

15 using similar protocols (200 ppb for 4 hours and 300 ppb for 1 hour) found that acute 

16 spirometric changes were not positively correlated with cellular and biochemical 

17 indicators of inflammation (Aris et al., 1993; Schelegle et al., 1991). However 

18 inflammation was correlated with changes in sRaw (Balmes et al., 1996). In another 

19 study, pretreatment with ibuprofen had no effect on neutrophilia although it blunted the 

20 spirometric response in exercising human subjects exposed for 2 hours to 400 ppb O3

21 (Hazucha et al., 1996). Taken together, results from these studies indicate different 

22 mechanisms underlying the spirometric and inflammatory responses to O3. 

23 A common mechanism underlying both inflammation and impaired pulmonary function 

24 was suggested by Krishna et al. (1997). This study, conducted in exercising humans 

25 exposed to 200 ppb O3 for 2 hours, demonstrated a correlation between loss of SP 

26 immunoreactivity from neurons in the bronchial mucosa and numbers of neutrophils and 

27 epithelial cells (shed epithelial cells are an index of injury) in the BALF 6-hours 

28 postexposure. Furthermore, the loss of SP immunoreactivity was correlated with the 

29 observed changes in FEV1. Another study found that SP was increased in lavage fluid of 

30 exercising human subjects immediately after O3 challenge (250 ppb, 1 hour) (Hazbun et 

31 al., 1993). SP is a neuropeptide released by sensory nerves which mediates neurogenic 

32 edema and bronchoconstriction (Krishna et al., 1997). Taken together, these findings 

33 suggest that O3-mediated stimulation of sensory nerves which leads to activation of 

34 central and local axon reflexes is s a common effector pathway leading to impaired 

35 pulmonary function and inflammation. 

36 Studies in animal models have confirmed many of these findings and provided evidence 

37 for additional mechanisms involved in O3-induced inflammation. A study in mice (2 ppm 
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1 O3, 3 hours) demonstrated that PAF may be important in neutrophil chemotaxis 

2 (Longphre et al., 1999), while ICAM-1 and macrophage inflammatory protein-2 (MIP-2), 

3 the rodent IL-8 homologue, have been implicated in a rat model (1 ppm O3, 3 hours)

4 (Bhalla and Gupta, 2000). Key roles for CXCR2, a receptor for keratinocyte-derived 

5 chemokine (KC) and MIP-2, and for IL-6 in O3-mediated neutrophil influx were 

6 demonstrated in mice (1 ppm O3, 3 hours) (Johnston et al., 2005a; Johnston et al., 

7 2005b). Activation of JNK and p38 pathways and cathepsin-S were also found to be 

8 important in this response (3 ppm O3, 3 hours) (Williams et al., 2009a; Williams et al., 

9 2008b; Williams et al., 2007a). Matrix metalloproteinase-9 (MMP-9) protected against 

10 O3-induced airways inflammation and injury in mice (0.3 ppm O3, 6-72 hours) (Yoon et 

11 al., 2007). Interleukin-10 (IL-10) was also found to be protective since IL-10 deficient 

12 mice responded to O3 exposure (0.3 ppm, 24-72 hours) with enhanced numbers of BAL 

13 neutrophils, enhanced NF-κB activation and MIP-2 levels compared with IL-10 sufficient 

14 mice (Backus et al., 2010).

15 In addition, lung epithelial cells may release ATP in response to O3 exposure (Ahmad et 

16 al., 2005). ATP and its metabolites (catalyzed by ecto-enzymes) can bind to cellular 

17 purinergic receptors resulting in activation of cell signaling pathways (Picher et al., 

18 2004). One such metabolite, adenine, is capable of undergoing oxidation leading to the 

19 formation of UA which, if present in high concentrations, could activate inflammasomes 

20 and result in caspase 1 activation and the maturation and secretion of IL-1β and IL-18 

21 (Dostert et al., 2008). A recent study in exercising human subjects exposed for 2 hours to 

22 400 ppb O3 demonstrated a correlation between ATP metabolites and inflammatory 

23 markers (Esther et al., 2011), which provides some support for this mechanism. 

24 Several recent studies have focused on the role of toll-like receptor (TLR) and its related 

25 adaptor protein MyD88 in mediating O3-induced neutrophilia. While Hollingsworth et al. 

26 (2004) demonstrated airways neutrophilia which was TLR4-independent following acute 

27 (2 ppm, 3 hours) and subchronic (0.3 ppm, 72 hours) O3 exposure in a mouse model, 

28 Williams et al. (2007b) found that MyD88 was important in mediating O3-induced 

29 neutrophilia in mice (3 ppm, 3 hours), with TLR4 and TLR2 contributing to the speed of 

30 the response. Moreover, MyD88, TLR2 and TLR4 contributed to inflammatory gene 

31 expression in this model and O3 upregulated MyD88, TLR4 and TLR4 gene expression 

32 (Williams et al., 2007a)

33 Hyaluronan was found to mediate a later phase (24 hours) of O3-induced inflammation in 

34 mice (Garantziotis et al., 2010; Garantziotis et al., 2009). Hyaluronan is an extracellular 

35 matrix component which is normally found in the ELF as a large polymer. Exposure to 

36 2 ppm O3 for 3 hours resulted in elevated levels of soluble low molecular weight 

37 hyaluronan in the BALF 24-hours postexposure (Garantziotis et al., 2010; Garantziotis et 
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1 al., 2009). Ozone may have caused the depolymerization of hyaluronan to soluble 

2 fragments which are known to be endogenous ligands of the CD44 receptor and TLR4 in 

3 the macrophage (Jiang et al., 2005). Binding of hyaluronan fragments to the CD44 

4 receptor activates hyaluronan clearance, while binding to TLR4 results in signaling 

5 through MyD88 to produce chemokines that stimulate the influx of inflammatory cells 

6 (Jiang et al., 2005). Activation of NF-κB occurred in both airway epithelia and alveolar 

7 macrophages 24-hours postexposure to O3. Increases in BALF pro-inflammatory factors 

8 KC, IL-1β, MCP-1, TNF-α and IL-6 observed 24 hours following O3 exposure were 

9 found to be partially dependent on TLR4 (Garantziotis et al., 2010) while increases in 

10 BAL inflammatory cells, which consisted mainly of macrophages, were dependent on 

11 CD44 (Garantziotis et al., 2009). BAL inflammatory cells number and injury markers 

12 following O3 exposure were similar in wild-type and TLR4-deficient animals 

13 (Garantziotis et al., 2010). 

14 Since exposure to O3 leads to airways inflammation characterized by neutrophilia, and 

15 since neutrophil-derived oxidants often scavenge ELF antioxidants, concentrations of 

16 ELF antioxidants have been examined during airways neutrophilia (Long et al., 2001; 

17 Gunnison and Hatch, 1999; Mudway et al., 1999b). In exercising humans exposed to 200 

18 ppb O3 for 2 hours, UA, GSH and α-TOH levels remained unchanged in BALF 6-hours 

19 postexposure while AH2 was decreased significantly in both BALF and plasma (Mudway 

20 et al., 1999b). A second study involving the same protocol reported a loss of AH2 from 

21 bronchial wash fluid and BALF, representing proximal and distal airway ELF 

22 respectively, as well as an increase in oxidized GSH in both compartments (Mudway et 

23 al., 2001). No change was observed in ELF UA levels in response to O3 (Mudway et al., 

24 2001). Further, O3 exposure (0.8 ppm, 4 hours) in female rats resulted in a 50% decrease 

25 in BALF AH2 immediately postexposure (Gunnison and Hatch, 1999). These studies 

26 suggested a role for AH2 and GSH in protecting against the oxidative stress associated 

27 with inflammation.

5.3.4 Alteration of epithelial barrier function

28 Following O3 exposure, injury and inflammation can lead to altered airway barrier 

29 function. Histologic analysis has demonstrated damage to tight junctions between 

30 epithelial cells, suggesting an increase in epithelial permeability. In addition, the presence 

31 of shed epithelial cells in the BALF and increased epithelial permeability, which is 

32 measured as the flux of small solutes, have been observed and are indicative of epithelial 

33 injury. Increases in vascular permeability, as measured by BALF protein and albumin, 

34 have also been demonstrated (Costa et al., 1985; Hu et al., 1982). 
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1 An early study in sheep measured changes in airway permeability as the flux of inhaled 

2 radiolabeled histamine into the plasma (Abraham et al., 1984). Exposure of sheep to 0.5 

3 ppm O3 for 2 hours via an endotracheal tube resulted in an increased rate of histamine 

4 appearance in the plasma at 1 day postexposure. Subsequently, numerous studies have 

measured epithelial permeability as the flux of the small solute 99m 5 TcDTPA which was 

6 introduced into the air spaces in different regions of the respiratory tract. Increased 

pulmonary epithelial permeability, measured as the clearance of 99m 7 Tc-DTPA, was 

8 demonstrated in humans 1-2 hours following a 2-hour exposure to 400 ppb O3 while 

9 exercising moderately (Kehrl et al., 1987). Another study in human subjects found

10 increased epithelial permeability 19-hours postexposure to 240 ppb O3 for 130 minutes 

11 while exercising (Foster and Stetkiewicz, 1996). Increased bronchial permeability was 

12 also observed in dogs 1-day postexposure (0.4 ppm O3 by endotracheal tube for 6 hours) 

13 and did not resolve for several days (Foster and Freed, 1999). 

14 A role for tachykinins in mediating airway epithelial injury and decreased barrier function 

15 has been suggested. Nishiyama et al. (1998) demonstrated that capsaicin, which depletes 

16 nerve fibers of substance P, blocked the O3-induced increase in permeability of guinea 

17 pig tracheal mucosa (0.5-3 ppm O3, 0.5 hours). Pretreatment with propranolol or atropine 

18 failed to inhibit this response, suggesting that adrenergic and cholinergic pathways were 

19 not involved. In another study, tachykinins working through NK-1 and CGRP receptors 

20 were found to contribute to airway epithelial injury in O3-exposed rats (1 ppm, 8 hours) 

21 (Oslund et al., 2009, 2008).

22 Kleeberger et al. (2000) evaluated genetic susceptibility to O3-induced altered barrier 

23 function in recombinant inbred strains of mice. Lung hyperpermeability, measured as 

24 BALF protein, was evaluated 72 hours after exposure to 0.3 ppm O3 and found to be 

25 associated with a functioning TLR4 gene. This study concluded that Tlr4 was a strong 

26 candidate gene for susceptibility to hyperpermeability in response to O3 (Kleeberger et 

27 al., 2000). A subsequent study by these same investigators found that Tlr4 modulated 

28 Nos2 mRNA levels and suggested that the gene product of Nos2, iNOS, plays an 

29 important role in O2-induced lung hyperpermeability (0.3 ppm, 72 hours) (Kleeberger et 

30 al., 2001). More recently, HSP70 was identified as part of the TLR4 signaling pathway 

31 (0.3 ppm, 6-72 hours) (Bauer et al., 2011). 

32 Antioxidants have been shown to confer resistance to O3-induced injury. In a recent 

33 study, lung hyperpermeability in response to O3 (0.3 ppm, 48 hours) was unexpectedly 

34 reduced in mice deficient in the glutamate-cysteine ligase modifier subunit gene 

35 compared with sufficient mice (Johansson et al., 2010). Since the lungs of these mice 

36 exhibited 70% glutathione depletion, protection against O3-induced injury was 

37 unexpected (Johansson et al., 2010). However it was found that several other antioxidant 
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1 defenses, including metallothionein, were upregulated in response to O3 to a greater 

2 degree in the glutathione-deficient mice compared with sufficient mice (Johansson et al., 

3 2010). The authors suggested that resistance to O3-induced lung injury was due to 

4 compensatory augmentation of antioxidant defenses (Johansson et al., 2010). Antioxidant 

5 effects have also been attributed to Clara cell secretory protein (CCSP) and surfactant 

6 protein A (SP-A). CCSP was found to modulate the susceptibility of airway epithelium to 

7 injury in mice exposed to O3 (0.2 or 1 ppm for 8 hours) by an unknown mechanism 

8 (Plopper et al., 2006). SP-A protected against O3-induced airways inflammation and

9 injury in mice (2 ppm, 3 hours), possibly by acting as a sacrificial substrate (Haque et al., 

10 2007).

11 Increased epithelial permeability has been proposed to play a role in allergic sensitization 

12 (Matsumura, 1970), in activation of neural reflexes and in stimulation of smooth muscle 

13 receptors (Dimeo et al., 1981). Abraham et al. (1984) reported a correlation between 

14 airway permeability and airways hyperresponsiveness (AHR) in O3-exposed sheep. 

15 However a recent study in human subjects exposed to 220 ppb O3 for 135 minutes while 

16 exercising did not find a relationship between O3-induced changes in airway permeability 

17 and AHR (Que et al.). 

5.3.5 Sensitization of bronchial smooth muscle

18 Bronchial reactivity is generally determined in terms of a response to a challenge agent. 

19 Non-specific bronchial reactivity in humans is assessed by measuring the effect of 

20 inhaling increasing concentrations of a bronchoconstrictive drug on lung mechanics 

21 (sRaw or FEV1). Methacholine is most commonly employed but histamine and other 

22 agents are also used. Specific bronchial reactivity is assessed by measuring effects in 

23 response to an inhaled allergen in individuals (or animals) already sensitized to that 

24 allergen. An increase in sRaw in response to non-specific or specific challenge agents 

25 indicates AHR.

26 In addition to causing mild airway obstruction as discussed above, acute O3 exposure 

27 results in reversible increases in bronchial reactivity by mechanisms which are not well 

28 understood. In one study, bronchial reactivity of healthy subjects was significantly 

29 increased 19-hours postexposure to O3 (120-240 ppb O3 for 2 hours with intermittent 

30 exercise) (Foster et al., 2000). These effects may be more significant in human subjects 

31 with already compromised airways (Section 5.4.2.2). 

32 Ozone may sensitize bronchial smooth muscle to stimulation through a direct effect on 

33 smooth muscle or through effects on the sensory nerves in the epithelium or on the motor 

34 nerves innervating the smooth muscle (O'Byrne et al., 1984; O'Byrne et al., 1983; 
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1 Holtzman et al., 1979). It is also recognized that increased bronchial reactivity can be 

2 both a rapidly occurring and a persistent response to O3 (Foster and Freed, 1999). 

3 Tachykinins and secondary oxidation products of O3 have been proposed as mediators of 

4 the early response and inflammation-derived products have been proposed as mediators 

5 of the later response (Foster and Freed, 1999). 

6 Ozone-induced increases in epithelial permeability, which could improve access of 

7 agonist to smooth muscle receptors, may be one mechanism of sensitization through a 

8 direct effect on bronchial smooth muscle (Holtzman et al., 1979). As noted above, a 

9 correlation between airway permeability and AHR has been reported in O3-exposed 

10 sheep (Abraham et al., 1984) but not in O3-exposed human subjects (Que et al.). 

11 Neurally-mediated sensitization has been demonstrated. In human subjects exposed for 2 

12 hours to 600 ppb O3 while exercising, pretreatment with atropine inhibited O3-induced 

13 AHR, suggesting the involvement of cholinergic postganglionic pathways (Holtzman et 

14 al., 1979). Animal studies have demonstrated that O3-induced AHR involved vagally15 mediated responses (rabbits, 0.2 ppm O3, 72 hours) (Freed et al., 1996) and local axon 

16 reflex responses through bronchopulmonary C-fiber-mediated release of SP (guinea pigs, 

17 0.8 ppm O3, 2 hours) (Joad et al., 1996). Further, pretreatment with capsaicin to deplete 

18 nerve fibers of SP blocked O3-mediated AHR (guinea pigs, 1-2 ppm O3, 2-2.25 hours) 

19 (Tepper et al., 1993). Other investigators demonstrated that SP released from airway 

20 nociceptive neurons in ferrets contributed to O3-induced AHR (2 ppm O3, 3 hours) (Wu 

21 et al., 2008b; Wu et al., 2003). 

22 Some evidence suggests the involvement of arachidonic acid metabolites and neutrophils 

23 in mediating O3-induced AHR (Seltzer et al., 1986; Fabbri et al., 1985). Increased BAL 

24 neutrophils and cyclooxygenase products were found in one study demonstrating AHR in 

25 exercising humans (600 ppb for 2 hours) immediately postexposure to (Seltzer et al., 

26 1986). Another study found that ibuprofen pretreatment had no effect on AHR in 

27 exercising humans following exposure to 400 ppb O3 for 2 hours, although spirometric 

28 responses were blunted (Hazucha et al., 1996). This study indicated that the arachidonic 

29 acid metabolites whose generation was blocked by ibuprofen, (i.e. prostaglandins, 

30 thromboxanes and some leukotrienes) did not play a role in AHR. Experiments in dogs 

31 exposed for 2 hours to 2.1 ppm O3 demonstrated a close correlation between O3-induced 

32 AHR and airways neutrophilic inflammation measured in tissue biopsies (Holtzman et 

33 al., 1983). Furthermore, the increased AHR observed in dogs following O3 exposure (3 

34 ppm, 2 hours) was inhibited by neutrophil depletion (O'Byrne et al., 1983) and by pre35 treatment with inhibitors of arachidonic acid metabolism. In one of these studies, 

36 indomethacin pre-treatment did not prevent airways neutrophilia in response to O3 (3 

37 ppm, 2 hours) providing evidence that the subset of arachidonic acid metabolites whose 
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1 generation was inhibitable by the cyclooxygenase inhibitor indomethacin (i.e., 

2 prostaglandins and thromboxanes) was not responsible for neutrophil influx (O'Byrne et 

3 al., 1984). Taken together, these findings suggest that arachidonic acid metabolites, but 

4 probably not prostaglandins or thromboxanes, may be involved in the AHR response 

5 following O3 exposure in dogs. Studies probing the role of neutrophils in mediating the 

6 AHR response have provided inconsistent results (Al-Hegelan et al., 2011). 

7 Evidence for cytokine and chemokine involvement in the AHR response to O3 has been 

8 described. Some studies have suggested a role for TNF-α (mice, 0.5 and 2 ppm O3, 3 

9 hours) (Cho et al., 2001; Shore et al., 2001) and IL-1 (mice and ferrets, 2 ppm O3, 3 

10 hours) (Wu et al., 2008b; Park et al., 2004). The latter study found that SP expression in 

11 airway neurons was upregulated by IL-1 which was released in response to O3. Other 

12 studies in mice have demonstrated a key role for CXCR2, the chemokine receptor for the 

13 neutrophil chemokines KC and MIP-2, but not for IL-6 in O3-mediated AHR ( 1 ppm O3, 

14 3 hours) (Johnston et al., 2005a; Johnston et al., 2005b). In contrast, CXCR2 and IL-6 

15 were both required for neutrophil influx in this model (Johnston et al., 2005a; Johnston et 

16 al., 2005b), as discussed above. Williams et al. (2008a) demonstrated that the Th2 

17 cytokine IL-13 contributed to AHR, as well as to airways neutrophilia, in mice (3 ppm 

18 O3, 3 hours).

19 Other studies have focused on the role of TLR4. Hollingsworth et al. (2004) measured 

20 AHR, as well as airways neutrophilia, in mice 6 and 24 hours following acute (2 ppm O3

21 for 3 hours) and subchronic (0.3 ppm for 3 days) exposure to O3. TLR4 is a key 

22 component of the innate immune system and is responsible for the immediate 

23 inflammatory response seen following challenge with endotoxin and other pathogen24 associated substances. In this study, a functioning TLR4 was required for the full AHR 

25 response following O3 exposure but not for airways neutrophilia (Hollingsworth et al., 

26 2004). These findings are complemented by an older study demonstrating that O3 effects 

27 on lung hyperpermeability required a functioning TLR4 (mice, 0.3 ppm O3, 72 hours) 

28 (Kleeberger et al., 2000). Williams et al. (2007b) found that TLR2, TLR4 and the TLR 

29 adaptor protein MyD88 contributed to AHR in mice (3 ppm O3, 3 hours). Ozone was also 

30 found to upregulate MyD88, TLR4 and TLR4 gene expression in this model (Williams et 

31 al., 2007b). 

32 A newly recognized mechanistic basis for O3-induced AHR is provided by studies 

33 focusing on the role of hyaluronan following O3 exposure in mice (Garantziotis et al., 

34 2010; Garantziotis et al., 2009). Hyaluronan is an extracellular matrix component which 

35 is normally found in the ELF as a large polymer. Briefly, TLR4 and CD44 were found to 

36 mediate AHR in response to O3 and hyaluronan. Exposure to 2 ppm O3 for 3 hours 

37 resulted in enhanced AHR and elevated levels of soluble low molecular weight 
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1 hyaluronan in the BALF 24-hours postexposure (Garantziotis et al., 2010; Garantziotis et 

2 al., 2009). Ozone may have caused the depolymerization of hyaluronan to soluble 

3 fragments which are known to be endogenous ligands of the CD44 receptor and TLR4 in 

4 the macrophage (Jiang et al., 2005). In the two recent studies, O3-induced AHR was 

5 attenuated in CD44 and TLR4-deficient mice (Garantziotis et al., 2010; Garantziotis et 

6 al., 2009). Hyaluronan fragment-mediated stimulation of AHR was found to require 

7 functioning CD44 receptor and TLR4 (Garantziotis et al., 2010; Garantziotis et al., 2009). 

8 In contrast, high-molecular-weight hyaluronan blocked AHR in response to O3

9 (Garantziotis et al., 2009). In another study high-molecular-weight hyaluronan enhanced 

10 repair of epithelial injury (Jiang et al., 2005). These studies provide a link between innate 

11 immunity and the development of AHR following O3 exposure, and indicate a role for 

12 TLR4 in increasing airways responsiveness. While TLR4-dependent responses usually 

13 involve activation of NF-κB and the upregulation of proinflammatory factors, the precise 

14 mechanisms leading to AHR are unknown (Al-Hegelan et al., 2011). 

15 In guinea pigs, AHR was found to be mediated by different pathways at 1- and 3-days 

16 postexposure to a single dose of O3 (2 ppm for 4 hours) (Verhein et al., 2011; Yost et al., 

17 2005). At 1 day, AHR was due to activation of airway parasympathetic nerves rather than 

18 to a direct effect on smooth muscle (Yost et al., 2005). This effect occurred as a result of 

19 O3-stimulated release of major basic protein from eosinophils (Yost et al., 2005). Major 

20 basic protein is known to block inhibitory M2 muscarinic receptors which normally 

21 dampen acetylcholine release from parasympathetic nerves (Yost et al., 2005). The 

22 resulting increase in acetylcholine release caused an increase in smooth muscle 

23 contraction following O3 exposure (Yost et al., 2005). Eosinophils played a different role 

24 3-days postexposure to O3 in guinea pigs (Yost et al., 2005). Ozone-mediated influx of 

25 eosinophils into lung airways resulted in a different population of cells present 3-days 

26 postexposure compared to those present at 1 day (Yost et al., 2005). At this time point, 

27 eosinophil-derived major basic protein increased smooth muscle responsiveness to 

28 acetylcholine which also contributed to AHR (Yost et al., 2005). However, the major 

29 effect of eosinophils was to protect against vagal hyperreactivity (Yost et al., 2005). The 

30 authors suggested that these beneficial effects were due to the production of nerve growth 

31 factor (Yost et al., 2005). Further work by these investigators demonstrated a key role for 

32 IL-1β in mediating AHR 3-days postexposure to O3 (Verhein et al., 2011). In this study, 

33 IL-1β increased nerve growth factor and SP which acted through the NK1 receptor to 

34 cause vagally-mediated bronchoconstriction (Verhein et al., 2011). The mechanism by 

35 which SP caused acetylcholine release from parasympathetic nerves following O3

36 exposure was not determined (Verhein et al., 2011). Taken together, the above study 

37 results indicate that mechanisms involved in O3-mediated AHR can vary over time 

38 postexposure and that eosinophils and SP can play a role. Results of this animal model 
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1 may provide some insight into allergic airways disease in humans which is characterized 

2 by eosinophilia (Section 5.4.2.2).

5.3.6 Modification of innate/adaptive immune system responses

3 Host defense depends on effective barrier function and on innate immunity and adaptive 

4 immunity (Al-Hegelan et al., 2011). Ozone’s effect on barrier function in the airways was 

5 discussed above (Section 5.3.4). This section focuses on the mechanisms by which O3

6 impacts innate and adaptive immunity. Both tissue damage and foreign pathogens are 

7 triggers for the activation of the innate immune system. This results in the influx of 

8 inflammatory cells such as neutrophils, mast cells, basophils, eosinophils, monocytes and 

9 dendritic cells and the generation of cytokines such as TNF-α, IL-1, IL-6, KC and IL-17 . 

10 Further, innate immunity encompasses the actions of complement and collectins and the 

11 phagocytic functions of macrophages, neutrophils and dendritic cells. Airway epithelium 

12 also contributes to innate immune responses. Innate immunity is highly dependent on cell 

13 signaling networks involving TLR4. Adaptive immunity provides immunologic memory 

14 through the actions of B and T cells. Important links between the two systems are 

15 provided by dendritic cells and antigen presentation. Recent studies demonstrate that 

16 exposure to O3 modifies cells and processes which are required for innate immunity, 

17 contributes to innate-adaptive immune system interaction and primes pulmonary immune 

18 responses to endotoxin. 

19 Ozone exposure of human subjects resulted in recruitment of activated innate immune 

20 cells to the airways. Healthy individuals were exposed to 80 ppb O3 for 6.6 hours with 

21 intermittent exercise and airways inflammation was characterized in induced sputum 18-

22 hours postexposure (Alexis et al., 2010). Previous studies demonstrated that induced 

23 sputum contains liquid and cellular constituents of the ELF from central conducting 

24 airways (Alexis et al., 2001b) and also identified these airways as a site of preferential O3

25 absorption during exercise (Hu et al., 1994). Ozone exposure resulted in increased 

26 numbers of neutrophils, airway monocytes and dendritic-like cells in sputum (Alexis et 

27 al., 2010). In addition, increased expression of cell surface markers characteristic of 

28 innate immunity and antigen presentation (i.e. CD-14 and HLA-DR) was demonstrated 

29 on airway monocytes (Alexis et al., 2010). Enhanced antigen presentation contributes to 

30 exaggerated T cell responses and promotes Th2 inflammation and an allergic phenotype 

31 (Lay et al., 2007). Upregulation of pro-inflammatory cytokines was also demonstrated in 

32 sputum of O3-exposed subjects (Alexis et al., 2010). One of these cytokines, IL-12p70, 

33 correlated with numbers of dendritic-like cells in the sputum, and is an indicator of 

34 dendritic cell activation (Alexis et al., 2010). These authors have previously reported that 

35 exposure of exercising human subjects to 400 ppb O3 for 2 hours resulted in activation of
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1 monocytes and macrophages (Lay et al., 2007), which could play a role in exacerbating 

2 existing asthma by activating allergen-specific memory T cells. The current study 

3 confirms these findings and extends them by suggesting a potential mechanism whereby 

4 O3-activated dendritic cells could stimulate naïve T-cells to promote the development of 

5 asthma (Alexis et al., 2010). A companion study by these same investigators (described in 

6 detail in Section 5.4.2.1) provides evidence of dendritic cell activation, measured as 

7 increased expression of HLA-DR, in a subset of the human subjects (GSTM1 null) 

8 exposed to 400 ppb O3 for 2 hours with intermittent exercise (Alexis et al., 2009). Since 

9 dendritic cells are a link between innate and adaptive immunity, these studies provide 

10 evidence for an O3-mediated interaction between the innate and adaptive immune 

11 systems. 

12 Another recent study linked O3-mediated activation of the innate immune system to the 

13 development of non-specific AHR in a mouse model (Pichavant et al., 2008). Repeated 

14 exposure to 1 ppm O3 for 3 hours (3 days over a 5 day period) induced non-specific AHR 

15 measured 24 hours following the last exposure (Pichavant et al., 2008). This response 

16 was found to require NKT cells, which are effector lymphocytes of innate immunity, as 

17 well as IL-17 and airways neutrophilia (Pichavant et al., 2008). Since glycolipids such as 

18 galactosyl ceramide are ligands for the invariant CD1 receptor on NKT cells and serve as 

19 endogenous activators of NKT cells, a role for O3-oxidized lipids in activating NKT cells 

20 was proposed (Pichavant et al., 2008). The authors contrasted this innate immunity 

21 pathway with that of allergen-provoked specific AHR which involves adaptive immunity, 

22 the cytokines IL-4, IL -13, IL-17, and airways eosinophilia (Pichavant et al., 2008). 

23 Interestingly, NKT cells were required for both the specific AHR provoked by allergen 

24 and the non-specific AHR provoked by O3 (Pichavant et al., 2008). Different cytokine 

25 profiles of the NKT cells from allergen and O3-exposed mice was proposed to account 

26 for the different pathways (Pichavant et al., 2008). More recently, NKT cells have been 

27 found to function in both innate and adaptive immunity (Vivier et al., 2011).

28 An interaction between allergen and O3 in the induction of nonspecific AHR was shown 

29 in another animal study (Larsen et al., 2010). Mice were sensitized with the aerosolized 

30 allergen OVA on 10 consecutive days followed by exposure to O3 (0.1-0.5 ppm for 3 

31 hours) (Larsen et al., 2010). While allergen sensitization alone did not alter airways 

32 responsiveness to a nonspecific challenge, O3 exposure of sensitized mice resulted in 

33 nonspecific AHR at 6- and 24-hours postexposure (Larsen et al., 2010). The effects of O3

34 on AHR were independent of airways eosinophilia and neutrophilia (Larsen et al., 2010). 

35 However, OVA pretreatment led to goblet cell metaplasia which was enhanced by O3

36 exposure (Larsen et al., 2010). It should be noted that OVA sensitization using only 

37 aerosolized antigen in this study is less common than the usual procedure for OVA 

38 sensitization achieved by one or more initial systemic injections of OVA and adjuvant 
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1 followed by repeated inhalation exposure to OVA. This study also points to an interaction 

2 between innate and adaptive immune systems in the development of the AHR response. 

3 Furthermore, O3 was found to act as an adjuvant for allergic sensitization (Hollingsworth 

4 et al., 2010). Oropharyngeal aspiration of OVA on day 0 and day 6 failed to lead to 

5 allergic sensitization unless mice were first exposed to 1 ppm O3 for 2 hours 

6 (Hollingsworth et al., 2010). The O3-mediated response involved Th2 (IL-4, IL-5 and IL7 9) and Th17 cytokines (IL-17) and was dependent on a functioning TLR4 (Hollingsworth 

8 et al., 2010). Ozone exposure also activated OVA-bearing dendritic cells in the thoracic 

9 lymph nodes, as measured by the presence of the CD86 surface marker, which suggests 

10 naïve T cell stimulation and the involvement of Th2 pathways (Hollingsworth et al., 

11 2010). Thus the adjuvant effects of O3 may be due to activation of both innate and 

12 adaptive immunity. 

13 Priming of the innate immune system by O3 was reported by Hollingsworth et al. (2007). 

14 In this study, exposure of mice to 2 ppm O3 for 3 hours led to nonspecific AHR at 24-

15 and 48-hours postexposure, an effect which subsided by 72 hours (Hollingsworth et al., 

16 2007). However, in mice treated with aerosolized endotoxin immediately following O3

17 exposure, AHR was greatly enhanced at 48-and 72-hours postexposure (Hollingsworth et 

18 al., 2007). In addition, O3 pre-exposure was found to reduce the number of inflammatory 

19 cells in the BALF, to increase cytokine production and total protein in the BALF and to 

20 increase systemic IL-6 following exposure to endotoxin (Hollingsworth et al., 2007). 

21 Furthermore, O3 stimulated the apoptosis of alveolar macrophages 24-hours 

22 postexposure, an effect which was greatly enhanced by endotoxin treatment. Apoptosis of 

23 circulating blood monocytes was also observed in response to the combined exposures 

24 (Hollingsworth et al., 2007). Ozone pre-exposure enhanced the response of lung 

25 macrophages to endotoxin (Hollingsworth et al., 2007). Taken together, these findings 

26 demonstrated that O3 exposure increased innate immune responsiveness to endotoxin. 

27 The authors attributed these effects to the increased surface expression of TLR4 and 

28 increased signaling in macrophages observed in the study (Hollingsworth et al., 2007). It 

29 was proposed that the resulting decrease in airway inflammatory cells could account for 

30 O3-mediated decreased clearance of bacterial pathogens observed in numerous animal 

31 models (Hollingsworth et al., 2007). 

32 More recently, these authors demonstrated that hyaluronan contributed to the O3-primed 

33 response to endotoxin (Li et al., 2010). In this study, exposure of mice to 1 ppm O3 for 3 

34 hours resulted in enhanced responses to endotoxin, which was mimicked by intratracheal 

35 instillation of hyaluronan fragments (Li et al., 2010). Hyaluronan, like O3, was also found 

36 to induce TLR4 receptor peripheralization in the macrophage membrane (Li et al., 2010; 

37 Hollingsworth et al., 2007), an effect which is associated with enhanced responses to 
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1 endotoxin. This study and previous ones by the same investigators showed elevation of 

2 BALF hyaluronan in response to O3 exposure (Garantziotis et al., 2010; Li et al., 2010; 

3 Garantziotis et al., 2009), providing evidence that the effects of O3 on innate immunity 

4 are at least in part mediated by hyaluronan fragments. The authors note that excessive 

5 TLR4 signaling can lead to lung injury and suggest that O3 may be responsible for an 

6 exaggerated innate immune response which may underlie lung injury and decreased host 

7 defense (Li et al., 2010). 

8 Activation or upregulation of the immune system has not been reported in all studies. 

9 Impaired antigen-specific immunity was demonstrated following subacute O3 exposure 

10 (0.6 ppm, 10 h/day for 15 days) in mice (Feng et al., 2006). Specifically, O3 exposure 

11 altered the lymphocyte subset and cytokine profile and impacted thymocyte early 

12 development leading to immune dysfunction. Further, recent studies demonstrated SP-A 

13 oxidation in mice exposed for 3-6 hours to 2 ppm O3. SP-A is an important innate 

14 immune protein which plays a number of roles in host defense including acting as

15 opsonin for the recognition of some pathogens (Haque et al., 2009). These investigations 

16 found that O3-mediated carbonylation of SP-A was associated with impaired macrophage 

17 phagocytosis in vitro (Mikerov et al., 2008b). Furthermore, O3 exposure (2 ppm for 3 

18 hours) in mice was found to increase susceptibility to pneumonia infection in mice 

19 through an impairment of SP-A dependent phagocytosis (Mikerov et al., 2008a; Mikerov 

20 et al., 2008c). 

21 Taken together, results of recent studies provide evidence that O3 alters host 

22 immunologic response and leads to immune system dysfunction through its effects on 

23 innate and adaptive immunity.

5.3.7 Airways remodeling

24 As noted above, the degree of airways inflammation due to O3 may have important long25 term consequences since airways inflammation is often accompanied by tissue injury 

26 (Balmes et al., 1996). The nasal airways, conducting airways and distal airways (i.e. 

27 respiratory bronchioles or centriacinar region depending on the species) have all been 

28 identified as sites of O3-mediated injury and inflammation (Mudway and Kelly, 2000). At 

29 all levels of the respiratory tract, loss of sensitive epithelial cells, degranulation of 

30 secretory cells, proliferation of resistant epithelial cells and neutrophilic influx have been 

31 observed as a result of O3 exposure (Mudway and Kelly, 2000; Cho et al., 1999). An 

32 important study (Plopper et al., 1998) conducted in adult rhesus monkeys (0.4 and 

33 1.0 ppm O3 for 2 hours) found that 1 ppm O3 resulted in the greatest epithelial injury in 

34 the respiratory bronchioles immediately postexposure although injury was observed at all 
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1 of the RT sites studied except for the lung parenchyma. Exposure to 0.4 ppm O3 resulted 

2 in epithelial injury only in the respiratory bronchioles.

3 Persistent inflammation and injury, observed in animal models of chronic and intermittent 

4 exposure to O3, are associated with airways remodeling, including mucous cell 

5 metaplasia of nasal transitional epithelium (Harkema et al., 1999; Hotchkiss et al., 1991)

6 and bronchiolar metaplasia of alveolar ducts (Mudway and Kelly, 2000). Fibrotic changes 

7 such as deposition of collagen in the airways and sustained lung function decrements 

8 especially in small airways have also been demonstrated as a response to chronic O3

9 exposure (Mudway and Kelly, 2000; Chang et al., 1992). These effects, described in 

10 detail in Section 7.2.3.1, have been demonstrated in rats exposed to levels of O3 as low as 

11 0.25 ppm. Mechanisms responsible for the resolution of inflammation and the repair of 

12 injury remain to be clarified and there is only a limited understanding of the biological 

13 processes underlying long-term morphological changes. However, a recent study in mice 

14 demonstrated a key role for the TGF-β signaling pathway in the deposition of collagen in 

15 the airways wall following chronic intermittent exposure to 0.5 ppm O3 (Katre et al., 

16 2011). 

17 It should be noted that repeated exposure to O3 results in attenuation of some O3-

18 induced responses, including those associated with the activation of neural reflexes (e.g. 

19 decrements in pulmonary function), as discussed in Section 5.3.2. However, numerous 

20 studies demonstrate that some markers of injury and inflammation remain increased 

21 during multi-day exposures to O3. Mechanisms responsible for attenuation, or the lack 

22 thereof, are incompletely understood. 

5.3.8 Systemic inflammation and oxidative/nitrosative stress

23 Extrapulmonary effects of O3 have been noted for decades (U.S. EPA, 2006b). It has 

24 been proposed that lipid oxidation products resulting from reaction of O3 with lipids 

25 and/or cellular membranes in the ELF are responsible for systemic effects, however it is 

26 not known whether they gain access to the vascular space (Chuang et al., 2009). 

27 Alternatively, extrapulmonary release of diffusible mediators may initiate or propagate 

28 inflammatory responses in the vascular or in systemic compartments (Cole and Freeman, 

29 2009). A role for O3 in modulating endothelin, a potent vasoconstrictor, has also been 

proposed. Studies in rats found that exposure to 0.4 and 0.8 ppm O3 30 induced endothelin 

31 system genes in the lung and increased circulating levels of endothelin (Thomson et al., 

32 2006; Thomson et al., 2005). Systemic oxidative stress is suggested by studies in humans 

33 which reported associations between O3 exposure and levels of plasma 8-isoprostanes 
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1 and the presence of peripheral blood lymphocyte micronuclei (Chen et al., 2007; Chen et 

2 al., 2006a).

3 Ozone-induced perturbations of the cardiovascular system were recently investigated in 

4 young mice and monkeys (Chuang et al., 2009) and in rats (Kodavanti et al., 2011; 

5 Perepu et al., 2010) (see Sections 6.3.3.2 and 7.3.1.2). These are the first studies to 

6 suggest that systemic oxidative stress and inflammation play a mechanistic role in O3-

7 induced effects on the systemic vascular and heart. Exposure to 0.5 ppm O3 for 5 days 

8 resulted in oxidative/nitrosative stress, vascular dysfunction and mitochondrial DNA 

9 damage in the aorta (Chuang et al., 2009). Chronic exposure to 0.8 ppm O3 resulted in an 

10 enhancement of inflammation and lipid peroxidation in the heart following an ischemia11 reperfusion challenge (Perepu et al., 2010). In addition, chronic intermittent exposure to 

12 0.4 ppm O3 increased aortic levels of mRNA for biomarkers of oxidative stress, 

13 thrombosis, vasoconstriction and proteolysis and aortic lectin-like oxidized-low density 

14 lipoprotein receptor-1(LOX-1) mRNA and protein levels (Kodavanti et al., 2011). The 

15 latter study suggests a role for circulating oxidized lipids in mediating the effects of O3. 

16 Systemic inflammation and oxidative/nitrosative stress may similarly affect other organ 

17 systems as well as the plasma compartment. Circulating cytokines have the potential to 

18 enter the brain through diffusion and active transport and to contribute to 

19 neuroinflammation, neurotoxicity, cerbrovascular damage and a break-down of the blood 

20 brain barrier (Block and Calderón-Garcidueñas, 2009) (see Sections 6.4 and 7.5). They 

21 can also activate neuronal afferents (Block and Calderón-Garcidueñas, 2009). Vagal 

22 afferent pathways originating in the respiratory tract may also be responsible for O3-

23 mediated activation of nucleus tractus solitarius neurons which resulted in neuronal 

24 activation in stress-responsive regions of the CNS in rats (0.5 or 2 ppm O3 for 1.5-120 

25 hours) (Gackière et al., 2011). Recent studies have demonstrated O3-induced brain lipid 

26 peroxidation, cytokine production in the brain and upregulated expression of VEGF in 

rats (0.5 ppm O3 27 , 3 hours or 0.25-0.5 ppm O3, 4 h/day, 15-60 days) (Guevara-Guzmán et 

28 al., 2009; Araneda et al., 2008; Pereyra-Muñoz et al., 2006). Further, O3-induced 

29 oxidative stress resulted in increased plasma lipid peroxides (0.25 ppm, 4h/day, 15-60 

30 days) (Santiago-López et al., 2010), which was correlated with damage to specific brain 

31 regions (Pereyra-Muñoz et al., 2006). 

32 Oxidative stress is one mechanism by which testicular and sperm function is disrupted 

33 (see Section 7.4.1). Oxidative stress may inhibit testicular steroidogenesis leading to 

34 decreased testosterone levels (Diemer et al., 2003). It may decrease sperm quality by lipid 

35 peroxidation of sperm plasma membrane which leads to impaired sperm mobility 

36 (Agarwal et al., 2003). Further, it may damage DNA in the sperm nucleus leading to 

37 apoptosis and a decline in sperm counts (Agarwal et al., 2003). Since oxidative stress is a 
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1 key event underlying many of the health effects of O3, it is possible that sperm quality 

2 and quantity may be impacted by this mechanism (Sokol et al., 2006). 

3 A role for plasma antioxidants in modulating O3-induced respiratory effects was 

4 suggested by a recent study (Aibo et al., 2010). In this study, pretreatment of rats with a 

5 high dose of acetaminophen resulted in increased levels of plasma cytokines and the 

6 influx of inflammatory cells into the lung following O3 exposure (0.25-0.5 ppm, 6 hours) 

7 (Aibo et al., 2010). These effects were not observed in response to O3 alone. 

8 Furthermore, acetaminophen-induced liver injury was exacerbated by O3 exposure. A 

9 greater increase in hepatic neutrophil accumulation and greater alteration in gene 

10 expression profiles was observed in mice exposed to O3 and acetaminophen compared 

11 with either exposure alone (Aibo et al., 2010). Although not measured in this study, 

12 glutathione depletion in the liver is known to occur in acetaminophen toxicity. Since liver 

13 glutathione is the source of plasma glutathione, acetaminophen treatment may have 

14 lowered plasma glutathione levels and altered the redox balance in the vascular 

15 compartment. These findings indicate interdependence between respiratory tract, plasma 

16 and liver responses to O3, possibly related to glutathione status. 

5.3.9 Impaired alveolar-arterial O2 transfer 

17 O3 may impair alveolar-arterial oxygen transfer and reduce the supply of arterial oxygen 

18 to the myocardium. This may have a greater impact in individuals with compromised 

19 cardiopulmonary systems. Gong et al. (1998) provided evidence of a small decrease in 

20 arterial oxygen saturation in human subjects exposed for 3 hours to 300 ppb O3 while 

21 exercising. In addition, Delaunois et al. (1998) demonstrated pulmonary vasoconstriction 

22 in O3-exposed rabbits (0.4 ppm, 4 hours). Although of interest, the contribution of this 

23 pathway to O3-induced cardiovascular effects remains uncertain. 

5.3.10 Summary

24 This section summarizes the modes of action and toxicity pathways resulting from O3

25 inhalation (Figure 5-9). These pathways provide a mechanistic basis for the health effects 

26 which are described in detail in Chapters 6 and 7. Three distinct short-term responses 

27 have been well-characterized in humans challenged with O3: decreased pulmonary 

28 function, airways inflammation, and increased bronchial reactivity. In addition, O3

29 exposure exacerbates, and possibly also causes, asthma and allergic airways disease in 

30 humans. Animal studies have demonstrated airways remodeling and fibrosis in response 

31 to chronic and intermittent O3 exposures and a wide range of other responses. While the 
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1 respiratory tract is the primary target tissue, cardiovascular and other organ effects occur 

2 following short- and long-term exposures of animals to O3. 

Figure 5-9 The modes of action/possible pathways underlying the health 

effects resulting from inhalation exposure to O3.

The initial key event in the toxicity pathway of O3 3 is the formation of secondary 

4 oxidation products in the respiratory tract. This involves direct reactions with components 

5 of the ELF and/or plasma membranes of cells residing in the respiratory tract. The 

6 resulting secondary oxidation products transmit signals to the epithelium, nociceptive 

7 sensory nerve fibers and, if present, dendritic cells, mast cells and eosinophils. Thus, O3

8 effects are mediated by components of ELF and by the multiple cell types found in the 

9 respiratory tract. Further, oxidative stress is an implicit part of this initial key event. 

Another key event in the toxicity pathway of O3 10 is the activation of neural reflexes which 

11 lead to decrements in pulmonary function (see Section 6.2.1). Evidence is accumulating 

12 that secondary oxidation products are responsible for this effect. Eicosanoids have been 

13 implicated in humans, while both eicosanoids and aldehydes are effective in animal 

14 models. Different receptors on bronchial C-fibers have been shown to mediate separate 

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 airways

Altered lung development 
Draft – Do Not Cite or Quote 5-48 September 2011

1 effects of O3 on pulmonary function. Nociceptive sensory nerves are involved in the 

2 involuntary truncation of respiration which results in decreases in FVC, FEV1, tidal 

3 volume and pain upon deep inspiration. Opioids block these responses while atropine has 

4 only a minimal effect. New evidence in an animal model suggests that TRPA1 receptors 

5 on bronchial C-fibers mediate this pathway. Ozone exposure also results in activation of 

6 vagal sensory nerves and a mild increase in airway obstruction measured as increased 

7 sRaw. Atropine and β-adrenergic agonists greatly inhibit this response in humans 

8 indicating that the airway obstruction is due to bronchoconstriction. Other studies in 

9 humans implicated SP release from bronchial C-fibers resulting in airway narrowing due 

10 to either neurogenic edema or bronchoconstriction. New evidence in an animal model 

11 suggests that the SP-NK receptor pathway caused bronchoconstriction following O3

12 exposure. 

13 Initiation of inflammation is also a key event in the toxicity pathway of O3. Secondary 

14 oxidation products, as well as chemokines and cytokines elaborated by airway epithelial 

15 cells and macrophages, have been implicated in the initiation of inflammation. Vascular 

16 endothelial adhesion molecules may also play a role. Work from several laboratories in 

17 using human subjects and animal models suggest that O3 triggers the release of 

18 tachykinins such as SP from airway sensory nerves which could contribute to 

19 downstream effects including inflammation (see Sections 6.2.3 and 7.2.4). Airways 

20 neutrophilia has been demonstrated in BALF, mucosal biopsy and induced sputum 

21 samples. Influx of mast cells, monocytes and macrophages also occur. Inflammation 

22 further contributes to O3-mediated oxidative stress. Recent investigations show that O3

23 exposure leads to the generation of hyaluronan fragments from high molecular weight 

24 polymers of hyaluronan normally found in the ELF in mice. Hyaluronan activates TLR4 

25 and CD44-dependent signaling pathways in macrophages, and results in an increased 

26 number of macrophages in the BALF. Activation of these pathways occurs later than the 

27 acute neutrophilic response suggesting that they may contribute to longer-term effects of 

28 O3. The mechanisms involved in clearing O3-provoked inflammation remain to be 

29 clarified. It should be noted that inflammation, as measured by airways neutrophilia, is 

30 not correlated with decrements in pulmonary function as measured by spirometry. 

31 A fourth key event in the toxicity pathway of O3 is alteration of epithelial barrier 

32 function. Increased permeability occurs as a result of damage to tight junctions between 

33 epithelial cells subsequent to O3-induced injury and inflammation. It may play a role in 

34 allergic sensitization and in AHR (see Sections 6.2.2, 6.2.6, and 7.2.5). Tachykinins 

35 mediate this response while antioxidants confer protection. Genetic susceptibility has 

36 been associated with a functioning TLR4 gene and with iNOS. 
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1 A fifth key event in the toxicity pathway of O3 is the sensitization of bronchial smooth 

2 muscle.

3 Increased bronchial reactivity can be both a rapidly occurring and a persistent response. 

4 The mechanisms responsible for early and later AHR are not well-understood (see 

5 Section 6.2.2). One proposed mechanism of sensitization, O3-induced increases in 

6 epithelial permeability, would improve access of agonist to smooth muscle receptors. The 

7 evidence for this mechanism is not consistent. Another proposed mechanism, for which 

8 there is greater evidence, is neurally-mediated sensitization. In humans exposed to O3, 

9 atropine blocked the early AHR response indicating the involvement of cholinergic 

10 postganglionic pathways. Animal studies demonstrated that O3-induced AHR involved 

11 vagally-mediated responses and local axon reflex responses through bronchopulmonary 

12 C-fiber-mediated release of SP. Later phases of increased bronchial reactivity may 

13 involve the induction of IL-1β which in turn upregulates SP production. In guinea pigs, 

14 eosinophil-derived major basic protein contributed to the stimulation of cholinergic 

15 postganglionic pathways. A novel role for hyaluronan in mediating the later phase effects 

16 O3-induced AHR has recently been demonstrated. Hyaluronan fragments stimulated AHR 

17 in a TLR4- and CD44 receptor-dependent manner. Tachykinins and secondary oxidation 

18 products of O3 have been proposed as mediators of the early response and inflammation19 derived products have been proposed as mediators of the later response. Inhibition of 

20 arachidonic acid metabolism was ineffective in blocking O3-induced AHR in humans 

21 while in animal models mixed results were found. Other cytokines and chemokines have 

22 been implicated in the AHR response to O3 in animal models. 

23 A sixth key event in the toxicity pathway of O3 is the modification of innate/adaptive 

24 immunity. While the majority of evidence for this key event comes from animal studies, 

25 there are several studies suggesting that this pathway may also be relevant in humans. O3

26 exposure of human subjects resulted in recruitment of activated innate immune cells to 

27 the airways. This included macrophages and monocytes with increased expression of cell 

28 surface markers characteristic of innate immunity and antigen presentation, the latter of 

29 which could contribute to exaggerated T cell responses and the promotion of an allergic 

30 phenotype. Evidence of dendritic cell activation was observed in GSTM1 null human 

31 subjects exposed to O3, suggesting O3-mediated interaction between the innate and 

32 immune systems. Animal studies further linked O3-mediated activation of the innate 

33 immune system to the development of nonspecific AHR, demonstrated an interaction 

34 between allergen and O3 in the induction of nonspecific AHR, and found that O3 acted as 

35 an adjuvant for allergic sensitization through the activation of both innate and adaptive 

36 immunity. Priming of the innate immune system by O3 was reported in mice. This 

37 resulted in an exaggerated response to endotoxin which included enhanced TLR4 

38 signaling in macrophages. Ozone-mediated impairment of the function of SP-A, an innate 
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1 immune protein, has also been demonstrated. Taken together these studies provide 

2 evidence that O3 can alter host immunologic response and lead to immune system 

3 dysfunction. These mechanisms may underlie the exacerbation and induction of asthma 

4 (see Sections 6.2.6 and 7.2.1), as well as decreases in host defense (see Sections 6.2.5 and 

5 7.2.6). 

6 Another key event in the toxicity pathway of O3 is airways remodeling. Persistent 

7 inflammation and injury, which are observed in animal models of chronic and 

8 intermittent exposure to O3, are associated with morphologic changes such as mucous 

9 cell metaplasia of nasal epithelium, bronchiolar metaplasia of alveolar ducts and fibrotic 

10 changes in small airways (see Section 7.2.3). Mechanisms responsible for these responses 

11 are not well-understood. However a recent study in mice demonstrated a key role for the 

12 TGF-β signaling pathway in the deposition of collagen in the airway wall following 

13 chronic intermittent exposure to O3. 

14 Systemic inflammation and vascular oxidative/nitrosative stress are also key events in the 

15 toxicity pathway of O3. Extrapulmonary effects of O3 occur in numerous organ systems, 

16 including the cardiovascular, central nervous, reproductive and hepatic systems (see 

17 Sections 6.3 to 6.5 and 7.3 to 7.5). It has been proposed that lipid oxidation products 

18 resulting from reaction of O3 with lipids and/or cellular membranes in the ELF are 

19 responsible for systemic responses, however it is not known whether they gain access to 

20 the vascular space. Alternatively, release of diffusible mediators from the lung into the 

21 circulation may initiate or propagate inflammatory responses in the vascular or in 

22 systemic compartments. Systemic oxidative stress is suggested by studies in humans 

23 which reported associations between O3 exposure and levels of plasma 8-isoprostanes 

24 and the presence of peripheral blood lymphocyte micronuclei. 

5.4 Interindividual Variability in Response

25 Responses to O3 exposure are variable within the population and the basis for this 

26 variability is not clear (Mudway and Kelly, 2000). Both dosimetric and mechanistic 

27 factors are likely to contribute to this variability and are discussed below. 

5.4.1 Dosimetric Considerations

28 Two studies have investigated the correlation of O3 uptake with the pulmonary function 

29 responses to O3 exposure (Reeser et al., 2005; Gerrity et al., 1994). These studies found 

30 that the large subject-to-subject variability in %ΔFEV1 response to O3 does not appear to 
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1 have a dosimetric explanation. Reeser et al. (2005) found no significant relationship 

2 between %ΔFEV1 and fractional absorption of O3 using the bolus method. Contrary to 

3 previous findings, the percent change in dead space volume of the respiratory tract 

4 (%ΔVD) did not correlate with O3 uptake, possibly due to the contraction of dead space 

5 caused by airway closure. Gerrity et al. (1994) found that intersubject variability in FEV1

6 and airway resistance was not related to differences in the O3 dose delivered to the lower 

7 airways, whereas minute ventilation was predictive of FEV1 decrement. No study has yet 

8 demonstrated that subjects show a consistent pattern of O3 retention when re-exposed 

9 over weeks of time, as has been shown to be the case for the FEV1 response, or that 

10 within-subject variation in FEV1 response is related to fluctuations in O3 uptake.

11 A delay in onset of O3-induced pulmonary function responses has been noted in 

12 numerous studies. Recently the delay was characterized in terms of changes in fB

13 (Schelegle et al., 2007). In humans exposed for 1-2 hours to 120-350 ppb O3 while 

14 exercising, no change in fB was observed until a certain cumulative inhaled dose of O3

15 had been reached. Subsequently, the magnitude of the change in fB was correlated with 

16 the inhaled dose rate (Schelegle et al., 2007). These investigators proposed that initial 

17 reactions of O3 with ELF resulted in a time-dependent depletion of ELF antioxidants, and 

18 that activation of neural reflexes occurred only after the antioxidant defenses were 

19 overwhelmed (Schelegle et al., 2007). 

20 Other studies investigated the relationship between O3 dose and cellular injury. In two 

21 studies, the initial cellular injury was found to correlate with the site-specific O3 dose. 

22 Contained within the CAR, the respiratory bronchioles were confirmed as the site 

receiving the greatest O3 dose (18 23 O mass/lung weight) and sustained the greatest initial 

24 cellular injury in O3 (0.4 and 1.0 ppm for 2 hours) exposed resting rhesus monkeys 

25 (Plopper et al., 1998). The respiratory bronchioles, having the highest concentration of 

26 local O3 dose, were also the site of significant GSH reduction. In addition, a study in 

27 isolated perfused rat lungs found greater injury in conducting airways downstream of 

28 bifurcations where local doses of O3 were higher (Postlethwait et al., 2000). 

Further, the degree of inflammation in rats has been correlated with 18 29 O-labeled O3 dose 

30 markers in the lower lung. In female rats exposed to 0.8 ppm O3 for 4 hours, BAL 

neutrophil number and 18 31 O reaction product were directly proportional (Gunnison and 

32 Hatch, 1999). Kari et al. (1997) observed that a 3-week caloric restriction (75%) in rats 

33 abrogated the toxicity of O3 (2 ppm, 2 hours), measured as BALF increases in protein, 

34 fibronectin and neutrophils, which was seen in normally fed rats. Accompanying this 

resistance to O3 toxicity was a reduction (30%) in the accumulation of 18 35 O reaction 

36 product in the lungs. These investigations also demonstrated an inverse relationship 

37 between AH2 levels and O3 dose and provided evidence for AH2 playing a protective 
Draft – Do Not Cite or Quote 5-52 September 2011

1 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 2 O reaction 

3 products compared with pre-pregnant rats in response to O3 exposure (Gunnison and 

4 Hatch, 1999). In the calorie restricted model, a 30% higher basal BALF AH2 

5 concentration and a rapid accumulation of AH2 into the lungs to levels 60% above 

6 normal occurred as result of O3 exposure (Kari et al., 1997). However, this relationship 

7 between AH2 levels and O3 dose did not hold up in every study. Aging rats (9 and 24 

8 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 9 O reaction 

10 products following O3 exposure (0.4-0.8 ppm, 2-6 hours) (Vincent et al., 1996a). 

11 Interindividual variability in the neutrophilic response has been noted in human subjects 

12 (Holz et al., 1999; Devlin et al., 1991; Schelegle et al., 1991). One study demonstrated a 

13 threefold difference in airways neutrophilia, measured as percent of total cells in 

14 proximal BALF, among human subjects exposed to 300 ppb O3 for 1 hour while 

15 exercising (Schelegle et al., 1991). Another study reported a 20-fold difference in BAL 

16 neutrophils following exposure to 80-100 ppb O3 for 6.6 hours in exercising human 

17 subjects (Devlin et al., 1991). Reproducibility of intra-individual responses to 1-hour 

18 exposure to 250 ppb O3, measured as sputum neutrophilia, was demonstrated by Holz 

19 (1999). Few studies have examined the dose- or concentration-responsiveness of airways 

20 neutrophilia in O3-exposed humans (Holz et al., 1999; Devlin et al., 1991). No 

21 concentration-responsiveness was observed in healthy human subjects exposed for 1 hour 

22 to 125-250 ppb O3 and a statistically significant increase in sputum neutrophilia was 

23 observed only at the higher dose (Holz et al., 1999). However, concentration-dependent 

24 and statistically significant increases in BAL neutrophils and the inflammatory mediator 

25 IL-6 were reported following exposure to 80 and 100 ppb O3 for 6.6 hours in exercising 

26 humans (Devlin et al., 1991). Additional evidence is provided by a meta-analysis of the 

27 O3 dose-inflammatory response in controlled human exposure studies involving exposure 

28 to 80-600 ppb O3 for 60-396 minutes (Mudway and Kelly, 2004b). Results demonstrated 

29 a linear relationship between inhaled O3 dose (determined as the product of 

30 concentration, ventilation and time) and BAL neutrophils at 0-6 hours and 18-24 hours 

31 following O3 exposure (Mudway and Kelly, 2004b). 

32 Collectively these studies demonstrate a correlation between dose and response for some 

33 O3-induced effects and suggest a role for ELF antioxidants in modulating the dose to 

34 tissue. The lack of correlation between O3-induced effects and calculated O3 dose may be 

35 a result of interindividual differences in TB volume. 
Draft – Do Not Cite or Quote 5-53 September 2011

5.4.2 Mechanistic Considerations

1 There was a large range of pulmonary function responses to O3 among healthy young 

2 adults exposed for 4 hours to 200 ppb O3 or for 1.5 hours to 420 ppb O3 while exercising 

3 (Hazucha et al., 2003; Balmes et al., 1996). Since individual responses were relatively 

4 consistent across time, it was thought that responsiveness reflected an intrinsic 

5 characteristic of the subject (Mudway and Kelly, 2000). Older adults were generally not 

6 responsive to O3 (Hazucha et al., 2003), while obese young women may have been more 

7 responsive than lean young women (420 ppb, 1.5 hours, while exercising) (Bennett et al., 

8 2007). The lack of spirometric responsiveness was not attributable to the presence of 

9 endogenous endorphins, which could potentially block C-fiber stimulation by O3, as 

10 demonstrated in a study involving intravenous administration of naloxone immediately 

11 following the O3 exposure (420 ppb, 2 hours, while exercising) to weak responders 

12 (Passannante et al., 1998). Inflammation and other responses to O3 were also 

13 characterized by a large degree of interindividual variability. Currently, the mechanisms 

14 underlying this variability are not known. It has been proposed that some of the variation 

15 in responses may be genetically determined (Yang et al., 2005a). The role of gene16 environment interactions, pre-existing diseases and conditions, nutritional status, 

17 lifestage, attenuation, and co-exposures in modulating responses to O3 are discussed 

18 below. 

5.4.2.1 Gene-Environment Interactions 

19 The significant interindividual variation in responses to O3 infers that genetic background 

20 is an important determinant of susceptibility to O3 (Cho and Kleeberger, 2007; 

21 Kleeberger et al., 1997) (see also Section 8.4). Strains of mice which are prone or 

22 resistant to O3-induced effects have been used to systematically identify candidate 

23 susceptibility genes. Genome wide linkage analyses (also known as positional cloning) 

24 demonstrated quantitative trait loci for O3-induced lung inflammation and 

25 hyperpermeability on chromosome 17 (Kleeberger et al., 1997) and chromosome 4 

26 (Kleeberger et al., 2000), respectively, using these recombinant inbred strains of mice and 

27 exposures to 0.3 ppm O3 for up to 72 hours. More specifically, these studies found that 

28 Tnf, whose protein product is the inflammatory cytokine TNF-α, and Tlr4, whose protein 

29 product is TLR4, were candidate susceptibility genes (Kleeberger et al., 2000; Kleeberger 

30 et al., 1997). Other studies, which used targeted deletion, identified genes encoding iNOS 

31 and heat shock proteins as TLR4 effector genes (Bauer et al., 2011; Kleeberger et al., 

32 2001) and found that IL-10 protects against O3-induced pulmonary inflammation 

33 (Backus et al., 2010). Investigations in inbred mouse strains found that differences in 

34 expression of certain proteins, such as CCSP (1.8 ppm O3 for 3 hours) (Broeckaert et al., 
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1 2003) and MARCO (0.3 ppm O3 for up to 48 hours) (Dahl et al., 2007), were responsible 

2 for phenotypic characteristics, such as epithelial permeability and scavenging of oxidized 

3 lipids, respectively, which confer sensitivity to O3. 

4 Genetic polymorphisms have received increasing attention as modulators of O3-mediated 

5 effects. Functionally relevant polymorphisms in candidate susceptibility genes have been 

6 studied at the individual and population level in humans, and also in animal models. 

7 Genes whose protein products are involved in antioxidant defense/oxidative stress and 

8 xenobiotic metabolism, such as glutathione-S-transferase M1 (GSTM1) and 

9 NADPH:quinone oxidoreductase 1 (NQO1), have also been a major focuses of these 

10 efforts. This is because oxidative stress resulting from O3 exposure is thought to 

11 contribute to the pathogenesis of asthma, and because xenobiotic metabolism detoxifies 

12 secondary oxidation products formed by O3 which contribute to oxidative stress (Islam et 

13 al., 2008). TNF-α is of interest since it is linked to a candidate O3 susceptibility gene and 

14 since it plays a key role in initiating airways inflammation (Li et al., 2006d). 

15 Polymorphisms of genes coding for GSTM1, NQO1 and TNF-α have been associated 

16 with altered susceptibility to O3-mediated effects (Li et al., 2006d; Yang et al., 2005a; 

17 Romieu et al., 2004a; Corradi et al., 2002; Bergamaschi et al., 2001). Additional studies 

18 have focused on functional variants in other genes involved in antioxidant defense such 

19 as catalase (CAT), myeloperoxidase, heme oxygenase (HMOX-1) and manganese 

20 superoxide dismutase (MnSOD) (Wenten et al., 2009; Islam et al., 2008). These studies 

21 are discussed below. 

22 GSTM1 is a phase II antioxidant enzyme which is transcriptionally regulated by NF-E2-

23 related factor 2-antioxidant response element (Nrf2-ARE) pathway. A large proportion 

24 (40-50%) of the general public (across ethnic populations) has the GSTM1-null genotype, 

25 which has been linked to an increased risk of health effects due to exposure to air 

26 pollutants (London, 2007). A role for GSTs in metabolizing electrophiles such as 4-

27 hydroxynonenal, which is a secondary oxidation product formed following O3 exposure, 

28 has been demonstrated (Awasthi et al., 2004). A recent study found that the GSTM1 

29 genotype modulated the time course of the neutrophilic inflammatory response following 

30 acute O3 exposure (400 ppb for 2 hours with intermittent exercise) in healthy adults 

31 (Alexis et al., 2009). In GSTM1-null and -sufficient subjects, O3-induced sputum

32 neutrophilia was similar at 4 hours. However, neutrophilia resolved by 24 hours in 

33 sufficient subjects but not in GSTM1-null subjects. In contrast, no differences in 24 hour 

34 sputum neutrophilia were observed between GSTM1-null and -sufficient human subjects 

35 exposed to 60 ppb O3 for 2 hours with intermittent exercise (Kim et al., 2011). It is not 

36 known whether the effect seen at the higher exposure level (Alexis et al., 2009) was due 

37 to the persistence of pro-inflammatory stimuli, impaired production of downregulators or 

38 impaired neutrophil apoptosis and clearance. However, a subsequent in vitro study by 
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1 these same investigators found that GSTM1 deficiency in airway epithelial cells 

2 enhanced IL-8 production in response to 0.4 ppm O3 for 4 hours (Wu et al., 2011). 

3 Furthermore, NF-κB activation was required for O3-induced IL-8 production (Wu et al., 

4 2011). Since IL-8 is a potent neutrophil activator and chemotaxin, this study provides 

5 additional evidence for the role of GSTM1 as a modulator of inflammatory responses due 

6 to O3 exposure. 

7 In addition, O3 exposure increased the expression of the surface marker CD14 in airway 

8 neutrophils of GSTM1-null subjects compared with sufficient subjects (Alexis et al., 

9 2009). Furthermore, differences in airway macrophages were noted between the GSTM1-

10 sufficient and -null subjects. Nnumbers of airway macrophages were decreased at 4 and 

11 24 hours following O3 exposure in GSTM1-sufficient subjects (Alexis et al., 2009). 

12 Airway macrophages in GSTM1-null subjects were greater in number and found to have 

13 greater oxidative burst and phagocytic capability than those of sufficient subjects. Airway 

14 macrophages and dendritic cells from GSTM1-null subjects exposed to O3 expressed 

15 higher levels of the surface marker HLA-DR, suggesting activation of the innate immune 

16 system (Alexis et al., 2009). These differences in inflammatory responses between the 

17 GSTM1-null and -sufficient subjects may provide biological plausibility for the 

18 differences in O3-mediated effects reported in controlled human exposure studies 

19 (Corradi et al., 2002; Bergamaschi et al., 2001). It should also be noted that GSTM1 

20 genotype did not affect the acute pulmonary function (i.e. spirometric) response to O3

21 which provides additional evidence for separate mechanisms underlying O3’s effects on 

22 pulmonary function and inflammation in adults (Alexis et al., 2009). However, GSTM1-

23 null asthmatic children were previously found to be more at risk of O3-induced effects on 

24 pulmonary function than GSTM1-sufficient asthmatic children (Romieu et al., 2004a).

25 Another enzyme involved in the metabolism of secondary oxidation products is NQO1.

26 NQO1 catalyzes the 2-electron reduction by NADPH of quinones to hydroquinones. 

27 Depending on the substrate, it is capable of both protective detoxification reactions and 

28 redox cycling reactions resulting in the generation of reactive oxygen species. A recent 

29 study using NQO1-null mice demonstrated that NQO1 contributes to O3-induced 

30 oxidative stress, AHR and inflammation following a 3-hour exposure to 1 ppm O3

31 (Voynow et al., 2009). These experimental results may provide biological plausibility for 

32 the increased biomarkers of oxidative stress and increased pulmonary function 

33 decrements observed in O3-exposed individuals bearing both the wild-type NQO1 gene 

34 and the null GSTM1 gene (Corradi et al., 2002; Bergamaschi et al., 2001). 

35 Besides enzymes, other mechanisms participate in the removal of secondary oxidation 

36 products formed as a result of O3 inhalation. One involves scavenging of oxidized lipids 

37 via the macrophage receptor with collagenous structure (MARCO) expressed on the cell 
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1 surface of alveolar macrophages. A recent study demonstrated increased gene expression 

2 of MARCO in the lungs of an O3-resistant C3H mouse strain (HeJ) but not in an O3-

3 sensitive, genetically similar strain (OuJ) (Dahl et al., 2007). Upregulation of MARCO 

4 occurred in mice exposed to 0.3 ppm O3 for 24-48 hours; inhalation exposure for 6 hours 

5 at this concentration was insufficient for this response. Animals lacking the MARCO 

6 receptor exhibited greater inflammation and injury, as measured by BAL neutrophils, 

7 protein and isoprostanes, following exposure to 0.3 ppm O3 (Dahl et al., 2007). MARCO 

8 also protected against the inflammatory effects of oxidized surfactant lipids (Dahl et al., 

9 2007). Scavenging of oxidized lipids may limit O3-induced injury since ozonized 

10 cholesterol species formed in the ELF (mice, 0.5-3 ppm O3, 3 hours) (Pulfer et al., 2005; 

11 Pulfer and Murphy, 2004) stimulated apoptosis and cytotoxicity in vitro (Gao et al., 

12 2009b; Sathishkumar et al., 2009; Sathishkumar et al., 2007a; Sathishkumar et al., 

13 2007b).

14 Two studies reported relationships between TNF promoter variants and O3-induced 

15 effects in humans. In one study, O3-induced change in lung function was significantly 

16 lower in adult subjects with TNF promoter variants -308A/A and -308G/A compared with 

17 adult subjects with the variant -308G/G (Yang et al., 2005a). This response was 

18 modulated by a specific polymorphism of LTA (Yang et al., 2005a), a previously 

19 identified candidate susceptibility gene whose protein product is lymphotoxin-α 

20 (Kleeberger et al., 1997). In the second study, an association between the TNF promoter 

21 variant -308G/G and decreased risk of asthma and lifetime wheezing in children was 

22 found (Li et al., 2006d). The protective effect on wheezing was modulated by ambient O3

23 levels and by GSTM1 and GSTP1 polymorphisms. The authors suggested that the 

24 TNF-308 G/G genotype may have a protective role in the development of childhood 

25 asthma (Li et al., 2006d).

26 Similarly, a promoter variant of the gene HMOX-1, consisting of a smaller number of 

27 (GT)n repeats, was associated with a reduced risk for new-onset asthma in non-Hispanic 

28 white children (Islam et al., 2008). The number of (GT)n repeats in this promoter has 

29 been shown to be inversely related to the inducibility of HMOX-1. A modulatory effect 

30 of O3 was demonstrated since the beneficial effects of this polymorphism were seen only 

31 in children living in low O3 communities (Islam et al., 2008). This study also identified 

32 an association between a polymorphism of the CAT gene and increased risk of new-onset 

33 asthma in Hispanic children; however no modulation by O3 was seen (Islam et al., 2008). 

34 No association was observed in this study between a MnSOD polymorphism and asthma 

35 (Islam et al., 2008).
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1 Studies to date indicate that some variability in individual responsiveness to O3 may be 

2 accounted for by functional genetic polymorphisms. Further, the effects of gene3 environment interactions may be different in children and adults.

5.4.2.2 Pre-existing Diseases and Conditions

4 Pre-existing diseases and conditions can alter the response to O3 exposure. For example, 

5 responsiveness to O3, as measured by spirometry, is decreased in smokers and individuals 

6 with COPD (U.S. EPA, 2006b). Asthma and allergic diseases are of major importance in 

7 this discussion. In individuals with asthma, there is increased responsiveness to 

8 bronchoconstrictor challenge. This results from a combination of structural and 

9 physiological factors including increased airway inner-wall thickness, smooth muscle 

10 responsiveness and mucus secretion. Although inflammation is likely to contribute, its 

11 relationship to AHR is not clear (U.S. EPA, 2006b). However, some asthmatics have

12 higher baseline levels of neutrophils, lymphocytes, eosinophils and mast cells in 

13 bronchial washes and bronchial biopsy tissue (Stenfors et al., 2002). It has been proposed 

14 that enhanced sensitivity to O3 is conferred by the presence of greater numbers of 

15 resident airway inflammatory cells in disease states such as asthma (Mudway and Kelly, 

16 2000).

17 In order to determine whether asthmatics exhibit greater responses to O3, several older 

18 studies compared pulmonary function in asthmatic and non-asthmatic subjects following 

19 O3 exposure. Some also probed mechanisms which could account for enhanced 

20 sensitivity. While the majority focused on measurements of FEV1 and FVC and found no 

21 differences between the two groups following exposures of 2-4 hours to 125-250 ppb O3

22 or to a 30-minute exposure to 120-180 ppb O3 by mouthpiece while exercising (Stenfors 

23 et al., 2002; Mudway et al., 2001; Holz et al., 1999; Scannell et al., 1996; Koenig et al., 

24 1987; Linn et al., 1978), there were notable exceptions. In one study, greater airway 

25 obstruction in asthmatics compared with non-asthmatic subjects was observed 

26 immediately following a 2-hour exposure to 400 ppb O3 with intermittent exercise (Kreit 

27 et al., 1989). These changes were measured as statistically significant greater decreases in 

28 FEV1 and in FEF25-75 (but not in FVC) in the absence of a bronchoconstrictor challenge 

29 (Kreit et al., 1989). These results suggest that this group of asthmatics responded to 

30 O3-exposure with a greater degree of vagally-mediated bronchoconstriction compared 

31 with the non-asthmatics. A second study demonstrated a statistically significant greater 

32 decrease in FEV1 and in FEV1/FVC (but not in FVC) in asthmatics compared with non33 asthmatics exposed to 160 ppb O3 for 7.6 hours with light exercise (Horstman et al., 

34 1995). These responses were accompanied by wheezing and inhaler use in the asthmatics 

35 (Horstman et al., 1995). Aerosol bolus dispersion measurements demonstrated a 
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1 statistically significant greater change in asthmatics compared with non-asthmatics, 

2 which was suggestive of O3-induced small airway dysfunction (Horstman et al., 1995). 

3 Furthermore, a statistically significant correlation was observed between the degree of 

4 baseline airway status and the FEV1 response to O3 in the asthmatic subjects (Horstman 

5 et al., 1995). A third study found similar decreases in FVC and FEV1 in both asthmatics 

6 and non-asthmatics exposed to 400 ppb O3 for 2 hours with mild exercise (Alexis et al., 

7 2000). However, a statistically significant decrease in FEF75, a measure of small airway 

8 function, was observed in asthmatics but not in non-asthmatics (Alexis et al., 2000). 

9 Taken together, these latter studies indicate that while the magnitude of restrictive type 

10 spirometric decline was similar in asthmatics and non-asthmatics, that obstructive type 

11 changes (i.e. bronchoconstriction) were greater in asthmatics. Further, asthmatics 

12 exhibited greater sensitivity to O3 in terms of small airways function. 

13 Since asthma exacerbations occur in response to allergens and/or other triggers, some 

14 studies have focused on O3-induced changes in AHR following a bronchoconstrictor 

15 challenge. No difference in sensitivity to methacholine bronchoprovocation was observed 

16 between asthmatics and non-asthmatics exposed to 400 ppb O3 for 2 hours with moderate 

17 exercise (Kreit et al., 1989). However, increased bronchial reactivity to inhaled allergens 

18 was demonstrated in mild allergic asthmatics exposed to 160 ppb for 7.6 hours, 250 ppb 

19 for 3 hours and 120 ppb for 1 hour while exercising (Kehrl et al., 1999; Jorres et al., 

20 1996; Molfino et al., 1991) and in allergen-sensitized guinea pigs following O3 exposure 

21 (1 ppm, 1 hour) (Sun et al., 1997). Similar, but modest, responses were reported for 

22 individuals with allergic rhinitis (Jorres et al., 1996). Further, the contractile response of 

23 isolated airways from human donor lung tissue, which were sensitized and challenged 

24 with allergen, was increased by pre-exposure to 1 ppm O3 for 20 (Roux et al., 1999). 

25 These studies provide support for O3-mediated enhancement of responses to allergens in 

26 allergic subjects. 

27 In terms of airways neutrophilia, larger responses were observed in asthmatics compared 

28 to non-asthmatics subjects exposed to O3 in some (Balmes et al., 1997; Scannell et al., 

29 1996; Basha et al., 1994) but not all (Mudway et al., 2001) of the older studies. While 

30 each of these studies involved exposure of exercising human subjects to 200 ppb O3, the 

31 duration of exposure was longer (i.e. 4-6 hours) in the former studies than in the latter 

32 study (2 hours). Further, statistically significantly increases in myeloperoxidase levels (an 

33 indicator of neutrophil activation) in bronchial washes was observed in mild asthmatics 

34 compared with non-asthmatics, despite no difference in O3-stimulated neutrophil influx 

35 between the 2 groups following exposure to 200 ppb O3 for 2 hours with mild exercise 

36 (Stenfors et al., 2002). A more recent study found that atopic asthmatic subjects exhibited 

37 an enhanced inflammatory response to O3 (400 ppb, 4 hours, with exercise) (Hernandez 

38 et al., 2010). This response was characterized by greater numbers of neutrophils, higher 
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1 levels of IL-6, IL-8 and IL-1β and greater macrophage cell-surface expression of TLR4 

2 and IgE receptors in induced sputum compared with healthy subjects. This study also 

3 reported a greater increase in hyaluronan in atopic subjects and atopic asthmatics 

4 compared with healthy subjects following O3 exposure. Animal studies have previously 

5 reported that hyaluronic acid activates TLR4 signaling and results in AHR (see Section 

6 5.3.5). Furthermore, levels of IL-10, a potent anti-inflammatory cytokine, were greatly 

7 reduced in atopic asthmatics compared to healthy subjects. These results provide 

8 evidence that innate immune and adaptive responses are different in asthmatics and 

9 healthy subjects exposed to O3. 

10 Eosinophils may be an important modulator of responses to O3 in asthma and allergic 

11 airways disease. Eosinophils and associated proteins are thought to affect muscarinic 

12 cholinergic receptors which are involved in vagally-mediated bronchoconstriction 

13 (Mudway and Kelly, 2000). Studies described in Section 5.3.5 which demonstrated a key 

14 role of eosinophils in O3-mediated AHR may be relevant to human allergic airways 

15 disease which is characterized by airways eosinophilia (Yost et al., 2005). Furthermore, 

16 O3 exposure sometimes results in airways eosinophilia in allergic subjects or animal 

17 models. For example, eosinophilia of the nasal and other airways was observed in 

18 individuals with pre-existing allergic disease following O3 inhalation (270 and 400 ppb 

19 O3, 2 hours, with exercise) (Vagaggini et al., 2002; Peden et al., 1995). Further, O3

20 exposure (0.5 ppm, 8 hours/day for 1-3 days) increased allergic responses, such as 

21 eosinophilia and augmented intraepithelial mucosubstances, in the nasal airways of 

22 ovalbumin (OVA)-sensitized rats (Wagner et al., 2002). In contrast, Stenfors (2002) found 

23 no stimulation of eosinophil influx measured in bronchial washes and BALF of mild 

24 asthmatics following exposure to a lower concentration (200 ppb, 2 hours, with exercise) 

25 of O3. 

26 The role of mast cells in O3-mediated asthma exacerbations has been investigated. Mast 

27 cells are thought to play a key role in O3-induced airways inflammation, since airways 

28 neutrophilia was decreased in mast cell-deficient mice exposed to O3 (Kleeberger et al., 

29 1993). However, another study found that mast cells were not involved in the 

30 development of increased bronchial reactivity in O3-exposed mice (Noviski et al., 1999). 

31 Nonetheless, mast cells release a wide variety of important inflammatory mediators 

32 which may lead to asthma exacerbations (Stenfors et al., 2002). A large increase in mast 

33 cell number in bronchial submucosa was observed in non-asthmatics and a significant 

34 decrease in mast cell number in bronchial epithelium was observed in mild asthmatics 6 

35 hours following exposure to 200 ppb O3 for 2 hours during mild exercise (Stenfors et al., 

36 2002). While these results point to an O3-mediated flux in bronchial mast cell 

37 populations which differed between the non-asthmatics and mild asthmatics, 
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1 interpretation of these findings is difficult. Furthermore, mast cell number did not change 

2 in airway lavages in either group in response to O3 (Stenfors et al., 2002)

3 Cytokine profiles in the airways have been investigated as an indicator of O3 sensitivity. 

4 Differences in epithelial cytokine expression were observed in bronchial biopsy samples 

5 in non-asthmatic and asthmatic subjects both at baseline and 6-hours postexposure to 200 

6 ppb O3 for 2 hours (Bosson et al., 2003). The asthmatic subjects had a higher baseline 

7 expression of IL-4 and IL-5 compared to non-asthmatics. In addition, expression of IL-5, 

8 IL-8, GM-CSF, and ENA-78 in asthmatics was increased significantly following O3

9 exposure compared to non-asthmatics (Bosson et al., 2003). Some of these (IL-4, IL-5 

10 and GM-CSF) are Th2-related cytokines or neutrophil chemoattractants, and play a role 

11 in IgE production, airways eosinophilia and suppression of Th1-cytokine production 

12 (Bosson et al., 2003). These findings suggest a link between adaptive immunity and 

13 enhanced responses of asthmatics to O3. 

14 A further consideration is the compromised status of ELF antioxidants in disease states 

15 such as asthma (Mudway and Kelly, 2000). This could possibly be due to ongoing 

16 inflammation which causes antioxidant depletion or to abnormal antioxidant transport or 

17 synthesis (Mudway and Kelly, 2000). For example, basal levels of AH2 were 

18 significantly lower and basal levels of oxidized GSH and UA were significantly higher in 

19 bronchial wash fluid and BALF of mild asthmatics compared with healthy control 

20 subjects (Mudway et al., 2001). Differences in ELF antioxidant content have also been 

21 noted between species. These observations have led to the suggestion that the amount and 

22 composition of ELF antioxidants, the capacity to replenish antioxidants in the ELF or the 

23 balance between beneficial and injurious interactions between antioxidants and O3 may 

24 contribute to O3 sensitivity, which varies between individuals and species (Mudway et 

25 al., 2006; Mudway and Kelly, 2000; Mudway et al., 1999a). The complexity of these 

26 interactions was demonstrated by a study in which a 2-hour exposure to 200 ppb O3, 

27 while exercising, resulted in similar increases in airway neutrophils and decreases in 

28 pulmonary function in both mild asthmatics and healthy controls, despite differences in 

29 ELF antioxidant concentrations prior to O3 exposure (Mudway et al., 2001). Further, the 

30 O3-induced increase in oxidized GSH and decrease in AH2 observed in ELF of healthy 

31 controls was not observed in mild asthmatics (Mudway et al., 2001). While the authors 

32 concluded that basal AH2 and oxidized GSH concentrations were not predictive of 

33 responsiveness to O3, they also suggested that the increased basal UA concentrations in 

34 the mild asthmatics may have played a protective role (Mudway et al., 2001). Thus 

35 compensatory mechanisms resulting in enhanced total antioxidant capacity may play a 

36 role in modulating responses to O3. 
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1 Collectively these older and more recent studies provide insight into mechanisms which 

2 may contribute to enhanced responses of asthmatic and atopic individuals following O3 

3 exposure. Greater airways inflammation and/or greater bronchial reactivity have been 

4 demonstrated in asthmatics compared to non-asthmatics. This pre-existing inflammation 

5 and altered baseline bronchial reactivity may contribute to the enhanced 

6 bronchoconstriction seen in asthmatics exposed to O3. Furthermore, O3-induced 

7 inflammation may contribute to O3-mediated AHR. An enhanced neutrophilic response 

8 has been demonstrated in some asthmatics. A recent study in humans provided evidence 

9 for differences in innate immune responses related to TLR4 signaling between asthmatics 

10 and healthy subjects. Animal studies have demonstrated a role for eosinophil-derived 

11 proteins in mediating the effects of O3. Since airways eosinophilia occurs in both allergic 

12 humans and allergic animal models, this pathway may underlie the exacerbation of 

13 allergic asthma by O3. In addition, differences have been noted in epithelial cytokine 

14 expression in bronchial biopsy samples of healthy and asthmatic subjects. A Th2 

15 phenotype, indicative of adaptive immune system activation and enhanced allergic 

16 responses, was observed before O3 exposure and was increased by O3 exposure in 

17 asthmatics. These findings support links between innate and adaptive immunity and 

18 sensitivity to O3-mediated effects in asthmatics and allergic airways disease. 

19 In addition to asthma and allergic diseases, obesity may alter responses to O3. While O3

20 is a trigger for asthma, obesity is a known risk factor for asthma (Shore, 2007). The 

21 relationship between obesity and asthma is not well understood but recent investigations 

22 have focused on alterations in endocrine function of adipose tissue in obesity. It is 

23 thought that the increases in serum levels of factors produced by adipocytes (i.e. 

24 adipokines) such as cytokines, chemokines, soluble cytokine receptors and energy 

25 regulating hormones, may contribute to the relationship between obesity and asthma. 

26 Some of these same mechanisms may be relevant to insulin resistant states such as 

27 metabolic syndrome. 

28 In a reanalysis of the data of Hazucha (2003), increasing body mass index in young 

29 women was associated with increased O3 responsiveness, as measured by spirometry 

30 following a 2-hour exposure to 500 ppb O3 while exercising (Bennett et al., 2007). In 

31 several mouse models of obesity, airways were found to be innately more 

32 hyperresponsive and responded more vigorously to acute O3 exposure than lean controls 

33 (Shore, 2007). Pulmonary inflammatory and injury in response to O3 were also enhanced 

34 (Shore, 2007). It was postulated that oxidative stress resulting from obesity-related 

35 hyperglycemia could account for these effects (Shore, 2007). However, responses to O3

36 in the different mouse models are somewhat variable and depend on whether exposures 

37 are acute or subacute. For example, diet-induced obesity augmented inflammation and 

38 injury, as measured by BALF markers, and enhanced AHR in mice exposed acutely to O3
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1 (2 ppm, 3 hours) (Johnston et al., 2008). In contrast, the inflammatory response following 

2 sub-acute exposure to O3 was dampened by obesity in a different mouse model (0.3 ppm, 

3 72 hours) (Shore et al., 2009). 

5.4.2.3 Nutritional Status

4 Many investigations have focused on antioxidant deficiency and supplementation as 

5 modulators of O3-mediated effects. One study in mice found that vitamin A deficiency 

6 enhanced lung injury induced by exposure to 0.3 ppm O3 for 72 hours (Paquette et al., 

7 1996). Ascorbate deficiency was shown to increase the effects of acute (0.5-1 ppm for 4 

8 hours), but not subacute (0.2-0.8 ppm for 7 days), O3 exposure in guinea pigs (Kodavanti 

9 et al., 1995; Slade et al., 1989). Supplementation with AH2 and α-TOH was protective in 

10 healthy adults who were on an AH2-deficient diet and exposed to 400 ppb O3 for 2 hours 

11 while exercising (Samet et al., 2001). In this study, the protective effect consisted of a 

12 smaller reduction in FEV1 following O3 exposure (Samet et al., 2001). However the 

13 inflammatory response (influx of neutrophils and levels of IL-6) measured in BALF 1 

14 hour after O3 exposure was not different between supplemented and non-supplemented 

15 subjects (Samet et al., 2001). Other investigators found that AH2 and α-TOH 

16 supplementation failed to ameliorate the pulmonary function decrements or airways 

17 neutrophilia observed in humans exposed to 200 ppb O3 for 2 hours (Mudway et al., 

18 2006). It was suggested that supplementation may be ineffective in the absence of 

19 antioxidant deficiency (Mudway et al., 2006).

20 In asthmatic adults, these same dietary antioxidants reduced O3-induced bronchial 

21 hyperresponsiveness (120 ppb, 45 min, with exercise) (Trenga et al., 2001). Furthermore, 

22 supplementation with AH2 and α-tocopherol protected against pulmonary function 

23 decrements and nasal inflammatory responses which were associated with high levels of 

24 ambient O3 in asthmatic children living in Mexico City (Sienra-Monge et al., 2004; 

25 Romieu et al., 2002). Similarly, supplementation with ascorbate, α-tocopherol and 

26 β-carotene improved pulmonary function in Mexico City street workers (Romieu et al., 

27 1998a). 

28 Protective effects of supplementation with α-tocopherol alone have not been observed in 

29 humans experimentally exposed to O3 (Mudway and Kelly, 2000). Alpha-TOH 

30 supplementation also failed to protect against O3-induced effects in animal models of 

31 allergic rhinosinusitis and lower airways allergic inflammation (rats, 1 ppm O3 for 2 

32 days) (Wagner et al., 2007). However, protection in these same animal models was 

33 reported using γ-TOH supplementation (Wagner et al., 2009; Wagner et al., 2007). Other

34 investigators found that α-TOH deficiency led to an increase in liver lipid peroxidation 
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1 (rats, 0.3 ppm 3 hours/day for 7 months) (Sato et al., 1980) and a drop in liver α-TOH 

2 levels following O3 exposure (mice, 0.5 ppm, 6 hours/day for 3 days) (Vasu et al., 2010). 

3 A recent study used α-TOH transfer protein null mice as a model of α-TOH deficiency 

4 and demonstrated an altered adaptive response of the lung genome to O3 exposure (Vasu 

5 et al., 2010). Taken together, these studies provide evidence that the tocopherol system 

6 modulates O3-induced responses.

5.4.2.4 Lifestage

7 Responses to O3 are modulated by factors associated with lifestage. On one end of the 

8 lifestage spectrum is aging. The spirometric response to O3 appears to be lost in humans 

9 as they age, as was demonstrated in two studies involving exposures of exercising human 

10 subjects to 420-450 ppb O3 for 1.5-2 hours (Hazucha et al., 2003; Drechsler-Parks, 

11 1995). In mice, physiological responses to O3 (600 ppb, 2 hours) were diminished with 

12 age (Hamade et al., 2010). Mechanisms accounting for this effect have not been well13 studied but could include altered number and sensitivity of receptors or altered signaling 

14 pathways involved in neural reflexes. 

15 On the other side of the lifestage spectrum is pre/postnatal development. Critical 

16 windows of development during the pre/postnatal period are associated with an enhanced 

17 sensitivity to environmental toxicants. Adverse birth outcomes and developmental 

18 disorders may occur as a result. 

19 Adverse birth outcomes may result from stressors which impact transplacental oxygen 

20 and nutrient transport by a variety of mechanisms including oxidative stress, placental 

21 inflammation and placental vascular dysfunction (Kannan et al., 2006). These 

22 mechanisms may be linked since oxidative/ nitrosative stress is reported to cause vascular 

23 dysfunction in the placenta (Myatt et al., 2000). As described in Section 7.4, systemic 

24 inflammation and oxidative/nitrosative stress and modification of innate and adaptive 

25 immunity are key events underlying the health effects of O3 and as such they may 

26 contribute to adverse birth outcomes. An animal toxicology study showing that exposure 

27 to 2 ppm O3 led to anorexia (Kavlock et al., 1979) (see Section 7.4.2) in exposed rat 

28 dams provide an additional mechanism by which O3 exposure could lead to diminished 

29 transplacental nutrient transport. Disturbances of the pituitary-adrenocortico-placental 

30 system (Ritz et al., 2000) may also impact normal intrauterine growth and development. 

31 Further, restricted fetal growth may result from pro-inflammatory cytokines which limit 

32 trophoblast invasion during the early stages of pregnancy (Hansen et al., 2008). Direct 

33 effects on maternal health, such as susceptibility to infection, and on fetal health, such as 

34 DNA damage, have also been proposed as mechanisms underlying adverse birth 
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1 outcomes (Ritz et al., 2000). In addition to restricted fetal growth, preterm birth may 

2 contribute to adverse birth outcomes. Preterm birth may result from the development of 

3 premature contractions and/or premature rupture of membranes as well as from disrupted 

4 implantation and placentation which results in suboptimal placental function (Darrow et 

5 al., 2009; Ritz et al., 2000). Genetic mutations are thought to be an important cause of 

6 placental abnormalities in the first trimester, while vascular alterations may be the main 

7 cause of placental abnormalities in later trimesters (Jalaludin et al., 2007). Ozone8 mediated systemic inflammation and oxidative stress/nitrosative stress may possibly be 

9 related to these effects although there is no firm evidence. 

10 Enhanced sensitivity to environmental toxicants during critical windows of development 

11 may also result in developmental disorders. For example, normal migration and 

12 differentiation of neural crest cells are important for heart development and are 

13 particularly sensitive to toxic insults (Ritz et al., 2002). Further, immune dysregulation 

14 and related pathologies are known to be associated with pre/postnatal environmental 

15 exposures (Dietert et al., 2010). Ozone exposure is associated with developmental effects 

16 in several organ systems. These include neurobehavioral changes which could reflect 

17 O3’s effects on CNS plasticity or the hypothalamic-pituitary axis (Auten and Foster, In 

18 Press) (see Section 7.4.9). 

19 The majority of developmental effects due to O3 have been described for the respiratory 

20 system (see Section 7.2.3 and 7.4.8). Since its growth and development take place during 

21 both the prenatal and early postnatal periods, both prenatal and postnatal exposures to O3

22 have been studied. Maternal exposure to 0.4-1.2 ppm O3 during gestation resulted in 

23 developmental health effects in the RT of mice (Sharkhuu et al., 2011). Recent studies 

24 involving postnatal exposure to O3 have focused on differences between developing and 

25 adult animals in antioxidant defenses, respiratory physiology and sensitivity to cellular 

26 injury (Auten and Foster, In Press). In particular, one set of studies in infant rhesus 

27 monkeys exposed to 0.5 ppm O3 intermittently over 5 months has identified numerous 

28 O3-mediated perturbations in the developing lung and immune system (Plopper et al., 

29 2007). These investigations were prompted by the dramatic rise in the incidence of 

30 childhood asthma and focused on the possible role of O3 and allergens in promoting 

31 remodeling of the epithelial-mesenchymal trophic unit during postnatal development of 

32 the tracheobronchial airway wall. These and other studies have focused on mechanisms, 

33 such as lung structural changes, antigen sensitization, interaction with nitric oxide 

34 signaling, altered airway afferent innervation and loss of alveolar repair capacity, by 

35 which early O3 exposure could lead to asthma pathogenesis or exacerbations in later life 

36 (Auten and Foster, In Press). Further, a recent study demonstrated that maternal exposure 

37 to particulate matter (PM) resulted in augmented lung inflammation, airway epithelial 

38 mucous metaplasia and AHR in young mice exposed chronically and intermittently to 1 
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1 ppm O3 (Auten et al., 2009). Early life exposure to O3 has also been found to modulate 

2 pulmonary and systemic innate immunity later in life in the infant rhesus monkey model 

3 (Maniar-Hew et al., 2011). 

5.4.2.5 Attenuation of Responses

4 In responsive individuals, a striking degree of response attenuation occurred following 

5 repeated daily exposures to O3. Generally, the young O3 responder was no longer 

6 responsive on the fourth or fifth day of consecutive daily O3 exposure (200-500 ppb O3

7 for 2-4 hours) and required days to weeks of non-exposure in order for the subject to 

8 regain O3 responsiveness (Christian et al., 1998; Devlin et al., 1997; Linn et al., 1982a; 

9 Horvath et al., 1981; Hackney et al., 1977). This phenomena has been reported for both 

10 lung function and symptoms such as upper airway irritation, nonproductive cough, 

11 substernal discomfort and pain upon deep inspiration (Linn et al., 1982a; Horvath et al., 

12 1981; Hackney et al., 1977). Repeated daily exposures also led to an attenuation of the 

13 sRaw response in exercising human subjects exposed for 4 hours to 200 ppb O3

14 (Christian et al., 1998) and to a dampened AHR response compared with a single day 

15 exposure in exercising human subjects exposed for 2 hours to 400 ppb O3 (Dimeo et al., 

16 1981). However, one group reported persistent small airway dysfunction despite 

17 attenuation of the FEV1 response on the third day of consecutive O3 exposure (250 ppb, 

18 2 hours, with exercise) (Frank et al., 2001). 

19 Studies in rodents also indicated an attenuation of the physiologic response measured by 

20 breathing patterns and tidal volume following five consecutive days of exposure to 0.35-1 

21 ppm O3 for 2.25 hours (Tepper et al., 1989). Attenuation of O3-induced bradycardic 

22 responses, which also result from activation of neural reflexes, has been reported in 

23 rodents (0.5-0.6 ppm O3, 2-6 h/dy, 3-5 days (Hamade and Tankersley, 2009; Watkinson et 

24 al., 2001). 

25 Multi-day exposure to O3 has been found to decrease some markers of inflammation 

26 compared with a single day exposure (Christian et al., 1998; Devlin et al., 1997). For 

27 example, in human subjects exposed for 4 hours to 200 ppb O3 during moderate exercise, 

28 decreased numbers of BAL neutrophils and decreased levels of BALF fibronectin and IL29 6 were observed after 4 days of consecutive exposure compared with responses after 1 

30 day (Christian et al., 1998). Results indicated an attenuation of the inflammatory response 

31 in both proximal airways and distal lung. However markers of injury, such as lactate 

32 dehydrogenase (LDH) and protein in the BALF, were not attenuated in this study 

33 (Christian et al., 1998). Other investigators found that repeated O3 exposure (200 ppb O3

34 for 4 hours on 4 consecutive days with intermittent exercise) resulted in increased 
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1 numbers of neutrophils in bronchial mucosal biopsies despite decreased BAL 

2 neutrophilia (Jorres et al., 2000). Other markers of inflammation, including BALF protein 

3 and IL-6 remained elevated following the multi-day exposure (Jorres et al., 2000).

4 In rats, the increases in BALF levels of proteins, fibronectin, IL-6 and inflammatory cells 

5 observed after one day of exposure to 0.4 ppm O3 for 12 hours were no longer observed 

6 after 5 consecutive days of exposure (Van Bree et al., 2002). A separate study in rats 

7 exposed to 0.35-1 ppm O3 for 2.25 hours for 5 consecutive days demonstrated a lack of 

8 attenuation of the increase in BALF protein, persistence of macrophages in the 

9 centriacinar region and histological evidence of progressive tissue injury (Tepper et al., 

10 1989). Findings that injury, measured by BALF markers or by histopathology, persist in 

11 the absence of BAL neutrophila or pulmonary function decrements suggested that 

12 repeated exposure to O3 may have serious long-term consequences such as airway 

13 remodeling. In particular, the small airways were identified as a site where cumulative 

14 injury may occur (Frank et al., 2001). 

15 Some studies examined the recovery of responses which were attenuated by repeated O3

16 exposure. In a study of humans undergoing heavy intermittent exercise who were 

17 exposed for 2 hours to 400 ppb O3 for five consecutive days (Devlin et al., 1997), 

18 recovery of the inflammatory responses which were diminished by repeated exposure 

19 required 10-20 days following the exposure (Devlin et al., 1997). In an animal study 

20 conducted in parallel (Van Bree et al., 2002), full susceptibility to O3 challenge following 

21 exposure to O3 for five consecutive days required 15-20 days recovery. 

22 Several mechanisms have been postulated to explain the attenuation of responses 

23 observed in human subjects and animal models following repeated exposure to O3. First, 

24 the upregulation of antioxidant defenses (or conversely, a decrease in critical O3-reactive 

25 substrates) may protect against O3-mediated adverse effects. Increases in antioxidant 

26 content of the BALF have been demonstrated in rats exposed to 0.25 and 0.5 ppm O3 for 

27 several hours on consecutive days (Devlin et al., 1997; Wiester et al., 1996a; Tepper et 

28 al., 1989). Second, IL-6 was demonstrated to be an important mediator of attenuation in 

29 rats exposed to 0.5 ppm for 4 hours on two consecutive days (McKinney et al., 1998). 

30 Third, a protective role for increases in mucus producing cells and mucus concentrations 

31 in the airways has been proposed (Devlin et al., 1997). Fourth, epithelial hyperplasia or 

32 metaplasia may decrease susceptibility to subsequent O3 challenge (Carey et al., 2007; 

33 Harkema et al., 1987a; Harkema et al., 1987b). These morphologic changes have been 

34 observed in nasal and lower airways in monkeys exposed chronically to 0.15-0.5 ppm O3. 

35 Although there is some evidence to support these possibilities, there is no consensus on 

36 mechanisms underlying response attenuation. Recent studies demonstrating that O3
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1 activates TRP receptors suggest that modulation of TRP receptor number or sensitivity by 

2 repeated O3 exposures may also contribute to the attenuation of responses. 

5.4.2.6 Co-Exposures with Particulate Matter 

3 Numerous studies have investigated the effects of co-exposure to O3 and PM because of 

4 the prevalence of these pollutants in ambient air. Results are highly variable and depend 

5 on whether exposures are simultaneous or sequential, the type of PM employed and the 

6 endpoint examined. Additive and interactive effects have been demonstrated. For 

7 example, simultaneous exposure to O3 (120 ppb for 2 hours at rest) and concentrated 

8 ambient particles (CAPs) in human subjects resulted in a diminished systemic IL-6 

9 response compared with exposure to CAPs alone (Urch et al., 2010). However, exposure 

10 to O3 alone did not alter blood IL-6 levels (Urch et al., 2010). The authors provided 

11 evidence that O3 mediated a switch to shallow breathing which may have accounted for 

12 the observed antagonism (Urch et al., 2010). Further, simultaneous exposure to O3 (114 

13 ppb for 2 hours at rest) and CAPs but not exposure to either alone, resulted in increased 

14 diastolic blood pressure in human subjects (Fakhri et al., 2009). Mechanisms underlying 

15 this potentiation of response were not explored. In some strains of mice, pre-exposure to 

16 O3 (0.5 ppm for 2 hours) modulated the effects of carbon black PM on heart rate, HRV 

17 and breathing patterns (Hamade and Tankersley, 2009). Another recent study in mice 

18 demonstrated that treatment with carbon nanotubes followed 12 hours later by O3

19 exposure (0.5 ppm for 3 hours) resulted in a dampening of some of the pulmonary effects 

20 of carbon nanotubes measured as markers of inflammation and injury in the BALF (Han 

21 et al., 2008). Lastly, Harkema et al. (2005) found that epithelial and inflammatory 

22 responses in the airways of rats were enhanced by co-exposure to O3 (0.5 ppm for 3 days) 

23 and LPS (used as a model of biogenic PM) or to O3 (1 ppm for 2 days) and OVA (used as 

24 a model of an aeroallergen). Many of the demonstrated responses were more-than25 additive. Overall, these findings are hard to interpret but demonstrate the complexity of 

26 responses following combined exposure to PM and O3. 

5.4.2.7 Summary

27 Collectively, these older and more recent studies provide evidence for mechanisms which 

28 may underlie the variability in responsiveness seen among individuals (Figure 5-10). 

29 Certain functional genetic polymorphisms, pre-existing conditions and diseases, 

30 nutritional status, lifestage and co-exposures contribute to altered risk of O3-induced 

31 effects. Attenuation of responses may also be important, but it is incompletely 

32 understood, both in terms of the pathways involved and the resulting consequences. 
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Figure 5-10 Factors which contribute to the interindividual variability in 

responses resulting from inhalation exposure to 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. Still, care must be taken when comparing quantitative dose-response 

7 relationships in animal models to humans due to obvious interspecies differences. This 

8 section will describe basic concepts in species homology concerning both dose and 

9 response to O3 exposure. This will not be a quantitative extrapolation of doses where O3

10 effects have been observed. Overall, there have been few new publications examining 

11 interspecies differences in dosimetry and response to O3 since the last AQCD. These 

12 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 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 and scales according 

9 to body mass. The difference in the cross-sectional shape and size of the nasal passages 

10 affects bulk airflow patterns such that major airflow streams are created. 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, significant 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 could affect the amount of O3 uptake. 

30 As O3 absorption and activity relies on ELF antioxidant substances as described in 

31 Section 5.2.3, variability in antioxidant concentrations and metabolism between species 

32 may affect dose and O3-induced health outcomes. The thickness of the ELF in the TB 

33 airways varies among species. Mercer et al. (1992) found that the human ELF thickness 

34 in bronchi and bronchioles was 6.9 and 1.8 μm, respectively, compared to 2.6 and 1.9 μm 

35 for the same locations in the rat. Guinea pigs and mice have a lower basal activity of 

36 GSH transferase and GSH peroxidase, and lower α-TOH levels in the lung compared to 
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1 rats (Ichinose et al., 1988; Sagai et al., 1987). Nasal lavage fluid analysis shows that 

2 humans have a higher proportion of their nasal antioxidants as UA and low levels of AH2

3 whereas mice, rats, or guinea pigs have high levels of AH2 and undetectable levels of UA 

4 (Figure 5-11a). GSH is not detected in the nasal lavage of most of these species, but is 

5 present in monkey nasal lavage. Guinea pigs and rats have a higher antioxidant to protein 

6 ratio in nasal lavage and BALF than humans (Hatch, 1992). The BALF profile differs 

7 from the nasal lavage (Figure 5-11b). Humans have a higher proportion of GSH and less 

8 AH2 making up their BALF content compared to the guinea pigs and rats (Slade et al., 

9 1993; Hatch, 1992). Similar to the nose, rats have the highest antioxidant to protein mass 

10 ratio found in BALF (Slade et al., 1993). Antioxidant defenses also vary with age 

11 (Servais et al., 2005) and exposure history (Duan et al., 1996). Duan et al. (1996; 1993)

12 reported that differences in antioxidant levels between species and lung regions did not 

13 appear to be the primary factor in O3 induced tissue injury. However, a close association 

14 between site-specific O3 dose, the degree of epithelial injury, and reduced glutathione 

15 depletion was later revealed in monkeys (Plopper et al., 1998).

16 Humans and animals are similar in the pattern of regional O3 dose distribution. As 

17 discussed for humans in Section 5.2.2, O3 flux to the air-liquid interface of the ELF 

18 slowly decreases distally in the TB region and then rapidly decreases distally in the 

19 alveolar region (Miller et al., 1985). Modeled tissue dose in the human RT, representing 

20 O3 flux to the liquid-tissue interface, is very low in the trachea, increases to a maximum 

21 in the CAR, and then rapidly decreases distally in the alveolar region (Figure 5-12). 

22 Similar patterns of O3 tissue dose profiles normalized to inhaled O3 concentration were 

23 predicted for rat, guinea pig, and rabbit (Miller et al., 1988; Overton et al., 1987) (Figure 

24 5-12a). Overton et al. (1987) modeled rat and guinea pig O3 dose distribution and found 

25 that after comparing two different morphometrically based anatomical models for each 

26 species, considerable difference in predicted percent RT and alveolar region uptakes were 

27 observed. This was due to the variability between the two anatomical models in airway 

28 path distance to the first alveolated duct. As a result, the overall dose profile was similar 

29 between species however the O3 uptake efficiency varied due to RT size and path length 

30 (Section 5.2.2). A similar pattern of O3 dose distribution was measured in monkeys 

exposed to 0.4 and 1.0 ppm 18O3 (Plopper et al., 1998) (Figure 5-12b). Less 18 31 O was 

32 measured in the trachea, proximal bronchus, and distal bronchus than was observed in the 

33 respiratory bronchioles. Again indicating the highest concentration of O3 tissue dose to 

34 be 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 the parenchyma (i.e. alveolar region), 

36 mimicking the rapid decrease in tissue O3 dose predicted by models for the alveolar 

37 regions of humans and other animals. 
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a.

b.

Source: Adapted with permission from CRC Press, Inc. (Slade et al., 1993; Hatch, 1992)

Figure 5-11 Species comparison of antioxidant / protein ratios of: (a) nasal 

lavage fluid and, (b) bronchoalveolar lavage fluid.

Rat

Guinea pig

Human

0 200 400 600 800 1000

Antioxidant / Protein, nanomoles / gram

Ascorbic acid

Uric acid

Glutathione

Guinea pig

Rat

Human

0 50 100 150 200 250

Antioxidant / Protein, nanomoles / gram

Ascorbic acid

Uric acid

Glutathione
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Source: Panel (a) U.S. EPA (1996a) (b) Plopper et al. (1998)

Figure 5-12 Humans and animals are similar in the regional pattern of O3 tissue 

dose distribution. 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.

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 

5 (0.4 ppm) (Figure 5-13). It was necessary to expose resting rats to 2 ppm O3 to achieve 

the same BALF accumulation of 18 6 O reaction product that was observed in humans 

7 exposed to 400 ppb with intermittent heavy exercise (MV ~60 L/min). The concentration 

of 18 8 O reaction product in BALF paralleled the accumulation of BALF protein and 

9 cellular effects of the O3 exposure observed such that these responses to 2.0 ppm O3 were 

10 similar to those of the 400 ppb O3 in exercising humans. This suggests that animal data 

11 obtained in resting conditions would underestimate the dose to the RT and presumably 

12 the resultant risk of effect for humans.

Source: Hatch et al. (1994)

Figure 5-13 Oxygen-18 incorporation into different fractions of BALF from 

humans and rats exposed to 0.4 and 2.0 ppm 18O3.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.
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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 model examined how interspecies anatomical and physiological differences 

5 affect intra-airway O3 concentrations and the amount of gas absorbed. The model was 

6 designed as cylindrical tubes with constant volume and one-dimensional gas movement 

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 

12 surface area had a significant impact on model results. The model is limited in that it did 

13 not account for chemical reactions in the ELF or consider gas diffusion as a driving force 

14 for O3 transport. Also, the model was run at a respiratory rate of 16/min simulating a 

15 resting individual, however exercise may cause a further deviation from animal models as 

16 was seen in Hatch et al. (1994).

17 Overall, animal models exhibit qualitatively similar patterns of O3 net and tissue dose 

18 distribution with the largest tissue dose delivered to the CAR. However, due to 

19 anatomical and biochemical RT differences the absolute values of O3 dose delivered 

20 differs. Past results suggest that animal data obtained in resting conditions would 

21 underestimate the dose to the RT and presumably the resultant risk of effect for humans, 

22 especially for humans during exercise.

5.5.2 Homology of Response

23 Risk of heath effects from O3 varies between and within species, as well as between 

24 endpoints. Rodents appear to have a slightly higher tachypneic response to O3 and are 

25 less sensitive to changes in pulmonary function test than humans (U.S. EPA, 1996a). 

26 However, rats experience attenuation of pulmonary function and tachypneic ventilatory 

27 responses, similar to humans (Wiester et al., 1996a). Hatch et al. (1986) reported that 

28 guinea pigs were the most responsive to O3-induced inflammatory cell and protein influx. 

29 Rabbits were the least responsive and rats, hamsters, and mice were intermediate 

30 responders. Further analysis of this study by Miller et al. (1988) found that the protein 

31 levels in guinea pigs increased more rapidly with predicted pulmonary tissue dose than in 

32 rats and rabbits. Alveolar macrophages isolated from guinea pigs and humans mounted 

33 similar qualitative and quantitative cytokine responses to in vitro O3 (0.1-1.0 ppm for 60 

34 minutes) exposure (Arsalane et al., 1995). 
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1 Also, because of their higher body surface to volume ratio, rodents can rapidly lower 

2 body temperature during exposure leading to lowered O3 dose and toxicity (Watkinson et 

3 al., 2003; Iwasaki et al., 1998; Slade et al., 1997). In addition to lowering the O3 dose to 

4 the lungs, this hypothermic response may cause: (1) lower metabolic rate, (2) altered 

5 enzyme kinetics, and (3) altered membrane function. The thermoregulatory mechanisms 

6 also may affect disruption of heart rate which may lead to: (1) decreased cardiac output, 

7 (2) lowered blood pressure, and (3) decreased tissue perfusion (Watkinson et al., 2003). 

8 These responses have not been observed in humans except at very high exposures, thus 

9 further complicating extrapolation of effects from animals to humans.

10 Recently, the three-dimensional detail of the nasal passages of immature Rhesus macaque 

11 monkeys was analyzed for developing predictive dosimetry models and exposure-dose12 response relationships (Carey et al., 2007). In doing so the authors reported that the 

13 relative amounts of the five epithelial cell types in the nasal airways of monkeys remains 

14 consistent between infancy and adulthood (comparing to (Gross et al., 1987; Gross et al., 

15 1982). Ozone exposures (0.5 ppm, 8 h/day under acute [5 days] and episodic conditions 

16 [5 replicates of the acute paradigm spaced a week apart]) confirmed that the ciliated 

17 respiratory and transitional epithelium were the most sensitive cell types in the nasal 

18 cavity to O3 exposure, showing 50-80% decreases in epithelial thickness and epithelial 

19 cell volume. The character and location of nasal lesions resulting from O3 exposure were 

20 similar between adult and infant monkeys similarly exposed. However, infant monkeys 

21 did not undergo nasal airway epithelial remodeling or adaptation that occurs in adult 

22 animals and they may develop persistent necrotizing rhinitis following episodic longer23 term exposures.

24 To further understand the genetic basis for age-dependent differential response to O3, 

25 adult (15 week old) and neonatal (15-16 day old) mice from 8 genetically diverse strains 

26 were examined for O3-induced (0.8 ppm for 5 hours) pulmonary injury and lung 

27 inflammation (Vancza et al., 2009). Ozone exposure increased polymorphonuclear 

28 leukocytes (PMN) influx in all strains of neonatal mice tested, but significantly greater 

29 PMNs occurred in neonatal compared to adult mice for only some sensitive strains, 

30 suggesting a genetic background effect. This strain difference was not due to differences 

in delivered dose of O3 to the lung, evidenced by 18 31 O lung enrichment. The sensitivity of 

32 strains for O3-induced increases in BALF protein and PMNs was different for different 

33 strains of mice suggesting that genetic factors contributed to heightened responses. 

Interestingly, adult mice accumulated more than twice the levels of 18 34 O reaction product

35 of O3 than corresponding strain neonates. Thus, it appeared that the infant mice showed a 

36 two- to threefold higher response than the adults when expressed relative to the 

37 accumulated O3 reaction product in their lungs. The apparent decrease in delivered O3
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1 dose in neonates could be a result of a more rapid loss of body temperature in infant 

2 rodents incident to maternal separation and chamber air flow. 

3 Further, O3-induced injury and inflammation responses are variable between species. For 

4 example, Dormans et al. (1999) found that rats, mice, and guinea pigs all exhibited 

5 O3-induced (0.2 - 0.4 ppm for 3-56 days) inflammation; however, guinea pigs were the 

6 most sensitive with respect to alveolar macrophage elicitation and pulmonary cell density 

7 in the centriacinar region. Mice were the most sensitive to bronchiolar epithelial 

8 hypertrophy and biochemical changes (e.g. LDH, glutathione reductase, glucose-6-

9 phosphate dehydrogenase activity), and had the slowest recovery from O3 exposure. All 

10 species displayed increased collagen in the ductal septa and large lamellar bodies in Type 

11 II pneumocytes at the longest exposure and highest concentration; whereas this response 

12 occurred in the rat and guinea pig at lower O3 levels (0.2 ppm) as well. Overall, the 

13 authors rated mice as most sensitive, followed by guinea pigs, then rats (Dormans et al., 

14 1999). Rats were also less sensitive to epithelial necrosis and inflammatory responses 

15 from O3 (1.0 ppm for 8 hours) than monkeys and ferrets, which manifested a similar 

16 response (Sterner-Kock et al., 2000). These data suggest that ferrets may be a good 

17 animal model for O3-induced airway effects due to the similarities in pulmonary structure 

18 between primates and ferrets. However, this study provided no dose metric and, it is 

19 possible that some of these differences may be attributable to disparate total inhaled dose 

20 or local organ dose. 

5.5.3 Summary 

21 In summary, for all species there are limitations that must be considered when attempting 

22 to extrapolate to human O3 exposures. Rats required 4-5 times higher exposure to O3 to 

23 achieve comparable increases in BALF protein and PMNs to exercising humans. New 

24 studies have shown that varied O3 response in different mouse strains was not due to 

25 differences in delivered dose of O3 to the lung but more likely genetic sensitivity, and that 

26 infant mice show greater toxicity relative to their smaller lung dose than adults. Even 

27 though interspecies differences limit quantitative comparison between species, the acute 

28 and chronic functional responses of laboratory animals to O3 appear qualitatively 

29 homologous to those of the human making them a useful tool in determining mechanistic 

30 and cause-effect relationships with O3 exposure.
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5.6 Chapter Summary

1 Ozone is a highly reactive gas and a powerful oxidant with a short half-life. Both O3

2 uptake and responses are dependent upon the formation of secondary reaction products in 

3 the ELF; however more complex interactions occur. Uptake in humans at rest is 80-95% 

4 efficient and it is influenced by a number of factors including RT morphology, breathing 

5 route, frequency, and volume, physicochemical properties of the gas, physical processes 

6 of gas transport, as well as the physical and chemical properties of the ELF and tissue 

7 layers. The primary uptake site of O3 delivery to the lung epithelium is believed to be the 

8 CAR, however changes in a number of factors (e.g. physical activity) can alter the 

9 distribution of O3 uptake in the RT. Ozone uptake is chemical reaction-dependent and the 

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

11 underlying tissues and to prevent penetration of O3 distally into the RT. Still, reactions of 

12 O3 with soluble ELF components or plasma membranes result in distinct products, some 

13 of which are highly reactive and can injure and/or transmit signals to RT cells.

14 Thus, in addition to contributing to the driving force for O3 uptake, formation of 

15 secondary oxidation products initiates pathways that provide the mechanistic basis for 

16 health effects which are described in detail in Chapters 6 and 7 and which involve the RT 

17 as well as extrapulmonary systems. These pathways include activation of neural reflexes, 

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

19 bronchial smooth muscle, modification of innate and adaptive immunity, airways 

20 remodeling, and systemic inflammation and oxidative/nitrosative stress. With the 

21 exception of airways remodeling, these pathways have been demonstrated in both 

22 animals and human subjects in response to the inhalation of O3. 

23 Both dosimetric and mechanistic factors contribute to the understanding of 

24 interindividual variability in responses to O3. Interindividual variability is influenced by 

25 variability in RT volume and thus surface area, certain genetic polymorphisms, pre26 existing conditions and disease, nutritional status, lifestages, attenuation, and co27 exposures. Some of these factors are also influential in understanding species homology 

28 and sensitivity. Qualitatively, animal models exhibit similar patterns of O3 net and tissue 

29 dose distribution with the largest tissue dose delivered to the CAR. However, due to 

30 anatomical and biochemical RT differences, the absolute value of delivered O3 dose 

31 differs, with animal data obtained in resting conditions underestimating the dose to the 

32 RT and presumably the resultant risk of effect for humans, especially humans during 

33 exercise. Even though interspecies differences limit quantitative comparison between 

34 species, the acute and chronic functional responses of laboratory animals to O3 appear 

35 qualitatively homologous to those of the human making them a useful tool in determining 

36 mechanistic and cause-effect relationships with O3 exposure.
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5.7 References

Abraham, WM; Delehunt, JC; Yerger, L; Marchette, B; Oliver W, J, r. (1984). Changes in airway permeability and 

responsiveness after exposure to ozone. Environ Res 34: 110-119. 

Agarwal, A; Saleh, RA; Bedaiwy, MA. (2003). Role of reactive oxygen species in the pathophysiology of human 

reproduction. Fertil Steril 79: 829-843. 

Ahmad, S; Ahmad, A; McConville, G; Schneider, BK; Allen, CB; Manzer, R; Mason, RJ; White, CW. (2005). Lung 

epithelial cells release ATP during ozone exposure: Signaling for cell survival. Free Radic Biol Med 39: 

213-226. http://dx.doi.org/10.1016/j.freeradbiomed.2005.03.009.

Aibo, DI; Birmingham, NP; Lewandowski, R; Maddox, JF; Roth, RA; Ganey, PE; Wagner, JG; Harkema, JR.

(2010). Acute exposure to ozone exacerbates acetaminophen-induced liver injury in mice. Toxicol Sci 

115: 267-285. http://dx.doi.org/10.1093/toxsci/kfq034.

Al-Hegelan, M; Tighe, RM; Castillo, C; Hollingsworth, JW. (2011). Ambient ozone and pulmonary innate 

immunity. Immunol Res 49: 173-191. http://dx.doi.org/10.1007/s12026-010-8180-z.

Alexis, N; Urch, B; Tarlo, S; Corey, P; Pengelly, D; O'Byrne, P; Silverman, F. (2000). Cyclooxygenase 

metabolites play a different role in ozone-induced pulmonary function decline in asthmatics compared 

to normals. Inhal Toxicol 12: 1205-1224. 

Alexis, N; Soukup, J; Nierkens, S; Becker, S. (2001b). Association between airway hyperreactivity and bronchial 

macrophage dysfunction in individuals with mild asthma. Am J Physiol Lung Cell Mol Physiol 280: 

L369-L375. 

Alexis, NE; Zhou, H; Lay, JC; Harris, B; Hernandez, ML; Lu, TS; Bromberg, PA; Diaz-Sanchez, D; Devlin, RB; 

Kleeberger, SR; Peden, DB. (2009). The glutathione-S-transferase Mu 1 null genotype modulates 

ozone-induced airway inflammation in human subjects. J Allergy Clin Immunol 124: 1222-1228. 

http://dx.doi.org/10.1016/j.jaci.2009.07.036.

Alexis, NE; Lay, JC; Hazucha, M; Harris, B; Hernandez, ML; Bromberg, PA; Kehrl, H; Diaz-Sanchez, D; Kim, C; 

Devlin, RB; Peden, DB. (2010). Low-level ozone exposure induces airways inflammation and modifies 

cell surface phenotypes in healthy humans. Inhal Toxicol 22: 593-600. 

http://dx.doi.org/10.3109/08958371003596587.

Alfaro, MF; Putney, L; Tarkington, BK; Hatch, GE; Hyde, DM; Schelegle, ES. (2004). Effect of rapid shallow 

breathing on the distribution of 18O-labeled ozone reaction product in the respiratory tract of the rat. 

Inhal Toxicol 16: 77-85. 

Alfaro, MF; Walby, WF; Adams, WC; Schelegle, ES. (2007). Breath condensate levels of 8-isoprostane and 

leukotriene B4 after ozone inhalation are greater in sensitive versus nonsensitive subjects. Exp Lung 

Res 33: 115-133. http://dx.doi.org/779284696 [pii]10.1080/01902140701364367.

Araneda, S; Commin, L; Atlagich, M; Kitahama, K; Parraguez, VH; Pequignot, JM; Dalmaz, Y. (2008). VEGF 

overexpression in the astroglial cells of rat brainstem following ozone exposure. Neurotoxicology 29: 

920-927. http://dx.doi.org/10.1016/j.neuro.2008.09.006.

Aris, RM; Christian, D; Hearne, PQ; Kerr, K; Finkbeiner, WE; Balmes, JR. (1993). Ozone-induced airway 

inflammation in human subjects as determined by airway lavage and biopsy. Am J Respir Crit Care 

Med 148: 1363-1372. 

Arito, H; Uchiyama, I; Arakawa, H; Yokoyama, E. (1990). Ozone-induced bradycardia and arrhythmia and their 

relation to sleep-wakefulness in rats. Toxicol Lett 52: 169-178. http://dx.doi.org/10.1016/0378-

4274(90)90151-B.

Arsalane, K; Gosset, P; Vanhee, D; Voisin, C; Hamid, Q; Tonnel, A, -B; Wallaert, B. (1995). Ozone stimulates 

synthesis of inflammatory cytokines by alveolar macrophages in vitro. Am J Respir Cell Mol Biol 13: 60-

68. 

Asplund, PT; Ben-Jebria, A; Rigas, ML; Ultman, JS. (1996). Longitudinal distribution of ozone absorption in the 

lung: Effect of continuous inhalation exposure. Arch Environ Occup Health 51: 431-438. 

Auten, RL; Foster, WM. (In Press) Biochemical effects of ozone on asthma during postnatal development. 

Biochim Biophys Acta. http://dx.doi.org/10.1016/j.bbagen.2011.01.008.

Auten, RL; Potts, EN; Mason, SN; Fischer, B; Huang, Y; Foster, WM. (2009). Maternal exposure to particulate 

matter increases postnatal ozone-induced airway hyperreactivity in juvenile mice. Am J Respir Crit 

Care Med 180: 1218-1226. http://dx.doi.org/10.1164/rccm.200901-0116OC.
Draft – Do Not Cite or Quote 5-79 September 2011

Awasthi, YC; Yang, Y; Tiwari, NK; Patrick, B; Sharma, A; Li, J; Awasthi, S. (2004). Regulation of 4-

hydroxynonenal-mediated signaling by gluathione S-transferases. Free Radic Biol Med 37: 607-619. 

http://dx.doi.org/10.1016/j.freeradbiomed.2004.05.033.

Backus, GS; Howden, R; Fostel, J; Bauer, AK; Cho, HY; Marzec, J; Peden, DB; Kleeberger, SR. (2010). 

Protective role of interleukin-10 in ozone-induced pulmonary inflammation. Environ Health Perspect 

118: 1721-1727. http://dx.doi.org/10.1289/ehp.1002182.

Ballinger, CA; Cueto, R; Squadrito, G; Coffin, JF; Velsor, LW; Pryor, WA; Postlethwait, EM. (2005). Antioxidantmediated augmentation of ozone-induced membrane oxidation. Free Radic Biol Med 38: 515-526. 

http://dx.doi.org/10.1016/j.freeradbiomed.2004.11.009.

Balmes, JR; Chen, LL; Scannell, C; Tager, I; Christian, D; Hearne, PQ; Kelly, T; Aris, RM. (1996). Ozoneinduced decrements in FEV1 and FVC do not correlate with measures of inflammation. Am J Respir 

Crit Care Med 153: 904-909. 

Balmes, JR; Aris, RM; Chen, LL; Scannell, C; Tager, IB; Finkbeiner, W; Christian, D; Kelly, T; Hearne, PQ; 

Ferrando, R; Welch, B. (1997). Effects of ozone on normal and potentially sensitive human subjects. 

Part I: Airway inflammation and responsiveness to ozone in normal and asthmatic subjects. Boston, 

MA: Health Effects Institute.

Bang, S; Kim, KY; Yoo, S; Kim, YG; Hwang, SW. (2007). Transient receptor potential A1 mediates 

acetaldehyde-evoked pain sensation. Eur J Neurosci 26: 2516-2523. http://dx.doi.org/10.1111/j.1460-

9568.2007.05882.x.

Basha, MA; Gross, KB; Gwizdala, CJ; Haidar, AH; Popovich, J, Jr. (1994). Bronchoalveolar lavage neutrophilia 

in asthmatic and healthy volunteers after controlled exposure to ozone and filtered purified air. Chest 

106: 1757-1765. 

Bastacky, J; Lee, CY; Goerke, J; Koushafar, H; Yager, D; Kenaga, L; Speed, TP; Chen, Y; Clements, JA.

(1995). Alveolar lining layer is thin and continuous: Low-temperature scanning electron microscopy of 

rat lung. J Appl Physiol 79: 1615-1628. 

Bates, ML; Brenza, TM; Ben-Jebria, A; Bascom, R; Ultman, JS. (2009). Longitudinal distribution of ozone 

absorption in the lung: Comparison of cigarette smokers and nonsmokers. Toxicol Appl Pharmacol 236: 

270-275. 

Bauer, AK; Rondini, EA; Hummel, KA; Degraff, LM; Walker, C; Jedlicka, AE; Kleeberger, SR. (2011). 

Identification of candidate genes downstream of TLR4 signaling after ozone exposure in mice: A role 

for heat shock protein 70. Environ Health Perspect 119: 1091-1097. 

http://dx.doi.org/10.1289/ehp.1003326.

Beckett, WS; McDonnell, WF; Horstman, DH; House, DE. (1985). Role of the parasympathetic nervous system 

in acute lung response to ozone. J Appl Physiol 59: 1879-1885. 

Belvisi, MG; Stretton, CD; Verleden, GM; Ledingham, SJ; Yacoub, MH; Barnes, PJ. (1992). Inhibition of 

cholinergic neurotransmission in human airways by opioids. J Appl Physiol 72: 1096-1100. 

Bennett, WD; Hazucha, MJ; Folinsbee, LJ; Bromberg, PA; Kissling, GE; London, SJ. (2007). Acute pulmonary 

function response to ozone in young adults as a function of body mass index. Inhal Toxicol 19: 1147-

1154. http://dx.doi.org/10.1080/08958370701665475.

Bergamaschi, E; De Palma, G; Mozzoni, P; Vanni, S; Vettori, MV; Broeckaert, F; Bernard, A; Mutti, A. (2001). 

Polymorphism of quinone-metabolizing enzymes and susceptibility to ozone-induced acute effects. Am 

J Respir Crit Care Med 163: 1426-1431. 

Bhalla, DK; Gupta, SK. (2000). Lung injury, inflammation, and inflammatory stimuli in rats exposed to ozone. J 

Toxicol Environ Health 59: 211-228. 

Block, ML; Calderón-Garcidueñas, L. (2009). Air pollution: Mechanisms of neuroinflammation and CNS disease. 

Trends Neurosci 32: 506-516. http://dx.doi.org/10.1016/j.tins.2009.05.009.

Blomberg, A; Mudway, IS; Nordenhall, C; Hedenstrom, H; Kelly, FJ; Frew, AJ; Holgate, ST; Sandstrom, T.

(1999). Ozone-induced lung function decrements do not correlate with early airway inflammatory or 

antioxidant responses. Eur Respir J 13: 1418-1428. 

Bosson, J; Stenfors, N; Bucht, A; Helleday, R; Pourazar, J; Holgate, ST; Kelly, FJ; Sandstrom, T; Wilson, S; 

Frew, AJ; Blomberg, A. (2003). Ozone-induced bronchial epithelial cytokine expression differs between 

healthy and asthmatic subjects. Clin Exp Allergy 33: 777-782. 
Draft – Do Not Cite or Quote 5-80 September 2011

Bosson, J; Blomberg, A; Pourazar, J; Mudway, IS; Frew, AJ; Kelly, FJ; Sandström, T. (2009). Early suppression 

of NFkappaB and IL-8 in bronchial epithelium after ozone exposure in healthy human subjects. Inhal 

Toxicol 21: 913-919. http://dx.doi.org/10.1080/08958370802657389.

Broeckaert, F; Clippe, A; Wattiez, R; Falmagne, P; Bernard, A. (2003). Lung hyperpermeability, Clara-cell 

secretory potein (CC16), and susceptibility to ozone of five inbred strains of mice. Inhal Toxicol 15: 

1209-1230. 

Bush, ML; Asplund, PT; Miles, KA; Ben-Jebria, A; Ultman, JS. (1996). Longitudinal distribution of O3 absorption 

in the lung: gender differences and intersubject variability. J Appl Physiol 81: 1651-1657. 

Bush, ML; Zhang, W; Ben-Jebria, A; Ultman, JS. (2001). Longitudinal distribution of ozone and chlorine in the 

human respiratory tract: Simulation of nasal and oral breathing with the single-path diffusion model. 

Toxicol Appl Pharmacol 173: 137-145. http://dx.doi.org/10.1006/taap.2001.9182.

Caceres, AI; Brackmann, M; Elia, MD; Bessac, BF; del Camino, D; D'Amours, M; Witek, JS; Fanger, CM; 

Chong, JA; Hayward, NJ; Homer, RJ; Cohn, L; Huang, X; Moran, MM; Jordt, SE. (2009). A sensory 

neuronal ion channel essential for airway inflammation and hyperreactivity in asthma. PNAS 106: 9099-

9104. http://dx.doi.org/10.1073/pnas.0900591106.

Carey, SA; Minard, KR; Trease, LL; Wagner, JG; Garcia, GJ; Ballinger, CA; Kimbell, JS; Plopper, CG; Corley, 

RA; Postlethwait, EM; Harkema, JR; Einstein, DR. (2007). Three-dimensional mapping of ozoneinduced injury in the nasal airways of monkeys using magnetic resonance imaging and morphometric 

techniques. Toxicol Pathol 35: 27-40. http://dx.doi.org/10.1080/01926230601072343.

Chang, L, -Y; Huang, Y; Stockstill, BL; Graham, JA; Grose, EC; Menache, MG; Miller, FJ; Costa, DL; Crapo, JD.

(1992). Epithelial injury and interstitial fibrosis in the proximal alveolar regions of rats chronically 

exposed to a simulated pattern of urban ambient ozone. Toxicol Appl Pharmacol 115: 241-252. 

http://dx.doi.org/10.1016/0041-008X(92)90329-Q.

Chen, C; Arjomandi, M; Qin, H; Balmes, J; Tager, I; Holland, N. (2006a). Cytogenetic damage in buccal epithelia 

and peripheral lymphocytes of young healthy individuals exposed to ozone. Mutagenesis 21: 131-137. 

http://dx.doi.org/10.1093/mutage/gel007.

Chen, C; Arjomandi, M; Balmes, J; Tager, I; N, H. (2007). Effects of chronic and acute ozone exposure on lipid 

peroxidation and antioxidant capacity in healthy young adults. Environ Health Perspect 115: 1732-

1737. http://dx.doi.org/10.1289/ehp.10294.

Cho, H, -Y; Zhang, L, -Y; Kleeberger, SR. (2001). Ozone-induced lung inflammation and hyperreactivity are 

mediated via tumor necrosis factor-"alpha" receptors. Am J Physiol 280: L537-L546. 

Cho, HY; Hotchkiss, JA; Harkema, JR. (1999). Inflammatory and epithelial responses during the development of 

ozone-induced mucous cell metaplasia in the nasal epithelium of rats. Toxicol Sci 51: 135-145. 

Cho, HY; Kleeberger, SR. (2007). Genetic mechanisms of susceptibility to oxidative lung injury in mice. Free 

Radic Biol Med 42: 433-445. http://dx.doi.org/10.1016/j.freeradbiomed.2006.11.021.

Christian, DL; Chen, LL; Scannell, CH; Ferrando, RE; Welch, BS; Balmes, JR. (1998). Ozone-induced 

inflammation is attenuated with multiday exposure. Am J Respir Crit Care Med 158: 532-537. 

Chuang, GC; Yang, Z; Westbrook, DG; Pompilius, M; Ballinger, CA; White, RC; Krzywanski, DM; Postlethwait, 

EM; Ballinger, SW. (2009). Pulmonary ozone exposure induces vascular dysfunction, mitochondrial 

damage, and atherogenesis. Am J Physiol Lung Cell Mol Physiol 297: L209-L216. 

http://dx.doi.org/10.1152/ajplung.00102.2009.

Cohen-Hubal, EA; Kimbell, JS; Fedkiw, PS. (1996). Incorporation of nasal-lining mass-transfer resistance into a 

CFD model for prediction of ozone dosimetry in the upper respiratory tract. Inhal Toxicol 8: 831-857. 

Cole, MP; Freeman, BA. (2009). Promotion of cardiovascular disease by exposure to the air pollutant ozone. 

Am J Physiol Lung Cell Mol Physiol 297: L209-L216. 

Coleridge, HM; Coleridge, JCG; Ginzel, KH; Baker, DG; Banzett, RB; Morrison, MA. (1976). Stimulation of 

`irritant' receptors and afferent C-fibers in the lungs by prostaglandins. Nature 264: 451-453. 

Coleridge, JCG; Coleridge, HM; Schelegle, ES; Green, JF. (1993). Acute inhalation of ozone stimulates 

bronchial C-fibers and rapidly adapting receptors in dogs. J Appl Physiol 74: 2345-2352. 

Corradi, M; Alinovi, R; Goldoni, M; Vettori, M; Folesani, G; Mozzoni, P; Cavazzini, S; Bergamaschi, E; Rossi, L; 

Mutti, A. (2002). Biomarkers of oxidative stress after controlled human exposure to ozone. Toxicol Lett 

134: 219-225. 

Costa, DL; Schafrank, SN; Wehner, RW; Jellett, E. (1985). Alveolar permeability to protein in rats differentially 

susceptible to ozone. J Appl Toxicol 5: 182-186. http://dx.doi.org/10.1002/jat.2550050309.
Draft – Do Not Cite or Quote 5-81 September 2011

Cross, CE; Motchnik, PA; Bruener, BA; Jones, DA; Kaur, H; Ames, BN; Halliwell, B. (1992). Oxidative damage 

to plasma constituents by ozone. FEBS Lett 298: 269-272. http://dx.doi.org/10.1016/0014-

5793(92)80074-Q.

Dahl, AR. (1990). Dose concepts for inhaled vapors and gases. Toxicol Appl Pharmacol 103: 185-197. 

Dahl, M; Bauer, AK; Arredouani, M; Soininen, R; Tryggvason, K; Kleeberger, SR; Kobzik, L. (2007). Protection 

against inhaled oxidants through scavenging of oxidized lipids by macrophage receptors MARCO and 

SR-AI/II. J Clin Invest 117: 757-764. http://dx.doi.org/10.1172/JCI29968.

Darrow, LA; Klein, M; Flanders, WD; Waller, LA; Correa, A; Marcus, M; Mulholland, JA; Russell, AG; Tolbert, PE.

(2009). Ambient air pollution and preterm birth: A time-series analysis. Epidemiology 20: 689-698. 

Delaunois, A; Segura, P; Montano, LM; Vargas, MH; Ansay, M; Gustin, P. (1998). Comparison of ozone-induced 

effects on lung mechanics and hemodynamics in the rabbit. Toxicol Appl Pharmacol 150: 58-67. 

Dempsey, J, . A.; Johnson, B, . D.; Saupe, K, . W. (1990). Adaptations and limitations in the pulmonary system 

during exercise. Chest 97: 81S-87S. 

Dempsey, J, . A.; McKenzie, D, . C.; Haverkamp, H, . C.; Eldridge, M, . W. (2008). Update in the understanding 

of respiratory limitations to exercise performance in fit, active adults. Chest 134: 613-622. 

http://dx.doi.org/10.1378/chest.07-2730.

Devlin, RB; McDonnell, WF; Mann, R; Becker, S; House, DE; Schreinemachers, D; Koren, HS. (1991). 

Exposure of humans to ambient levels of ozone for 6.6 hours causes cellular and biochemical changes 

in the lung. Am J Respir Cell Mol Biol 4: 72-81. 

Devlin, RB; Folinsbee, LJ; Biscardi, F; Hatch, G; Becker, S; Madden, MC; Robbins, M; Koren, HS. (1997). 

Inflammation and cell damage induced by repeated exposure of humans to ozone. Inhal Toxicol 9: 211-

235. 

Diemer, T; Allen, JA; Hales, KH; Hales, DB. (2003). Reactive oxygen disrupts mitochondria in MA-10 tumor 

Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis. 

Endocrinology 144: 2882-2891. http://dx.doi.org/10.1210/en.2002-0090.

Dietert, RR; DeWitt, JC; Germolec, DR; Zelikoff, JT. (2010). Breaking patterns of environmentally influenced 

disease for health risk reduction: Immune perspectives. Environ Health Perspect 118: 1091-1099. 

http://dx.doi.org/10.1289/ehp.1001971.

Dimeo, MJ; Glenn, MG; Holtzman, MJ; Sheller, JR; Nadel, JA; Boushey, HA. (1981). Threshold concentration of 

ozone causing an increase in bronchial reactivity in humans and adaptation with repeated exposures. 

Am Rev Respir Dis 124: 245-248. 

Dormans, JAM, A; Van Bree, L; Boere, AJF; Marra, M; Rombout, PJA. (1999). Interspecies differences in time 

course of pulmonary toxicity following repeated exposure to ozone. Inhal Toxicol 11: 309-329. 

Dostert, C; Petrilli, V; Van Bruggen, R; Steele, C; Mossman, BT; Tschopp, J. (2008). Innate immune activation 

through Nalp3 inflammasome sensing of asbestos and silica. Science 320: 674-677. 

Drechsler-Parks, DM. (1995). The dose-response relationship in older men exposed to ozone. Exp Gerontol 30: 

65-75. 

Duan, X; Buckpitt, AR; Plopper, CG. (1993). Variation in antioxidant enzyme activities in anatomic 

subcompartments within rat and rhesus monkey lung. Toxicol Appl Pharmacol 123: 73-82. 

Duan, X; Buckpitt, AR; Pinkerton, KE; Ji, C; Plopper, CG. (1996). Ozone-induced alterations in glutathione in 

lung subcompartments of rats and monkeys. Am J Respir Cell Mol Biol 14: 70-75. 

Emmons, K; Foster, WM. (1991). Smoking cessation and acute airway response to ozone. Arch Environ Occup 

Health 46: 288-295. http://dx.doi.org/10.1080/00039896.1991.9934389.

Enami, S; Hoffmann, MR; Colussi, AJ. (2008a). Acidity enhances the formation of a persistent ozonide at 

aqueous ascorbate/ozone gas interfaces. PNAS 105: 7365-7369. 

http://dx.doi.org/10.1073/pnas.0710791105.

Enami, S; Hoffmann, MR; Colussi, AJ. (2008b). Ozonolysis of uric acid at the air/water interface. J Phys Chem 

B 112: 4153–4156. http://dx.doi.org/10.1021/jp712010k.

Enami, S; Hoffmann, MR; Colussi, AJ. (2009a). How phenol and alpha-tocopherol react with ambient ozone at 

gas/liquid interfaces. J Phys Chem A 113: 7002-7010. http://dx.doi.org/10.1021/jp901712k.

Enami, S; Hoffmann, MR; Colussi, AJ. (2009b). Ozone oxidizes glutathione to a sulfonic acid. Chem Res Toxicol 

22: 35-40. http://dx.doi.org/10.1021/tx800298j10.1021/tx800298j.
Draft – Do Not Cite or Quote 5-82 September 2011

Enami, S; Hoffmann, MR; Colussi, AJ. (2009c). Simultaneous detection of cysteine sulfenate, sulfinate, and 

sulfonate during cysteine interfacial ozonolysis. J Phys Chem B 113: 9356-9358. 

http://dx.doi.org/10.1021/jp904316n.

Engel, LA. (1985). Intraregional gas mixing and distribution. In Gas Mixing and Distribution in the Lung (pp. 287-

358). New York: Marcel Dekker.

Esther, CR; Peden, DB; Alexis, NE; Hernandez, ML. (2011). Airway purinergic responses in healthy, atopic 

nonasthmatic, and atopic asthmatic subjects exposed to ozone. Inhal Toxicol 23: 324-330. 

http://dx.doi.org/10.3109/08958378.2011.572096.

Fabbri, LM; Aizawa, H; O'Byrne, PM; Bethel, RA; Walters, EH; Holtzman, MJ; Nadel, JA. (1985). An antiinflammatory drug (BW755C) inhibits airway hyperresponsiveness induced by ozone in dogs. J Allergy 

Clin Immunol 76: 162-166. http://dx.doi.org/10.1016/0091-6749(85)90695-5.

Fakhri, AA; Ilic, LM; Wellenius, GA; Urch, B; Silverman, F; Gold, DR; Mittleman, MA. (2009). Autonomic effects 

of controlled fine particulate exposure in young healthy adults: Effect modification by ozone. Environ 

Health Perspect 117: 1287-1292. http://dx.doi.org/10.1289/ehp.0900541.

Feng, R; He, W; Ochi, H; Castranova, V. (2006). Ozone exposure impairs antigen-specific immunity but 

activates IL-7-induced proliferation of CD4-CD8- thymocytes in BALB/c mice. J Toxicol Environ Health 

A 69: 1511-1526. http://dx.doi.org/10.1080/15287390500468696.

Foster, WM; Stetkiewicz, PT. (1996). Regional clearance of solute from the respiratory epithelia: 18-20 h 

postexposure to ozone. J Appl Physiol 81: 1143-1149. 

Foster, WM; Freed, AN. (1999). Regional clearance of solute from peripheral airway epithelia: Recovery after 

sublobar exposure to ozone. J Appl Physiol 86: 641-646. 

Foster, WM; Brown, RH; Macri, K; Mitchell, CS. (2000). Bronchial reactivity of healthy subjects: 18-20 h 

postexposure to ozone. J Appl Physiol 89: 1804-1810. 

Frampton, MW; Morrow, PE; Torres, A; Voter, KZ; Whitin, JC; Cox, C; Speers, DM; Tsai, Y; Utell, MJ. (1997a). 

Effects of ozone on normal and potentially sensitive human subjects Part II: airway inflammation and 

responsiveness to ozone in nonsmokers and smokers. Boston, MA: Health Effects Institute.

Frampton, MW; Morrow, PE; Torres, A; Cox, C; Voter, KZ; Utell, MJ; Gibb, FR; Speers, DM. (1997b). Ozone 

responsiveness in smokers and nonsmokers. Am J Respir Crit Care Med 155: 116-121. 

Frampton, MW; Pryor, WA; Cueto, R; Cox, C; Morrow, PE; Utell, MJ. (1999). Ozone exposure increases 

aldehydes in epithelial lining fluid in human lung. Am J Respir Crit Care Med 159: 1134-1137. 

Frank, R; Liu, MC; Spannhake, EW; Mlynarek, S; Macri, K; Weinmann, GG. (2001). Repetitive ozone exposure 

of young adults: Evidence of persistent small airway dysfunction. Am J Respir Crit Care Med 164: 

1253-1260. 

Freed, AN; Chou, CL; Fuller, SD; Croxton, TL. (1996). Ozone-induced vagal reflex modulates airways reactivity 

in rabbits. Respir Physiol Neurobiol 105: 95-102. 

Freed, AN; Cueto, R; Pryor, WA. (1999). Antioxidant transport modulates peripheral airway reactivity and 

inflammation during ozone exposure. J Appl Physiol 87: 1595-1603. 

Fujita, M; Sasayama, S; Ohno, A; Nakajima, H; Asanoi, H. (1987). Importance of angina for development of 

collateral circulation. Heart 57: 139-143. 

Gackière, F; Saliba, L; Baude, A; Bosler, O; Strube, C. (2011). Ozone inhalation activates stress-responsive 

regions of the CNS. J Neurochem 117: 961-972. http://dx.doi.org/10.1111/j.1471-4159.2011.07267.x.

Gao, X; Raghavamenon, AC; D'Auvergne, O; Uppu, RM. (2009b). Cholesterol secoaldehyde induces apoptosis 

in J774 macrophages via mitochondrial pathway but not involving reactive oxygen species as 

mediators. Biochem Biophys Res Commun 389: 382-387. http://dx.doi.org/10.1016/j.bbrc.2009.09.005.

Garantziotis, S; Li, Z; Potts, EN; Kimata, K; Zhuo, L; Morgan, DL; Savani, RC; Noble, PW; Foster, WM; 

Schwartz, DA; Hollingsworth, JW. (2009). Hyaluronan mediates ozone-induced airway 

hyperresponsiveness in mice. J Biol Chem 284: 11309-11317. 

http://dx.doi.org/10.1074/jbc.M802400200.

Garantziotis, S; Li, Z; Potts, EN; Lindsey, JY; Stober, VP; Polosukhin, VV; Blackwell, TS; Schwartz, DA; Foster, 

WM; Hollingsworth, JW. (2010). TLR4 is necessary for hyaluronan-mediated airway 

hyperresponsiveness after ozone inhalation. Am J Respir Crit Care Med 181: 666-675. 

http://dx.doi.org/10.1164/rccm.200903-0381OC.

Gerrity, TR; Weaver, RA; Berntsen, J; House, DE; O'Neil, JJ. (1988). Extrathoracic and intrathoracic removal of 

O3 in tidal-breathing humans. J Appl Physiol 65: 393-400. 
Draft – Do Not Cite or Quote 5-83 September 2011

Gerrity, TR; McDonnell, WF; House, DE. (1994). The relationship between delivered ozone dose and functional 

responses in humans. Toxicol Appl Pharmacol 124: 275-283. 

Gerrity, TR; Biscardi, F; Strong, A; Garlington, AR; Brown, JS; Bromberg, PA. (1995). Bronchoscopic 

determination of ozone uptake in humans. J Appl Physiol 79: 852-860. 

Giamalva, D; Church, DF; Pryor, WA. (1985). A comparison of the rates of ozonation of biological antioxidants 

and oleate and linoleate esters. Biochem Biophys Res Commun 133: 773-779. 

Gong, H, Jr; Wong, R; Sarma, RJ; Linn, WS; Sullivan, ED; Shamoo, DA; Anderson, KR; Prasad, SB. (1998). 

Cardiovascular effects of ozone exposure in human volunteers. Am J Respir Crit Care Med 158: 538-

546. 

Graham, DE; Koren, HS. (1990). Biomarkers of inflammation in ozone-exposed humans: Comparison of the 

nasal and bronchoalveolar lavage. Am J Respir Crit Care Med 142: 152-156. 

Gross, EA; Swenberg, JA; Fields, S; Popp, JA. (1982). Comparative morphometry of the nasal cavity in rats and 

mice. J Anat 135: 83-88. 

Gross, EA; Starr, TB; Randall, HW; Morgan, KT. (1987). Morphometric analysis of the primate nasal cavity 

[Abstract]. Toxicologist 7: 193. 

Guevara-Guzmán, R; Arriaga, V; Kendrick, KM; Bernal, C; Vega, X; Mercado-Gómez, OF; Rivas-Arancibia, S.

(2009). Estradiol prevents ozone-induced increases in brain lipid peroxidation and impaired social 

recognition memory in female rats. Neuroscience 159: 940-950. 

http://dx.doi.org/10.1016/j.neuroscience.2009.01.047.

Gunnison, AF; Hatch, GE; Crissman, K; Bowers, A. (1996). Comparative sensitivity of lactating and virgin 

female rats to ozone-induced pulmonary inflammation. Inhal Toxicol 8: 607-623. 

Gunnison, AF; Hatch, GE. (1999). O3-induced inflammation in prepregnant, pregnant, and lactating rats 

correlates with O3 dose estimated by 18O. Am J Physiol 276: L332-L340. 

Hackney, JD; Linn, WS; Mohler, JG; Collier, CR. (1977). Adaptation to short-term respiratory effects of ozone in 

men exposed repeatedly. J Appl Physiol 43: 82-85. 

Hamade, AK; Tankersley, CG. (2009). Interstrain variation in cardiac and respiratory adaptation to repeated 

ozone and particulate matter exposures. Am J Physiol Regul Integr Comp Physiol 296: R1202-R1215. 

http://dx.doi.org/10.1152/ajpregu.90808.2008.

Hamade, AK; Misra, V; Rabold, R; Tankersley, CG. (2010). Age-related changes in cardiac and respiratory 

adaptation to acute ozone and carbon black exposures: Interstrain variation in mice. Inhal Toxicol 22: 

84-94. http://dx.doi.org/10.3109/08958378.2010.503974.

Hamilton, RF; Li, L; Eschenbacher, WL; Szweda, L; Holian, A. (1998). Potential involvement of 4-

hydroxynonenal in the response of human lung cells to ozone. Am J Physiol 274: L8-L16. 

Han, SG; Andrews, R; Gairola, CG; Bhalla, DK. (2008). Acute pulmonary effects of combined exposure to 

carbon nanotubes and ozone in mice. Inhal Toxicol 20: 391-398. 

http://dx.doi.org/10.1080/08958370801904014.

Hansen, CA; Barnett, AG; Pritchard, G. (2008). The effect of ambient air pollution during early pregnancy on 

fetal ultrasonic measurements during mid-pregnancy. Environ Health Perspect 116: 362-369. 

Haque, R; Umstead, TM; Ponnuru, P; Guo, X; Hawgood, S; Phelps, DS; Floros, J. (2007). Role of surfactant 

protein-A (SP-A) in lung injury in response to acute ozone exposure of SP-A deficient mice. Toxicol 

Appl Pharmacol 220: 72-82. http://dx.doi.org/10.1016/j.taap.2006.12.017.

Haque, R; Umstead, TM; Freeman, WM; Floros, J; Phelps, DS. (2009). The impact of surfactant protein-A on 

ozone-induced changes in the mouse bronchoalveolar lavage proteome. Proteome Science 7: 12. 

http://dx.doi.org/10.1186/1477-5956-7-12.

Harkema, JR; Plopper, CG; Hyde, DM; StGeorge, JA; Dungworth, DL. (1987a). Effects of an ambient level of 

ozone on primate nasal epithelial mucosubstances: quantitative histochemistry. Am J Pathol 127: 90-

96. 

Harkema, JR; Plopper, CG; Hyde, DM; StGeorge, JA; Wilson, DW; Dungworth, DL. (1987b). Response of the 

macaque nasal epithelium to ambient levels of ozone: A morphologic and morphometric study of the 

transitional and respiratory epithelium. Am J Pathol 128: 29-44. 

Harkema, JR; Hotchkiss, JA; Barr, EB; Bennett, CB; Gallup, M; Lee, JK; Basbaum, C. (1999). Long-lasting 

effects of chronic ozone exposure on rat nasal epithelium. Am J Respir Cell Mol Biol 20: 517-529. 
Draft – Do Not Cite or Quote 5-84 September 2011

Harkema, JR; Wagner, JG. (2005). Epithelial and inflammatory responses in the airways of laboratory rats 

coexposed to ozone and biogenic substances: Enhancement of toxicant-induced airway injury. Exp 

Toxicol Pathol 57: 129-141. http://dx.doi.org/10.1016/j.etp.2005.05.013.

Hatch, GE; Slade, R; Stead, AG; Graham, JA. (1986). Species comparison of acute inhalation toxicity of ozone 

and phosgene. J Toxicol Environ Health 19: 43-53. http://dx.doi.org/10.1080/15287398609530905.

Hatch, GE; Wiester, MJ; Overton, JH, Jr; Aissa, M. (1989). Respiratory tract dosimetry of [18]O-labeled ozone in 

rats: Implications for a rat-human extrapolation of ozone dose. In Atmospheric ozone research and its 

policy implications (Vol. 35, pp. 553-560). Nijmegend, the Netherlands: Elsevier Science Publishers 

B.V.

Hatch, GE. (1992). Comparative biochemistry of airway lining fluid. In RA Parent (Ed.), Comparative biology of 

the normal lung (Vol. 1, pp. 617-632). Boca Raton, FL: CRC Press, Inc.

Hatch, GE; Slade, R; Harris, LP; McDonnell, WF; Devlin, RB; Koren, HS; Costa, DL; McKee, J. (1994). Ozone 

dose and effect in humans and rats: A comparison using oxygen-18 labeling and bronchoalveolar 

lavage. Am J Respir Crit Care Med 150: 676-683. 

Hazbun, ME; Hamilton, R; Holian, A; Eschenbacher, WL. (1993). Ozone-induced increases in substance P and 

8-epi-prostaglandin F2 alpha in the airways of human subjects. Am J Respir Cell Mol Biol 9: 568-572. 

http://dx.doi.org/10.1165/ajrcmb/9.5.568.

Hazucha, MJ; Bates, DV; Bromberg, PA. (1989). Mechanism of action of ozone on the human lung. J Appl 

Physiol 67: 1535-1541. 

Hazucha, MJ; Madden, M; Pape, G; Becker, S; Devlin, R; Koren, HS; Kehrl, H; Bromberg, PA. (1996). Effects of 

cyclo-oxygenase inhibition on ozone-induced respiratory inflammation and lung function changes. Eur J 

Appl Physiol 73: 17-27. 

Hazucha, MJ; Folinsbee, LJ; Bromberg, PA. (2003). Distribution and reproducibility of spirometric response to 

ozone by gender and age. J Appl Physiol 95: 1917-1925. 

Hernandez, ML; Lay, JC; Harris, B; Esther, CR; Brickey, WJ; Bromberg, PA; Diaz-Sanchez, D; Devlin, RB; 

Kleeberger, SR; Alexis, NE; Peden, DB. (2010). Atopic asthmatic subjects but not atopic subjects 

without asthma have enhanced inflammatory response to ozone. J Allergy Clin Immunol 126: 537-544. 

http://dx.doi.org/10.1016/j.jaci.2010.06.043.

Hoigné, J; Bader, H. (1983). Rate constants of reactions of ozone with organic and inorganic compounds in 

water - II: Dissociating organic compounds. Water Res 17: 185-194. http://dx.doi.org/10.1016/0043-

1354(83)90099-4.

Hollingsworth, JW; Maruoka, S; Li, Z; Potts, EN; Brass, DM; Garantziotis, S; Fong, A; Foster, WM; Schwartz, 

DA. (2007). Ambient ozone primes pulmonary innate immunity in mice. J Immunol 179: 4367-4375. 

Hollingsworth, JW; Free, ME; Li, Z; Andrews, LN; Nakano, H; Cook, DN. (2010). Ozone activates pulmonary 

dendritic cells and promotes allergic sensitization through a Toll-like receptor 4-dependent mechanism. 

J Allergy Clin Immunol 125: 1167-1170. 

Hollingsworth, JW, II; Cook, DN; Brass, DM; Walker, JKL; Morgan, DL; Foster, WM; Schwartz, DA. (2004). The 

role of Toll-like receptor 4 in environmental airway injury in mice. Am J Respir Crit Care Med 170: 126-

132. 

Holtzman, MJ; Cunningham, JH; Sheller, JR; Irsigler, GB; Nadel, JA; Boushey, HA. (1979). Effect of ozone on 

bronchial reactivity in atopic and nonatopic subjects. Am Rev Respir Dis 120: 1059-1067. 

Holtzman, MJ; Fabbri, LM; O'Byrne, PM; Gold, BD; Aizawa, H; Walters, EH; Alpert, SE; Nadel, JA. (1983). 

Importance of airway inflammation for hyperresponsiveness induced by ozone. Am Rev Respir Dis 127: 

686-690. 

Holz, O; Jorres, RA; Timm, P; Mucke, M; Richter, K; Koschyk, S; Magnussen, H. (1999). Ozone-induced airway 

inflammatory changes differ between individuals and are reproducible. Am J Respir Crit Care Med 159: 

776-784. 

Horstman, DH; Ball, BA; Brown, J; Gerrity, T; Folinsbee, LJ. (1995). Comparison of pulmonary responses of 

asthmatic and nonasthmatic subjects performing light exercise while exposed to a low level of ozone. 

Toxicol Ind Health 11: 369-385. 

Horvath, SM; Gliner, JA; Folinsbee, LJ. (1981). Adaptation to ozone: Duration of effect. Am Rev Respir Dis 123: 

496-499. 

Hotchkiss, JA; Harkema, JR; Henderson, RF. (1991). Effect of cumulative ozone exposure on ozone-induced 

nasal epithelial hyperplasia and secretory metaplasia in rats. Exp Lung Res 15: 589-600. 
Draft – Do Not Cite or Quote 5-85 September 2011

Hu, PC; Miller, FJ; Daniels, MJ; Hatch, G. (1982). Protein accumulation in lung lavage fluid following ozone 

exposure. Environ Res 29: 377-388. http://dx.doi.org/10.1016/0013-9351(82)90039-1.

Hu, S, -C; Ben-Jebria, A; Ultman, JS. (1994). Longitudinal distribution of ozone absorption in the lung: Effects of 

respiratory flow. J Appl Physiol 77: 574-583. 

Hu, SC; Ben-Jebria, A; Ultman, JS. (1992). Longitudinal distribution of ozone absorption in the lung: Quiet 

respiration in healthy subjects. J Appl Physiol 73: 1655-1667. 

Ichinose, T; Arakawa, K; Shimojo, N; Sagai, M. (1988). Biochemical effects of combined gases of nitrogen 

dioxide and ozone: II Species differences in lipid peroxides and antioxidative protective enzymes in the 

lungs. Toxicol Lett 42: 167-176. 

ICRP. (International Commission on Radiological Protection). (1994). Human respiratory tract model for 

radiological protection: A report of a task group of the International Commission on Radiological 

Protection. Ann ICRP 24: 1-482. 

Ignatenko, AV; Cherenkevich, SN. (1985). Reactivity of amino-acids and proteins in reactions with ozone. Kinet 

Catal 26: 1145-1148. 

Islam, T; McConnell, R; Gauderman, WJ; Avol, E; Peters, JM; Gilliland, FD. (2008). Ozone, oxidant defense 

genes and risk of asthma during adolescence. Am J Respir Crit Care Med 177: 388-395. 

http://dx.doi.org/10.1164/rccm.200706-863OC.

Iwasaki, T; Takahashi, M; Saito, H; Arito, H. (1998). Adaptation of extrapulmonary responses to ozone exposure 

in conscious rats. Ind Health 36: 57-60. 

Jalaludin, B; Mannes, T; Morgan, G; Lincoln, D; Sheppeard, V; Corbett, S. (2007). Impact of ambient air 

pollution on gestational age is modified by season in Sydney, Australia. Environ Health 6: 16. 

Jiang, D; Liang, J; Fan, J; Yu, S; Chen, S; Luo, Y; Prestwich, GD; Mascarenhas, MM; Garg, HG; Quinn, DA; 

Homer, RJ; Goldstein, DR; Bucala, R; Lee, PJ; Medzhitov, R; Noble, PW. (2005). Regulation of lung 

injury and repair by Toll-like receptors and hyaluronan. Nat Med 11: 1173-1179. 

http://dx.doi.org/10.1038/nm1315.

Joad, JP; Kott, KS; Bric, JM. (1996). The local C-fiber contribution to ozone-induced effects on the isolated 

guinea pig lung. Toxicol Appl Pharmacol 141: 561-567. 

Johansson, E; Wesselkamper, SC; Shertzer, HG; Leikauf, GD; Dalton, TP; Chen, Y. (2010). Glutathione 

deficient C57BL/6J mice are not sensitized to ozone-induced lung injury. Biochem Biophys Res 

Commun 396: 407-412. http://dx.doi.org/10.1016/j.bbrc.2010.04.105.

Johnston, RA; Mizgerd, JP; Shore, SA. (2005a). CXCR2 is essential for maximal neutrophil recruitment and 

methacholine responsiveness after ozone exposure. Am J Physiol Lung Cell Mol Physiol 288: L61-L67. 

http://dx.doi.org/10.1152/ajplung.00101.2004 00101.2004.

Johnston, RA; Schwartzman, IN; Flynt, L; Shore, SA. (2005b). Role of interleukin-6 in murine airway responses 

to ozone. Am J Physiol Lung Cell Mol Physiol 288: L390-L397. 

http://dx.doi.org/10.1152/ajplung.00007.2004.

Johnston, RA; Theman, TA; Lu, FL; Terry, RD; Williams, ES; Shore, SA. (2008). Diet-induced obesity causes 

innate airway hyperresponsiveness to methacholine and enhances ozone-induced pulmonary 

inflammation. J Appl Psychol 104: 1727-1735. http://dx.doi.org/10.1152/japplphysiol.00075.2008.

Jorres, R; Nowak, D; Magnussen, H; Speckin, P; Koschyk, S. (1996). The effect of ozone exposure on allergen 

responsiveness in subjects with asthma or rhinitis. Am J Respir Crit Care Med 153: 56-64. 

Jorres, RA; Holz, O; Zachgo, W; Timm, P; Koschyk, S; Muller, B; Grimminger, F; Seeger, W; Kelly, FJ; Dunster, 

C; Frischer, T; Lubec, G; Waschewski, M; Niendorf, A; Magnussen, H. (2000). The effect of repeated 

ozone exposures on inflammatory markers in bronchoalveolar lavage fluid and mucosal biopsies. Am J 

Respir Crit Care Med 161: 1855-1861. 

Kabel, JR; Ben-Jebria, A; Ultman, JS. (1994). Longitudinal distribution of ozone absorption in the lung: 

Comparison of nasal and oral quiet breathing. J Appl Physiol 77: 2584-2592. 

Kafoury, RM; Pryor, WA; Squadrito, GL; Salgo, MG; Zou, X; Friedman, M. (1998). Lipid ozonation products 

activate phospholipases A2, C, and D. Toxicol Appl Pharmacol 150: 338-349. 

Kannan, S; Misra, DP; Dvonch, T; Krishnakumar, A. (2006). Exposures to airborne particulate matter and 

adverse perinatal outcomes: A biologically plausible mechanistic framework for exploring potential 

effect modification by nutrition. Environ Health Perspect 114: 1636-1642. 

Kanofsky, JR; Sima, PD. (1995). Reactive absorption of ozone by aqueous biomolecule solutions: Implications 

for the role of sulfhydryl compounds as targets for ozone. Arch Biochem Biophys 316: 52-62. 
Draft – Do Not Cite or Quote 5-86 September 2011

Kari, F; Hatch, G; Slade, R; Crissman, K; Simeonova, PP; Luster, M. (1997). Dietary restriction mitigates ozoneinduced lung inflammation in rats: A role for endogenous antioxidants. Am J Respir Cell Mol Biol 17: 

740-747. 

Katre, A; Ballinger, C; Akhter, H; Fanucchi, M; Kim, DK; Postlethwait, E; Liu, RM. (2011). Increased transforming 

growth factor beta 1 expression mediates ozone-induced airway fibrosis in mice. Inhal Toxicol 23: 486-

494. http://dx.doi.org/10.3109/08958378.2011.584919.

Kavlock, R; Daston, G; Grabowski, CT. (1979). Studies on the developmental toxicity of ozone. I. Prenatal 

effects. Toxicol Appl Pharmacol 48: 19-28. http://dx.doi.org/10.1016/S0041-008X(79)80004-6.

Kehrl, HR; Vincent, LM; Kowalsky, RJ; Horstman, DH; O'Neil, JJ; McCartney, WH; Bromberg, PA. (1987). 

Ozone exposure increases respiratory epithelial permeability in humans. Am Rev Respir Dis 135: 1124-

1128. 

Kehrl, HR; Peden, DB; Ball, BA; Folinsbee, LJ; Horstman, DH. (1999). Increased specific airway reactivity of 

persons with mild allergic asthma after 7.6 hours of exposure to 0.16 ppm ozone. J Allergy Clin 

Immunol 104: 1198-1204. 

Kermani, S; Ben-Jebria, A; Ultman, JS. (2006). Kinetics of ozone reaction with uric acid, ascorbic acid, and 

glutathione at physiologically relevant conditions. Arch Biochem Biophys 451: 8-16. 

http://dx.doi.org/10.1016/j.abb.2006.04.015.

Kim, CS; Alexis, NE; Rappold, AG; Kehrl, H; Hazucha, MJ; Lay, JC; Schmitt, MT; Case, M; Devlin, RB; Peden, 

DB; Diaz-Sanchez, D. (2011). Lung function and inflammatory responses in healthy young adults 

exposed to 0.06 ppm ozone for 6.6 hours. Am J Respir Crit Care Med 183: 1215-1221. 

http://dx.doi.org/10.1164/rccm.201011-1813OC.

Kleeberger, SR; Seiden, JE; Levitt, RC; Zhang, L, -Y. (1993). Mast cells modulate acute ozone-induced 

inflammation of the murine lung. Am J Respir Crit Care Med 148: 1284-1291. 

Kleeberger, SR; Levitt, RC; Zhang, L, -Y; Longphre, M; Harkema, J; Jedlicka, A; Eleff, SM; DiSilvestre, D; 

Holroyd, KJ. (1997). Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred 

mice. Nat Genet 17: 475-478. 

Kleeberger, SR; Reddy, S; Zhang, L, -Y; Jedlicka, AE. (2000). Genetic susceptibility to ozone-induced lung 

hyperpermeability: Role of toll-like receptor 4. Am J Respir Cell Mol Biol 22: 620-627. 

Kleeberger, SR; Reddy, SP; Zhang, L, -Y; Cho, H, -Y; Jedlicka, AE. (2001). Toll-like receptor 4 mediates ozoneinduced murine lung hyperpermeability via inducible nitric oxide synthase. Am J Physiol 280: L326-

L333. 

Kodavanti, UP; Costa, DL; Dreher, KL; Crissman, K; Hatch, GE. (1995). Ozone-induced tissue injury and 

changes in antioxidant homeostasis in normal and ascorbate-deficient guinea pigs. Biochem 

Pharmacol 50: 243-251. http://dx.doi.org/10.1016/0006-2952(95)00122-G.

Kodavanti, UP; Thomas, R; Ledbetter, AD; Schladweiler, MC; Shannahan, JH; Wallenborn, JG; Lund, AK; 

Campen, MJ; Butler, EO; Gottipolu, RR; Nyska, A; Richards, JE; Andrews, D; Jaskot, RH; McKee, J; 

Kotha, SR; Patel, RB; Parianandi, NL. (2011). Vascular and cardiac impairments in rats Inhaling ozone 

and diesel exhaust particles. Environ Health Perspect 119: 312-318. 

http://dx.doi.org/10.1289/ehp.1002386.

Koenig, JQ; Covert, DS; Marshall, SG; Van Belle, G; Pierson, WE. (1987). The effects of ozone and nitrogen 

dioxide on pulmonary function in healthy and in asthmatic adolescents. Am J Respir Crit Care Med 136: 

1152-1157. 

Kreit, JW; Gross, KB; Moore, TB; Lorenzen, TJ; D'Arcy, J; Eschenbacher, WL. (1989). Ozone-induced changes 

in pulmonary function and bronchial responsiveness in asthmatics. J Appl Physiol 66: 217-222. 

Krishna, MT; Springall, D; Meng, Q, -H; Withers, N; Macleod, D; Biscione, G; Frew, A; Polak, J; Holgate, S.

(1997). Effects of ozone on epithelium and sensory nerves in the bronchial mucosa of healthy humans. 

Am J Respir Crit Care Med 156: 943-950. 

Larsen, ST; Matsubara, S; McConville, G; Poulsen, SS; Gelfand, EW. (2010). Ozone increases airway 

hyperreactivity and mucus hyperproduction in mice previously exposed to allergen. J Toxicol Environ 

Health A 73: 738-747. http://dx.doi.org/10.1080/15287391003614034.

Lay, JC; Alexis, NE; Kleeberger, SR; Roubey, RA; Harris, BD; Bromberg, PA; Hazucha, MJ; Devlin, RB; Peden, 

DB. (2007). Ozone enhances markers of innate immunity and antigen presentation on airway 

monocytes in healthy individuals. J Allergy Clin Immunol 120: 719-722. 

http://dx.doi.org/10.1016/j.jaci.2007.05.005.
Draft – Do Not Cite or Quote 5-87 September 2011

Li, YF; Gauderman, WJ; Avol, E; Dubeau, L; Gilliland, FD. (2006d). Associations of tumor necrosis factor G308A with childhood asthma and wheezing. Am J Respir Crit Care Med 173: 970-976. 

http://dx.doi.org/10.1164/rccm.200508-1256OC.

Li, Z; Potts, EN; Piantadosi, CA; Foster, WM; Hollingsworth, JW. (2010). Hyaluronan fragments contribute to the 

ozone-primed immune response to lipopolysaccharide. J Immunol 185: 6891-6898. 

http://dx.doi.org/10.4049/jimmunol.1000283.

Linn, WS; Buckley, RD; Spier, CE; Blessey, RL; Jones, MP; Fischer, DA; Hackney, JD. (1978). Health effects of 

ozone exposure in asthmatics. Am Rev Respir Dis 117: 835-843. 

Linn, WS; Medway, DA; Anzar, UT; Valencia, LM; Spier, CE; FS-D, T; Fischer, DA; Hackney, JD. (1982a). 

Persistence of adaptation to ozone in volunteers exposed repeatedly for six weeks. Am Rev Respir Dis 

125: 491-495. 

London, SJ. (2007). Gene-air pollution interactions in asthma. Proc Am Thorac Soc 4: 217-220. 

http://dx.doi.org/10.1513/pats.200701-031AW.

Long, NC; Suh, J; Morrow, JD; Schiestl, RH; Krishna Murthy, GG; Brain, JD; Frei, B. (2001). Ozone causes lipid 

peroxidation but little antioxidant depletion in exercising and nonexercising hamsters. J Appl Physiol 91: 

1694-1700. 

Longphre, M; Zhang, L, -Y; Harkema, JR; Kleeberger, SR. (1999). Ozone-induced pulmonary inflammation and 

epithelial proliferation are partially mediated by PAF. J Appl Physiol 86: 341-349. 

Maniar-Hew, K; Postlethwait, EM; Fanucchi, MV; Ballinger, CA; Evans, MJ; Harkema, JR; Carey, SA; 

McDonald, RJ; Bartolucci, AA; Miller, LA. (2011). Postnatal episodic ozone results in persistent 

attenuation of pulmonary and peripheral blood responses to LPS challenge. Am J Physiol Lung Cell 

Mol Physiol 300: L462-L471. http://dx.doi.org/10.1152/ajplung.00254.2010.

Matsumura, Y. (1970). The effects of ozone, nitrogen dioxide, and sulfur dioxide on the experimentally induced 

allergic respiratory disorder in guinea pigs: II. The effects of ozone on the absorption and the retention 

of antigen in the lung. Am Rev Respir Dis 102: 438-443. 

McBride, JT. (1992). Architecture of the tracheobronchial tree. In RA Parent (Ed.), Comparative biology of the 

normal lung (pp. 49-61). Boca Raton, FL: CRC Press.

McDonnell, WF; Horstman, DH; Hazucha, MJ; Seal, E, Jr; Haak, ED; Salaam, SA; House, DE. (1983). 

Pulmonary effects of ozone exposure during exercise: Dose-response characteristics. J Appl Physiol 

54: 1345-1352. 

McKinney, WJ; Jaskot, RH; Richards, JH; Costa, DL; Dreher, KL. (1998). Cytokine mediation of ozone-induced 

pulmonary adaptation. Am J Respir Cell Mol Biol 18: 696-705. 

Mercer, RR; Anjilvel, S; Miller, FJ; Crapo, JD. (1991). Inhomogeneity of ventilatory unit volume and its effects on 

reactive gas uptake. J Appl Physiol 70: 2193-2205. 

Mercer, RR; Russell, ML; Crapo, JD. (1992). Mucous lining layers in human and rat airways [Abstract]. Am Rev 

Respir Dis 145: A355. 

Mikerov, AN; Haque, R; Gan, X; Guo, X; Phelps, DS; Floros, J. (2008a). Ablation of SP-A has a negative impact 

on the susceptibility of mice to Klebsiella pneumoniae infection after ozone exposure: Sex differences. 

Respir Res 9: 77. http://dx.doi.org/10.1186/1465-9921-9-77.

Mikerov, AN; Umstead, TM; Gan, X; Huang, W; Guo, X; Wang, G; Phelps, DS; Floros, J. (2008b). Impact of 

ozone exposure on the phagocytic activity of human surfactant protein A (SP-A) and SP-A variants. Am 

J Physiol Lung Cell Mol Physiol 294: L121-L130. http://dx.doi.org/10.1152/ajplung.00288.2007.

Mikerov, AN; Gan, X; Umstead, TM; Miller, L; Chinchilli, VM; Phelps, DS; Floros, J. (2008c). Sex differences in 

the impact of ozone on survival and alveolar macrophage function of mice after Klebsiella pneumoniae 

infection. Respir Res 9: 24. http://dx.doi.org/10.1186/1465-9921-9-24.

Miller, FJ; McNeal, CA; Kirtz, JM; Gardner, DE; Coffin, DL; Menzel, DB. (1979). Nasopharyngeal removal of 

ozone in rabbits and guinea pigs. Toxicology 14: 273-281. http://dx.doi.org/10.1016/0300-

483X(79)90009-X.

Miller, FJ; Overton, JH, Jr; Jaskot, RH; Menzel, DB. (1985). A model of the regional uptake of gaseous 

pollutants in the lung: I. The sensitivity of the uptake of ozone in the human lung to lower respiratory 

tract secretions and exercise. Toxicol Appl Pharmacol 79: 11-27. http://dx.doi.org/10.1016/0041-

008X(85)90364-3.
Draft – Do Not Cite or Quote 5-88 September 2011

Miller, FJ; Overton, JH; Gerrity, TR; Graham, RC. (1988). Interspecies dosimetry of reactive gases. In U Mohr; D 

Dungworth; R McClellan; G Kimmerle; W Stober; J Lewkowski (Eds.), Inhalation toxicology: The design 

and interpretation of inhalation studies and their use in risk assessment (pp. 139-155). New York, NY: 

Springer-Verlag.

Miller, FJ; Kimbell, JS. (1995). Regional dosimetry of inhaled reactive gases. In RO McClellan; RF Henderson 

(Eds.), Concepts in inhalation toxicology (2nd ed., pp. 257-287). Washington, DC: Taylor & Francis.

Miller, FJ. (1995). Uptake and fate of ozone in the respiratory tract. Toxicol Lett 82-83: 277-285. 

Molfino, NA; Wright, SC; Katz, I; Tarlo, S; Silverman, F; McClean, PA; Szalai, JP; Raizenne, M; Slutsky, AS; 

Zamel, N. (1991). Effect of low concentrations of ozone on inhaled allergen responses in asthmatic 

subjects. Lancet 338: 199-203. 

Mudway, IS; Housley, D; Eccles, R; Richards, RJ; Datta, AK; Tetley, TD; Kelly, FJ. (1996). Differential depletion 

of human respiratory tract antioxidants in response to ozone challenge. Free Radic Res 25: 499-513. 

Mudway, IS; Kelly, FJ. (1998). Modeling the interactions of ozone with pulmonary epithelial lining fluid 

antioxidants. Toxicol Appl Pharmacol 148: 91-100. 

Mudway, IS; Blomberg, A; Frew, AJ; Holgate, ST; Sandstrom, T; Kelly, FJ. (1999a). Antioxidant consumption 

and repletion kinetics in nasal lavage fluid following exposure of healthy human volunteers to ozone. 

Eur Respir J 13: 1429-1438. 

Mudway, IS; Krishna, MT; Frew, AJ; MacLeod, D; Sandstrom, T; Holgate, ST; Kelly, FJ. (1999b). Compromised 

concentrations of ascorbate in fluid lining the respiratory tract in human subjects after exposure to 

ozone. Occup Environ Med 56: 473-481. 

Mudway, IS; Kelly, FJ. (2000). Ozone and the lung: A sensitive issue. Mol Aspects Med 21: 1-48. 

Mudway, IS; Stenfors, N; Blomberg, A; Helleday, R; Dunster, C; Marklund, SL; Frew, AJ; Sandstrom, T; Kelly, 

FJ. (2001). Differences in basal airway antioxidant concentrations are not predictive of individual 

responsiveness to ozone: A comparison of healthy and mild asthmatic subjects. Free Radic Biol Med 

31: 962-974. 

Mudway, IS; Kelly, FJ. (2004b). An investigation of inhaled ozone dose and the magnitude of airway 

inflammation in healthy adults: Online data supplement. Am J Respir Crit Care Med 169: 1089-1095. 

http://dx.doi.org/10.1164/rccm.200309-1325PP.

Mudway, IS; Behndig, AF; Helleday, R; Pourazar, J; Frew, AJ; Kelly, FJ; Blomberg, A. (2006). Vitamin 

supplementation does not protect against symptoms in ozone-responsive subjects. Free Radic Biol 

Med 40: 1702-1712. 

Murphy, RC; Johnson, KM. (2008). Cholesterol, reactive oxygen species, and the formation of biologically active 

mediators. J Biol Chem 283: 15521-15525. http://dx.doi.org/10.1074/jbc.R700049200.

Myatt, L; Kossenjans, W; Sahay, R; Eis, A; Brockman, D. (2000). Oxidative stress causes vascular dysfunction 

in the placenta. J Matern Fetal Med 9: 79-82. http://dx.doi.org/10.1002/(SICI)1520-

6661(200001/02)9:1<79::AID-MFM16>3.0.CO;2-O.

Nishiyama, H; Ikeda, H; Kaneko, T; Fu, L; Kudo, M; Ito, T; Okubo, T. (1998). Neuropeptides mediate the ozoneinduced increase in the permeability of the tracheal mucosa in guinea pigs. Am J Physiol 275: L231-

L238. 

Nodelman, V; Ultman, JS. (1999). Longitudinal distribution of chlorine absorption in human airways: A 

comparison to ozone absorption. J Appl Physiol 87: 2073-2080. 

Noviski, N; Brewer, JP; Skornik, WA; Galli, SJ; Drazen, JM; Martin, TR. (1999). Mast cell activation is not 

required for induction of airway hyperresponsiveness by ozone in mice. J Appl Physiol 86: 202-210. 

O'Byrne, P; Walters, E; Gold, B; Aizawa, H; Fabbri, L; Alpert, S; Nadel, J; Holtzman, M. (1983). Neutrophil 

depletion inhibits airway hyperresponsiveness induced by ozone exposure. Am Rev Respir Dis 130: 

214-219. 

O'Byrne, PM; Walters, EH; Aizawa, H; Fabbri, LM; Holtzman, MJ; Nadel, JA. (1984). Indomethacin inhibits the 

airway hyperresponsiveness but not the neutrophil influx induced by ozone in dogs. Am Rev Respir Dis 

130: 220-224. 

Oslund, KL; Hyde, DM; Putney, LF; Alfaro, MF; Walby, WF; Tyler, NK; Schelegle, ES. (2008). Activation of 

neurokinin-1 receptors during ozone inhalation contributes to epithelial injury and repair. Am J Respir 

Cell Mol Biol 39: 279-288. http://dx.doi.org/10.1165/rcmb.2008-0009OC.
Draft – Do Not Cite or Quote 5-89 September 2011

Oslund, KL; Hyde, DM; Putney, LF; Alfaro, MF; Walby, WF; Tyler, NK; Schelegle, ES. (2009). Activation of 

calcitonin gene-related peptide receptor during ozone inhalation contributes to airway epithelial injury 

and repair. Toxicol Pathol 37: 805-813. http://dx.doi.org/10.1177/0192623309345691.

Overton, JH; Graham, RC; Miller, FJ. (1987). A model of the regional uptake of gaseous pollutants in the lung: II. 

The sensitivity of ozone uptake in laboratory animal lungs to anatomical and ventilatory parameters. 

Toxicol Appl Pharmacol 88: 418-432. http://dx.doi.org/10.1016/0041-008X(87)90216-X.

Overton, JH; Graham, RC. (1989). Predictions of ozone absorption in human lungs from newborn to adult. 

Health Phys 1: 29-36. 

Overton, JH; Graham, RC; Menache, MG; Mercer, RR; Miller, FJ. (1996). Influence of tracheobronchial region 

expansion and volume on reactive gas uptake and interspecies dose extrapolations. Inhal Toxicol 8: 

723-745. 

Paquette, NC; Zhang, L, -Y; Ellis, WA; Scott, AL; Kleeberger, SR. (1996). Vitamin A deficiency enhances ozoneinduced lung injury. Am J Physiol 270: L475-L482. 

Park, JW; Taube, C; Swasey, C; Kodama, T; Joetham, A; Balhorn, A; Takeda, K; Miyahara, N; Allen, CB; 

Dakhama, A; Kim, SH; Dinarello, CA; Gelfand, EW. (2004). Interleukin-1 receptor antagonist attenuates 

airway hyperresponsiveness following exposure to ozone. Am J Respir Cell Mol Biol 30: 830-836. 

http://dx.doi.org/10.1165/rcmb.2003-0373OC.

Passannante, AN; Hazucha, MJ; Bromberg, PA; Seal, E; Folinsbee, L; Koch, G. (1998). Nociceptive 

mechanisms modulate ozone-induced human lung function decrements. J Appl Physiol 85: 1863-1870. 

Pearson, AC; Bhalla, DK. (1997). Effects of ozone on macrophage adhesion in vitro and epithelial and 

inflammatory responses in vivo: The role of cytokines. J Toxicol Environ Health 50: 143-157. 

Peden, DB; Setzer, RW, Jr; Devlin, RB. (1995). Ozone exposure has both a priming effect on allergen-induced 

responses and an intrinsic inflammatory action in the nasal airways of perennially allergic asthmatics. 

Am J Respir Crit Care Med 151: 1336-1345. 

Peden, DB. (2011). The role of oxidative stress and innate immunity in O(3) and endotoxin-induced human 

allergic airway disease. Immunol Rev 242: 91-105. http://dx.doi.org/10.1111/j.1600-065X.2011.01035.x.

Perepu, RS; Garcia, C; Dostal, D; Sethi, R. (2010). Enhanced death signaling in ozone-exposed ischemicreperfused hearts. Mol Cell Biochem 336: 55-64. http://dx.doi.org/10.1007/s11010-009-0265-4.

Pereyra-Muñoz, N; Rugerio-Vargas, C; Angoa-Pérez, M; Borgonio-Pérez, G; Rivas-Arancibia, S. (2006). 

Oxidative damage in substantia nigra and striatum of rats chronically exposed to ozone. J Chem 

Neuroanat 31: 114-123. http://dx.doi.org/10.1016/j.jchemneu.2005.09.006.

Perez-Gil, J. (2008). Structure of pulmonary surfactant membranes and films: The role of proteins and lipidprotein interactions. Biochim Biophys Acta 1778: 1676-1695. 

http://dx.doi.org/10.1016/j.bbamem.2008.05.003.

Pichavant, M; Goya, S; Meyer, EH; Johnston, RA; Kim, HY; Matangkasombut, P; Zhu, M; Iwakura, Y; Savage, 

PB; DeKruyff, RH; Shore, SA; Umetsu, DT. (2008). Ozone exposure in a mouse model induces airway 

hyperreactivity that requires the presence of natural killer T cells and IL-17. J Exp Med 205: 385-393. 

http://dx.doi.org/10.1084/jem.20071507.

Picher, M; Burch, LH; Boucher, RC. (2004). Metabolism of P2 receptor agonists in human airways: Implications 

for mucociliary clearance and cystic fibrosis. J Biol Chem 279: 20234-20241. 

http://dx.doi.org/10.1074/jbc.M400305200.

Plopper, CG; Hatch, GE; Wong, V; Duan, X; Weir, AJ; Tarkington, BK; Devlin, RB; Becker, S; Buckpitt, AR.

(1998). Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, sitespecific ozone dose and glutathione depletion in rhesus monkeys. Am J Respir Cell Mol Biol 19: 387-

399. 

Plopper, CG; Mango, GW; Hatch, GE; Wong, VJ; Toskala, E; Reynolds, SD; Tarkington, BK; Stripp, BR. (2006). 

Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in 

Clara cell secretory protein. Toxicol Appl Pharmacol 213: 74-85. 

http://dx.doi.org/10.1016/j.taap.2005.09.003.

Plopper, CG; Smiley-Jewell, SM; Miller, LA; Fanucchi, MV; Evans, MJ; Buckpitt, AR; Avdalovic, M; Gershwin, 

LJ; Joad, JP; Kajekar, R; Larson, S; Pinkerton, KE; Van Winkle, LS; Schelegle, ES; Pieczarka, EM; Wu, 

R; Hyde, DM. (2007). Asthma/allergic airways disease: Does postnatal exposure to environmental 

toxicants promote airway pathobiology? Toxicol Pathol 35: 97-110. 

http://dx.doi.org/10.1080/01926230601132030.
Draft – Do Not Cite or Quote 5-90 September 2011

Postlethwait, EM; Langford, SD; Bidani, A. (1994). Determinants of inhaled ozone absorption in isolated rat 

lungs. Toxicol Appl Pharmacol 125: 77-89. 

Postlethwait, EM; Cueto, R; Velsor, LW; Pryor, WA. (1998). O3-induced formation of bioactive lipids: Estimated 

surface concentrations and lining layer effects. Am J Physiol 274: L1006-L1016. 

Postlethwait, EM; Joad, JP; Hyde, DM; Schelegle, ES; Bric, JM; Weir, AJ; Putney, LF; Wong, VJ; Velsor, LW; 

Plopper, CG. (2000). Three-dimensional mapping of ozone-induced acute cytotoxicity in 

tracheobronchial airways of isolated perfused rat lung. Am J Respir Cell Mol Biol 22: 191-199. 

Postlethwait, EM; Ultman, JS. (2001). Airspace surface chemistry mediates O3-induced lung injury. Hum Ecol 

Risk Assess 7: 1145-1159. http://dx.doi.org/10.1080/20018091094907.

Pryor, WA. (1976). Free radical reactions in biology: Initiation of lipid autoxidation by ozone and nitrogen 

dioxide. Environ Health Perspect 16: 180-181. 

Pryor, WA; Giamalva, DH; Church, DF. (1984). Kinetics of ozonation. 2. Amino acids and model compounds in 

water and comparisons to rates in nonpolar solvents. J Am Chem Soc 106: 7094-7100. 

Pryor, WA; Das, B; Church, DF. (1991). The ozonation of unsaturated fatty acids: Aldehydes and hydrogen 

peroxide as products and possible mediators of ozone toxicity. Chem Res Toxicol 4: 341-348. 

Pryor, WA. (1992). How far does ozone penetrate into the pulmonary air/tissue boundary before it reacts? Free 

Radic Biol Med 12: 83-88. http://dx.doi.org/10.1016/0891-5849(92)90060-T.

Pryor, WA. (1994). Mechanisms of radical formation from reactions of ozone with target molecules in the lung.

Free Radic Biol Med 17: 451-465. 

Pryor, WA; Bermudez, E; Cueto, R; Squadrito, GL. (1996). Detection of aldehydes in bronchoalveolar lavage of 

rats exposed to ozone. Toxicol Sci 34: 148-156. 

Pulfer, MK; Murphy, RC. (2004). Formation of biologically active oxysterols during ozonolysis of cholesterol 

present in lung surfactant. J Biol Chem 279: 26331-26338. 

Pulfer, MK; Taube, C; Gelfand, E; Murphy, RC. (2005). Ozone exposure in vivo and formation of biologically 

active oxysterols in the lung. J Pharmacol Exp Ther 312: 256-264. 

Que, LG; Stiles, JV; Sundy, JS; Foster, WM. (2011). Pulmonary function, bronchial reactivity, and epithelial 

permeability are response phenotypes to ozone and develop differentially in healthy humans. J Appl 

Physiol 111: 679-687. http://dx.doi.org/10.1152/japplphysiol.00337.2011.

Rashba-Step, J; Tatoyan, A; Duncan, R; Ann, D; Pushpa-Rehka, TR; Sevanian, A. (1997). Phospholipid 

peroxidation induces cytosolic phospholipase A2 activity: Membrane effects versus enzyme 

phosphorylation. Arch Biochem Biophys 343: 44-54. http://dx.doi.org/10.1006/abbi.1997.0134.

Reeser, WH; Lee, GM; Taylor, A; Wang, L; Arnold, SF; Ultman, JS; Ben-Jebria, A. (2005). Uptake of ozone in 

human lungs and its relationship to local physiological response. Inhal Toxicol 17: 699-707. 

http://dx.doi.org/10.1080/08958370500224433.

Rigas, ML; Ben-Jebria, A; Ultman, JS. (1997). Longitudinal distribution of ozone absorption in the lung: Effects 

of nitrogen dioxide, sulfur dioxide, and ozone exposures. Arch Environ Occup Health 52: 173-178. 

Rigas, ML; Catlin, SN; Ben-Jebria, A; Ultman, JS. (2000). Ozone uptake in the intact human respiratory tract: 

Relationship between inhaled dose and actual dose. J Appl Physiol 88: 2015-2022. 

Ritz, B; Yu, F; Chapa, G; Fruin, S. (2000). Effect of air pollution on preterm birth among children born in 

Southern California between 1989 and 1993. Epidemiology 11: 502-511. 

Ritz, B; Yu, F; Fruin, S; Chapa, G; Shaw, GM; Harris, JA. (2002). Ambient air pollution and risk of birth defects in 

Southern California. Am J Epidemiol 155: 17-25. 

Romieu, I; Meneses, F; Ramirez, M; Ruiz, S; Padilla, RP; Sienra, JJ; Gerber, M; Grievink, L; Dekker, R; Walda, 

I; Brunekreef, B. (1998a). Antioxidant supplementation and respiratory functions among workers 

exposed to high levels of ozone. Am J Respir Crit Care Med 158: 226-232. 

Romieu, I; Sienra-Monge, JJ; Ramirez-Aguilar, M; Tellez-Rojo, MM; Moreno-Macias, H; Reyes-Ruiz, NI; Del 

Rio-Navarro, BE; Ruiz-Navarro, MX; Hatch, G; Slade, R; Hernandez-Avila, M. (2002). Antioxidant 

supplementation and lung functions among children with asthma exposed to high levels of air 

pollutants. Am J Respir Crit Care Med 166: 703-709. 

Romieu, I; Sienra-Monge, JJ; Ramirez-Aguilar, M; Moreno-Macias, H; Reyes-Ruiz, NI; Estela del Rio-Navarro, 

B; Hernandez-Avila, M; London, SJ. (2004a). Genetic polymorphism of GSTM1 and antioxidant 

supplementation influence lung function in relation to ozone exposure in asthmatic children in Mexico 

City. Thorax 59: 8-10. 
Draft – Do Not Cite or Quote 5-91 September 2011

Roux, E; Hyvelin, J, -M; Savineau, J, -P; Marthan, R. (1999). Human isolated airway contraction: Interaction 

between air pollutants and passive sensitization. Am J Respir Crit Care Med 160: 439-445. 

Rutkowski, JM; Santiag, LY; Ben-Jebria, A; Ultman, JS. (2011). Comparison of ozone-specific (OZAC) and 

oxygen radical (ORAC) antioxidant capacity assays for use with nasal lavage fluid. Toxicol In Vitro 25: 

1406-1413. http://dx.doi.org/10.1016/j.tiv.2011.04.008.

Sagai, M; Arakawa, K; Ichinose, T; Shimojo, N. (1987). Biochemical effects on combined gases of nitrogen 

dioxide and ozone: I. Species differences of lipid peroxides and phospholipids in lungs. Toxicology 46: 

251-265. http://dx.doi.org/10.1016/0300-483X(87)90207-1.

Samet, JM; Hatch, GE; Horstman, D; Steck-Scott, S; Arab, L; Bromberg, PA; Levine, M; McDonnell, WF; Devlin, 

RB. (2001). Effect of antioxidant supplementation on ozone-induced lung injury in human subjects. Am 

J Respir Crit Care Med 164: 819-825. 

Santiago-López, D; Bautista-Martínez, JA; Reyes-Hernandez, CI; Aguilar-Martínez, M; Rivas-Arancibia, S.

(2010). Oxidative stress, progressive damage in the substantia nigra and plasma dopamine oxidation, 

in rats chronically exposed to ozone. Toxicol Lett 197: 193-200. 

http://dx.doi.org/10.1016/j.toxlet.2010.05.020.

Santiago, LY; Hann, MC; Ben-Jebria, A; Ultman, JS. (2001). Ozone absorption in the human nose during 

unidirectional airflow. J Appl Physiol 91: 725-732. 

Sarangapani, R; Gentry, PR; Covington, TR; Teeguarden, JG; Clewell HJ, III. (2003). Evaluation of the potential 

impact of age- and gender-specific lung morphology and ventilation rate on the dosimetry of vapors. 

Inhal Toxicol 15: 987-1016. 

Sathishkumar, K; Xi, X; Martin, R; Uppu, RM. (2007a). Cholesterol secoaldehyde, an ozonation product of 

cholesterol, induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons. J 

Alzheimers Dis 11: 261-274. 

Sathishkumar, K; Murthy, SN; Uppu, RM. (2007b). Cytotoxic effects of oxysterols produced during ozonolysis of 

cholesterol in murine GT1-7 hypothalamic neurons. Free Radic Res 41: 82-88. 

http://dx.doi.org/10.1080/10715760600950566.

Sathishkumar, K; Gao, X; Raghavamenon, AC; Parinandi, N; Pryor, WA; Uppu, RM. (2009). Cholesterol 

secoaldehyde induces apoptosis in H9c2 cardiomyoblasts through reactive oxygen species involving 

mitochondrial and death receptor pathways. Free Radic Biol Med 47: 548-558. 

http://dx.doi.org/10.1016/j.freeradbiomed.2009.05.020.

Sato, S; Shimura, S; Hirose, T; Maeda, S; Kawakami, M; Takishima, T; Kimura, S. (1980). Effects of long-term 

ozone exposure and dietary vitamin E in rats. Tohoku J Exp Med 130: 117-128. 

Sawyer, K; Brown, J; HazuchaM; Bennett, WD. (2007). The effect of exercise on nasal uptake of ozone in 

healthy human adults. J Appl Physiol 102: 1380-1386. 

http://dx.doi.org/10.1152/japplphysiol.00269.2006.

Scannell, C; Chen, L; Aris, RM; Tager, I; Christian, D; Ferrando, R; Welch, B; Kelly, T; Balmes, JR. (1996). 

Greater ozone-induced inflammatory responses in subjects with asthma. Am J Respir Crit Care Med 

154: 24-29. 

Schelegle, ES; Adams, WC; Siefkin, AD. (1987). Indomethacin pretreatment reduces ozone-induced pulmonary 

function decrements in human subjects. Am Rev Respir Dis 136: 1350-1354. 

Schelegle, ES; Siefkin, AD; McDonald, RJ. (1991). Time course of ozone-induced neutrophilia in normal 

humans. Am J Respir Crit Care Med 143: 1353-1358. 

Schelegle, ES; Carl, ML; Coleridge, HM; Coleridge, JCG; Green, JF. (1993). Contribution of vagal afferents to 

respiratory reflexes evoked by acute inhalation of ozone in dogs. J Appl Physiol 74: 2338-2344. 

Schelegle, ES; Walby, WF; Adams, WC. (2007). Time course of ozone-induced changes in breathing pattern in 

healthy exercising humans. J Appl Physiol 102: 688-697. 

http://dx.doi.org/10.1152/japplphysiol.00141.2006.

Schroter, RC; Sudlow, MF. (1969). Flow patterns in models of the human bronchial airways. Respir Physiol 

Neurobiol 7: 341-355. 

Seltzer, J; Bigby, BG; Stulbarg, M; Holtzman, MJ; Nadel, JA; Ueki, IF; Leikauf, GD; Goetzl, EJ; Boushey, HA.

(1986). O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans. 

J Appl Physiol 60: 1321-1326. 
Draft – Do Not Cite or Quote 5-92 September 2011

Servais, S; Boussouar, A; Molnar, A; Douki, T; Pequignot, JM; Favier, R. (2005). Age-related sensitivity to lung 

oxidative stress during ozone exposure. Free Radic Res 39: 305-316. 

http://dx.doi.org/10.1080/10715760400011098.

Sharkhuu, T; Doerfler, DL; Copeland, C; Luebke, RW; Gilmour, MI. (2011). Effect of maternal exposure to ozone 

on reproductive outcome and immune, inflammatory, and allergic responses in the offspring. J 

Immunotoxicol 8: 183-194. http://dx.doi.org/10.3109/1547691X.2011.568978.

Shore, SA; Schwartzman, IN; Le Blanc, B; Krishna Murthy, GG; Doerschuk, CM. (2001). Tumor necrosis factor 

receptor 2 contributes to ozone-induced airway hyperresponsiveness in mice. Am J Respir Crit Care 

Med 164: 602-607. 

Shore, SA. (2007). Obesity and asthma: lessons from animal models. J Appl Physiol 102: 516-528. 

Shore, SA; Lang, JE; Kasahara, DI; Lu, FL; Verbout, NG; Si, H; Williams, ES; Terry, RD; Lee, A; Johnston, RA.

(2009). Pulmonary responses to subacute ozone exposure in obese vs. lean mice. J Appl Physiol 107: 

1445-1452. http://dx.doi.org/10.1152/japplphysiol.00456.2009.

Sienra-Monge, JJ; Ramirez-Aguilar, M; Moreno-Macias, H; Reyes-Ruiz, NI; Del Rio-Navarro, BE; Ruiz-Navarro, 

MX; Hatch, G; Crissman, K; Slade, R; Devlin, RB; Romieu, I. (2004). Antioxidant supplementation and 

nasal inflammatory responses among young asthmatics exposed to high levels of ozone. Clin Exp 

Immunol 138: 317-322. http://dx.doi.org/10.1111/j.1365-2249.2004.02606.x.

Slade, R; Highfill, JW; Hatch, GE. (1989). Effects of depletion of ascorbic acid or nonprotein sulfhydryls on the 

acute inhalation toxicity of nitrogen dioxide, ozone, and phosgene. Inhal Toxicol 1: 261-271. 

Slade, R; Crissman, K; Norwood, J; Hatch, G. (1993). Comparison of antioxidant substances in bronchoalveolar 

lavage cells and fluid from humans, guinea pigs, and rats. Exp Lung Res 19: 469-484. 

Slade, R; Watkinson, WP; Hatch, GE. (1997). Mouse strain differences in ozone dosimetry and body 

temperature changes. Am J Physiol 272: L73-L77. 

Sokol, RZ; Kraft, P; Fowler, IM; Mamet, R; Kim, E; Berhane, KT. (2006). Exposure to environmental ozone alters

semen quality. Environ Health Perspect 114: 360-365. 

Stenfors, N; Pourazar, J; Blomberg, A; Krishna, MT; Mudway, I; Helleday, R; Kelly, FJ; Frew, AJ; Sandstrom, T.

(2002). Effect of ozone on bronchial mucosal inflammation in asthmatic and healthy subjects. Respir 

Med 96: 352-358. 

Sterner-Kock, A; Kock, M; Braun, R; Hyde, DM. (2000). Ozone-induced epithelial injury in the ferret is similar to 

nonhuman primates. Am J Respir Crit Care Med 162: 1152-1156. 

Sun, J; Koto, H; Chung, KF. (1997). Interaction of ozone and allergen challenges on bronchial responsiveness 

and inflammation in sensitised guinea pigs. Int Arch Allergy Immunol 112: 191-195. 

Taylor-Clark, TE; McAlexander, MA; Nassenstein, C; Sheardown, SA; Wilson, S; Thornton, J; Carr, MJ; Undem, 

BJ. (2008). Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal 

bronchopulmonary C-fibres by the endogenous autacoid 4-oxononenal. J Physiol 586: 3447-3459. 

http://dx.doi.org/10.1113/jphysiol.2008.153585.

Taylor-Clark, TE; Undem, BJ. (2010). Ozone activates airway nerves via the selective stimulation of TRPA1 ion 

channels. J Physiol 588: 423-433. http://dx.doi.org/10.1113/jphysiol.2009.183301.

Taylor, AB; Lee, GM; Nellore, K; Ben-Jebria, A; Ultman, JS. (2006). Changes in the carbon dioxide expirogram 

in response to ozone exposure. Toxicol Appl Pharmacol 213: 1-9. 

http://dx.doi.org/10.1016/j.taap.2005.09.009.

Taylor, AB; Borhan, A; Ultman, JS. (2007). Three-dimensional simulations of reactive gas uptake in single 

airway bifurcations. Ann Biomed Eng 35: 235-249. http://dx.doi.org/10.1007/s10439-006-9195-4.

Tepper, JS; Costa, DL; Lehmann, JR; Weber, MF; Hatch, GE. (1989). Unattenuated structural and biochemical 

alterations in the rat lung during functional adaptation to ozone. Am J Respir Crit Care Med 140: 493-

501. 

Tepper, JS; Costa, DL; Fitzgerald, S; Doerfler, DL; Bromberg, PA. (1993). Role of tachykinins in ozone-induced 

acute lung injury in guinea pigs. J Appl Physiol 75: 1404-1411. 

Thomson, E; Kumarathasan, P; Goegan, P; Aubin, RA; Vincent, R. (2005). Differential regulation of the lung 

endothelin system by urban particulate matter and ozone. Toxicol Sci 88: 103-113. 

Thomson, E; Kumarathasan, P; Vincent, R. (2006). Pulmonary expression of preproET-1 and preproET-3 

mRNAs is altered reciprocally in rats after inhalation of air pollutants. Exp Biol Med 231: 979-984. 

Trenga, CA; Koenig, JQ; Williams, PV. (2001). Dietary antioxidants and ozone-induced bronchial 

hyperresponsiveness in adults with asthma. Arch Environ Occup Health 56: 242-249. 
Draft – Do Not Cite or Quote 5-93 September 2011

Trevisani, M; Siemens, J; Materazzi, S; Bautista, DM; Nassini, R; Campi, B; Imamachi, N; Andrè, E; Patacchini, 

R; Cottrell, GS; Gatti, R; Basbaum, AI; Bunnett, NW; Julius, D; Geppetti, P. (2007). 4-Hydroxynonenal, 

an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant 

receptor TRPA1. PNAS 104: 13519-13524. http://dx.doi.org/10.1073/pnas.0705923104.

Tsujino, I; Kawakami, Y; Kaneko, A. (2005). Comparative simulation of gas transport in airway models of rat, 

dog, and human. Inhal Toxicol 17: 475-485. http://dx.doi.org/10.1080/08958370590964476.

U.S. EPA. (U.S. Environmental Protection Agency). (1996a). Air quality criteria for ozone and related 

photochemical oxidants. (EPA/600/P-93/004AF). Research Triangle Park, NC.

U.S. EPA. (U.S. Environmental Protection Agency). (2005). Guidelines for carcinogen risk assessment. 

(EPA/630/P-03/001F). Washington, DC. http://www.epa.gov/cancerguidelines/.

U.S. EPA. (U.S. Environmental Protection Agency). (2006b). Air quality criteria for ozone and related 

photochemical oxidants. (EPA/600/R-05/004AF). Research Triangle Park, NC: U.S. Environmental 

Protection Agency, Office of Research and Development. 

http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=149923.

U.S. EPA. (U.S. Environmental Protection Agency). (2009f). The U.S. Environmental Protection Agency’s 

strategic plan for evaluating the toxicity of chemicals. (EPA/100/K-09/001). Washington, DC. 

http://www.epa.gov/osa/spc/toxicitytesting/docs/toxtest_strategy_032309.pdf.

Ultman, JS. (1985). Gas transport in the conducting airways. In LA Engel; M Paiva (Eds.), Gas mixing and 

distribution in the lung (pp. 63-136). New York, NY: Marcel Dekker.

Ultman, JS; Anjilvel, S. (1990). Monte Carlo simulation of ozone uptake in an asymmetric lung model. In DJ 

Schneck; CL Lucas (Eds.), Biofluid mechanics 3: Proceedings of the third Mid-Atlantic Conference on 

Biofluid Mechanics; October; Blacksburg, VA (pp. 45-52). New York, NY: New York University Press.

Ultman, JS; Ben-Jebria, A; Hu, S, -C. (1994). Noninvasive determination of respiratory ozone absorption: The 

bolus-response method. (HEI Research Report 69). Cambridge, MA: Health Effects Institute.

Ultman, JS; Ben-Jebria, A; Arnold, SF. (2004). Uptake distribution of ozone in human lungs: Intersubject 

variability in physiologic response. (HEI Research Report 125). Boston, MA: Health Effects Institute. 

http://pubs.healtheffects.org/view.php?id=70.

Uppu, RM; Cueto, R; Squadrito, GL; Pryor, WA. (1995). What does ozone react with at the air/lung interface? 

Model studies using human red blood cell membranes. Arch Biochem Biophys 319: 257-266. 

Urch, B; Speck, M; Corey, P; Wasserstein, D; Manno, M; Lukic, KZ; Brook, JR; Liu, L; Coull, B; Schwartz, J; 

Gold, DR; Silverman, F. (2010). Concentrated ambient fine particles and not ozone induce a systemic 

interleukin-6 response in humans. Inhal Toxicol 22: 210-218. 

http://dx.doi.org/10.3109/08958370903173666.

Vagaggini, B; Taccola, M; Clanchetti, S; Carnevali, S; Bartoli, ML; Bacci, E; Dente, FL; Di Franco, A; Giannini, D; 

Paggiaro, PL. (2002). Ozone exposure increases eosinophilic airway response induced by previous 

allergen challenge. Am J Respir Crit Care Med 166: 1073-1077. 

Van Bree, L; Dormans, JAM, A; Koren, HS; Devlin, RB; Rombout, PJA. (2002). Attenuation and recovery of 

pulmonary injury in rats following short-term, repeated daily exposure to ozone. Inhal Toxicol 14: 883-

900. 

Vancza, EM; Galdanes, K; Gunnison, A; Hatch, G; Gordon, T. (2009). Age, strain, and gender as factors for 

increased sensitivity of the mouse lung to inhaled ozone. Toxicol Sci 107: 535-543. 

http://dx.doi.org/10.1093/toxsci/kfn253.

Vasu, VT; Oommen, S; Lim, Y; Valacchi, G; Hobson, B; Eiserich, JP; Leonard, SW; Traber, MG; Cross, CE; 

Gohil, K. (2010). Modulation of ozone-sensitive genes in alpha-tocopherol transfer protein null mice. 

Inhal Toxicol 22: 1-16. http://dx.doi.org/10.3109/08958370902838145.

Verhein, KC; Hazari, MS; Moulton, BC; Jacoby, IW; Jacoby, DB; Fryer, AD. (2011). Three days after a single 

exposure to ozone the mechanism of airway hyperreactivity is dependent upon substance P and nerve 

growth factor. Am J Physiol Lung Cell Mol Physiol 300: L176-L184. 

http://dx.doi.org/10.1152/ajplung.00060.2010.

Vesely, KR; Schelegle, ES; Stovall, MY; Harkema, JR; Green, JF; Hyde, DM. (1999). Breathing pattern 

response and epithelial labeling in ozone-induced airway injury in neutrophil-depleted rats. Am J Respir 

Cell Mol Biol 20: 699-709.
Draft – Do Not Cite or Quote 5-94 September 2011

Vincent, R; Vu, D; Hatch, G; Poon, R; Dreher, K; Guenette, J; Bjarnason, S; Potvin, M; Norwood, J; McMullen, 

E. (1996a). Sensitivity of lungs of aging Fischer 344 rats to ozone: Assessment by bronchoalveolar 

lavage. Am J Physiol 271: L555-L565. 

Vivier, E; Raulet, DH; Moretta, A; Caligiuri, MA; Zitvogel, L; Lanier, LL; Yokoyama, WM; Ugolini, S. (2011). 

Innate or adaptive immunity? The example of natural killer cells. Science 331: 44-49. 

http://dx.doi.org/10.1126/science.1198687.

Voynow, JA; Fischer, BM; Zheng, S; Potts, EN; Grover, AR; Jaiswal, AK; Ghio, AJ; Foster, WM. (2009). 

NAD(P)H quinone oxidoreductase 1 is essential for ozone-induced oxidative stress in mice and 

humans. Am J Respir Cell Mol Biol 41: 107-113. http://dx.doi.org/10.1165/rcmb.2008-0381OC.

Wagner, JG; Hotchkiss, JA; Harkema, JR. (2002). Enhancement of nasal inflammatory and epithelial responses 

after ozone and allergen coexposure in brown Norway rats. Toxicol Sci 67: 284-294. 

Wagner, JG; Jiang, Q; Harkema, JR; Illek, B; Patel, DD; Ames, BN; Peden, DB. (2007). Ozone enhancement of 

lower airway allergic inflammation is prevented by gamma-tocopherol. Free Radic Biol Med 43: 1176-

1188. http://dx.doi.org/10.1016/j.freeradbiomed.2007.07.013.

Wagner, JG; Harkema, JR; Jiang, Q; Illek, B; Ames, BN; Peden, DB. (2009). Gamma-tocopherol attenuates 

ozone-induced exacerbation of allergic rhinosinusitis in rats. Toxicol Pathol 37: 481-491. 

http://dx.doi.org/10.1177/0192623309335630.

Watkinson, WP; Campen, MJ; Nolan, JP; Costa, DL. (2001). Cardiovascular and systemic responses to inhaled 

pollutants in rodents: Effects of ozone and particulate matter. Environ Health Perspect 109: 539-546. 

Watkinson, WP; Campen, MJ; Wichers, LB; Nolan, JP; Costa, DL. (2003). Cardiac and thermoregulatory 

responses to inhaled pollutants in healthy and compromised rodents: Modulation via interaction with 

environmental factors. Environ Res 92: 35-47. 

Weibel, ER. (1980). Design and structure of the human lung. In AP Fishman (Ed.), Assessment of pulmonary 

function. New York, NY: McGraw-Hill.

Weinmann, GG; Weidenbach-Gerbase, M; Foster, WM; Zacur, H; Frank, R. (1995a). Evidence for ozoneinduced small-airway dysfunction: Lack of menstrual-cycle and gender effects. Am J Respir Crit Care 

Med 152: 988-996. 

Weinmann, GG; Liu, MC; Proud, D; Weidenbach-Gerbase, M; Hubbard, W; Frank, R. (1995b). Ozone exposure 

in humans: Inflammatory, small and peripheral airway responses. Am J Respir Crit Care Med 152: 

1175-1182. 

Welch, RW; Wang, Y; Crossman, A, Jr; Park, JB; Kirk, KL; Levine, M. (1995). Accumulation of vitamin C 

(ascorbate) and its oxidized metabolite dehydroascorbic acid occurs by separate mechanisms. J Biol 

Chem 270: 12584-12592. http://dx.doi.org/10.1074/jbc.270.21.12584.

Wenten, M; Gauderman, WJ; Berhane, K; Lin, PC; Peters, J; Gilliland, FD. (2009). Functional variants in the 

catalase and myeloperoxidase genes, ambient air pollution, and respiratory-related school absences: 

An example of epistasis in gene-environment interactions. Am J Epidemiol 170: 1494-1501. 

http://dx.doi.org/10.1093/aje/kwp310.

Wiester, MJ; Williams, TB; King, ME; Menache, MG; Miller, FJ. (1987). Ozone uptake in awake Sprague-Dawley 

rats. Toxicol Appl Pharmacol 89: 429-437. http://dx.doi.org/10.1016/0041-008X(87)90162-1.

Wiester, MJ; Tepper, JS; King, ME; Menache, MG; Costa, DL. (1988). Comparative study of ozone (O3) uptake 

in three strains of rats and in the guinea pig. Toxicol Appl Pharmacol 96: 140-146. 

Wiester, MJ; Tepper, JS; Winsett, DW; Crissman, KM; Richards, JH; Costa, DL. (1996a). Adaptation to ozone in 

rats and its association with ascorbic acid in the lung. Toxicol Sci 31: 56-64. 

Wiester, MJ; Stevens, MA; Menache, MG; McKee, JL, Jr; Gerrity, TR. (1996c). Ozone uptake in healthy adult 

males during quiet breathing. Toxicol Sci 29: 102-109. 

Williams, AS; Issa, R; Leung, SY; Puneeta, N; Gregory, D; Ferguson, D; Brydon, L; Bennett, I; Adcock, M; 

Chung, KF. (2007a). Attenuation of ozone-induced airway inflammation and hyper-responsiveness by 

c-Jun NH2 terminal kinase inhibitor SP600125. J Pharmacol Exp Ther 322: 351-359. 

http://dx.doi.org/10.1124/jpet.107.121624.

Williams, AS; Leung, SY; Nath, P; Khorasani, NM; Bhavsar, P; Issa, R; Mitchell, JA; Adcock, IM; Chung, KF.

(2007b). Role of TLR2, TLR4, and MyD88 in murine ozone-induced airway hyperresponsiveness and 

neutrophilia. J Appl Physiol 103: 1189-1195. http://dx.doi.org/10.1152/japplphysiol.00172.2007.
Draft – Do Not Cite or Quote 5-95 September 2011

Williams, AS; Nath, P; Leung, SY; Khorasani, N; McKenzie, ANJ; Adcock, IM; Chung, KF. (2008a). Modulation 

of ozone-induced airway hyperresponsiveness and inflammation by interleukin-13. Eur Respir J 32: 

571-578. http://dx.doi.org/10.1183/09031936.00121607.

Williams, AS; Issa, R; Durham, A; Leung, SY; Kapoun, A; Medicherla, S; Higgins, LS; Adcock, IM; Chung, KF.

(2008b). Role of p38 mitogen-activated protein kinase in ozone-induced airway hyperresponsiveness 

and inflammation. Eur J Pharmacol 600: 117-122. http://dx.doi.org/10.1016/j.ejphar.2008.09.031.

Williams, AS; Eynott, PR; Leung, SY; Nath, P; Jupp, R; De Sanctis, GT; Resnick, R; Adcock, IM; Chung, KF.

(2009a). Role of cathepsin S in ozone-induced airway hyperresponsiveness and inflammation. Pulm 

Pharmacol Ther 22: 27-32. http://dx.doi.org/10.1016/j.pupt.2008.11.002.

Wu, W; Doreswamy, V; Diaz-Sanchez, D; Samet, JM; Kesic, M; Dailey, L; Zhang, W; Jaspers, I; Peden, DB.

(2011). GSTM1 modulation of IL-8 expression in human bronchial epithelial cells exposed to ozone. 

Free Radic Biol Med 51: 522-529. http://dx.doi.org/10.1016/j.freeradbiomed.2011.05.006.

Wu, ZX; Satterfield, BE; Dey, RD. (2003). Substance P released from intrinsic airway neurons contributes to 

ozone-enhanced airway hyperresponsiveness in ferret trachea. J Appl Physiol 95: 742-750. 

Wu, ZX; Barker, JS; Batchelor, TP; Dey, RD. (2008b). Interleukin (IL)-1 regulates ozone-enhanced tracheal 

smooth muscle responsiveness by increasing substance P (SP) production in intrinsic airway neurons 

of ferret. Respir Physiol Neurobiol 164: 300-311. http://dx.doi.org/10.1016/j.resp.2008.07.019.

Yang, IA; Holz, O; Jorres, RA; Magnussen, H; Barton, SJ; Rodriguez, S; Cakebread, JA; Holloway, JW; Holgate, 

ST. (2005a). Association of tumor necrosis factor alpha polymorphisms and ozone-induced change in 

lung function. Am J Respir Crit Care Med 171: 171-176. 

Yokoyama, E; Frank, R. (1972). Respiratory uptake of ozone in dogs. Arch Environ Occup Health 25: 132-138. 

Yoon, HK; Cho, HY; Kleeberger, SR. (2007). Protective role of matrix metalloproteinase-9 in ozone-induced 

airway inflammation. Environ Health Perspect 115: 1557-1563. http://dx.doi.org/10.1289/ehp.10289.

Yost, BL; Gleich, GJ; Jacoby, DB; Fryer, AD. (2005). The changing role of eosinophils in long-term 

hyperreactivity following a single ozone exposure. Am J Physiol Lung Cell Mol Physiol 289: L627-L635. 

http://dx.doi.org/10.1152/ajplung.00377.2004.