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Chapter 5. Dosimetry and Mode of Action

5.1. Hu man an d Animal Ozo n e Do s imetry

5.1.1. Introd uc tion

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. The measurement of 

3 the dose of reactive gases such as O3 can range in refinement from their concentration in the ambient 

4 exposure atmosphere to the “effective” dose of the chemical or its reaction products that actively 

5 participate in toxic reactions (Dahl, 1990, 094536). Thus, the units for the expression of the dose of 

O3 might range from the concentration of gas in the air (units of ppm or mg/m3 6 ), to the quantity of 

7 gas inhaled as the product of gas concentration × minute ventilation × time (units of ppm × L × h), to 

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

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

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

11 Ozone is a highly reactive though poorly water soluble gas. The latter feature is believed to be 

12 the reason why it is able to penetrate into targets in the lower respiratory tract. The fact that it is so 

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

14 of secondary oxidation products such as aldehydes and peroxides. Reaction products are formed 

15 when O3 interacts with components of the extracellular lining fluid (ELF) such as lipids and 

16 antioxidants. Ozone toxicity is observed to some extent in the nasal cavity, however further toxicity 

17 exists in the deep lung where the ELF thickness narrows allowing O3 to react directly with cells 

18 protruding from the ELF and surface macrophages. Ozone uptake relates directly to these ELF 

19 substrate reactions and is termed “reactive absorption.” Thus the uptake of O3 is related to both the 

20 concentration of O3 as well as the availability of substrates within the ELF.

21 Two types of measurement have been used to arrive at the O3 dose to target sites during 

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

23 measurement of chemical reactions or the product of those reactions with tissues or with 

24 biomolecules known to be present in tissues (termed “reactions”). The results of the above 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments such as the Integrated Science Assessments (ISAs) and the Integrated Risk Information System (IRIS).
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1 measurements have been incorporated into mathematical models for the purpose of explaining, 

2 predicting, and extrapolating O3 dose in different exposure scenarios of interest. 

3 This chapter is intended as an update of the past O3 AQCDs (U.S. EPA, 1996, 017831; 

4 U.S. EPA, 2006, 088089) and restates the basic concepts derived from O3 dosimetry literature 

5 presented in previous documents as well as introduces the recent relevant literature. Particular 

6 attention is given to dosimetric factors influencing individual susceptibility to adverse effects from 

7 O3 and factors that affect the ability to extrapolate between species (e.g., experimental animal to

8 human). As there have been few O3 dosimetry studies since the last AQCD, the reader is referred to 

9 previous documents (U.S. EPA, 1996, 017831; U.S. EPA, 2006, 088089) for more detailed 

10 discussion of the literature. Evaluation of the progress in the interpretation of past dosimetry studies, 

11 as well as studies published since 2005, in the areas of reactions, uptake, and models for O3

12 dosimetry, is discussed in the following sections. 

5.1.2. Ozone Reactions and Reaction Products

5.1.2.1. Summary o f Findings fro m 2006 Ozone AQCD

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

14 result from O3 exposure. Since O3 is chemically reactive with a wide spectrum of biomolecules, it is 

15 not feasible to delineate its many reaction products. Measurements of reaction formation have 

16 included either the loss of a specific molecule and appearance of plausible products, or the addition 

17 of O3-derived oxygen to biomolecules through the use of oxygen-18 labeling. In vitro exposure of 

18 ELF shows that O3 disappearance from the gas phase depends on the characteristics of the ELF 

19 substrates (Hu et al., 1994, 041323; Postlethwait et al., 1998, 086754). 

20 To gain access to the underlying cellular compartments, O3 must dissolve at the air-liquid 

21 interface of the airway surface and travel through the ELF layer. The ELF is comprised of the airway 

22 surface lining that includes the periciliary layer and overlying mucus layer, and the alveolar surface 

23 lining that includes the subphase of liquid and vesicular surfactant and the surfactant monolayer. 

24 There is a progressive decrease in ELF thickness and increase in interfacial surface with progression 

25 from the large airways to the alveolus, with the mucus coating becoming patchy in the distal 

26 conducting airways (Figure 5-1). Some cells, such as macrophages, may protrude into the gas phase, 

27 allowing for direct contact between O3 and cell membranes. The progressive thinning in the ELF 

28 decreases the distance O3 must travel to reach the cellular tissue layer. A computational fluid 

29 dynamics (CFD) model was able to predict experimentally measured O3 uptake, but only with nasal 

30 mucus layer thickness considered (Cohen-Hubal et al., 1996, 043785), reaffirming the importance of 

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

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

33 that O3 will not penetrate ELF layers greater than 0.1 μm without being transformed to other more 

34 long-lived reactive species, thus initiating a reaction cascade (Pryor, 1992, 042725). It follows that 
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1 the ELF should be considered an important target for O3 toxicity in the airways. Experimental 

2 support for this concept comes from several studies which measure the total oxygen-addition product 

3 of O3 reactions in the airways through the use of oxygen-18 labeled O3. High concentrations of O3

4 reaction products are found in the nasal lavage cells, bronchoalveolar lavage (BAL), mucus, 

5 surfactant, and cells, and in the epithelial cells of the lower airways, providing evidence that O3

6 reacts at the air-liquid interface. Model calculations of the nasal cavity based on diffusion equations 

7 and reaction rates of O3 with model substrates predict an O3 penetration distance (0.5 μm) less than 

8 the thickness of the mucus layer (10 μm) (Santiago et al., 2001, 019841). Thus, O3 may cause injury 

9 both by direct reaction with constituents of the lining layer and cells protruding from it, and by 

10 initiating a reaction cascade that carries the oxidative burden deeper into the tissues. 

Source: Panel (a) reproduced with permission from McGraw-Hill (Weibel, 1980, 079848). 

Figure 5-1. Structure of lower airways with progression from the large airways to the alveolus.﷒

Panel (a) illustrates basic airway anatomy. Structures are epithelial cells, EP; 

basement membrane, BM; smooth muscle cells, SM; and fibrocartilaginous coat, FC. 

Panel (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. 
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1 Ozone may interact with many of the components that make up the ELF including 

2 phospholipids, neutral lipids, free fatty acids, proteins, and low molecular weight antioxidants 

3 (Perez-Gil, 2008, 198890; Uppu et al., 1995, 076212). The composition of ELF will vary as a 

4 function of anatomic location, species, strain, and likely exposure due to substrate depletion. 

5 Reduced substrates enter the ELF from the cellular layer or are transported across the cell layer from 

6 blood plasma, or can be regenerated from previously oxidized moieties that are reduced by other

7 reduction reactions.

8 Ozone reacts with the double bond of lipids such as unsaturated fatty acids, a large component 

9 of ELF, to form stable and less reactive ozonide, aldehyde, and hydroperoxide reaction products via 

10 chemical reactions such as the Criegee ozonolysis mechanism (Figure 5-2) (Pryor et al., 1991, 

11 042482). Lipid ozonation products, such as the aldehydes hexanal, heptanal, and nonanal, have been 

12 recovered after O3 exposure in human bronchial alveolar lavage fluid (BALF), rat BALF, isolated rat 

13 lung, and in vitro systems (Frampton et al., 1999, 040757; Postlethwait et al., 1998, 086754; Pryor et 

14 al., 1996, 082697). It was estimated that 88% of the O3 that does not come in contact with 

15 antioxidants will react with unsaturated fatty acids including phospholipids or neutral lipids in the 

16 ELF (Uppu et al., 1995, 076212). Nonanal has been suggested as a relatively specific biomarker for 

17 O3 exposure since the monounsaturated fatty acid parent compound, oleic acid, does not undergo 

18 autoxidation (Pryor et al., 1996, 082697). Adducts of the aldehyde 4-hydroxynonenal were found in 

19 human alveolar macrophages after O3 exposure (Hamilton et al., 1998, 086157). Polyunsaturated 

20 fatty acid (PUFA) reactions are limited by the availability of O3 since lipids are so abundant in the 

21 ELF. Yields of O3-induced aldehydes were increased by the decrease in other substrates such as 

22 ascorbic acid (AH2) (Postlethwait et al., 1998, 086754). PUFA reactions may not generate sufficient 

23 bioactive materials to account for acute cell injury, however only modest amounts of products may 

24 be necessary to induce cytotoxicity (Postlethwait and Ultman, 2001, 196805; Postlethwait et al., 

25 1998, 086754). 
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Source: (U.S. EPA, 2006, 088089) 

Figure 5-2. Schematic overview of ozone interaction with PUFA in ELF and lung cells. It should 

be noted that not all secondary reaction products are shown.

1 Cholesterol is the most abundant neutral lipid in human ELF. Reaction of cholesterol with O3

2 results in biologically active cholesterol products such as the oxysterols, β-epoxide and 6-oxo-3,5-

3 diol (Murphy and Johnson, 2008, 197792; Pulfer and Murphy, 2004, 076673; Pulfer et al., 2005, 

4 076663). Product yields will depend on ozonolysis conditions, however cholesterol ozonolysis 

5 products were formed in similar abundance to phospholipid-derived ozonolysis products in rat ELF 

6 (Pulfer and Murphy, 2004, 076673). 

7 Antioxidant substances appear to be an important cellular defense against O3. The level and 

8 type of antioxidant present in ELF varies between species, regions of the respiratory tract, and can be 

9 altered by O3 exposure. Endogenous antioxidants such as urate (UA), ascorbate (AH2), and reduced 

10 glutathione (GSH) display high intrinsic reactivities toward O3, but do not possess equal O3

11 reactivity. In individual and in limited composite mixtures, UA was the most reactive antioxidant 

12 tested, followed by AH2 (Mudway and Kelly, 1998, 000273). In human BALF samples, the mean 

13 consumption of AH2 was greater than UA (Mudway et al., 1996, 080730). GSH was consistently less 

14 reactive than UA or AH2 (Kanofsky and Sima, 1995, 075973; Mudway and Kelly, 1998, 000273; 

15 Mudway et al., 1996, 080730). In a red cell based system, AH2 augmented the in vitro uptake of O3

16 by sixfold as computed by the mass balance across the exposure chamber (Ballinger et al., 2005, 

17 076649). In addition, O3-induced cell membrane oxidation required interactions with AH2 and GSH, 

18 but not UA or the vitamin E analog Trolox, however UA could block reactions of O3 with AH2

19 (Ballinger et al., 2005, 076649). The consumption of antioxidants by O3 was linear with time and 

20 positively correlated with initial substrate concentration and chamber O3 concentration (Mudway and 

21 Kelly, 1998, 000273; Mudway et al., 1996, 080730). However, estimated in vitro O3 uptake was not 

22 proportional to the production of O3-derived aldehydes from red cell membrane exposure (Ballinger 

23 et al., 2005, 076649). Studies with rats exposed to O3 show consumption of ascorbate that correlates 
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1 with O3 exposure (Gunnison and Hatch, 1999, 087204; Gunnison et al., 1996, 080803; Vincent et al., 

2 1996, 080778). 

3 ELF also contains proteins present in blood plasma as well as proteins secreted by surface 

4 epithelial cells. Ozone reactions with proteins have been studied by their in vitro reactions as well as 

5 reactions of their constituent amino acids (the most reactive of which are cysteine, histidine, 

6 methionine, tyrosine, and tryptophan). Ozone reaction with S-containing biomolecules has been 

7 shown to follow the following order: thiosulfate > ascorbate > cysteine ≈ methionine > glutathione 

8 (Kanofsky and Sima, 1995, 075973). Rate constants for the reaction of amino acids with O3 vary 

9 between investigations due to differing reaction conditions and assumptions; however aliphatic 

10 amino acids consistently are very slow to react with O3 (e.g., alanine: 25-100 moles/L/sec) (Hoigné 

11 and Bader, 1983, 625266; Ignatenko and Cherenkevich, 1985, 625265; Kanofsky and Sima, 1995, 

12 075973; Pryor et al., 1984, 595130). Uppu et al. (1995, 076212) predicted that 12% of inhaled O3

13 that does not react with antioxidants will react with proteins in the ELF, whereas 88% will react with 

14 PUFAs. 

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

16 mixtures. Individual antioxidant consumption rates decreased as the substrate mixture complexity 

17 increased (e.g., antioxidant mixtures and albumin addition) (Mudway and Kelly, 1998, 000273). 

18 However, O3 reactions with AH2 predominated over the reaction with lipids, when exposed to 

19 substrate solution mixtures (Postlethwait et al., 1998, 086754). It was suggested that O3 may react 

20 with other substrates once AH2 concentrations within the reaction plane fall sufficiently. 

21 Additionally, once AH2 was consumed, the absorption efficiency diminished, allowing inhaled O3 to 

22 be distributed to more distal airways (Postlethwait et al., 1998, 086754). Multiple studies have 

23 concluded O3 is more reactive with AH2 and UA than with the weakly reacting GSH (or cysteine or 

24 methionine) or with amino acid residues and protein thiols (Cross et al., 1992, 625299; Kanofsky 

25 and Sima, 1995, 075973). 

5.1.2.2. Recent Publications

26 Further experiments on the reaction kinetics between O3 and the antioxidants present in the 

27 ELF have been conducted since the last review. To quantify these reactions, Kermani, et al. (2006, 

28 195643) evaluated the interfacial exposure of aqueous solutions of UA, AH2, and GSH (50-200 μM) 

29 with O3 (1-5 ppm). Similar to the results of Mudway and Kelly (1998, 000273), this study found the 

30 hierarchy in reactivity between O3 and these antioxidants to be UA>AH2>>GSH. UA and AH2

31 shared a 1:1 stoichiometry with O3, whereas 2.5 moles of GSH were consumed per mole of O3. 

Using these stoichiometries, reaction rate constants were derived (5.8×104 32 moles/L/sec, 

5.5×104 moles/L/sec, and 57.5/M0.75 33 /sec for the reaction of O3 with UA, AH2, and GSH, 

34 respectively). These values are similar to those derived from data presented in Mudway and Kelly 

35 (1998, 000273). Other studies reported reactive rate constants that are two to three orders of 

36 magnitude larger, however these studies used higher concentrations of O3 and antioxidants under less 
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1 physiologically relevant experimental conditions (Giamalva et al., 1985, 595129; Kanofsky and 

2 Sima, 1995, 075973; Pryor et al., 1984, 595130). 

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

4 initial air-liquid interface interactions of O3 with ELF components (e.g., antioxidants and proteins) in 

5 ~1 millisecond (Enami et al., 2008, 195834; Enami et al., 2008, 195833; Enami et al., 2009, 197791; 

6 Enami et al., 2009, 195835; Enami et al., 2009, 195621). Solutions of aqueous UA, AH2, GSH, 

7 α-tocopherol (α-TOH), and protein cysteines (CyS) were sprayed as microdroplets in O3(g)/N2

8 mixtures at atmospheric pressure and analyzed by electrospray mass spectrometry. These recent 

9 studies demonstrated different reactivity toward AH2, UA, and GSH by O3 in the gas phase 

10 compared to the liquid phase thus supporting the relevance of reactions between gas phase O3 and 

11 ELF.

12 As was seen in previous studies (Kanofsky and Sima, 1995, 075973; Kermani et al., 2006, 

13 195643), the hierarchy of reactivity of these ELF components with O3(g) was determined to be AH2

14 ≈ UA > CyS > GSH. There was some variance between the reaction rates and product formation of 

15 UA, AH2, and GSH with O3(g) as investigated by Enami et al. versus O3(aq) as described previously. 

16 UA was more reactive than AH2 toward O3(aq), but in reactions with O3(g), these antioxidants have 

17 equivalent reactivity (Enami et al., 2008, 195834). As O3 is a kinetically slow one-electron acceptor 

18 but very reactive O-atom donor, products of the interaction of O3 with UA, AH2, GSH, CyS, and αTOH result from addition of n O-atoms (n = 1-4). These products included epoxides (e.g., U-O- 19 ), 

peroxides (e.g. U-O2

-

), and ozonides (e.g., U-O3

- 20 ). For instance, GSH was oxidized to sulfonates 

(GSO3

-

/GSO3

2- 21 ), not glutathione disulfide (GSSG) by O3(g) (Enami et al., 2009, 197791). However, 

22 it is possible that other oxidative species are oxidizing GSH in vivo, since sulfonates are not detected 

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

24 with GSH than other antioxidants, such that < 3% of O3 will be scavenged by GSH when in 

25 equimolar amounts with AH2 (Enami et al., 2009, 197791). 

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

27 3-4), such as those that may result from acidic particulate exposure or pathological conditions like 

28 asthma (pH ≈ 6), decreased the scavenging ability of UA and GSH for O3; such that at low pH, the 

29 scavenging of O3 must be taken over by other antioxidants, such as AH2 (Enami et al., 2008, 195834; 

30 Enami et al., 2009, 197791). Also, under acidic conditions (pH ≈ 5), the ozonolysis products of AH2

31 shifted from the innocuous dehydroascorbic acid to the more persistent products, ascorbate ozonide 

32 and threonic acid (Enami et al., 2008, 195833). It is possible that the acidification of the ELF by 

33 acidic co-pollutant exposure will increase the toxicity of O3 by preventing some antioxidant 

34 reactions and shifting the reaction products to more persistent compounds.

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

36 barrier and target for inhaled O3. The thickness of the lining fluid and mucus layer is an important 

37 determinant of the dose of O3 to the tissues. The antioxidant substances present in the ELF appear in 

38 most cases to limit interaction of O3 with underlying tissues and to prevent penetration of O3 deeper 
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1 into the lung. However, new findings indicate that in some cases, the antioxidants might themselves 

2 be participating in harmful reactions. The formation of toxic reaction products is likely related to the 

3 concentration of antioxidants present and the quenching ability of the lining fluid. New findings also 

4 emphasize the importance of gaseous O3 acting at the air-liquid interface in initiating a reaction 

5 cascade, and point out important distinctions in the reaction rates and product formation between 

6 gaseous and aqueous O3 reactivity toward airway antioxidants. In addition to the highly reactive O3, 

7 secondary oxidation products formed in the aqueous phase might penetrate into the cells and cause 

8 injury (Section 5.2).

5.1.3. Ozone Uptake 

5.1.3.1. Sum ma ry o f Findings fro m the 2006 Ozone AQCD

9 Past AQCDs (U.S. EPA, 1996, 017831; U.S. EPA, 2006, 088089) provide information on the 

10 majority of literature relevant to understanding the state of the science in O3 dosimetry. One method 

11 of addressing the question of O3 dosimetry is to measure the amount of O3 removed from the air 

12 stream during breathing (termed “uptake”). The percentage of the O3 in the air stream that is 

13 removed is termed “uptake efficiency.” Uptake studies have utilized both bolus and continuous O3

14 exposure techniques as well as dosimetry modeling to investigate uptake efficiency and distribution 

15 of O3 uptake between upper and lower respiratory tract regions. A number of studies that have 

16 measured the fractional O3 uptake efficiency of the human respiratory tract (FRT), upper airways 

17 (FUA), and lower respiratory tract (FLRT) are presented in Table 5-1 as a review. 

Table 5-1. Human respiratory tract uptake efficiency data

Reference Mouth/Nosea Inspiratory Flow 

(mL/s) VT (mL) fB (bpm)b

 FRT FUA FLRT

CONTINUOUS EXPOSURE

Gerrity et al. (1988, 040899) 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, 041314)

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, 042785) Mouthpiece 330 825 12 0.91 0.27 0.95

Wiester et al. (1996, 041280) M 539 631 16 0.76

N 514 642 16 0.73

Santiago et al. (2001, 019841) N 50 0.80d

N 250 0.33

Rigas et al. (2000, 010454) Face mask 480 1,100 27.6 0.86

BOLUS EXPOSURE

Hu et al. (1992, 042794) Mouthpiece 250 0.96 0.46
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Target Sites for Ozone Dose

1 A primary uptake site of O3 delivery to the lungs is believed to be the centriacinar region 

2 (CAR). The CAR refers to the zone at the junction of the conducting airways and the gas exchange 

3 region. This area is also considered the proximal alveolar region (PAR) and is defined as the first 

4 generation distal to the terminal bronchioles. Contained within the CAR, the respiratory bronchioles 

were confirmed as the site receiving the greatest O3 dose (18 5 O mass/lung weight) in resting O3

6 exposed rhesus monkeys, when not considering the nose (Plopper et al., 1998, 087203). 

7 Furthermore, the greatest cellular injury occurred in the vicinity of the respiratory bronchioles and 

was dependent on the delivered O3 dose to these tissues. However, 18 8 O label was detected to a lesser 

9 extent in other regions of the tracheobronchial airway tree, showing that O3 is delivered to these 

10 compartments as well, resulting in a smaller dose. Models predict that the net O3 dose (O3 flux to air11 liquid interface) gradually decreases distally from the trachea toward the end of the tracheobronchial 

12 region (TB) and then rapidly decreases in the pulmonary region (Miller et al., 1985, 040307). 

13 However, the tissue dose (O3 flux to liquid-tissue interface) is low in the trachea, increases to a 

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

15 pulmonary region. These models are limited by the exclusion of the upper respiratory tract as well as 

16 reactions occurring between ELF constituents and O3 after the 16th generation, representing the 

17 CAR region (Miller et al., 1985, 040307). 

Nasopharyngeal Removal and Dose of Ozone 

18 In both animals and humans, about 50% of the absorbed O3 is removed in the head (nose, 

19 mouth, and pharynx), about 7% in the larynx/trachea, and about 43% in the lungs (Hatch et al., 1989, 

20 041799; Hu et al., 1992, 042794). The nasopharyngeal region provides a defense against O3 entering 

21 the lungs by removing half of the inhaled O3 from the airstream. The limiting factors in nasal O3

22 uptake are simultaneous diffusion and chemical reaction of O3 in the nasal ELF layer (Santiago et al., 

23 2001, 019841). The lining layer in the nose is thicker than in the lungs, but, like for the lungs, 

Ultman et al. (1994, 041746) Mouthpiece 250 500e 15 0.30 

Mouthpiece 250 500 15 0.47

Ultman et al. (2004, 057197) M 490 450e 32.7 0.87

M 517 574 27 0.91

Nodelman and Ultman (1999, 015112) 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 by natural breathing; N = nasal exposure by natural breathing; M/N = pooled data from mouth and nasal exposure; mouthpiece = 

exposure by mouthpiece; FRT = total RT uptake; FUA = upper airway 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, 040899) and Gerrity et al. (1994, 041314) did not include the contribution from UA uptake efficiency during 

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

d

FUA from Santiago et al. (2001, 019841) represents nasal absorption (Fnose). e

VT is computed from flow and fB. 
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1 mathematical estimates predict that O3 penetrates less than the thickness of the ELF layer leaving the 

2 reaction products as likely the agents damaging the nasal tissue. The percentage of O3 taken up is 

3 inversely related to flow rate and weakly related to inlet O3 concentration. It was hypothesized that 

4 the nonlinear reaction kinetics of the nose could result from the depleting substrates in the nasal ELF 

5 becoming the limiting factor of the reaction (Santiago et al., 2001, 019841). 

6 Uptake efficiencies have been calculated for various segments of the upper airways 

7 (Table 5-1). Gerrity et al. (1995, 042785) reported unidirectional uptake efficiencies of O3 inhaled 

8 from a mouthpiece of 17.6% from the mouth to vocal cords, 12.8% from the vocal cords to the upper 

9 trachea (totaling 27.0%), 11.5% from the upper trachea to the main bifurcation carina (totaling 

10 35.5%), and essentially zero between the carina and the bronchus intermedius (totaling 32.5%). 

11 These values are lower than those calculated by Hu et al. (1992, 042794) that reported uptake 

12 efficiencies of 21, 36, 44, and 46% between the mouth and the vocal cords, the upper trachea, the 

13 main bifurcation carina, and the bronchus intermedius, respectively. The lower efficiencies seen in 

14 Gerrity et al. (1995, 042785) may have resulted from the mouthpiece scrubbing O3 from the breath 

15 during inhalation. Overall, the nasopharyngeal region removes half of the inhaled O3 by reactions in 

16 the nasal ELF. The exact uptake efficiency will change due to variations in flow rate and inhaled 

17 concentration. 

Pulmonary Ozone Uptake and Dose

18 O3 uptake in rats is approximately 54% efficient (Hatch et al., 1989, 041799), while in humans 

19 at rest it ranges from 80-95% efficient (Hu et al., 1992, 042794). Approximately 43% of inhaled O3

20 is absorbed in the lungs of both humans and animals. Uptake efficiency is affected by changes in a 

21 number of variables, including tidal volume (VT), breathing frequency (fB), exposure time, minute 

22 volume, and O3 concentration. Simulations from the Overton et al. (1996, 080733) single-path 

23 anatomical respiratory tract model, where the upper and lower respiratory tracts were modeled but 

24 uptake by the upper airways was not considered, predicted that fractional uptake and PAR O3 dose 

25 increased with VT. Likewise, experimental studies found that O3 uptake is positively correlated with 

26 changes in VT (Gerrity et al., 1988, 040899; Ultman et al., 2004, 057197). Also, O3 exposure leads to 

27 a reflex mediated increase in fB and reduction in VT, hypothesized to be protective by decreasing the 

28 dose delivered in the lung (Gerrity et al., 1994, 041314). While maintaining a constant minute 

29 volume, a decrease in VT will result in an increase in fB. Nasal flow rate (Santiago et al., 2001, 

30 019841) and fB are inversely related to O3 uptake, such that an increase in fB will decrease uptake 

31 efficiency (Figure 5-3) (Gerrity et al., 1988, 040899; Ultman et al., 2004, 057197; Wiester et al., 

32 1996, 041280). Modeling also predicted a decrease in fractional uptake with increased fB, but an 

33 increase in PAR dose with increased fB (Overton et al., 1996, 080733). Similarly, increased fB (80 - 

160 bpm) and rapid shallow breathing in rats resulted in a decrease in midlevel tracheal 18 34 O content 

and an increase in 18 35 O content in the mainstem bronchi (Alfaro et al., 2004, 053551). This 

36 dependence may be a result of frequency-induced alterations in contact time that affects the first-
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1 order absorption rate for O3 (Postlethwait et al., 1994, 044219). Also, an association with O3 uptake 

2 efficiency was found with minute volume and exposure time. 

Source: Used with permission from Health Effects Institute, Ultman et al. (2004, 057197) 

Figure 5-3. Total ozone uptake efficiency as a function of breathing frequency at a minute 

ventilation of 30 L/min. Subjects breathed 0.25 ppm ozone oronasally through 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).

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

4 O3 is absorbed in the upper airways and shifts uptake to the central and lower airways and 

5 respiratory airspaces (Hu et al., 1994, 041323; Nodelman and Ultman, 1999, 015112; Ultman et al., 

6 1994, 041746). Hu et al. (1994, 041323; Ultman et al., 1994, 041746) found that O3 absorption 

7 increases relative to the respiratory tract penetration volume (Vp) of a bolus O3 dose (Figure 5-4). 

8 Ozone uptake efficiency and Vp are not affected by bolus O3 concentration (Gerrity et al., 1988, 

9 040899; Hu et al., 1992, 042794; Kabel et al., 1994, 095597), indicating that O3 uptake is a linear 

10 absorption process, where the diffusion and chemical reaction rates of O3 are proportional to the O3

11 concentration. This relationship was not true for nasal cavity uptake, which was proportional to O3

12 concentration in the inlet air (Santiago et al., 2001, 019841), or in exercising individuals, where 

13 uptake efficiency had a weak but significant negative dependence on O3 concentration (Rigas et al., 

14 2000, 010454). Overall O3 inhalation uptake is over 80% efficient, but the exact efficiency that 

15 determines how much O3 is available at longitudinally distributed compartments in the lung is 

16 sensitive to changes in VT, fB, and exposure time. Increased fB will shift the O3 uptake from the upper 

17 airways to the central airways and respiratory airspaces.
March 2011 5-12 DRAFT – DO NOT CITE OR QUOTE 

Source: Adapted with permission from Health Effects Institute, Ultman et al. (2004, 057197) 

Figure 5-4. Ozone uptake fraction as a function of volumetric penetration (Vp) in a representative 

subject. ﷒Each point represents the ozone 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, 

057197) found that Vp50% was well correlated with VD and better correlated with the 

volume of the conducting airways, i.e., VD minus VUA. 

Mode of Breathing

1 Ozone uptake and distribution is highly sensitive to the mode of breathing. Variability in 

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

3 compared to oral breathing. This could be a result of O3 scrubbing in the nasal passageways that are 

4 bypassed by oral breathing. Studies by Ultman and colleagues using bolus inhalation demonstrate 

5 that O3 uptake fraction is greater during nasal breathing than during oral breathing at each Vp (Kabel 

6 et al., 1994, 095597; Nodelman and Ultman, 1999, 015112; Ultman et al., 1994, 041746). However, 

7 oral breathing results in deeper penetration of O3 into the lung with a higher absorbed fraction in the 

8 pharyngeal, lower airways, and respiratory airways (Nodelman and Ultman, 1999, 015112). Similar 

9 results were obtained from O3 uptake studies in dogs (Yokoyama and Frank, 1972, 039756); 

10 however earlier human studies suggest that oral or oronasal breathing results in a higher O3 uptake 

11 efficiency than nasal breathing (Gerrity et al., 1988, 040899; Wiester et al., 1996, 041280). These 

12 human studies measured total respiratory tract absorption after continuous O3 exposure using a 

13 pharyngeal sampling tube, which may decrease sensitivity and lead to measurement errors.

Interindividual Variability 

14 Similarly exposed individuals vary in the amount of actual dose received with intersubject 

15 differences in fractional O3 uptake by the respiratory tract accounting for the majority of total 
March 2011 5-13 DRAFT – DO NOT CITE OR QUOTE 

1 variation in O3 uptake efficiency (Bush et al., 1996, 080763; Rigas et al., 2000, 010454; Santiago et 

2 al., 2001, 019841). Interindividual variability accounted for between 10-50% of the absolute 

3 variability in O3 uptake measurements (Rigas et al., 2000, 010454; Santiago et al., 2001, 019841). 

4 When concentration, time, and minute ventilation are held constant, fractional absorption ranges 

5 from 0.80 to 0.91 (Rigas et al., 2000, 010454). It has been hypothesized that interindividual variation 

6 in O3 uptake is the result of substituting a dose surrogate, such as exposure concentration or inhaled 

7 dose, for the actual O3 dose delivered to the tissues. 

8 Variability in local dose may be attributed to differences in the pulmonary physiology. Since 

9 the conducting airways (CA) remove the majority of inhaled O3 before it reaches the gas exchange 

10 region, the volume and surface area of the upper airways will influence O3 uptake. Models predict 

11 that fractional O3 uptake and PAR dose (flux of O3 to the PAR surfaces divided by exposure 

12 concentration) increase with decreasing TB volume and decreasing TB region expansion. On the 

13 contrary, alveolar expansion had minimal effect on uptake efficiency as little O3 reaches the 

14 peripheral lung (Bush et al., 2001, 016665; Overton et al., 1996, 080733). Ozone uptake is virtually 

15 complete by the time O3 reaches the alveolar spaces of the lung (Postlethwait et al., 1994, 044219). 

16 Experimental studies have found that differences in CA volumes may account for 75% of the

17 variation in absorption between subjects (Ultman et al., 2004, 057197). In support of this concept, 

18 regression analysis showed that O3 absorption is positively correlated with anatomical dead space 

19 (VD) and CA volume (i.e., VD minus VUA), but not total lung capacity (TLC), forced vital capacity 

20 (FVC), or functional residual capacity (FRC) (Bush et al., 1996, 080763; Hu et al., 1994, 041323; 

21 Postlethwait et al., 1994, 044219; Ultman et al., 2004, 057197). Variability in VD is correlated more 

22 with the variability in the CA than the upper airways. Similarly, uptake was correlated with changes 

23 in individual bronchial cross-sectional area, indicating that changes in cross-sectional area available 

24 for gas diffusion are related to overall O3 retention (Reeser et al., 2005, 195718; Ultman et al., 2004, 

25 057197). These studies provide support to the pulmonary physiology, especially the CA volume and 

26 surface area, playing a key role in variability of O3 uptake between individuals.

27 When absorption data between genders is normalized to Vp/VD, then the variability attributed 

28 to gender differences is no longer distinguishable (Bush et al., 1996, 080763). A physiologically 

29 based pharmacokinetic (PBPK) model simulating O3 uptake indicates that regional extraction of O3

30 is relatively insensitive to age, but extraction per unit surface area is two- to eightfold higher in 

31 infants compared to adults, due to the fact that children under age 5 have much a much smaller 

32 airway surface area in the extrathoracic (nasal) and pulmonary regions (Sarangapani et al., 2003, 

33 054581). 

Correlation of Dose and Response

34 Two studies have investigated the correlation of O3 uptake with the pulmonary function 

35 responses to O3 exposure (Gerrity et al., 1994, 041314; Reeser et al., 2005, 195718). These studies 

36 found that the large subject-to-subject variability in %ΔFEV1 response to O3 does not appear to have 
March 2011 5-14 DRAFT – DO NOT CITE OR QUOTE 

1 a dosimetric explanation. Reeser et al. (2005, 195718) found no significant relationship between 

2 %ΔFEV1 and fractional absorption of O3 using the bolus method. Contrary to previous findings, the 

3 percent change in dead space volume of the respiratory tract (%ΔVD) did not correlate with O3

4 uptake, possibly due to the contraction of dead space caused by airway closure. Gerrity et al. (1994, 

5 041314) found that intersubject variability in FEV1 and airway resistance was not related to 

6 differences in the O3 dose delivered to the lower airways, whereas minute ventilation was predictive 

7 of FEV1 decrement. No study has yet demonstrated that subjects show a consistent pattern of O3

8 retention when re-exposed over weeks of time, as has been shown to be the case for the FEV1

9 response, or that within-subject variation in FEV1 response is related to fluctuations in O3 uptake.

10 On the contrary, cellular injury and inflammation have been found to correlate with the site11 specific O3 dose. Contained within the CAR, the respiratory bronchioles were confirmed as the site 

receiving the greatest O3 dose (18 12 O mass/lung weight) and sustained the greatest cellular injury in O3

13 (0.4 and 1.0 ppm) exposed resting rhesus monkeys (Plopper et al., 1998, 087203). The respiratory 

14 bronchioles, having the highest concentration of local O3 dose, were also the site of significant GSH 

15 reduction. 

Co-Pollutant and Sequential Ozone Exposure

16 Previous continuous O3 exposure (0.12 or 0.36 ppm) decreased bolus O3 uptake, possibly due 

17 to depletion of compounds able to react with O3 (Asplund et al., 1996, 082505; Rigas et al., 1997, 

18 083602). Conversely, O3 (0.36 ppm) bolus uptake was increased with prior NO2 (0.36 or 0.72 ppm) 

19 or SO2 (0.36 ppm) exposure (Rigas et al., 1997, 083602). It was hypothesized that this increased 

20 fractional absorption could be due to increased production of reactive substrates in the ELF due to 

21 oxidant-induced airway inflammation. 

Physical Activity

22 Exercise increases the overall exposure of the lung due in most part to the increased volume of 

23 air passing through the lung. Exercise increases breathing frequency and flow rate. According to 

24 present thinking, doubling minute ventilation is assumed to lead to a doubling of dose, however, the 

25 linearity of the dose relative to ventilation relationship has not been carefully studied. A recent study 

26 by Sawyer et al. (2007, 195142) showed that doubling minute ventilation led to only a 1.6-fold 

27 higher dose of O3 in the lower airway. In addition to increasing the quantity of O3 in the lung, 

28 exercise also has been shown to lead to a switch to oronasal breathing. By increasing flow to what is 

29 common in moderate exercise, the upper airways absorbed a smaller fraction of the O3 (~0.50 at 

30 quiet breathing to 0.10 at exercise); however, the trachea and more distal conducting airways 

31 received higher doses than during quiet breathing (0.65 absorbed in the lower conducting airways, 

32 and 0.25 absorbed in the respiratory zone) (Hu et al., 1994, 041323). The same shift in the O3 dose 

33 distribution to deeper into the lung occurred in other studies mimicking the effects of exercise 

34 (Nodelman and Ultman, 1999, 015112). 
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5.1.3.2. Recent Publications

1 Few new studies have investigated the uptake of O3 in the respiratory tract since the end of the 

2 last O3 assessment (U.S. EPA, 2006, 088089). The studies that have been conducted agree with the 

3 results presented above and do not change the dosimetry conclusions of the last document. 

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

5 reproducible pattern of severity between daughter branches of individual bifurcations that is 

6 dependent on the O3 concentration-time profile of the inhaled gas. A 3-dimensional computational 

7 fluid dynamics model was created to investigate the dose-response relationship leading to the 

8 distribution of damage in a single airway bifurcation (Taylor et al., 2007, 195717). The model 

9 consisted of one parent branch and two symmetrical daughter branches with a branching angle of 90° 

10 and a sharp carinal ridge. Various flow scenarios were simulated using Reynolds numbers (Re) 

11 ranging from 100 to 500. The Re that corresponds to a certain airway generation is dependent upon 

12 both lung size and minute ventilation, such that the range in Re from 100-500 would encompass 

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

14 respectively, whereas the same Re range corresponds to generations 0-4, 1-6, and 4-8 for a 4-year15 old child. Consistent with early physical models of Schroter and Sudlow (1969, 071359), the model 

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

17 throughout the parent branch, but skewed towards the inner wall within the daughter branches. 

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

19 slightly skewed towards the outer walls of the daughter branches. Hot spots of wall flux existed at 

20 the carina during inspiration and expiration with Re >100. Additional hot spots were found during 

21 expiration on the parent branch wall downstream of the branching region. 

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

23 rest of the respiratory tract from damage from inspired O3 (Gerrity et al., 1988, 040899; Santiago et 

24 al., 2001, 019841). Sawyer et al. (2007, 195142) further investigated nasal uptake of O3 in healthy 

25 adults during exercise. Fractional O3 uptake, acoustic rhinometry (AR), and nasal NO measurements 

26 were taken on ten adults (8 W, 2 M) exposed to 0.2 ppm O3 before and after moderate exercise at 

27 two flow rates (10 and 20 L/min). The percent nasal uptake of O3 was ~50% greater at 10 L/min 

28 compared to 20 L/min both pre- and postexercise. However, the inhaled O3 delivery rate to the lung 

29 (i.e., flow rate X [O3 ppm] X nasal O3 penetration) was 1.6-fold greater at the higher flow than at the 

30 lower flow (2.5 compared to 0.9 ppm·L/min). Exercise did not affect O3 uptake at either flow rate, 

31 but did significantly increase nasal volume (Vn) and AR measurements of nasal cross-sectional area 

32 (MCA, CSA2, and CSA3) (p ≤ 0.05). Conversely, exercise decreased nasal resistance (Rn) (p < 0.01) 

33 and NO production (p > 0.05). The change in Vn and CSA2:MCA ratio was correlated with the 

34 percent change in nasal uptake, however the overall effect was small and sensitive to elimination of 

35 outliers and gender segregation. 

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

37 affect the fractional uptake of a bolus dose of O3 in apparently healthy smokers with limited smoking 
March 2011 5-16 DRAFT – DO NOT CITE OR QUOTE 

1 history (Bates et al., 2009, 195727). Despite similar internal O3 dose distribution, the smokers 

2 exhibited greater pulmonary responses to O3 bolus exposures, measured as FEV1 decrements and 

3 increases in the normalized slope of the alveolar plateau (SN). This is contrary to previous studies 

4 conducted in smokers with a greater smoking history that found decreased O3 induced decrements in 

5 FEV1 in smokers (Emmons and Foster, 1991, 042430; Frampton et al., 1997, 082692). 

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

7 intersubject variability in nasal O3 uptake determined by Sawyer et al. (2007, 195142) ranged from 

8 26.8 to 65.4% (pre- and postexercise). A second study investigating the use of the CO2 expirogram to 

9 quantify pulmonary responses to O3 found that intersubject variability accounted for 50% of the 

10 overall variance in the study (Taylor et al., 2006, 195731). 

11 In summary, O3 uptake efficiency is sensitive to a number of factors. As discussed before, the 

12 characteristics of the ELF layer is a key determinant in the dose of O3 that reaches the tissue layer. 

13 Fractional absorption will decrease with increased flow and increase proportional to VT. Decreased 

14 uptake efficiency due to increased fB and oronasal breathing, as occurs during exercise, will shift the 

15 O3 dose distribution deeper and lead to a greater dose to the lower respiratory tract. Individual total 

16 airway O3 uptake efficiency is also sensitive to large changes in O3 concentration, exposure time, and 

17 VE. Major sources of variability in absorption of O3 include O3 concentration, exposure time, 

18 breathing frequency, minute volume, and tidal volume, but the interindividual variation is the 

19 greatest source of variability uptake efficiency. However, to this date, studies have failed to show 

20 that the large differences in biological response between subjects (FEV1, BAL cell inflammatory 

21 response, etc.) are explainable by the differences in O3 uptake. Recent studies have provided 

22 evidence for hot spots of O3 flux around bifurcations in the airways. 

5.1.4. Sp e cie s Ho molo gy, Se ns itivity, a nd Animal-to-Human Dos e 

Extrapolation

5.1.4.1. Sum ma ry o f Findings fro m 2006 Ozone AQCD

23 The 2006 O3 AQCD (U.S. EPA, 2006, 088089) discussed the suitability of animal models for 

24 comparison with human O3 exposure and concluded that the acute and chronic functional responses 

25 of laboratory animals to O3 appear qualitatively homologous to human responses. Thus, animal 

26 studies can provide important data in determining cause-effect relationships between exposure and 

27 health outcome that would be impossible to collect in human studies. Still, care must be taken when 

28 comparing quantitative dose-response relationships in animal models to humans due to obvious 

29 interspecies differences. 

30 Physiological and anatomical differences exist between experimental species. Primates are 

31 oronasal breathers with a dichotomous branching lung structure, while rodents are obligate nasal 

32 breathers with a monopodial branching lung structure. In addition, rodents have fewer terminal 

33 bronchioles, the major site of O3 uptake, compared to humans or canines (McBride, 1992, 078532). 
March 2011 5-17 DRAFT – DO NOT CITE OR QUOTE 

1 Past studies of the effect of body size on resting oxygen consumption also suggest that rodents inhale 

2 more volume of air per lung mass than primates. These distinctions as well as differences in nasal 

3 structure between primates and rodents could affect the site and amount of O3 uptake. Also, because 

4 of their higher body surface to volume ratio, rodents can rapidly lower body temperature during 

5 exposure leading to lowered O3 dose and toxicity (Iwasaki et al., 1998, 086165; Slade et al., 1997, 

6 082708; Watkinson et al., 2003, 050547). In addition to lowering the O3 dose to the lungs, this 

7 hypothermic response may cause: (1) lower metabolic rate, (2) altered enzyme kinetics, and (3) 

8 altered membrane function. The thermoregulatory mechanisms also may affect disruption of heart 

9 rate which may lead to: (1) decreased cardiac output (CO), (2) lowered blood pressure (BP), and (3) 

10 decreased tissue perfusion (Watkinson et al., 2003, 050547). These responses have not been 

11 observed in humans except at very high exposures, thus further complicating extrapolation of effects 

12 from animals to humans.

13 Sensitivity to heath effects from O3 varies between and within species, as well as between 

14 endpoints. Rodents appear to have a slightly higher tachypneic response to O3 and are less sensitive 

15 to changes in pulmonary function test than humans (U.S. EPA, 1996, 017831). However, rats do 

16 experience attenuation of pulmonary function and tachypneic ventilatory responses, similar to 

17 humans (Wiester et al., 1996, 080829). Hatch et al. (1986, 040472) reported that guinea pigs were 

18 the most responsive to O3-induced inflammatory cell and protein influx. Rabbits were the least 

19 responsive and rats, hamsters, and mice were intermediate responders. Further analysis of this study 

20 by Miller et al. (1988, 041545) found that the protein levels in guinea pigs increased more rapidly 

21 with predicted pulmonary tissue dose than in rats and rabbits. Alveolar macrophages isolated from 

22 guinea pigs and humans mounted similar qualitative and quantitative cytokine responses to in vitro

23 O3 (0.1-1.0 ppm for 60 minutes) exposure (Arsalane et al., 1995, 077430). 

24 Humans and animal models are similar in the pattern of regional O3 dose, but absolute values 

25 differ. Hatch et al. (1994, 038953) reported that exercising humans exposed to oxygen-18 labeled O3

26 (0.4 ppm) accumulated 4-5 times higher concentrations of O3 reaction product in BAL cells, 

27 surfactant and protein fractions compared to resting rats similarly exposed (0.4 ppm). It was 

necessary to expose resting rats to 2 ppm O3 to achieve the same BAL accumulation of 18 28 O reaction 

29 product that was observed in humans exposed to 0.4 ppm with intermittent heavy exercise. The 

concentration of 18 30 O reaction product in BAL paralleled the accumulation of BAL protein and 

31 cellular effects of the O3 exposure observed such that these responses to 2.0 ppm O3 were similar to 

32 those of the 0.4 ppm O3 in exercising humans.

33 As O3 absorption and activity relies on ELF antioxidant substances as described in Section 5.2, 

34 variability in antioxidant concentrations and metabolism between species may affect dose and O3-

35 induced health outcomes. Guinea pigs and mice have a lower basal activity of GSH transferase and 

36 GSH peroxidase, and lower vitamin E levels in the lung compared to rats (Ichinose et al., 1988, 

37 041805; Sagai et al., 1987, 041704). Nasal lavage fluid analysis shows that humans have a higher 

38 proportion of their nasal antioxidants as UA and low levels of AH2 whereas mice, rats, or guinea pigs 
March 2011 5-18 DRAFT – DO NOT CITE OR QUOTE 

1 have high levels of AH2 and undetectable levels of UA (Figure 5-5a). GSH is not detected in the 

2 nasal lavage of most of these species, but is present in monkey nasal lavage. Guinea pigs and rats 

3 have a higher antioxidant to protein ratio in nasal lavage and BAL fluid than humans (Hatch, 1992, 

4 043901). The BALF profile differs from the nasal lavage (Figure 5-5b). Humans have a higher 

5 proportion of GSH and less AH2 making up their BALF content compared to the guinea pigs and rats 

6 (Hatch, 1992, 043901; Slade et al., 1993, 042865). Similar to the nose, rats have the highest 

7 antioxidant to protein mass ratio found in BALF (Slade et al., 1993, 042865). Antioxidant defenses 

8 also vary with age (Servais et al., 2005, 195667) and exposure history (Duan et al., 1996, 080791). 

9 Duan et al. (1993, 086326; 1996, 080791) reported that differences in antioxidant levels between 

10 species and lung regions did not appear to be the primary factor in O3 induced tissue injury. 

11 However, a close association between site-specific O3 dose, the degree of epithelial injury, and 

12 reduced glutathione depletion was later revealed in monkeys (Plopper et al., 1998, 087203). 
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 a. 

Rat

Guinea pig

Human

0 200 400 600 800 1000

Antioxidant / Protein, nanomoles / gram

Ascorbic acid

Uric acid

Glutathione

 

b.

 

Guinea pig

Rat

Human

0 50 100 150 200 250

Antioxidant / Protein, nanomoles / gram

Ascorbic acid

Uric acid

Glutathione

Source: Adapted with permission from CRC Press, Inc., Hatch (1992, 043901) and with permission from Slade et al. (1993, 042865) 

Figure 5-5. Species comparison of antioxidant / protein ratios of: (a) nasal lavage fluid and, 

(b) bronchoalveolar lavage fluid. 

5.1.4.2. Recent Publications

1 There have been few new publications examining interspecies differences in dosimetry and 

2 response to O3 since the last AQCD. These studies do not overtly change the conclusions discussed 

3 above from the previous document (U.S. EPA, 2006, 088089). 
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1 A quantitative comparison of O3 transport in the airways of rats, dogs, and humans was 

2 conducted using a three-compartment airways model, based on upper and lower airway casts and 

3 mathematical calculation for alveolar parameters (Tsujino et al., 2005, 195842). The model was 

4 designed as cylindrical tubes with constant volume and one-dimensional gas movement and no 

5 airway branching patterns. It used data for solubility of O3 as well as measured nasopharyngeal 

6 removal rates of O3 published previously. This model examined how interspecies anatomical and 

7 physiological differences affect intra-airway O3 concentrations and the amount of gas absorbed (10% 

8 O3 exposure). Peak, real-time, and mean O3 concentrations were higher in the upper and lower 

9 airways of humans compared to rats and dogs, but lowest in the alveoli of humans. The amount of O3

absorbed was lowest in humans when normalized by body weight (8.47 × 10-8 10 g/kg compared to 

1.1 × 10-7 in rats and 1.46 × 10-7 11 in dogs). The intra-airway concentration decreased distally in all 

12 species. Sensitivity analysis demonstrated that VT, fB, and upper and lower airways surface area had 

13 a significant impact on model results. The model is limited in that it did not account for chemical 

14 reactions in the ELF or consider gas diffusion as a driving force for O3 transport. Also, the model 

15 was run at a respiratory rate of 16/min simulating a resting individual, however exercise may cause a 

16 further deviation from animal models as was seen in Hatch et al. (1994, 038953). 

17 To further understand the genetic basis for age-dependent differential response to O3, adult 

18 (15 weeks old) and neonatal (15-16 days old) mice from 8 genetically diverse strains were examined 

19 for O3-induced (0.8 ppm for 5 hours) pulmonary injury and lung inflammation (Vancza et al., 2009, 

20 596419). Ozone exposure increased polymorphonuclear leukocytes (PMN) influx in all strains of 

21 mice tested, but significantly in only some sensitive strains, suggesting a genetic background effect. 

22 This strain difference was not due to differences in delivered dose of O3 to the lung, evidenced by 

18 23 O lung enrichment. The sensitivity of strains for O3-induced increases in BAL protein and PMNs 

24 was different for different strains of rats suggesting that genetic factors contribute to heightened 

responses. Interestingly, adult mice accumulated more than twice the levels of 18 25 O reaction product 

26 of O3 than corresponding strain neonates. Thus, it appeared that the infant mice showed a two- to 

27 threefold higher response than the adults when expressed relative to the accumulated O3 reaction 

28 product in their lungs. The apparent decrease in delivered O3 dose in neonates could be a result of a 

29 more rapid loss of body temperature in infant rats incident to maternal separation and chamber air 

30 flow. 

31 The three-dimensional detail of the nasal passages of immature Rhesus macaque monkeys

32 were analyzed for developing predictive dosimetry models and exposure-dose-response relationships

33 (Carey et al., 2007, 195752). In doing so the authors report that the relative amounts of the five 

34 epithelial cell types in the nasal airways of monkeys remains consistent between infancy and 

35 adulthood (comparing to (Gross et al., 1982, 040121; Gross et al., 1987, 625447)). Ozone exposures 

36 (0.5 ppm, 8 h/day under acute [5 days] and episodic conditions [5 replicates of the acute paradigm 

37 spaced a week apart]) confirmed that the ciliated respiratory and transitional epithelium were the 

38 most sensitive cell types in the nasal cavity, showing 50-80% decreases in epithelial thickness and 
March 2011 5-21 DRAFT – DO NOT CITE OR QUOTE 

1 epithelial cell volume. The character and location of nasal lesions resulting from O3 exposure are 

2 similar between adult and infant monkeys similarly exposed. However, infant monkeys did not 

3 undergo nasal airway epithelial remodeling or adaptation that occurs in adult animals and they may 

4 develop persistent necrotizing rhinitis following episodic longer-term exposures. 

5 In summary, for all species there are limitations that must be considered when attempting to 

6 extrapolate to human O3 exposures. Rats required 4-5 times higher exposure to O3 to achieve 

7 comparable increases in BAL protein and PMNs to exercising humans. New studies have shown that 

8 varied O3 response in different mouse strains was not due to differences in delivered dose of O3 to 

9 the lung but more likely genetic sensitivity, and that infant mice show greater toxicity relative to the 

10 their smaller lung dose than adults. Even though interspecies differences limit quantitative 

11 comparison between species, the acute and chronic functional responses of laboratory animals to O3

12 appear qualitatively homologous to those of the human making them a useful tool in determining 

13 mechanistic and cause-effect relationships with O3 exposure.

5.2. P o s s ible P a th wa ys /Mo d e s o f Ac tio n

5.2.1. Introd uc tion

14 As described in the previous section, O3 is a highly reactive oxidant gas with low water 

15 solubility. Its diffusion into the fluid/tissue compartment of the respiratory tract occurs by reactive 

16 absorption. This process depends on the availability of substrates such as antioxidants, lipids, 

17 proteins, and carbohydrates and results in their oxidative modification. Because of its chemical 

18 reactivity, inhaled O3 directly targets components residing on the airways and alveolar surfaces, 

19 including ELF and surface macrophages. Although the O3 molecule is consumed and may not reach 

20 the apical plasma membrane of airways and alveolar epithelium, secondary oxidation products 

21 transmit signals to the epithelium, nociceptive sensory nerve fibers and, if present, dendritic cells, 

22 mast cells and eosinophils. Thus, O3 effects are mediated by components of ELF and by the multiple 

23 cell types found in the respiratory tract.

24 Three distinct short-term responses have been well-characterized in humans challenged with 

25 O3: decreased pulmonary function, airways inflammation, and increased bronchial reactivity. In 

26 addition, evidence has been accumulating that O3 exposure exacerbates, and possibly causes, asthma 

27 and allergic airways disease in humans. Effects on the nasal airways and distal lung of humans, 

28 including inflammation and injury, have also been described. Animal studies have demonstrated a 

29 wide range of respiratory system effects. While the respiratory tract is the primary target tissue, 

30 cardiovascular and other organ effects occur following short- and long-term exposures of animals to 

31 O3. Mechanisms responsible for these effects are incompletely understood. 

32 This section of the ISA highlights findings of studies published since the last O3 AQCD 

33 (U.S. EPA, 2006, 088089) which provide insight into the biological pathways underlying the effects 
March 2011 5-22 DRAFT – DO NOT CITE OR QUOTE 

1 of O3. Older studies which represent the current state of the science are also discussed. Studies 

2 conducted at more environmentally-relevant concentrations of O3 are of greater interest, since 

3 mechanisms responsible for effects at low O3 concentrations may not be identical to those occurring 

4 at high O3 concentrations. In fact, some evidence suggests a concentration-dependent hierarchy of 

5 effects. The following subsections describe the current understanding of potential pathways and 

6 modes of action responsible for the pulmonary and extrapulmonary effects of O3 exposure. 

5.2.2. Fo rma tio n o f S e co n da ry Oxid ation P ro d uc ts in the Re s pira to ry Tra c t

7 Since O3 does not diffuse far into the aqueous layer of the ELF without reacting, it is not likely 

8 to directly impact the underlying cells of the respiratory tract (Pryor, 1992, 042725). This does not 

9 preclude direct reactions with the plasma membranes of cells extending beyond the ELF such as 

10 surface macrophages. The secondary oxidation products formed in the ELF following O3 exposure 

11 are primarily responsible for ozone’s effects at the molecular, cellular and tissue level. The amount 

12 and type of secondary oxidation product formed are important determinants of the anatomic sites of 

13 reaction and injury due to O3 exposure, as will be discussed below. 

14 Although not itself a free radical, ozone’s effects are primarily mediated through free radical 

15 reactions. Free radicals are generated during O3-mediated oxidation reactions (Pryor, 1994, 075987). 

16 Subsequent reactions of these radical species produce cytotoxic nonradicals such as ozonides and 

17 aldehydes (Cueto et al., 1992, 042770; Pryor, 1976, 038940). These effects are reduced by the 

18 presence of the lipid-soluble free radical scavenger alpha-tocopherol (Fujita et al., 1987, 004280; 

19 Pryor, 1976, 038940; Pryor, 1994, 075987). Although O3 can react with all hydrocarbons, its 

20 reactivity towards specific groups varies greatly (Pryor, 1992, 042725). Polyunsaturated fatty acids 

21 are one preferred target of O3. Following reaction of O3 with unsaturated fatty acids in the ELF, 

22 measurable amounts of aldehydes were found in human bronchoalveolar lavage (BAL) fluid 

23 (Frampton et al., 1999, 040757; Mudway and Kelly, 2000, 010452). Peroxidation of membrane 

24 lipids is an important mechanism underlying O3-induced injury (Mudway and Kelly, 2000, 010452; 

25 Pryor, 1976, 038940). This could occur by free-radical reactions initiated by O3 in the ELF or by 

26 direct effects of O3 on membranes of cells, like surface macrophages, which extend beyond the ELF. 

27 Markers of lipid peroxidation have been demonstrated in lung tissue and BAL fluid following O3

28 exposure and are enhanced in alpha-tocopherol deficient animals (Mudway and Kelly, 2000, 

29 010452). Ozone-mediated lipid peroxidation leads to the rapid formation of eicosanoids, another 

30 class of secondary oxidation products (discussed below). Ozonized cholesterol species have been 

31 measured in BAL fluid (Pulfer et al., 2005, 076663) and in isolated surfactant (Pulfer and Murphy, 

32 2004, 076673), indicating that O3 reacts with the cholesterol found in surfactant. In addition, O3

33 attacks ELF proteins through reactions with cysteine, methionine, tryptophan and tyrosine residues 

34 (Mudway and Kelly, 2000, 010452). This results in protein oxidation and carbonylation (Mudway 

35 and Kelly, 2000, 010452). 
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1 The ELF contains numerous antioxidants including alpha-tocopherol, albumin, ascorbate, 

2 ceruloplasmin, glutathione, lactoferrin, mucins, urate and transferrin (Freed et al., 1999, 011829; 

3 Mudway et al., 2006, 196536). Ascorbate, glutathione and urate are present in relatively high 

4 concentrations in the surface liquid of human conducting airways and are known to be preferred 

5 targets of O3. These antioxidants are thought to be the first line of defense against inhaled O3, 

6 preventing free radical reactions with cellular proteins and lipids (Mudway and Kelly, 2000, 

7 010452). In vitro studies have demonstrated consumption of water-soluble antioxidants and the 

8 formation of oxidation products by O3 as well as a reactive hierarchy with O3 (Cross et al., 1992, 

9 625299; Mudway and Kelly, 1998, 000273). When examined as a single antioxidant in solution, 

10 urate exhibited the greatest reactivity, followed by ascorbate and glutathione (Mudway and Kelly, 

11 1998, 000273). Results using mixtures have demonstrated greater complexity (Mudway and Kelly, 

12 2000, 010452). 

13 Although ELF constituents such as antioxidants may protect against the deleterious effects of 

14 O3, there is some evidence that antioxidants may paradoxically facilitate O3-mediated damage. This 

15 apparent contradiction should be viewed in terms of the concentration-dependent role of the ELF 

16 antioxidants. Studies in vitro using red cell ghosts as a target showed that aqueous phase reactions 

17 between O3 and the low molecular weight antioxidants ascorbate and glutathione generated 

18 secondary oxidation products capable of perturbing membrane proteins and lipids (Ballinger et al., 

19 2005, 076649). Reactions between O3 and these antioxidant species exhibited a biphasic 

20 concentration response, with oxidation of protein and lipid occurring at lower, but not higher, 

21 concentrations of antioxidant. In this way, endogenous reactants led to the formation of secondary 

22 oxidation products which were injurious and also led to quenching reactions which were protective. 

23 Aqueous phase reactions between O3 and urate or bovine serum albumin did not result in membrane 

24 oxidation (Ballinger et al., 2005, 076649). Further, the presence of urate or bovine serum albumin 

25 protected against lipid and protein oxidation resulting from the reaction of O3 and ascorbate 

26 (Ballinger et al., 2005, 076649). Thus, the formation of secondary oxidation products mediated by 

27 some antioxidants was opposed by quenching reactions involving other antioxidants. 

28 Local scavenging of inhaled O3 by antioxidants in specific respiratory regions has been 

29 demonstrated in vivo (Gunnison and Hatch, 1999, 087204; Mudway et al., 1999, 001270). Urate, but 

30 not ascorbate or glutathione, was depleted in nasal lavage fluid during exposure of human subjects to 

31 0.2 ppm O3 for 2 hours indicating that urate is the predominant antioxidant with respect to O3

32 reactivity in the nasal cavity (Mudway et al., 1999, 001270). In addition, depletion of urate during O3

33 exposure was associated with a small but significant increase in plasma urate levels (Mudway et al., 

34 1999, 001270). Efforts to identify the predominant antioxidant(s) in other respiratory tract regions 

35 and in other species have failed to yield definitive results. In one study, glutathione was increased, 

36 rather than decreased, in BAL fluid and bronchial wash fluid 1.5 h following a 2 h exposure of 

37 human subjects to 0.2 ppm O3 (Blomberg et al., 1999, 001267). 
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1 Since exposure to O3 often leads to airway inflammation characterized by neutrophilia and 

2 since neutrophil-derived oxidants often scavenge ELF antioxidants, concentrations of ELF 

3 antioxidants were examined during airways neutrophilia which generally occurs 4-6 hours 

4 postexposure to O3 (Gunnison and Hatch, 1999, 087204; Long et al., 2001, 057301; Mudway et al., 

5 1999, 011833). In humans exposed to 0.2 ppm O3 for 2 hours, urate, glutathione and alpha6 tocopherol levels remained unchanged in BAL fluid 6 hours postexposure while ascorbate was 

7 decreased significantly in both BAL fluid and plasma (Mudway et al., 1999, 011833). A second 

8 study involving the same protocol reported a loss of ascorbate from bronchial wash fluid and BAL 

9 fluid, representing proximal and distal airway ELF respectively, as well as an increase in oxidized 

10 glutathione in both compartments (Mudway et al., 2001, 025327), No change was observed in ELF 

11 urate levels in response to O3 (Mudway et al., 2001, 025327). Further, O3 exposure (0.8 ppm, 

12 4 hours) in female rats resulted in a 50% decrease in BAL fluid ascorbate immediately postexposure 

13 (Gunnison and Hatch, 1999, 087204). These studies suggested a role for ascorbate and glutathione in 

14 protecting against oxidative stress associated with inflammation. On the other hand, a study in 

15 hamsters exposed to 3 ppm O3 for 6 hours found no depletion of ascorbate, glutathione or alpha16 tocopherol in BAL fluid (Long et al., 2001, 057301). Instead an increase in BAL fluid urate and a 

17 decrease in plasma ascorbate were observed (Long et al., 2001, 057301). 

18 Although it is known that ELF antioxidants are variably distributed among regions of the 

19 respiratory tract, mechanisms underlying this variability are not well-understood. It is thought that 

20 both plasma ultrafiltrate and locally secreted substances contribute to the antioxidant content of the 

21 ELF (Freed et al., 1999, 011829; Mudway et al., 2006, 196536). In the case of urate, the major 

22 source appears to be the plasma (Peden et al., 1995, 076189). Repletion of urate in nasal lavage fluid 

23 was demonstrated during sequential nasal lavage in human subjects (Mudway et al., 1999, 001270). 

24 When these subjects were exposed to O3, nasal lavage urate was significantly decreased while 

25 plasma urate levels was significantly increased (Mudway et al., 1999, 001270). In addition, 

26 concentrations of urate were increased by cholinergic stimulation of the airways which suggests that 

27 increased mucosal gland secretions can be an important source (Peden et al., 1995, 076189). 

28 Regulation of ascorbate, glutathione and alpha-tocopherol concentrations within the ELF is less clear 

29 than that of urate (Mudway et al., 2006, 196536). In a sequential nasal lavage study in humans, 

30 wash-out of ascorbate and glutathione occurred, indicating the absence of rapidly acting repletion 

31 mechanisms (Mudway et al., 1999, 001270). Other studies discussed above demonstrated increases 

32 in BALF glutathione and decreases in BALF and plasma ascorbate levels several hours following O3

33 exposure (Blomberg et al., 1999, 001267; Mudway et al., 1999, 011833; Mudway et al., 2001, 

34 025327). Furthermore, high levels of dehydroascorbate, the oxidized form of ascorbate, have been 

35 reported in human ELF (Mudway et al., 2006, 196536). Other investigators have demonstrated 

36 cellular uptake of oxidized ascorbate by several cell types leading to intracellular reduction and 

37 export of reduced ascorbate (Welch et al., 1995, 644675). 
March 2011 5-25 DRAFT – DO NOT CITE OR QUOTE 

1 A further consideration is the compromised status of ELF antioxidants in disease states such as 

2 asthma (Mudway and Kelly, 2000, 010452). This could possibly be due to ongoing inflammation 

3 which causes antioxidant depletion or to abnormal antioxidant transport or synthesis (Mudway and 

4 Kelly, 2000, 010452). For example, basal ascorbate levels were significantly lower and basal levels 

5 of oxidized glutathione and urate were significantly higher in bronchial wash fluid and BAL fluid of 

6 mild asthmatics compared with healthy control subjects (Mudway et al., 2001, 025327). Differences 

7 in ELF antioxidant content have also been noted between species. These observations have led to the 

8 suggestion that the amount and composition of ELF antioxidants, the capacity to replenish 

9 antioxidants in the ELF or the balance between beneficial and injurious interactions between 

10 antioxidants and O3 may contribute to O3 sensitivity which varies between individuals and species 

11 (Mudway and Kelly, 2000, 010452; Mudway et al., 1999, 001270; Mudway et al., 2006, 196536). 

12 The complexity of these interactions was demonstrated by a study in which O3 exposure resulted in 

13 similar increases in airway neutrophils and decreases in pulmonary function in both mild asthmatics 

14 and healthy controls, despite differences in ELF antioxidant concentrations prior to O3 exposure 

15 (Mudway et al., 2001, 025327). Further, the O3-induced increase in oxidized glutathione and 

16 decrease in ascorbate observed in ELF of healthy controls was not observed in mild asthmatics 

17 (Mudway et al., 2001, 025327). While the authors concluded that basal ascorbate and oxidized 

18 glutathione concentrations were not predictive of responsiveness to O3, they also suggested that the 

19 increased basal urate concentrations in the mild asthmatics may have played a protective role 

20 (Mudway et al., 2001, 025327). Thus compensatory mechanisms resulting in enhanced total 

21 antioxidant capacity may play a role in modulating responses to O3. 

Several studies in animals evaluated the relationships between 18 22 O-labeled O3 dose markers, 

23 injury markers and ascorbate concentrations following O3 exposure. In female rats exposed to 

24 0.8 ppm O3 for 4 hours, BAL indicators of injury and inflammation (protein and neutrophil number) 

and 18 25 O reaction product were increased inversely with the reduction in ascorbate (Gunnison and 

26 Hatch, 1999, 087204). In another study, aging rats (9 and 24 months old) were shown to have 49% 

27 and 64% lower ascorbate in lung tissue, respectively, than 2-month-old rats (Vincent et al., 1996, 

080778). However, aging-induced ascorbate loss did not increase the accumulation of 18 28 O reaction 

29 products following O3 exposure (0.4-0.8 ppm, 2-6 hours). Pregnancy and lactation also caused lower 

30 ascorbate content in BAL and nasal lavage fluid and was associated with an increase in accumulation 

of 18 31 O reaction products following O3 exposure (Gunnison and Hatch, 1999, 087204). Kari et al. 

32 (1997, 086171) observed that a 3-week caloric restriction (75%) in rats abrogated the toxicity of O3

33 (2 ppm, 2 hours), measured as BAL fluid increases in protein, fibronectin and neutrophils, which 

34 was seen in normally fed rats. Accompanying this resistance to O3 toxicity, was a 30% higher basal 

35 BAL fluid ascorbate concentration, a rapid accumulation of ascorbate into the lungs to levels 60% 

above normal and reduction (30%) in the accumulation of 18 36 O reaction product in the lungs. These 

37 investigations demonstrated an inverse relationship between ascorbate levels and O3 dose and 

38 provide evidence for ascorbate playing a protective role following O3 exposure in these studies.
March 2011 5-26 DRAFT – DO NOT CITE OR QUOTE 

1 Many investigations have focused on antioxidant deficiency and supplementation as 

2 modulators of O3-mediated effects. Ascorbate deficiency has been shown to increase the effects of 

3 acute, but not chronic, O3 exposure in guinea pigs and humans (Kodavanti et al., 1995, 077440; 

4 Slade et al., 1989, 059465). Supplementation with ascorbate and alpha-tocopherol was protective in 

5 healthy adults who were on an ascorbate-deficient diet and exposed to 0.4 ppm O3 for 2 hours while 

6 exercising (Samet et al., 2001, 019034). In this study, the protective effect consisted of a smaller 

7 reduction in forced expiratory volume in one second (FEV1) following O3 exposure (Samet et al., 

8 2001, 019034). However the inflammatory response (influx of neutrophils and levels of IL-6) 

9 measured in BAL fluid 1 hour after O3 exposure was not different between supplemented and non10 supplemented subjects (Samet et al., 2001, 019034). Supplementation with ascorbate and alpha11 tocopherol also protected against pulmonary function decrements and nasal inflammatory responses 

12 which were associated with high levels of ambient O3 in asthmatic children living in Mexico City

13 (Romieu et al., 2002, 034711; Sienra-Monge et al., 2004, 196422). Similarly, supplementation with 

14 ascorbate, alpha-tocopherol and beta-carotene improved pulmonary function in Mexico City

15 streetworkers (Romieu et al., 1998, 086756). However, ascorbate and alpha-tocopherol 

16 supplementation failed to ameliorate the pulmonary function decrements or airways neutrophilia 

17 observed in humans exposed to 0.2 ppm O3 for 2 hours (Mudway et al., 2006, 196536). It was 

18 suggested that supplementation may be ineffective in the absence of antioxidant deficiency (Mudway 

19 et al., 2006, 196536). Furthermore, protective effects of supplementation with alpha-tocopherol 

20 alone have not been observed in humans (Mudway and Kelly, 2000, 010452). 

21 Recent studies in animals demonstrated protection against O3-induced effects using gamma22 tocopherol supplementation in models of allergic rhinosinusitis (Wagner et al., 2009, 201574) and 

23 lower airway allergic inflammation (Wagner et al., 2007, 596420). Previous studies demonstrated 

24 that supplementation with alpha-tocopherol was ineffective in these models (Wagner et al., 2007, 

25 596420). Other investigators found that alpha-tocopherol deficiency led to an increase in liver lipid 

26 peroxidation (Sato et al., 1980, 039738) and a drop in liver alpha-tocopherol levels following O3

27 exposure (Vasu et al., 2010, 201561). A recent study used alpha-tocopherol transfer protein null mice 

28 as a model of alpha-tocopherol deficiency and demonstrated an altered adaptive response of the lung 

29 genome to O3 exposure (Vasu et al., 2010, 201561). Taken together, these studies provide evidence 

30 that the tocopherol system modulates O3-induced responses.

31 Other antioxidants have been shown to confer resistance to O3-induced injury. In a recent 

32 study, lung hyperpermeability in response to O3 was unexpectedly reduced in mice deficient in the 

33 glutamate-cysteine ligase modifier subunit gene compared with sufficient mice (Johansson et al., 

34 2010, 644476). Since the lungs of these mice exhibited 70% glutathione depletion, protection against 

35 O3-induced injury was unexpected (Johansson et al., 2010, 644476). However it was found that 

36 several other antioxidant defenses, including metallothionein, were upregulated in response to O3 to 

37 a greater degree in the glutathione-deficient mice compared with sufficient mice (Johansson et al., 

38 2010, 644476). The authors suggested that resistance to O3-induced lung injury was due to 
March 2011 5-27 DRAFT – DO NOT CITE OR QUOTE 

1 compensatory augmentation of antioxidant defenses (Johansson et al., 2010, 644476). Antioxidant 

2 effects have also been attributed to Clara cell secretory protein (CCSP) and surfactant protein A 

3 (SP-A). CCSP was found to modulate the susceptibility of airways epithelium to injury in mice 

4 exposed to O3 (0.2 or 1 ppm for 8 hours) by an unknown mechanism (Plopper et al., 2006, 596410). 

5 SP-A protected against O3-induced airways inflammation and injury, possibly by acting as a 

6 sacrificial substrate (Haque et al., 2007, 597606). 

7 A role for plasma antioxidants in modulating O3-induced respiratory effects has also been 

8 suggested (Aibo et al., 2010, 378559). In this study, pretreatment of rats with a high dose of 

9 acetaminophen resulted in increased levels of plasma cytokines and the influx of inflammatory cells 

10 into the lung following 6 h exposure to 0.25 and 0.5 ppm O3 (Aibo et al., 2010, 378559). These 

11 effects were not observed in response to O3 alone. Although not measured in this study, glutathione 

12 depletion in the liver is known to occur in acetaminophen toxicity. Since liver glutathione is the 

13 source of plasma glutathione, acetaminophen treatment may have lowered plasma glutathione levels 

14 and altered the redox balance in the vascular compartment. These findings indicate an 

15 interdependence between respiratory tract, plasma and liver responses to O3, possibly related to 

16 glutathione status.

17 Another important consideration is the non-uniformity of the injury response to O3 throughout 

18 the respiratory tract. Several mechanisms have been proposed to explain this phenomenon. First, 

19 dosimetry may be a key determinant since the sites receiving the largest dose might be expected to 

20 exhibit the greatest injury or inflammation (Plopper et al., 1998, 087203; Postlethwait et al., 2000, 

21 003000). An important corollary is that the uneven distribution of mucus in the respiratory tract 

22 airways may influence the capacity of O3 to reach the aqueous layer (Mudway and Kelly, 2000, 

23 010452). Secondly, the non-homogeneous formation of cytotoxic products in the ELF may account 

24 for the variable response (Postlethwait et al., 2000, 003000). The thickness of the ELF varies along 

25 the respiratory tract, being greater in the upper airways and less more distally. Further the 

26 composition of the ELF varies along the respiratory tract. Thus, the amount of protective 

27 antioxidants and other scavengers in various respiratory tract regions is likely to limit the formation 

28 of cytotoxic products. Similarly, the availability of reactants which are precursors of potent cytotoxic 

29 products may facilitate their formation in a particular region. For example, the formation of highly 

30 electrophilic aldehydes from unsaturated fatty acids may occur in all respiratory tract regions while 

31 the formation of oxidized surfactant lipids is likely restricted to the alveolar region and respiratory 

32 bronchioles where surfactant is found. Thus, region-specific formation of particular oxidation 

33 products may dictate patterns of epithelial injury in the respiratory tract.

34 The relationship between site-specific O3 dose, epithelial injury and glutathione concentration 

35 was investigated by Plopper (1998, 087203). Adult rhesus monkeys were exposed for 2 hours to 0.4 

and 1.0 ppm O3, which was labeled with 18 36 O, and tissues were analyzed immediately postexposure

37 for dose, epithelial injury and glutathione levels. Results indicated that exposure to 1 ppm O3

38 resulted in the greatest epithelial injury in the respiratory bronchioles although injury was observed 
March 2011 5-28 DRAFT – DO NOT CITE OR QUOTE 

1 at all of the airway sites but not in the lung parenchyma. Exposure to 0.4 ppm O3 resulted in 

2 epithelial injury only in the respiratory bronchioles. Local O3 dose was found to be highly variable 

3 among the different sites with the greatest levels found in the respiratory bronchioles and lowest 

4 levels found in the parenchyma following 1 ppm O3. Glutathione levels varied in the different 

5 airways sites in monkeys exposed to filtered air. Exposure to 1 ppm O3 decreased glutathione levels 

6 only in the respiratory bronchioles. This study demonstrated a close relationship between the 

exposure dose of O3 (uptake of 18 7 O) and the degree of initial epithelial injury at a particular site in 

8 the respiratory tract. Glutathione depletion observed only at that site suggests that glutathione played 

9 a protective role during O3 exposure. 

10 While the formation of secondary oxidation products is the key event leading to O3-mediated 

11 effects, scavenging and/or metabolism of those products is likely to be an important determinant of 

12 outcomes. One such mechanism may be scavenging of oxidized lipids via the macrophage receptor 

13 with collagenous structure (MARCO) expressed on the cell surface of alveolar macrophages. A 

14 recent study demonstrated increased gene expression of MARCO in the lungs of an O3-resistant C3H 

15 mouse strain (HeJ) but not in an O3-sensitive, genetically nearly identical strain (OuJ) (Dahl et al., 

16 2007, 196986). Upregulation of MARCO occurred in mice exposed to 0.3 ppm O3 for 24-48 hours; 

17 inhalation exposure for 6 hours at this concentration was insufficient for this response. Animals 

18 lacking the MARCO receptor exhibited greater inflammation and injury, as measured by BAL 

19 neutrophils, protein and isoprostanes, following exposure to 0.3 ppm O3 (Dahl et al., 2007, 196986). 

20 MARCO also protected against the inflammatory effects of oxidized surfactant lipids (Dahl et al., 

21 2007, 196986). Scavenging of oxidized lipids may limit O3-induced injury since ozonized 

22 cholesterol species formed in the ELF (Pulfer and Murphy, 2004, 076673; Pulfer et al., 2005, 

23 076663) stimulate apoptosis and cytotoxicity (Gao et al., 2009, 200764; Sathishkumar et al., 2007, 

24 097758; Sathishkumar et al., 2007, 197785; Sathishkumar et al., 2009, 201549) in vitro. While these 

25 studies have focused on the alveolar compartment (alveolar macrophages, surfactant lipids), 

26 comparable pathways have yet to be elucidated in the conducting airways. A second mechanism 

27 likely to impact O3-mediated effects is the metabolism of secondary oxidation products catalyzed by 

28 antioxidant enzymes such as glutathione peroxidase, glutathione S-transferases (GST) and 

29 NADPH:quinone oxidoreductase 1 (NQO1). Evidence for the importance of GST and NQO1 in 

30 modulating the effects of O3 is presented below (see Gene-Environment Interactions). 

31 Secondary oxidation products formed as a result of O3 exposure initiate numerous responses at 

32 the cellular, tissue and whole organ level of the respiratory system. These responses include the 

33 activation of neural reflexes, injury and inflammation, hyperpermeability, increased bronchial 

34 reactivity and altered host defenses, as will be discussed below. In addition, the enhancement of 

35 asthma and allergic responses demonstrated as a result of O3 exposure is likely due to secondary 

36 oxidation products. Exposure to O3 also results in effects on other organ systems such as the 

37 cardiovascular, hepatic and central nervous systems. Mechanisms underlying these extrapulmonary 

38 responses are not well understood. It is unlikely that lipid ozonides and other secondary oxidation 
March 2011 5-29 DRAFT – DO NOT CITE OR QUOTE 

1 products, which are bioactive and cytotoxic in the respiratory system, gain access to the vascular 

2 space (Chuang et al., 2009, 197202). However O3 exposure may result in systemic oxidative stress, 

3 as suggested by studies in humans reporting an association between O3 exposure and both levels of 

4 plasma 8-isoprostanes and the presence of peripheral blood lymphocyte micronuclei (Chen et al., 

5 2006, 196504; Chen et al., 2007, 145956). 

5.2.2.1. Summary

6 The initial key event in ozone’s toxicity pathway is the formation of secondary oxidation 

7 products in the respiratory tract. Pathways for the removal of those products are also of great 

8 importance. Due to the highly reactive nature of O3, direct reactions most likely involve components 

9 of the ELF and/or plasma membranes of surface macrophages which extend beyond the ELF.

10 Reaction products likely mediate ozone’s effects on respiratory tract epithelium. 

5.2.3. Ac tiva tio n o f Ne ural Re fle xe s

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

12 of neural reflexes. The involvement of bronchial C-fibers, a type of nociceptive sensory nerve, has 

13 been demonstrated in dogs (Coleridge et al., 1993, 038695; Schelegle et al., 1993, 039203) and the 

14 involvement of nociceptive sensory nerves has been demonstrated in humans (Passannante et al., 

15 1998, 030114). Furthermore there is evidence that substance P (SP), a tachykinin which is known to 

16 be released from C-fibers, plays a role in O3-mediated effects (Hazbun et al., 1993, 043914; Krishna 

17 et al., 1997, 084262). 

18 The response to O3 in humans is characterized by substernal discomfort, especially on deep 

19 inspiration, accompanied by involuntary truncation of inspiration (Hazucha et al., 1989, 041909). 

20 This leads to decreased inspiratory capacity and to decreased forced vital capacity (FVC) and forced 

21 expiratory volume in one second (FEV1), as measured by spirometry, and is accompanied by a 

22 decreased tidal volume and increased respiratory frequency in human subjects during exercise 

23 (Hazucha et al., 1989, 041909). For example, these pulmonary function responses have been noted 

24 immediately after a 1-h exposure to 0.3 ppm O3, resolving by 6 hours after exposure (Schelegle et 

25 al., 1991, 042491), and during and immediately after a 4-h exposure to 0.2 ppm O3 in exercising 

26 humans (Aris et al., 1993, 038275; Balmes et al., 1996, 080830). Although spirometric changes 

27 began to return to baseline shortly after exposure, small residual spirometric decrements were 

28 reported at 24 hours postexposure (Hazucha et al., 1996, 043923). Spirometric changes in FEV1 and 

29 FVC were not due to changes in respiratory muscle strength (Hazucha et al., 1989, 041909). 

30 Changes in FVC or symptoms were not modified by treatment with bronchodilators such as atropine

31 (Beckett et al., 1985, 039758). Thus, parasympathetic involvement in the O3-mediated decreases in 

32 lung volume was not significant (Mudway and Kelly, 2000, 010452). However, the loss of vital 

33 capacity was reversible with intravenous administration of the rapid-acting opioid agonist, 

34 sufentanyl, indicating an involvement of opioid receptor-containing nerve fibers and/or more central 
March 2011 5-30 DRAFT – DO NOT CITE OR QUOTE 

1 neurons (Passannante et al., 1998, 030114). The effects of sufentanyl may be attributed to blocking 

2 C-fiber stimulation by O3 since activation of opioid receptors downregulates C-fiber function 

3 (Belvisi et al., 1992, 644681). There is some evidence that eicosanoids play a role in the neural 

4 reflex since cyclooxygenase inhibition with indomethacin (Alexis et al., 2000, 013072; Schelegle et 

5 al., 1987, 041706) or ibuprofen, which also blocks some lipoxygenase activity (Hazucha et al., 1996, 

6 043923), before exposure to O3 significantly blunted the spirometric responses. In the latter study, 

7 ibuprofen treatment resulted in measurable decreases in BAL levels of PGE2 and TXB2 at 1 hour 

8 postexposure (Hazucha et al., 1996, 043923). Although an earlier study demonstrated that PGE2 

9 stimulated bronchial C-fibers (Coleridge et al., 1976, 038612; Coleridge et al., 1993, 038695) and 

10 suggested that PGE2 mediated O3-induced decreases in pulmonary function, no correlation was 

11 observed between the degree of ibuprofen-induced inhibition of BAL PGE2 levels and blunting of 

12 the spirometric response to O3 (Hazucha et al., 1996, 043923). Nonetheless, recent studies continue 

13 to provide evidence that arachidonic acid metabolites, as well as oxidative stress, contribute to 

14 human responsiveness to O3 (Alfaro et al., 2007, 196567). 

15 A delay in onset of O3-induced pulmonary function responses has been noted in numerous 

16 studies. Recently the delay was characterized in terms of changes in breathing frequency (Schelegle 

17 et al., 2007, 195841). In humans exposed to O3, no change in breathing frequency was observed until 

18 a certain cumulative inhaled dose of O3 had been reached. Subsequently, the magnitude of the 

19 change in breathing frequency was correlated with the inhaled dose rate (Schelegle et al., 2007, 

20 195841). These investigators proposed that initial reactions of O3 with ELF resulted in a time21 dependent depletion of ELF antioxidants, and that activation of neural reflexes occurred only after 

22 the antioxidant defenses were overwhelmed (Schelegle et al., 2007, 195841). 

23 There is a large range of pulmonary function responses to O3 among healthy young adults 

24 (Balmes et al., 1996, 080830; Hazucha et al., 2003, 048168). Since individual responses are 

25 relatively consistent across time, it is thought that responsiveness reflects an intrinsic characteristic 

26 of the subject (Mudway and Kelly, 2000, 010452). Older adults are generally not responsive to O3

27 (Hazucha et al., 2003, 048168), while obese young women may be more responsive than lean young 

28 women (Bennett et al., 2007, 418827). The lack of spirometric responsiveness is not attributable to 

29 the presence of endogenous endorphins, which could potentially block C-fiber stimulation by O3, as 

30 demonstrated in a study involving intravenous administration of naloxone immediately following the 

31 O3 exposure to weak responders (Passannante et al., 1998, 030114). Currently, the mechanisms 

32 underlying the inter-individual variation in responsiveness to O3 are not known. It has been proposed 

33 that some of the variation in response may be genetically determined (Yang et al., 2005, 077211). 

34 More discussion on this topic is found below (Section 5.2.9.1).

35 In addition to the spirometric changes, mild airways obstruction occurs as a result of O3

36 exposure. This pulmonary function decrement is generally measured as specific airway resistance 

37 (sRaw) which is the product of airway resistance and thoracic gas volume. Increased sRaw occurred 

38 fairly rapidly in exercising humans, peaking at 1-6 hours and resolving by 24 hours following O3
March 2011 5-31 DRAFT – DO NOT CITE OR QUOTE 

1 exposure (Aris et al., 1993, 038275; Balmes et al., 1996, 080830; Hazucha et al., 1996, 043923; 

2 Schelegle et al., 1991, 042491). Small but statistically significant increases in sRaw during O3

3 exposure (0.2 ppm for 4 hours with intermittent exercise) and immediately following O3 exposure 

4 (0.3 ppm for 1 hour, moderate exercise or 0.4 ppm for 2 hours, intermittent exercise) were observed 

5 in several studies (Aris et al., 1993, 038275; Balmes et al., 1996, 080830; Hazucha et al., 1996, 

6 043923; Schelegle et al., 1991, 042491). These changes in sRaw correlated with changes in 

7 inflammatory and injury endpoints measured 18 hours postexposure, as will be discussed below, but 

8 not with the time course or degree of spirometric change measured during exposure (Aris et al., 

9 1993, 038275; Balmes et al., 1996, 080830; Schelegle et al., 1991, 042491). In addition, a small but 

10 persistent increase in airways resistance associated with narrowing of small peripheral airways 

11 (measured as changes in isoV FEF25-75) was demonstrated in O3-exposed humans (0.35 ppm for 

12 130 minutes with intermittent exercise) (Weinmann et al., 1995, 077206; Weinmann et al., 1995, 

13 038645). A similar study (0.4 ppm O3 for 2 hours with intermittent exercise) found decreases in 

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

15 dysfunction (Kreit et al., 1989, 041817). In separate studies, a statistically significant increase in 

16 residual volume (Hazucha et al., 1989, 041909) and a statistically significant decrease in FEF25-75

17 (Horstman et al., 1995, 075834) were observed following O3 exposure.

18 Mechanisms underlying the rapid increase in airways resistance following O3 exposure are

19 incompletely understood. However pretreatment with atropine was found to decrease baseline sRaw

20 and prevent O3-induced increases in sRaw (Beckett et al., 1985, 039758), indicating the involvement 

21 of muscarinic cholinergic receptors of the parasympathetic nervous system. Interestingly, atropine 

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

23 breathing rate, tidal volume or respiratory symptoms (Beckett et al., 1985, 039758). Thus pulmonary 

24 function decrements measured as FEV1 may reflect both restrictive and obstructive type changes in 

25 airways responses. Using a beta-adrenergic agonist, it was shown that smooth muscle contraction, 

26 not increased airways mucus section, was responsible for O3-induced increases in airways resistance 

27 (Beckett et al., 1985, 039758). Furthermore, tachykinins may contribute to O3-mediated increases in 

28 airways resistance. Bronchopulmonary C fibers mediate local axon responses by releasing 

29 tachykinins such as SP. Tachykinins bind to neurokinin (NK) receptors resulting in responses such as 

30 bronchoconstriction. In one study in which bronchial biopsies were performed and studied by 

31 immunohistochemistry, SP was substantially diminished in submucosal sensory nerves 6 hours 

32 following O3 exposure (0.2 ppm O3 for 2 hours with exercise) (Krishna et al., 1997, 084262). A 

33 statistically significant correlation was observed between loss of SP immunoreactivity from neurons 

34 in the bronchial mucosa and changes in FEV1 measured 1 hour postexposure (Krishna et al., 1997, 

35 084262). Another study found that SP was increased in lavage fluid of human subjects immediately 

36 after O3 challenge (0.25 ppm O3 for 1 hour with heavy exercise) (Hazbun et al., 1993, 043914) These 

37 results provide evidence that the increased airways resistance observed following O3 exposure is due 
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1 to vagally-mediated responses and possibly by local axon reflex responses through 

2 bronchopulmonary C fiber-mediated release of SP. 

3 In responsive individuals, a striking degree of response attenuation occurs following repeated 

4 daily exposures to O3. This phenomena has been reported for both lung function and symptoms such 

5 as upper airway irritation, nonproductive cough and substernal discomfort and pain upon deep 

6 inspiration (Folinsbee et al., 1980, 038880; Hackney et al., 1977, 038282; Horvath et al., 1981, 

7 039221). Repeated daily exposures also led to an attenuation of the sRaw response in exercising

8 humans (Christian et al., 1998, 029925). It is well-established that a young O3 responder will no 

9 longer be responsive on the fourth or fifth day of consecutive daily O3 exposure (0.4 ppm O3 for 

10 4 hours or 0.2 ppm O3 for 4 hours) and that after developing this tolerance it takes up to 7-10 days of 

11 non-exposure in order for the subject to regain O3 responsiveness (Christian et al., 1998, 029925; 

12 Devlin et al., 1997, 083577; Folinsbee et al., 1980, 038880; Hackney et al., 1977, 038282; Horvath 

13 et al., 1981, 039221; Linn et al., 1982, 039646). One group reported persistent small airway 

14 dysfunction despite attenuation of the FEV1 response on the third day of consecutive O3 exposure 

15 (Frank et al., 2001, 093491). Studies in animals also indicate an attenuation of the physiologic 

16 response as measured by breathing patterns and tidal volume following five consecutive days of O3

17 exposure (Tepper et al., 1989, 041991). The mechanisms underlying this attenuation in humans and 

18 animal models are not well understood (Devlin et al., 1997, 083577), although some studies have 

19 implicated an alteration in lung antioxidant capacity, enhanced mucus production or factors related 

20 to epithelial hyperplasia following O3 exposure (Devlin et al., 1997, 083577). These potential 

21 mechanisms and others are discussed below. Adaptation of O3-induced bradycardic responses, which 

22 also result from activation of neural reflexes, have also been reported in animal studies (Hamade and 

23 Tankersley, 2009, 596386; Watkinson et al., 2001, 016245). 

24 Finally, the degree of acute decrease of vital capacity in young, healthy adults does not 

25 correlate (positively) with the degree of neutrophilic inflammation observed in their airways or the 

26 degree of airways obstruction elicited by O3 exposure (Aris et al., 1993, 038275; Aris et al., 1995, 

27 075945; Balmes et al., 1996, 080830; Schelegle et al., 1991, 042491), implying that different 

28 mechanisms are at work. Further evidence is provided by the finding that pretreatment with 

29 ibuprofen attenuated O3-induced lung function changes and increases in BAL fluid PGE2 levels, but 

30 had no effect on neutrophilia (Hazucha et al., 1996, 043923). 

5.2.3.1. Ne w Ce llula r a n d Mole c ula r Ins igh ts

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

32 responses mediated by bronchopulmonary C-fibers, a type of nociceptive sensory nerve. Ozone

33 exposure in mice was found to selectively activate a subset of receptors on bronchopulmonary C34 fibers which are TRPA1 ion channels (Taylor-Clark and Undem, 2010, 377143). TRPA1 ion 

35 channels are members of the TRP family of ion channels, which are known to mediate the responses 

36 of sensory neurons to inflammatory mediators (Caceres et al., 2009, 628549). In addition to TRPA1 
March 2011 5-33 DRAFT – DO NOT CITE OR QUOTE 

1 ion channels possibly playing a key role in O3-induced decrements in pulmonary function, they may 

2 mediate allergic asthma (Caceres et al., 2009, 628549). Activation of TRPA1 ion channels following 

3 O3 exposure is likely due to the formation of secondary products such as aldehydes and 

4 prostaglandins (Taylor-Clark and Undem, 2010, 377143). Ozonation of unsaturated fatty acids in the 

5 ELF results in the generation of aldehydes (Frampton et al., 1999, 040757). For example

6 4-hydroxynonenal and 4-oxononenal are derived from the peroxidation of omega-6 unsaturated fatty 

7 acids (Taylor-Clark et al., 2008, 628565; Trevisani et al., 2007, 628590). 4-oxononenal is a stronger 

8 electrophile than 4-hydroxynonenal and exhibits greater potency towards the TRPA1 channels 

9 (Taylor-Clark et al., 2008, 628565). TRPA1 channels can be activated by aldehydes and other 

10 electrophiles through covalent modification of cysteine and lysine residues (Trevisani et al., 2007, 

11 628590). In addition, PGE2 is known to sensitize TRPA1 channels (Bang et al., 2007, 628545) and, 

12 as noted previously, PGE2 (or other products of cyclooxygenase-catalyzed reactions) has been 

13 proposed to mediate the pulmonary function changes observed following O3 exposure in humans. 

14 In addition to stimulating central nervous system reflexes, bronchopulmonary C fibers mediate 

15 local axon responses by releasing neuropeptides such as SP, neurokinin (NK) A and calcitonin gene16 related peptide (CGRP). Oslund et al. (2008, 195654) demonstrated that NK-1 receptor blockade had 

17 no effect on O3-stimulated physiologic responses such as tidal volume and breathing frequency in 

18 rats over the 8-h exposure period. However, SP and NK receptors contributed to vagally-mediated 

19 bronchoconstriction in guinea pigs 3 days after a single exposure to O3 (2 ppm for 4 hours) (Verhein 

20 et al., 2011, 670295). 

5.2.3.2. Summary

21 A key event in ozone’s toxicity pathway is the activation of neural reflexes which leads to 

22 decrements in pulmonary function. Evidence is accumulating that secondary oxidation products are 

23 responsible for this effect. Eicosanoids have been implicated in humans while eicosanoids and 

24 aldehydes are effective in animal models. Different receptors on bronchial C-fibers have been shown 

25 to mediate separate effects of O3 on pulmonary function. Nociceptor sensory nerves are involved in 

26 the involuntary truncation of respiration which results in decreases in FVC, FEV1, tidal volume and 

27 an increase in respiratory frequency and pain upon deep inspiration. Opioids block these responses 

28 while atropine does not. New evidence in an animal model suggests that TRPA1 receptors on 

29 bronchial C-fibers mediate this pathway. Ozone exposure also results in activation of vagal sensory 

30 nerves and a mild increase in airways obstruction measured as increased sRaw. Atropine and beta31 adrenergic agonists blocked this response in one study indicating that the airway obstruction was due 

32 to bronchoconstriction. Other studies in humans implicated SP release from bronchial C-fibers 

33 resulting in airway narrowing due to either neurogenic edema or bronchoconstriction. New evidence 

34 in an animal model suggests that the SP-NK receptor pathway caused bronchoconstriction following 

35 O3 exposure. Considerable inter-individual variability exists in O3 responsiveness measured by 

36 decrements in pulmonary function. Further, attenuation of these pulmonary function decrements 
March 2011 5-34 DRAFT – DO NOT CITE OR QUOTE 

1 occurs following O3 exposure for several consecutive days. Mechanisms responsible for these effects 

2 are not known but may be related to inherent differences in neural sensitivity. 

5.2.4. Re s pirato ry Tra c t Inju ry and Infla mma tio n

3 As described above, O3 reacts with components of the ELF resulting in the generation of 

4 secondary oxidation products. Higher concentrations of these products may directly injure 

5 respiratory tract epithelium. Lower concentrations may initiate cellular responses including cytokine 

6 generation, adhesion molecule expression and modification of tight junctions leading to 

7 inflammation and increased permeability across airways epithelium (Dahl et al., 2007, 196986; 

8 Mudway and Kelly, 2000, 010452). Subsequent epithelial remodeling may also occur (Mudway and 

9 Kelly, 2000, 010452). 

10 Injury and inflammation have been observed in many different regions of the respiratory tract 

11 following O3 exposure (Plopper et al., 1998, 087203; Postlethwait et al., 2000, 003000). The nasal 

12 airways, conducting airways and distal airways (i.e. respiratory bronchioles or centriacinar region 

13 depending on the species) have all been identified as sites of O3-mediated injury and inflammation 

14 (Mudway and Kelly, 2000, 010452). One study found greater injury in conducting airways 

15 downstream of bifurcations where local doses of O3 were higher (Postlethwait et al., 2000, 003000). 

16 Although the extent of O3-induced injury is variable along the respiratory tract, common features of 

17 the injury response have been noted (Mudway and Kelly, 2000, 010452). In the conducting airways, 

18 necrosis of ciliated cells and degranulation of secretory cells has been observed and in the alveolar 

19 region, necrosis of Type 1 pneumocytes occurs. 

20 Further, O3-induced injury and inflammation responses are variable between species. For 

21 example, Dormans et al. (1999, 040766) found that rats, mice, and guinea pigs all exhibited 

22 O3-induced (0.2 - 0.4 ppm for 3-56 days) inflammation; however, guinea pigs were the most 

23 sensitive with respect to alveolar macrophage elicitation and pulmonary cell density in the 

24 centriacinar region. Mice were the most sensitive to bronchiolar epithelial hypertrophy and 

25 biochemical changes (e.g. lactate dehydrogenase, glutathione reductase, glucose-6-phosphate 

26 dehydrogenase activity), and had the slowest recovery from O3 exposure. All species displayed 

27 increased collagen in the ductal septa and large lamellar bodies in Type II pneumocytes at the longest 

28 exposure and highest concentration, whereas this response occurred in the rat and guinea pig at 

29 lower O3 levels (0.2 ppm) as well. Since no dose metric was measured, it is possible that some of 

30 these differences may be attributable to disparate total inhaled dose or local organ dose. Overall, the 

31 authors rated mice as most susceptible, followed by guinea pigs, then rats (Dormans et al., 1999, 

32 040766). Rats were also less sensitive to epithelial necrosis and inflammatory responses from O3

33 (1.0 ppm for 8 h) than monkeys and ferrets, which manifested a similar response (Sterner-Kock et 

34 al., 2000, 013033). These data suggest that ferrets may be a good animal model for O3-induced 

35 airway effects due to the similarities in pulmonary structure between primates and ferrets.
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1 While injury and inflammation often accompany each other, in many cases epithelial injury 

2 precedes observable inflammatory effects and in other cases inflammation leads to injury of the 

3 surrounding cells and tissues. In addition to being species-dependent, the acute injury response is 

4 focal, site-specific and dependent on exposure parameters and the time that elapsed since exposure 

5 (Postlethwait et al., 2000, 003000). The presence of shed epithelial cells in the BAL, increases in 

6 levels of BAL lactate dehydrogenase and protein and increased epithelial permeability have been 

7 observed and are indicative of epithelial injury. In addition, histologic analysis has demonstrated 

8 damage to tight junctions between epithelial cells, suggesting an increase in epithelial permeability. 

9 Several studies have measured epithelial permeability as the flux of the small solute 99mTc10 DTPA which was introduced into the air spaces in different regions of the respiratory tract. An early 

11 study demonstrated increased pulmonary epithelial permeability, measured as the clearance of

12 99mTc-DTPA, in humans exposed for 2 h to 0.4 ppm O3 while exercising moderately (Kehrl et al., 

13 1987, 040824). Another study found that increased epithelial permeability occurred at 1-3 hours and 

14 18-20 hours postexposure and did not resolve for several days (Foster and Stetkiewicz, 1996, 

15 079920). Increased bronchial permeability was also observed in dogs immediately after and 18 hours

16 postexposure to 0.2 ppm O3 for 6 hours (Freed et al., 1996, 080798). Increased epithelial 

17 permeability has been proposed to play a role in allergic sensitization (Matsumura, 1970, 050626), in 

18 activation of neural reflexes and in stimulation of smooth muscle receptors (Dimeo et al., 1981, 

19 039662). Studies in animals have also demonstrated increased vascular permeability, as measured by 

20 BAL protein and albumin (Costa et al., 1985, 040273; Hu et al., 1982, 039418). 

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

22 inflammation. Although neutrophil influx into nasal airways has been demonstrated in human 

23 subjects (Graham and Koren, 1990, 042299), most studies of neutrophil influx have focused on the 

24 lower airways (Aris et al., 1993, 038275; Hazucha et al., 1996, 043923). The time course of this 

25 response and its resolution is slower than that of the decrements in pulmonary function (Hazucha et 

26 al., 1996, 043923). In general, airways neutrophilia is observable within 1-2 hours, peaks at 

27 4-6 hours and is returning to baseline levels at 24 h following exposure to O3 in exercising humans 

28 involving 0.4 ppm for 2 hours (Devlin et al., 1991, 040359) or 0.3 ppm for 1 hour (Schelegle et al., 

29 1991, 042491). Since the influx and persistence of neutrophils in airways following O3 exposure 

30 correlates with the temporal profile of epithelial injury (Hu et al., 1982, 039418), neutrophils are 

31 likely to be injurious. However, neutrophils can contribute to the repair of O3-injured epithelium by 

32 removing necrotic epithelial cells (Mudway and Kelly, 2000, 010452; Vesely et al., 1999, 051045). 

33 The degree of airways inflammation due to O3 is thought to have more important long-term 

34 consequences than the more quickly resolving changes in pulmonary function since airways 

35 inflammation is often accompanied by tissue injury (Balmes et al., 1996, 080830). 

36 The influx of inflammatory cells in the airways of human subjects has been assessed by 

37 bronchoscopy and by morphometric measurements in bronchial mucosal biopsies. Bronchoscopy can 

38 be used to sample fluid from all of the airways and from the lung parenchyma distal to the wedged 
March 2011 5-36 DRAFT – DO NOT CITE OR QUOTE 

1 bronchoscope tip or just the more proximal portion. While many studies have documented a distal 

2 lung inflammatory response using conventional (pooled) BAL, a lesser number of studies have 

3 documented inflammation in the proximal airways using “proximal” BAL sampling (i.e., liquid 

4 aspirated from a 20-30 mL initial lavage aliquot after wedging the bronchoscope or from the left 

5 main bronchus transiently isolated by inflation of proximal and distal balloons) or by using bronchial 

6 mucosal biopsy (Aris et al., 1993, 038275; Schelegle et al., 1991, 042491). Airways neutrophilia was 

7 observed at 1 and 6 hours postexposure in proximal airways BAL but only at 6 hours postexposure 

8 in BAL from all of the airways combined (Schelegle et al., 1991, 042491). This result demonstrated 

9 that measurements made in proximal airways BAL better reflected the earliest phase of airways 

10 inflammation than measurements made in conventional multi-aliquot (pooled) BAL.

11 Inter-individual variability in the neutrophilic response has been noted (Devlin et al., 1991, 

12 040359; Holz et al., 1999, 058731; Schelegle et al., 1991, 042491). One study demonstrated a 

13 threefold difference in airways neutrophilia, measured as percent of total cells in proximal BAL, 

14 among human subjects exposed to 0.3 ppm O3 for 1 hour while exercising (Schelegle et al., 1991, 

15 042491), while a 20-fold difference was demonstrated in BAL neutrophils following exposure to 

16 0.08-0.10 ppm O3 for 6.6 hours while exercising (Devlin et al., 1991, 040359). Reproducibility of 

17 intra-individual responses to 0.25 ppm O3, measured as sputum neutrophilia, was demonstrated by 

18 Holz (1999, 058731). Few studies have examined the dose-responsiveness of airways neutrophilia in 

19 O3-exposed humans (Devlin et al., 1991, 040359; Holz et al., 1999, 058731). No dose20 responsiveness was observed in healthy human subjects exposed for 1 hour to 0.125 and 0.25 ppm 

21 O3 and a statistically significant increase in sputum neutrophilia was observed only at the higher 

22 dose (Holz et al., 1999, 058731). However, dose-dependent and statistically significant increases in 

23 BAL neutrophils and the inflammatory mediator IL-6 were reported following exposure to 0.08 and 

24 0.1 ppm O3 for 6.6 hours in exercising humans (Devlin et al., 1991, 040359). Additional evidence is 

25 provided by a meta-analysis of the O3 dose-inflammatory response in controlled human exposure 

26 studies involving exposure to 0.08-0.6 ppm O3 for 60-396 minutes (Mudway and Kelly, 2004, 

27 399328). Results demonstrated a linear relationship between inhaled O3 dose (determined as the 

28 product of concentration, ventilation and time) and BAL neutrophils at 0-6 hours and 18-24 hours 

29 following O3 exposure (Mudway and Kelly, 2004, 399328). 

30 Ozone exposure results in alterations in other airways inflammatory cells besides neutrophils. 

31 Numbers of lymphocytes and total cells in BAL fluid were decreased early after O3 exposure, 

32 preceding the neutrophil influx (Blomberg et al., 1999, 001267; Krishna et al., 1997, 084262; 

33 Mudway and Kelly, 2000, 010452). The decrease in total cells was thought to reflect decreases in 

34 airway macrophages, although it was not clear whether the cells were necrotic or whether membrane 

35 adhesive properties were altered making them more difficult to obtain by lavage (Blomberg et al., 

36 1999, 001267; Frampton et al., 1997, 086111; Mudway and Kelly, 2000, 010452; Mudway et al., 

37 1999, 011833; Pearson and Bhalla, 1997, 082686). Recent studies have demonstrated increases in 

38 numbers of sputum monocytes and dendritic-like cells (Alexis et al., 2010, 628538) (discussed 
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1 further in section 5.2.7). Increases in submucosal mast cells were observed 1.5 hours after a 2-h 

2 exposure of healthy human subjects to 0.2 ppm O3 (Blomberg et al., 1999, 001267) and increases in 

3 BAL mast cell number were observed 18 hours after O3 exposure (Frampton et al., 1997, 086111). 

4 Mast cells may play an important role in mediating neutrophil influx since they are an important 

5 source of several pro-inflammatory cytokines and since their influx precedes that of the neutrophils 

6 (Blomberg et al., 1999, 001267; Stenfors et al., 2002, 030473). Further, a study using mast cell7 deficient mice demonstrated decreased neutrophilic inflammation in response to O3 compared with 

8 wild type mice (Kleeberger et al., 1993, 044203). The mechanisms involved in clearing O3-provoked 

9 inflammation remain to be clarified.

10 The cellular and molecular signals involved in injury and inflammatory responses following 

11 O3 exposure have been extensively evaluated (U.S. EPA, 2006, 088089). Eicosanoids are one class 

12 of secondary oxidation products which may be formed rapidly following O3 exposure and which 

13 may mediate injury and inflammation. Eicosanoids are metabolites of arachidonic acid, a 20-carbon 

14 polyunsaturated fatty acid, which is released from membrane phospholipids by phospholipase 

15 A2-mediated catalysis. Activation of phospholipase A2 occurs by several cell signaling pathways 

16 and may be triggered by O3-mediated lipid peroxidation of cellular membranes (Rashba-Step et al., 

17 1997, 628562). Additionally, cellular phospholipases A2, C and D may be activated by lipid 

18 ozonation products (Kafoury et al., 1998, 016913). While the conversion of arachidonic acid to 

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

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

21 leading to the generation of a wide variety of eicosanoids and reactive oxygen species. Further, the 

22 release of arachidonic acid from phospholipids is accompanied by the formation of 

23 lysophospholipids which are precursors for platelet activating factors. Thus, formation of 

24 eicosanoids, reactive oxygen species and platelet activating factors accompanies O3-mediated lipid 

25 peroxidation. 

26 Additional cell signaling mediators are generated subsequent to O3 exposure. Secondary 

27 reaction products may stimulate airway macrophages to produce cytokines such as IL-1, IL-6 and 

28 TNF-α which in turn activate IL-8 production by epithelial cells. Ozone exposure is also known to 

29 upregulate the vascular endothelial adhesion molecules P-selectin and ICAM-1 (Blomberg et al., 

30 1999, 001267; Krishna et al., 1997, 084262) and to increase the expression of pro-inflammatory 

31 mediators GM-CSF, Gro-α and IL-8 (Mudway and Kelly, 2000, 010452). In addition, lung epithelial 

32 cells may release ATP in response to O3 exposure (Ahmad et al., 2005, 196429). ATP and its 

33 metabolites (catalyzed by ecto-enzymes) can bind to cellular purinergic receptors resulting in 

34 activation of cell signaling pathways (Picher et al., 2004, 644780). One such metabolite, adenine, is 

35 capable of undergoing oxidation leading to the formation of urate which, if present in high 

36 concentrations, could activate inflammasomes and result in caspase 1 activation and the maturation 

37 and secretion of IL-1beta and IL-18 (Dostert et al., 2008, 155753). 
March 2011 5-38 DRAFT – DO NOT CITE OR QUOTE 

1 Many studies have focused on cell signaling pathways leading to airways neutrophilia in 

2 humans or animal models which generally peaks 4-6 hours after exposure. Although IL-8 has been 

3 proposed to play a role in neutrophil chemotaxis, measurements of IL-8 in lavage fluid from humans 

4 exposed to O3 show increases that are too late to account for this effect (Mudway and Kelly, 2000, 

5 010452). However, the profiles of PGE2 and IL-6 responses suggest that they may play a role in 

6 neutrophil chemotaxis. A study in mice demonstrated that PAF may be important in this response 

7 (Longphre et al., 1999, 001199), while macrophage inflammatory protein-2 (MIP-2) and ICAM-1 

8 have also been implicated in a rat model (Bhalla and Gupta, 2000, 015036). Other studies have 

9 investigated mechanisms involved in earlier or later phases of neutrophilic airways inflammation and 

10 inflammation occurring in the nasal airways and more distal lung (U.S. EPA, 2006, 088089). 

11 One set of studies in humans focused on the earliest phase of airways inflammation (1-2 hours 

12 following exposure). Exercising subjects were exposed to 0.2 ppm O3 for 2 hours and bronchial 

13 biopsy tissues were obtained 1.5 and 6 hours after exposure (Blomberg et al., 1999, 001267; Bosson 

14 et al., 2003, 051687; Bosson et al., 2009, 399331; Stenfors et al., 2002, 030473). Results 

15 demonstrated upregulation of vascular endothelial adhesion molecules P-selectin and ICAM-1 at 

16 both 1.5 and 6 hours (Blomberg et al., 1999, 001267; Stenfors et al., 2002, 030473). Submucosal 

17 mast cell numbers were increased at 1.5 hours in the biopsy samples without an accompanying 

18 increase in neutrophil number (Blomberg et al., 1999, 001267). Pronounced neutrophil infiltration 

19 was observed at 6 h in the bronchial mucosa (Stenfors et al., 2002, 030473). Surprisingly, 

20 suppression of the NFkB and AP-1 pathways at 1.5 hours and a lack of increased IL-8 at 1.5 or 

21 6 hours in bronchial epithelium was observed (Bosson et al., 2009, 399331). The authors suggested 

22 that vascular endothelial adhesion molecules, rather than redox sensitive transcription factors, are 

23 key to early neutrophil recruitment in response to O3. 

24 Multi-day exposure to O3 has been found to dampen the inflammatory response, but not the 

25 injury response, compared with a single day exposure (Christian et al., 1998, 029925; Devlin et al., 

26 1997, 083577). In human subjects exposed for 4 hours to 0.2 ppm O3 during moderate exercise, 

27 decreased numbers of BAL neutrophils were observed after 4 days of consecutive exposure 

28 compared with responses after 1 day (Christian et al., 1998, 029925). Results indicated an 

29 attenuation of the inflammatory response in both proximal airways and distal lung. However 

30 repeated exposure did not result in attenuation of the injury markers lactate dehydrogenase and 

31 protein in the BAL. Similar results were found in a study of humans undergoing heavy intermittent 

32 exercise who were exposed for 2 hours to 0.4 ppm O3 for 5 consecutive days (Devlin et al., 1997, 

33 083577). In this latter study, partial recovery of the inflammatory response was noted 10 days 

34 following the exposure (Devlin et al., 1997, 083577). In an animal study conducted in parallel (Van 

35 Bree et al., 2002, 035452), full susceptibility to O3 challenge following exposure to O3 for 5 

36 consecutive days required 15-20 days recovery. Further, no attenuation of cellular proliferation in 

37 terminal bronchioles was observed during the 5 consecutive days of O3 exposure. In a separate study 

38 in rats involving repeated O3 exposures, a lack of attenuation of the injury marker lavagable protein, 
March 2011 5-39 DRAFT – DO NOT CITE OR QUOTE 

1 the persistence of macrophages in the centriacinar region, and histological evidence of progressive 

2 tissue injury was demonstrated (Tepper et al., 1989, 041991). Thus, the inflammatory response 

3 resembled that of the pulmonary function response which was attenuated with repeated short-term 

4 O3 exposure in both human subjects and animals (Christian et al., 1998, 029925; Hackney et al., 

5 1977, 038282; Horvath et al., 1981, 039221). Findings that injury, measured by BAL markers or by 

6 histopathology, persisted in the absence of inflammation or pulmonary function decrements suggests 

7 that, despite adaptation, repeated exposure to O3 may have serious long-term consequences such as 

8 airway remodeling. The mechanisms involved in clearing O3-provoked inflammation remain to be 

9 clarified.

10 Increases in markers of inflammation and of injury occurred to a comparable degree in 

11 subjects with mild (least sensitive) and more remarkable (more sensitive) spirometric responses to 

12 O3 (Balmes et al., 1996, 080830). Two other studies using similar exposure protocols found that 

13 acute spirometric changes were not positively correlated with cellular and biochemical indicators of 

14 inflammation (Aris et al., 1993, 038275; Schelegle et al., 1991, 042491). However inflammation was 

15 correlated with changes in sRaw (Balmes et al., 1996, 080830). In another study, pretreatment with 

16 ibuprofen had no effect on neutrophilia although it blunted the spirometric response (Hazucha et al., 

17 1996, 043923). Taken together, results from these studies indicate different mechanisms underlying

18 the spirometric and inflammatory responses to O3. 

19 In contrast, a common mechanism underlying both inflammation and impaired pulmonary 

20 function was suggested by (Krishna et al., 1997, 084262). This study, conducted in exercising 

21 humans exposed to 0.2 ppm O3 for 2 hours, demonstrated a correlation between loss of SP 

22 immunoreactivity from neurons in the bronchial mucosa and numbers of neutrophils and epithelial 

23 cells (shed epithelial cells are an index of injury) in the BAL 6 h postexposure. Furthermore, the loss 

24 of SP immunoreactivity was correlated with the observed changes in FEV1. SP is a neuropeptide 

25 released by sensory nerves which mediates neurogenic edema and bronchoconstriction (Krishna et 

26 al., 1997, 084262). Further, another study found that SP was increased in lavage fluid of human 

27 subjects immediately after O3 challenge (Hazbun et al., 1993, 043914). Taken together, these 

28 findings suggest O3-mediated stimulation of sensory nerves leading to activation of central and local 

29 axon reflexes as a common effector pathway leading to impaired pulmonary function and 

30 inflammation. 

31 Chronic exposure to O3 has been studied in animal models. In the nasal airways, exposure to 

32 O3 for days or weeks results in mucous cell metaplasia of nasal transitional epithelium (Harkema et 

33 al., 1999, 001209; Hotchkiss et al., 1991, 042441). This remodeling effect was characterized by 

34 neutrophilic infiltration, a loss of sensitive nasal epithelial cells, the proliferation of resistant 

35 epithelial cells and mucin gene overexpression (Cho et al., 1999, 011985). Bronchiolitis, bronchiolar 

36 metaplasia of alveolar ducts, proliferation of Type 2 pneumocytes and fibrosis were reported 

37 following chronic O3 exposure (Mudway and Kelly, 2000, 010452). It was suggested that remodeling 

38 of respiratory epithelium may lead to enhanced resistance or tolerance to O3 (Mudway and Kelly, 
March 2011 5-40 DRAFT – DO NOT CITE OR QUOTE 

1 2000, 010452). Deposition of collagen in the airways and sustained lung function decrements 

2 especially in small airways have also been demonstrated as a response to chronic O3 exposure 

3 (Chang et al., 1992, 042387; Mudway and Kelly, 2000, 010452). 

5.2.4.1. New Cellular an d Mole c ula r Ins igh ts

4 Recent investigations in animal models have elucidated additional mechanisms involved in 

5 O3-induced inflammation and injury. In one study, tachykinins working through NK-1 and CGRP 

6 receptors were found to contribute to airways epithelial injury, but not to neutrophil influx, in O3-

7 exposed rats (Oslund et al., 2008, 195654; Oslund et al., 2009, 201539). Key roles for CXCR2, a 

8 receptor for the cytokines KC and MIP-2, and for IL-6 in O3-mediated neutrophil influx were

9 demonstrated in mice (Johnston et al., 2005, 596393; Johnston et al., 2005, 596394). Activation of 

10 JNK and p38 pathways and cathepsin-S were also found to be important in this response (Williams 

11 et al., 2007, 628609; Williams et al., 2008, 628607; Williams et al., 2009, 628605). Furthermore, 

12 matrix metalloproteinase-9 (MMP-9) protected against O3-induced airways inflammation and injury 

13 in mice (Yoon et al., 2007, 596422). 

14 Williams et al. (2007, 597545) found that the toll-like receptor (TLR) adaptor protein MyD88 

15 was important in mediating O3-induced neutrophilia in mice exposed to 3 ppm O3 for 3 hours, with 

16 TLR4 and TLR2 contributing to the speed of the response. Moreover, MyD88, TLR2 and TLR4 

17 contributed to inflammatory gene expression in this model and O3 upregulated MyD88, TLR4 and 

18 TLR4 gene expression. These results complement those of Hollingsworth et al. (2004, 097816) who 

19 demonstrated airways neutrophilia following acute (2 ppm O3 for 3 hours) and subchronic O3

20 exposure (0.3 ppm for 3 days) in a mouse model (Hollingsworth et al., 2004, 097816). In this study, 

21 airways neutrophilia was not dependent on TLR4 (Hollingsworth et al., 2004, 097816). Ozone

22 effects on lung hyperpermeability, which is often correlated with neutrophil influx, were previously 

23 found to require a functioning TLR4 (Kleeberger et al., 2000, 014895). 

24 Other studies focused on the role of hyaluronan in mediating a later phase (24 hours) of 

25 O3-induced inflammation in mice (Garantziotis et al., 2009, 597603; Garantziotis et al., 2010, 

26 624947). Hyaluronan is an extracellular matrix component which is normally found in the ELF as a 

27 large polymer. Exposure to 2 ppm O3 for 3 hours resulted in elevated levels of soluble low molecular 

28 weight hyaluronan in the BAL fluid 24 hours postexposure (Garantziotis et al., 2009, 597603; 

29 Garantziotis et al., 2010, 624947). Ozone may have caused the depolymerization of hyaluronan to 

30 soluble fragments which are known to be endogenous ligands of the CD44 receptor and TLR4 in the 

31 macrophage (Jiang et al., 2005, 628556). Binding of hyaluronan fragments to the CD44 receptor 

32 activates hyaluronan clearance, while binding to TLR4 results in signaling through MyD88 to 

33 produce chemokines that stimulate the influx of inflammatory cells (Jiang et al., 2005, 628556). 

34 Activation of NFkB occurred in both airway epithelia and alveolar macrophages 24 hours 

35 postexposure to O3. Increases in BAL pro-inflammatory factors KC, IL-1beta, MCP-1, tumor 

36 necrosis factor-α (TNF-α) and IL-6 observed 24 hours following O3 exposure were found to be 
March 2011 5-41 DRAFT – DO NOT CITE OR QUOTE 

1 partially dependent on TLR4 while increases in BAL inflammatory cells, which consisted mainly of 

2 macrophages, were dependent on CD44. BAL inflammatory cells number and injury markers 

3 following O3 exposure were similar in wild-type and TLR4-deficient animals. 

5.2.4.2. Summary

4 Injury and inflammation are key events in ozone’s toxicity pathway. Secondary oxidation 

5 products have been implicated in a number of these processes. Although there may be inter-species 

6 differences with respect to specific mediators, mechanisms involved in the acute responses to O3

7 include epithelial injury and airways neutrophilia. Longer-term exposures may result in mucus cell 

8 metaplasia of nasal epithelium or airways remodeling and fibrosis. Work from several laboratories in 

9 humans and animal models suggest that O3 triggers the release of tachykinins such as SP from 

10 airway sensory nerves which could contribute to downstream effects including injury and 

11 inflammation. New investigations show that O3 exposure leads to the generation of hyaluronan 

12 fragments which activate TLR4 and CD44-dependent signaling pathways in macrophages and result 

13 in a greater turnover of macrophage populations in the lung. Activation of these pathways occurs 

14 later than the acute neutrophilic response suggesting that they may contribute to longer-term effects 

15 of O3. The mechanisms involved in clearing O3-provoked inflammation remain to be clarified.

16 Similar to the pulmonary function responses discussed in the previous section, considerable 

17 inter-individual variability exists in O3 responsiveness as measured by airways neutrophilia. Further, 

18 attenuation of the inflammatory response occurs following O3 exposure for several consecutive days. 

19 However evidence suggests that injury may continue despite the dampening of the inflammatory 

20 response during repeated exposures. Mechanisms responsible for inter-individual variability and 

21 response attenuation, or the lack thereof, are not known. It should be noted that inflammation, as 

22 measured by airways neutrophilia, is not correlated with decrements in pulmonary function as 

23 measured by spirometry. Consequently, spirometric measures are not a good surrogate for the degree 

24 of inflammation in any given individual following O3 exposure. Furthermore, airways neutrophilia 

25 may not be a good indicator of O3-mediated lung injury.

5.2.5. Inc re a s e d Bron c hial Rea c tivity

26 In addition to causing mild airways obstruction as discussed above, acute O3 exposure results 

27 in reversible increases in bronchial reactivity by mechanisms which are not well understood. These 

28 effects may be more significant in human subjects with already compromised airways 

29 (Section 5.2.6). Bronchial reactivity is generally determined in terms of a response to a challenge 

30 agent. Non-specific bronchial reactivity in humans is assessed by measuring the effect of inhaling 

31 increasing concentrations of a bronchoconstrictive drug on lung mechanics (sRaw or FEV1). 

32 Methacholine is most commonly employed but histamine and other agents are also used. Specific 

33 bronchial reactivity is assessed by measuring effects in response to an inhaled allergen in individuals 
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1 (or animals) already sensitized to that allergen. An increase in sRaw in response to non-specific or 

2 specific challenge agents indicates airways hyperresponsiveness (AHR).

3 Ozone may sensitize bronchial smooth muscle to stimulation through a direct effect on smooth 

4 muscle or through effects on the sensory nerves in the epithelium or on the motor nerves innervating 

5 the smooth muscle (Holtzman et al., 1979, 039220; O'Byrne et al., 1983, 041366; O'Byrne et al., 

6 1984, 040066). One possibility may be related to O3-mediated increases in epithelial permeability, 

7 which would improve access of agonist to smooth muscle receptors (Holtzman et al., 1979, 039220). 

8 Neurally-mediated effects have been demonstrated in several studies. In one, pretreatment with 

9 atropine was found to block O3-induced AHR, suggesting the involvement of cholinergic 

10 postganglionic pathways (Holtzman et al., 1979, 039220). Other studies in animals demonstrated 

11 that O3-induced AHR involved vagally-mediated responses (Freed et al., 1996, 080798) and local 

12 axon reflex responses through bronchopulmonary C fiber-mediated release of SP (Joad et al., 1996, 

13 082711). Further, pretreatment with capsaicin to deplete nerve fibers of SP blocked O3-mediated 

14 bronchial reactivity measured as AHR (Tepper et al., 1993, 628570). 

15 Some evidence suggested the involvement of arachidonic acid metabolites (Fabbri et al., 1985,

16 040276; Seltzer et al., 1986, 040383). An early study found AHR in exercising humans immediately 

17 postexposure to 0.6 ppm O3 for 2 hours, which was associated with increases in BAL neutrophils and 

18 cyclooxygenase products (Seltzer et al., 1986, 040383). Other investigators found that ibuprofen 

19 pretreatment had no effect on AHR following exposure to 0.4 ppm O3 for 2 hours, although 

20 spirometric responses were blunted (Hazucha et al., 1996, 043923). This study indicated that the 

21 arachidonic acid metabolites whose generation was blocked by ibuprofen, an inhibitor of 

22 cyclooxygenase and some lipoxygenase activity, (i.e. prostaglandins, thromboxanes and some 

23 leukotrienes) did not play a role in AHR. Experiments in dogs demonstrated a close correlation 

24 between O3-induced AHR and airway neutrophilic inflammation measured in tissue biopsies 

25 (Holtzman et al., 1983, 039745). Furthermore, the increased AHR observed in dogs following O3

26 exposure was inhibited by neutrophil depletion (O'Byrne et al., 1983, 041366) and by pre-treatment 

27 with inhibitors of arachidonic acid metabolism. In one of these studies, indomethacin pre-treatment 

28 did not prevent airway neutrophilia in response to O3 providing evidence that the subset of 

29 arachidonic acid metabolites whose generation was inhibitable by the cyclooxygenase inhibitor 

30 indomethacin (i.e., prostaglandins and thromboxanes) was not responsible for neutrophil influx 

31 (O'Byrne et al., 1984, 040066). Taken together, these findings suggest that arachidonic acid 

32 metabolites, but probably not prostaglandins or thromboxanes, may be involved in the AHR response 

33 following O3 exposure in dogs. 

34 Later it was recognized that increased bronchial reactivity can be both a rapidly occurring and 

35 persistent response to O3 (Foster and Freed, 1999, 001202). Secondary oxidation products of O3 and 

36 tachykinins have been proposed as early mediators of the response and inflammation-derived 

37 products have been proposed as mediators of the later response (Foster and Freed, 1999, 001202). 

38 Some studies have suggested an involvement of IL-1 (Park et al., 2004, 644864) and TNF-α (Cho et 
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1 al., 2001, 016160; Shore et al., 2001, 018993). Furthermore, multiday exposure to O3 has been found 

2 to dampen the AHR response compared with a single day exposure (Dimeo et al., 1981, 039662). 

5.2.5.1. Ne w Ce llula r a n d Mole c ula r Ins igh ts 

3 Recent studies in animal models provide new evidence for mechanisms underlying increased 

4 bronchial reactivity. In guinea pigs, AHR was found to be mediated by different pathways at 1 and 

5 3 days postexposure to a single dose of O3 (2 ppm for 4 hours) (Verhein et al., 2011, 670295; Yost et 

6 al., 2005, 597549). At 1 day, airway hyperreactivity was due to activation of airway parasympathetic 

7 nerves rather than to a direct effect on smooth muscle (Yost et al., 2005, 597549). This effect 

8 occurred as a result of O3-stimulated release of major basic protein from eosinophils (Yost et al., 

9 2005, 597549). Major basic protein is known to block inhibitory M2 muscarinic receptors which 

10 normally dampen acetylcholine release from parasympathetic nerves (Yost et al., 2005, 597549). The 

11 resulting increase in acetylcholine release caused an increase in smooth muscle contraction 

12 following O3 exposure (Yost et al., 2005, 597549). Eosinophils played a different role 3 days 

13 postexposure to O3 in guinea pigs (Yost et al., 2005, 597549). Ozone-mediated influx of eosinophils 

14 into lung airways resulted in a different population present 3 days postexposure compared to those 

15 present at one day (Yost et al., 2005, 597549). At this point, eosinophil-derived major basic protein 

16 increased smooth muscle responsiveness to acetylcholine which also contributed to AHR (Yost et al., 

17 2005, 597549). However, the major effect of eosinophils was to protect against vagal hyperreactivity 

18 (Yost et al., 2005, 597549). The authors suggested that these beneficial effects were due to the 

19 production of nerve growth factor (Yost et al., 2005, 597549). Further work by these investigators 

20 demonstrated a key role for IL-1beta in mediating AHR three days postexposure to O3 (Verhein et 

21 al., 2011, 670295). In this study, IL-1beta increased nerve growth factor and SP which acted through 

22 the NK1 receptor to cause vagally-mediated bronchoconstriction (Verhein et al., 2011, 670295). The 

23 mechanism by which SP caused acetylcholine release from parasympathetic nerves following O3

24 exposure was not determined (Verhein et al., 2011, 670295). Two studies by other investigators 

25 demonstrated that SP released from airways nociceptive neurons contributed to O3-induced AHR 

26 measured 3 hours postexposure to 2 ppm O3 in ferrets (Wu et al., 2003, 628616; Wu et al., 2008, 

27 597548). These authors further suggested that SP expression in airway neurons was upregulated by 

28 IL-1 which was released in response to O3 (Wu et al., 2008, 597548). Taken together, the above 

29 study results indicate that mechanisms involved in O3-mediated AHR can vary over time 

30 postexposure and that eosinophils and SP can play a role. 

31 Evidence for cytokine and chemokine involvement in the AHR response to O3 has recently 

32 been described. Williams et al. (2008, 597546) demonstrated that the Th2 cytokine IL-13 contributed 

33 to AHR, as well as to airways neutrophilia, following exposure of mice to 3 ppm O3 for 3 hours. 

34 Other studies in mice have demonstrated a key role for CXCR2, the chemokine receptor for the 

35 neutrophil chemokines KC and MIP-2, but not for IL-6 in O3-mediated AHR (Johnston et al., 2005, 

36 596394; Johnston et al., 2005, 596393). In contrast, CXCR2 and IL-6 were both required for 
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1 neutrophil influx in this model (Johnston et al., 2005, 596393; Johnston et al., 2005, 596394), as 

2 discussed above. 

3 Other studies have focused on the role of TLR4. Hollingsworth measured AHR, as well as 

4 airways neutrophilia, in mice 6 and 24 hours following acute (2 ppm O3 for 3 hours) and subchronic 

5 (0.3 ppm for 3 days) exposure to O3 (Hollingsworth et al., 2004, 097816). TLR4 is a key component 

6 of the innate immune system and is responsible for the immediate inflammatory response seen 

7 following challenge with endotoxin and other pathogen-associated substances. In the 2004 study, a 

8 functioning TLR4 was required for the full AHR response following O3 exposure but not for airways 

9 neutrophilia (Hollingsworth et al., 2004, 097816). These findings are complemented by an older 

10 study demonstrating that O3 effects on lung hyperpermeability required a functioning TLR4 

11 (Kleeberger et al., 2000, 014895). Williams et al. (2007, 597545) found that TLR2, TLR4 and the 

12 TLR adaptor protein MyD88 contributed to AHR in mice exposed to 3 ppm O3 for 3 hours. Ozone

13 was also found to upregulate MyD88, TLR4 and TLR4 gene expression in this model (Williams et 

14 al., 2007, 597545). 

15 A newly recognized mechanistic basis for O3-induced AHR is provided by studies focusing on 

16 the role of hyaluronan following O3 exposure in mice (Garantziotis et al., 2010, 624947) 

17 (Garantziotis et al., 2009, 597603). Briefly, TLR4 and CD44 were found to mediate AHR in response 

18 to O3 and hyaluronan. Hyaluronan is an extracellular matrix component which is normally found in 

19 the ELF as a large polymer. Exposure to 2 ppm O3 for 3 hours resulted in enhanced AHR and 

20 elevated levels of soluble low molecular weight hyaluronan in the BAL fluid 24 hours postexposure 

21 (Garantziotis et al., 2009, 597603; Garantziotis et al., 2010, 624947). Ozone may have caused the 

22 depolymerization of hyaluronan to soluble fragments which are known to be endogenous ligands of 

23 the CD44 receptor and TLR4 in the macrophage (Jiang et al., 2005, 628556). In the two recent 

24 studies, O3-induced AHR was attenuated in CD44 and TLR4-deficient mice (Garantziotis et al., 

25 2009, 597603; Garantziotis et al., 2010, 624947). Hyaluronan fragment-mediated stimulation of 

26 AHR was found to require functioning CD44 receptor and TLR4 (Garantziotis et al., 2009, 597603; 

27 Garantziotis et al., 2010, 624947). In contrast, high-molecular-weight hyaluronan blocked AHR in 

28 response to O3 (Garantziotis et al., 2009, 597603). In another study high-molecular-weight 

29 hyaluronan enhanced repair of epithelial injury (Jiang et al., 2005, 628556). These studies provide a 

30 link between innate immunity and the development of AHR following O3 exposure, and indicate a 

31 role for TLR4 in increasing bronchial smooth muscle reactivity. 

5.2.5.2. Summary

32 Increased bronchial reactivity is a key event in the toxicity pathway of O3. It can be both a 

33 rapidly occurring and persistent response, although adaptation can also occur during multi-day 

34 exposures. Both direct effects on smooth muscle and neurally-mediated effects on smooth muscle 

35 have been proposed to contribute to AHR following O3 exposure. Currently, more evidence has 

36 accumulated for the latter mechanism. In humans exposed to O3, atropine was found to block the 
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1 early AHR response indicating the involvement of cholinergic postganglionic pathways. Inhibition 

2 of arachidonic acid metabolism was ineffective in blocking this response in humans while mixed 

3 results were found in animal models. Studies in O3-exposed animals have demonstrated a role for SP 

4 release from bronchial C fibers in mediating neurally-mediated effects on smooth muscle. Later 

5 phases of increased bronchial reactivity may involve the induction of IL-1beta which in turn 

6 upregulates SP production. In guinea pigs, eosinophil-derived major basic protein contributed to the 

7 stimulation of cholinergic postganglionic pathways. A novel role for hyaluronan in mediating the 

8 later phase effects of O3 has recently been demonstrated. High molecular weight polymers of 

9 hyaluronan normally found in the ELF were degraded following O3 exposure in mice. The resulting 

10 hyaluronan fragments stimulated AHR in a TLR4- and CD44 receptor-dependent manner. Previous 

11 work has shown that O3-mediated increases in lung permeability required a functioning TLR4 

12 suggesting a possible relationship between increased epithelial permeability and AHR in this model.

13 Other cytokines and chemokines have been implicated in the AHR response to O3 in animals models. 

5.2.6. Exa ce rba tio n a n d Ind u ction of As thma and Allergic Res po n s e s

14 In individuals with asthma, there is increased responsiveness to bronchoconstrictor challenge. 

15 This results from a combination of structural and physiological factors including increased inner16 wall thickness, smooth muscle responsiveness and mucus secretion. Although inflammation is likely 

17 to contribute, its relationship to AHR is not clear (U.S. EPA, 2006, 088089). However, some 

18 asthmatics have higher baseline levels of neutrophils, lymphocytes, eosinophils and mast cells in 

19 bronchial washes and bronchial biopsy tissue (Stenfors et al., 2002, 030473). Evidence is 

20 accumulating that O3 exposure exacerbates asthmatic and allergic responses in sensitive individuals. 

21 Further, some studies suggest that O3 exposure leads to the development of asthmatic and allergic 

22 responses. 

23 In order to determine whether asthmatics exhibit greater sensitivity to O3, several older studies 

24 compared pulmonary function responses in asthmatic and non-asthmatic subjects following O3

25 exposure. While the majority focused on measurements of FEV1 and FVC and found no differences 

26 between the two groups (Holz et al., 1999, 058731; Koenig et al., 1987, 041521; Linn et al., 1978, 

27 038874; Mudway et al., 2001, 025327; Scannell et al., 1996, 080755; Stenfors et al., 2002, 030473), 

28 there were notable exceptions. In one study, greater airways obstruction in asthmatics compared with 

29 non-asthmatic subjects was observed immediately following a 2-h exposure to 0.4 ppm O3 with 

30 intermittent exercise (Kreit et al., 1989, 041817). These changes were measured as statistically 

31 significant greater decreases in FEV1 and FEF25-75 in the absence of a bronchoconstrictor challenge 

32 (Kreit et al., 1989, 041817). These results suggest that this group of asthmatics responded to 

33 O3-exposure with a greater degree of vagally-mediated bronchoconstriction compared with the non34 asthmatics. A second study demonstrated a statistically significant greater decrease in FEV1 and 

35 FEV1/FVC in asthmatics compared with non-asthmatics exposed to 0.12 ppm O3 for 7.6 hours with 

36 light exercise (Horstman et al., 1995, 075834). These responses were accompanied by wheezing and 
March 2011 5-46 DRAFT – DO NOT CITE OR QUOTE 

1 inhaler use in the asthmatics (Horstman et al., 1995, 075834). Aerosol bolus dispersion 

2 measurements demonstrated a statistically significant greater change in asthmatics compared with 

3 non-asthmatics which was suggestive of O3-induced small airway dysfunction (Horstman et al., 

4 1995, 075834). Furthermore, a statistically significant correlation was observed between the degree 

5 of baseline airway status and the FEV1 response to O3 in the asthmatic subjects (Horstman et al.,

6 1995, 075834). A third study found similar decreases in FVC and FEV1 in both asthmatics and non7 asthmatics exposed to 0.4 ppm O3 for 2 hours with mild exercise (Alexis et al., 2000, 013072). 

8 However, a statistically significant decrease in FEF75, a measure of small airway function, was 

9 observed in asthmatics but not in non-asthmatics (Alexis et al., 2000, 013072). Taken together these 

10 latter studies indicate that while the magnitude of restrictive type spirometric decline was similar in 

11 asthmatics and non-asthmatics, that obstructive type changes (i.e. bronchoconstriction) were greater 

12 in asthmatics. Further, asthmatics exhibited greater sensitivity to O3 in terms of small airways 

13 function. 

14 Since asthma exacerbations occur in response to allergens and/or other triggers, some studies 

15 have focused on O3-induced changes in AHR following a bronchoconstrictor challenge. No 

16 difference in sensitivity to methacholine bronchoprovocation was observed between asthmatics and 

17 non-asthmatics exposed to 0.4 ppm O3 for 2 hours with moderate exercise (Kreit et al., 1989, 

18 041817). However, increased bronchial reactivity to inhaled allergens was demonstrated in mild 

19 allergic asthmatics (Jorres et al., 1996, 078122; Kehrl et al., 1999, 022101; Molfino et al., 1991, 

20 042379) and in allergen-sensitized guinea pigs following O3 exposure (Sun et al., 1997, 082724). 

21 Similar, but modest, responses were reported for individuals with allergic rhinitis (Jorres et al., 1996, 

22 078122). Further, the contractile response of isolated airways was increased by pre-exposure to O3 in 

23 human subjects sensitized and challenged with allergen (Roux et al., 1999, 001264). 

24 In terms of airways neutrophilia, larger responses were observed in asthmatics compared to 

25 non-asthmatics subjects exposed to O3 in some (Balmes et al., 1997, 086092; Basha et al., 1994, 

26 075950; Scannell et al., 1996, 080755) but not all (Mudway et al., 2001, 025327) of the older 

27 studies. Further, statistically significantly increases in myeloperoxidase levels (an indicator of 

28 neutrophil activation) in bronchial washes was observed in mild asthmatics compared with non29 asthmatics, despite no difference in O3-stimulated neutrophil influx between the 2 groups following 

30 exposure to 0.2 ppm O3 for 2 hours with mild exercise (Stenfors et al., 2002, 030473). 

31 Eosinophils and associated proteins are thought to affect muscarinic cholinergic receptors 

32 which are involved in vagally-mediated bronchoconstriction (Mudway and Kelly, 2000, 010452). 

33 Studies described in Section 5.2.5.1 which demonstrated a key role of eosinophils in O3-mediated 

34 AHR may not be entirely relevant to humans given the large numbers of eosinophils normally 

35 present in guinea pig airways compared with humans (Yost et al., 2005, 597549). However airways 

36 eosinophilia often occurs in human allergic airways disease, suggesting greater plausibility of this

37 mechanism in allergic asthmatics. Furthermore, O3 exposure sometimes often results in airways 

38 eosinophilia in allergic subjects or animal models. For example, eosinophilia of the nasal and other 
March 2011 5-47 DRAFT – DO NOT CITE OR QUOTE 

1 airways was observed in individuals with preexisting allergic disease following O3 inhalation (Peden 

2 et al., 1995, 076189; Vagaggini et al., 2002, 035191). Further, O3 exposure increased allergic

3 responses, such as eosinophilia and augmented intraepithelial mucosubstances, in the nasal airways 

4 of ovalbumin (OVA)-sensitized rats (Wagner et al., 2002, 026079). In contrast, Stenfors (2002, 

5 030473) found no stimulation of eosinophil influx measured in bronchial washes and BAL fluid of 

6 mild asthmatics following exposure to a lower concentration of O3. 

7 The role of mast cells in O3-mediated asthma exacerbations has been investigated. Mast cells 

8 are thought to play a key role in O3-induced airways inflammation, since airways neutrophilia was 

9 decreased in mast cell-deficient mice exposed to O3 (Kleeberger et al., 1993, 044203). However, 

10 another study found that mast cells were not involved in the development of increased bronchial 

11 reactivity in O3-exposed mice (Noviski et al., 1999, 001198). Nonetheless, mast cells release a wide 

12 variety of important inflammatory mediators which may lead to asthma exacerbations (Stenfors et 

13 al., 2002, 030473). A large increase in mast cell number in bronchial submucosa was observed in 

14 non-asthmatics and a significant decrease in mast cell number in bronchial epithelium was observed 

15 in mild asthmatics 6 hours following exposure to 0.2 ppm O3 for 2 hours during mild exercise 

16 (Stenfors et al., 2002, 030473). While these results point to an O3-mediated flux in bronchial mast 

17 cell populations which differed between the non-asthmatics and mild asthmatics, interpretation of 

18 these findings is difficult. Furthermore, mast cell number did not change in airway lavages in either 

19 group in response to O3 (Stenfors et al., 2002, 030473) 

20 Cytokine profiles in the airways have been investigated as an indicator of O3 sensitivity. 

21 Differences in epithelial cytokine expression were observed in bronchial biopsy samples in non22 asthmatic and asthmatic subjects both at baseline and 6 h postexposure to 0.2 ppm O3 for 2 hours 

23 (Bosson et al., 2003, 051687). The asthmatic subjects had a higher baseline expression of IL-4 and 

24 IL-5 compared to non-asthmatics. In addition, expression of IL-5, IL-8, GM-CSF, and ENA-78 in 

25 asthmatics was increased significantly following O3 exposure compared to non-asthmatics (Bosson 

26 et al., 2003, 051687). Some of these (IL-4, IL-5 and GM-CSF) are T helper type 2 (Th2)-related 

27 cytokines or neutrophil chemoattractants, and play a role in IgE production, airway eosinophilia and 

28 suppression of Th1-cytokine production (Bosson et al., 2003, 051687). These findings suggest a link 

29 between adaptive immunity and enhanced sensitivity of asthmatics to O3. 

5.2.6.1. New Cellular an d Mole c ula r Ins igh ts

30 Since asthmatics may have enhanced sensitivity to O3, one recent study investigated whether 

31 O3 exposure exacerbated asthmatic responses in persistent asthmatics. These subjects had been 

32 therapeutically treated with inhaled corticosteroids for several months prior to the study. Exposure of 

33 these subjects to 0.2 ppm O3 for 2 hours with mild exercise resulted in decrements in FEV1 and FVC 

34 and an increase in sRaw immediately postexposure (Stenfors et al., 2010, 386512). In addition, large 

35 increases in neutrophil number and myeloperoxidase levels (an indicator of neutrophil activation) in 

36 airway lavages and in mast cell number in bronchial submucosa tissue obtained by biopsy was 
March 2011 5-48 DRAFT – DO NOT CITE OR QUOTE 

1 observed 18 hours postexposure (Stenfors et al., 2010, 386512). No change in bronchial wash or 

2 BAL fluid mast cell number or eosinophil number in any compartment was observed. (Stenfors et 

3 al., 2010, 386512). These results suggest that some standard treatments for asthma may not protect 

4 against the effects of environmental O3 and that this group may be particularly prone to asthma 

5 exacerbations given that airways neutrophilia occurred despite the inhaled corticosteroid treatment 

6 (Stenfors et al., 2010, 386512). 

7 Other recent studies in humans and animals provide evidence that O3 causes the development 

8 of allergic responses and/or asthma. Several of these suggested that O3-mediated activation of innate 

9 immunity contributes to O3-induced, as well as to O3-amplified, allergic responses. In one study 

10 involving human subjects, the authors hypothesized that O3 exposure would result in recruitment of 

11 activated innate immune cells to the airways. Healthy individuals were exposed to 0.08 ppm O3 for 

12 6.6 hours with intermittent exercise and airways inflammation was characterized in induced sputum 

13 18 h postexposure (Alexis et al., 2010, 628538). Previous studies demonstrated that induced sputum 

14 contains liquid and cellular constituents of the lining fluid from central conducting airways (Alexis 

15 et al., 2001, 190013) and also identified these airways as a site of preferential O3 absorption during 

16 exercise (Hu et al., 1994, 041323). Ozone exposure resulted in sputum neutrophilia and increased 

17 numbers of airways monocytes and dendritic-like cells (Alexis et al., 2010, 628538). In addition, 

18 increased expression of cell surface markers characteristic of innate immunity and antigen 

19 presentation (i.e. CD-14 and HLA-DR) was demonstrated on airways monocytes (Alexis et al., 2010, 

20 628538). Enhanced antigen presentation contributes to exaggerated T cell responses and promotes 

21 Th2 inflammation and an allergic phenotype (Lay et al., 2007, 196610). Upregulation of pro22 inflammatory cytokines in sputum was also demonstrated in O3-exposed subjects (Alexis et al., 

23 2010, 628538). One of these cytokines, IL-12p70, correlated with numbers of dendritic-like cells in 

24 the sputum, and is an indicator of dendritic cell activation (Alexis et al., 2010, 628538). These 

25 authors have previously reported that O3 activates monocytes and macrophages (Lay et al., 2007, 

26 196610) which could play a role in exacerbating existing asthma by activating allergen-specific 

27 memory T-cells. The current study confirms these findings and extends them by suggesting a 

28 potential mechanism whereby O3-activated dendritic cells could stimulate naïve T-cells to promote 

29 the development of asthma (Alexis et al., 2010, 628538). A companion study by these same 

30 investigators (described in detail in Section 5.2.9.1) provides evidence of dendritic cell activation, 

31 measured as increased expression of HLA-DR, in a subset of the human subjects (GSTM1 null) 

32 exposed to 0.4 ppm O3 for 2 hours with intermittent exercise (Alexis et al., 2009, 628542) 

33 Another recent study demonstrated O3-mediated activation of the innate immune system and 

34 linked it to the development of non-specific AHR in a mouse model (Pichavant et al., 2008, 596409). 

35 Repeated exposure to 1 ppm O3 for 3 hours induced non-specific AHR measured 24 hours following 

36 the last exposure (Pichavant et al., 2008, 596409). This response was found to require NKT cells, 

37 which are effector lymphocytes of innate immunity, as well as IL-17 and airways neutrophilia 

38 (Pichavant et al., 2008, 596409). Since glycolipids such as galactosyl ceramide are ligands for the 
March 2011 5-49 DRAFT – DO NOT CITE OR QUOTE 

1 invariant CD1 receptor on NKT cells and serve as endogenous activators of NKT cells, a role for 

2 O3-oxidized lipids in activating NKT cells was proposed (Pichavant et al., 2008, 596409). The 

3 authors contrasted this innate immunity pathway with that of allergen-provoked specific AHR which 

4 involves adaptive immunity, the cytokines IL-4, IL -13, IL-17, and airways eosinophilia (Pichavant 

5 et al., 2008, 596409). Interestingly, NKT cells were required for both the specific AHR provoked by 

6 allergen and the non-specific AHR provoked by O3 (Pichavant et al., 2008, 596409). Different 

7 cytokine profiles of the NKT cells from allergen and O3-exposed mice in mediating was proposed to 

8 account for the different pathways (Pichavant et al., 2008, 596409). More recently, NKT cells have 

9 been found to function in both innate and adaptive immunity (Vivier et al., 2011, 676697). 

10 Priming of the innate immune system by O3 was reported by Hollingsworth et al., (2007, 

11 597609). In this study, exposure of mice to 2 ppm O3 for 3 hours led to nonspecific AHR at 24 and 

12 48 hours postexposure, an effect which subsided by 72 hours (Hollingsworth et al., 2007, 597609). 

13 However in mice treated with aerosolized endotoxin immediately following O3 exposure, AHR was 

14 greatly enhanced at 48 and 72 hours postexposure (Hollingsworth et al., 2007, 597609). Ozone pre15 exposure was found to reduce the number of inflammatory cells, to increase cytokine production and 

16 total protein in the BAL fluid and to increase systemic IL-6 following exposure to endotoxin 

17 (Hollingsworth et al., 2007, 597609). Furthermore, O3 stimulated the apoptosis of alveolar 

18 macrophages 24 hours postexposure, an effect which was greatly enhanced by endotoxin treatment. 

19 Apoptosis of blood monocytes was also observed in response to the combined exposures 

20 (Hollingsworth et al., 2007, 597609). Ozone pre-exposure enhanced the response of lung 

21 macrophages to endotoxin (Hollingsworth et al., 2007, 597609). Taken together, these findings 

22 demonstrated that O3 exposure increased innate immune responsiveness to endotoxin. The authors 

23 proposed that this effect was mediated by TLR4-dependent pathways since O3 increased surface 

24 expression of TLR4 on macrophages (Hollingsworth et al., 2007, 597609). More recently, these 

25 authors demonstrated that hyaluronan contributed to the O3-primed response to endotoxin (Li et al., 

26 2010, 670282). In this study, exposure of mice to 1 ppm O3 for 3 h resulted in enhanced responses to 

27 endotoxin, which was mimicked by intratracheal instillation of hyaluronan fragments (Li et al., 2010, 

28 670282). Hyaluronan, like O3, was also found to induce TLR4 receptor peripheralization in the 

29 membrane (Hollingsworth et al., 2007, 597609; Li et al., 2010, 670282), an effect which is 

30 associated with enhanced responses to endotoxin. This study and previous ones by the same 

31 investigators showed elevation of BAL hyaluronan in response to O3 exposure (Garantziotis et al., 

32 2009, 597603; Garantziotis et al., 2010, 624947; Li et al., 2010, 670282), providing evidence that

33 ozone’s effects on innate immunity are at least in part mediated by hyaluronan fragments. The 

34 authors note that excessive TLR4 signaling can lead to lung injury and suggest that O3 may be 

35 responsible for an exaggerated innate immune response which may underlie lung injury and 

36 decreased host defense (Li et al., 2010, 670282) (Section 5.2.7.1). 

37 An interaction between allergen and O3 in the induction of nonspecific AHR was shown in 

38 another animal study (Larsen et al., 2010, 628560). Mice were sensitized with the aerosolized 
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1 allergen OVA on 10 consecutive days followed by exposure to O3 (0.1-0.5 ppm for 3 hours) (Larsen 

2 et al., 2010, 628560). While allergen sensitization alone did not alter airway responsiveness to a 

3 nonspecific challenge, O3 exposure of sensitized mice resulted in nonspecific AHR at 6 and 24 hours

4 postexposure (Larsen et al., 2010, 628560). The effects of O3 on AHR were independent of airways 

5 eosinophilia and neutrophilia (Larsen et al., 2010, 628560). However, OVA pretreatment led to 

6 goblet cell metaplasia which was enhanced by O3 exposure (Larsen et al., 2010, 628560). It should 

7 be noted that OVA sensitization using only aerosolized antigen in this study is less common than the 

8 usual procedure for OVA sensitization achieved by one or more initial systemic injections of OVA 

9 and adjuvant followed by repeated inhalation exposure to OVA. 

10 Furthermore, O3 was found to act as an adjuvant for allergic sensitization (Hollingsworth et 

11 al., 2010, 635786). In this study in mice, oropharyngeal aspiration of OVA on day 0 and day 6 failed 

12 to lead to allergic sensitization unless mice were first exposed to 1 ppm O3 for 2 hours 

13 (Hollingsworth et al., 2010, 635786). The O3-mediated response involved Th2 (IL-4, IL-5 and IL-9) 

14 and Th17 cytokines (IL-17) and was dependent on a functioning TLR4 (Hollingsworth et al., 2010, 

15 635786). Ozone exposure also activated OVA-bearing dendritic cells in the thoracic lymph nodes, as 

16 measured by the presence of the CD86 surface marker, which suggests naïve T-cell stimulation and 

17 the involvement of Th2 pathways (Hollingsworth et al., 2010, 635786). Thus ozone’s adjuvant 

18 effects may be due to activation of both innate and adaptive immunity. 

19 Results of recent studies demonstrate participation of innate immune pathways and also 

20 suggest involvement of adaptive immune pathways in both the induction and the exacerbation of 

21 allergic responses and AHR by O3. 

5.2.6.2. Summary

22 Collectively these older and more recent studies provide insight into ozone’s ability to provoke 

23 asthma exacerbations in humans. Greater airways inflammation and/or greater bronchial reactivity 

24 have been demonstrated in asthmatics compared to non-asthmatics. This pre-existing inflammation 

25 and altered baseline bronchial reactivity may contribute to the enhanced bronchoconstriction seen in 

26 asthmatics exposed to O3. Furthermore, inflammation may contribute to O3-mediated AHR. Animal 

27 studies have demonstrated a role for eosinophil-derived proteins in mediating these effects. Since 

28 airways eosinophilia occurs in both allergic humans and allergic animal models, this pathway may 

29 underlie the exacerbation of allergic asthma by O3. In addition, differences have been noted in 

30 epithelial cytokine expression in bronchial biopsy samples of healthy and asthmatic subjects. A Th2 

31 phenotype, indicative of adaptive immune system activation and enhanced allergic responses, was

32 observed before O3 exposure and was increased by O3 exposure in asthmatics. Since eosinophilia is a 

33 hallmark of a Th2 phenotype, these findings support links between allergic asthma, sensitivity to O3

34 and adaptive immunity. Studies in humans and animal models also provide evidence for activation of 

35 innate immunity by O3. In humans, O3 exposure resulted in increased numbers of airways monocytes 

36 and dendritic-like cells. Altered expression of cell surface markers characteristic of innate immunity 
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1 and antigen presentation was observed on monocytes and macrophages. Recruitment of these 

2 activated immune cells could lead to activation of allergen-specific memory T-cells in allergic 

3 individuals and result in the exacerbation of existing asthma in response to an allergen trigger. In 

4 animal studies, O3 exposure primed the innate immune system and led to increased endotoxin5 induced AHR by a mechanism involving hyaluronan and TLR4. The exaggerated immune response 

6 to O3 + endotoxin could lead to a more pronounced lung injury response to a bacterial trigger. 

7 Enhanced bronchial reactivity, airways eosinophilia, Th2 phenotype, recruitment of activated innate 

8 immune cells, and enhanced responsiveness to endotoxin all provide biological plausibility for 

9 epidemiologic evidence of asthma exacerbations associated with exposure to O3. Thus, the influx of 

10 immunomodulatory cells and the activation of innate and adaptive immunity lead to the exacerbation 

11 of asthma and allergic responses which is emerging as a key event in the toxicity pathway of O3. 

12 Recent studies in humans and animal models also provide evidence that O3 exposure causes 

13 induction of AHR and allergic responses. Both activation of innate immunity and promotion of 

14 adaptive immunity have been implicated. In humans, O3 exposure resulted in increased numbers of 

15 dendritic-like cells and levels of a cytokine associated with dendritic cell activation in the sputum, 

16 suggesting the presence of a population of activated dendritic cells which could stimulate naïve 

17 T-cells to promote the development of asthma. Evidence for activated dendritic cells was also found

18 in GSTM1 null human subjects (Section 5.2.9.1) and in allergen-sensitized animals exposed to O3. In 

19 the latter study, O3 acted as an adjuvant for allergic sensitization and the development of AHR by a

20 mechanism involving TLR4. In a different animal model, O3-induced AHR required the presence of

21 NKT cells and IL-17, both of which indicate innate immune system activation. Ozone-induced 

22 goblet cell metaplasia has also been demonstrated. These findings suggest that O3 may be capable of 

23 causing new onset asthma and allergic responses in humans. Thus, promotion of adaptive immunity 

24 and activation of innate immunity leads to the induction of AHR and allergic responses which is 

25 emerging as a key event in the toxicity pathway of O3. 

5.2.7. Impaire d Hos t De fen s e

26 O3 impacts host defense by a variety of different mechanisms. Animal models have 

27 demonstrated decreased mucociliary particle clearance and effects on alveolar macrophages, 

28 including inhibited phagocytosis and production of reactive oxygen intermediates, and altered 

29 chemotaxis and adhesion in response to O3 exposure (U.S. EPA, 2006, 088089). Ozone has been 

30 shown to target SP-A, resulting in a decrease in its function (U.S. EPA, 2006, 088089). In addition, 

31 reduced clearance of bacterial pathogens and enhanced susceptibility to bacterial lung infections 

32 were observed in rodents exposed acutely to O3 (Gilmour et al., 1993, 039620; U.S. EPA, 2006, 

33 088089). Further, O3-induced alterations in immune function have been demonstrated (Jakab et al., 

34 1995, 039548; U.S. EPA, 2006, 088089). 
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5.2.7.1. New Cellular and Molec ular Ins igh ts

1 As described above, priming of the innate immune system by O3 was reported by 

2 Hollingsworth et al. (2007, 597609). Besides effects on AHR, exposure of mice to 2 ppm O3 for 

3 3 hours reduced inflammatory cell influx in the airways in response to endotoxin (Hollingsworth et 

4 al., 2007, 597609). In addition, O3 exposure stimulated the apoptosis of alveolar macrophages 

5 24 hours postexposure, an effect which was enhanced by a subsequent treatment with endotoxin 

6 (Hollingsworth et al., 2007, 597609). Apoptosis of circulating blood monocytes was also observed in 

7 response to O3 and endotoxin (Hollingsworth et al., 2007, 597609). The authors attributed these 

8 effects to the increased surface expression of TLR4 and increased signaling in macrophages 

9 observed in the study (Hollingsworth et al., 2007, 597609). It was proposed that the resulting 

10 decrease in airways inflammatory cells could account for O3-mediated decreased clearance of 

11 bacterial pathogens observed in numerous animal models (Hollingsworth et al., 2007, 597609). A 

12 more recent study by these investigators provided evidence that hyaluronan mediates O3-priming of 

13 innate immunity and suggested that exaggerated innate immune responses may underlie lung injury 

14 and decreased host defense (Li et al., 2010, 670282). 

15 Recent studies also demonstrated SP-A oxidation by O3. SP-A is an important innate immune 

16 protein which plays a number of roles in host defense including acting as opsonin for the recognition 

17 of some pathogens (Haque et al., 2009, 200767). These investigations demonstrated that O3-

18 mediated carbonylation of SP-A was associated with impaired macrophage phagocytosis in vitro 

19 (Mikerov et al., 2008, 596405). Furthermore, O3 exposure in mice was found to increase 

20 susceptibility to pneumonia infection in mice through an impairment of SP-A dependent

21 phagocytosis (Mikerov et al., 2008, 201537; Mikerov et al., 2008, 597493). 

22 Another recent study demonstrated impaired antigen-specific immunity following subacute O3

23 exposure (0.6 ppm, 10 h/day for 15 days) in mice (Feng et al., 2006, 596381). Specifically, O3

24 exposure altered the lymphocyte subset and cytokine profile and impacted thymocyte early 

25 development leading to immune dysfunction. 

5.2.7.2. Summary

26 Collectively these older and more recent studies in animal models provide several mechanisms 

27 by which O3 exposure could enhance susceptibility to lung infections. Both decreased mucociliary 

28 particle clearance and decreased numbers and function of alveolar macrophage have been 

29 implicated. Recent studies suggest that O3-mediated oxidation of SP-A oxidation and priming of the 

30 innate immune system may contribute to decreased pathogen clearance. Immune dysfunction outside 

31 of the lung has also been demonstrated. Thus, immune system modulation resulting in impaired host 

32 defense is emerging as a key event in ozone’s toxicity pathway. 
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5.2.8. Extrapulmonary Effects

1 Extrapulmonary effects of O3 have been noted for decades (U.S. EPA, 2006, 088089). One 

2 such effect is hypothermia, which in rodents occurs subsequent to the activation of neural reflexes 

3 and involves the parasympathetic nervous system (Watkinson et al., 2001, 016245). Other 

4 mechanisms are likely to be involved in extrapulmonary effects. It has been proposed that lipid 

5 oxidation products resulting from reaction of O3 with lipids in the ELF are responsible for systemic

6 effects, however it is not known whether they gain access to the vascular space (Chuang et al., 2009, 

7 197202). Alternatively, extrapulmonary release of diffusible mediators may initiate or propagate 

8 inflammatory responses in the vascular or in systemic compartments (Cole and Freeman, 2009, 

9 597507). For example, one such mediator, the cytokine IL-6, is known to have pleitropic effects such 

10 as inducing the acute phase response, activating the hypothalamus-pituitary-adrenal axis and altering 

11 serum cholesterol levels (Tarrant, 2010, 644810). 

5.2.8.1. Ca rdiova s c ula r Effe c ts

12 Effects of O3 on the cardiovascular system have been demonstrated in both humans and animal 

13 models (U.S. EPA, 2006, 088089). Several mechanisms have been proposed to account for these 

14 responses (Perepu et al., 2010, 385020). First, O3 may impair alveolar-arterial oxygen transfer and 

15 reduce the supply of arterial oxygen to the myocardium. This may have a greater impact in 

16 individuals with compromised cardiopulmonary systems. Gong et al. (1998, 029938) provided 

17 evidence of a small decrease in arterial oxygen saturation in human subjects exposed to O3. In 

18 addition, Delaunois et al. (1998, 015779) demonstrated pulmonary vasoconstriction in O3-exposed 

19 rabbits. Although of interest, the contribution of this pathway to O3-induced cardiovascular effects 

20 remains uncertain. Secondly, O3 may trigger neural reflexes which stimulate the autonomic nervous 

21 system and alter electrophysiologic responses of the heart. For example, bradycardia, altered HRV

22 and arrhythmia have been demonstrated in animals exposed to O3 (Arito et al., 1990, 042285; 

23 Hamade and Tankersley, 2009, 596386; Watkinson et al., 2001, 016245). Third, O3-induced 

24 pulmonary inflammation may lead to inflammatory or injury responses in the cardiovascular system 

25 (Cole and Freeman, 2009, 597507). 

26 Some recent studies have suggested that O3 may alter the systemic vasculature. In resting 

27 humans, exposure to fine particulate matter (PM) + O3 resulted in arterial vasoconstriction and 

28 increased diastolic blood pressure during the 2-h exposure (Brook et al., 2002, 024987). However, a 

29 recent study in humans observed no changes in HRV or blood pressure in healthy nonsmokers 

30 exposed at rest to 0.12 ppm O3 for 2 hours (Brook et al., 2009, 195611). Thus, the previously 

31 observed effects of the combined O3 and PM exposure may have been attributable to PM alone. A 

32 role for O3 in modulating endothelin, a potent vasoconstrictor, has also been proposed. Studies in 

33 animals found that O3-induced endothelin system genes in the lung and increased circulating levels 

34 of endothelin (Thomson et al., 2005, 087554; Thomson et al., 2006, 097483). 
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Recent Cellular and Molecular Insights

1 Ozone-induced perturbations of the cardiovascular system were recently investigated in young 

2 mice and monkeys (Chuang et al., 2009, 197202). Young mice exposed to 0.5 ppm O3 for 5 days 

3 demonstrated increased heart rate and blood pressure. Decreases in endothelial-dependent 

4 vasorelaxation and NO homeostasis were observed in arterial tissue. Oxidative and nitrosative stress 

5 were demonstrated in lung and aortic tissue following O3 exposure. Mitochondrial DNA was 

6 damaged in lung and aortas from young mice and infant rhesus monkeys similarly exposed to O3. 

7 Since altered NO homeostasis, mitochondrial DNA damage and oxidative stress are known to 

8 contribute to the development of atherosclerosis, young Apo E null mice were exposed 

9 subchronically to O3 (0.5 ppm O3 for 5 days/week over 8 weeks). Ozone exposure augmented the 

10 aortic lesion areas compared with controls exposed to filtered air, suggesting an acceleration of 

11 atherogenesis. This is the first study to definitively identify the systemic vasculature as a target of 

12 O3-induced effects. 

13 A second recent study in animals demonstrated effects on the heart due to chronic O3 exposure 

14 (Perepu et al., 2010, 385020). Rats were exposed to 0.8 ppm O3 for 28 and 56 days and isolated 

15 hearts were subjected to ischemia-reperfusion injury. Ozone exposure enhanced the sensitivity to 

16 injury in this model, as demonstrated by decreased cardiac function compared with control rats that 

17 were exposed to filtered air. Further, markers of lipid peroxidation and inflammation were greater in 

18 the hearts of O3-exposed rats. This study is the first to definitively identify the heart as a target for 

19 O3-induced effects. 

20 Further evidence for O3-induced effects in the systemic vasculature and heart is provided by a 

21 recent chronic study in rats (Kodavanti et al., In Press, 666323). Episodic exposure to 0.4 ppm O3 for 

22 16 weeks (5 h/day for 1 day/week) resulted in increased aortic levels of mRNA for biomarkers of 

23 oxidative stress, thrombosis, vasoconstriction and proteolysis. Ozone exposure also increased lectin24 like oxidized-low density lipoprotein receptor-1(LOX-1) mRNA and protein levels in the aorta. 

25 Depletion of cardiac mitochondrial phospholipid fatty acids was also observed. Taken together, these 

26 results suggest a role for circulating oxidized lipids in mediating the effects of O3. 

5.2.8.2. Hepa tic Effe c ts

27 Changes in hepatic gene expression have also been noted following O3 exposure. Specifically, 

28 downregulation of gene families related to lipid, fatty acid and carbohydrate metabolism was 

29 demonstrated in the liver of O3-exposed mice (Last et al., 2005, 596400). In addition, transcription of 

30 enzymes involved in xenobiotic metabolism was decreased. Impairment of hepatic drug metabolism 

31 was suggested in an older study which found that mice exposed to O3 had prolonged pentobarbital 

32 sleeping time (Graham et al., 1985, 040289). Further evidence of hepatic effects is provided by a 

33 recent study in which exposure to 0.25 and 0.5 ppm O3 for 6 hours resulted in exacerbation of drug34 induced liver injury in mice pre-exposed to acetaminophen. This included a greater increase in 
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1 hepatic neutrophil accumulation and greater alteration in gene expression profiles in mice exposed to 

2 O3 and acetaminophen compared with either exposure alone (Aibo et al., 2010, 378559). 

5.2.8.3. Summary

3 Collectively, these older and more recent studies in animal models provide evidence for 

4 extrapulmonary effects of O3. Although it was suggested that these effects are directly mediated by 

5 secondary oxidation products formed in the lung as a result of O3 exposure, there is no evidence that 

6 these species enter the circulation. Alternatively, extrapulmonary effects may be due to activation of 

7 neural reflexes or to release of diffusible mediators which may initiate or propagate inflammatory 

8 responses in the vascular or systemic compartments. Recent studies suggest that oxidative/nitrosative 

9 stress contributes to O3-induced cardiovascular effects. Thus, systemic inflammation and vascular 

10 oxidative/nitrosative stress are emerging as key events in the toxicity pathway of O3. 

5.2.9. Fa c to rs Affe c tin g Res po n s e s to Ozone

11 Responses to O3 are variable within the population and the basis for this variability is not clear 

12 (Mudway and Kelly, 2000, 010452). Research has focused on the role of gene-environment 

13 interactions, preexisting conditions, adaptive mechanisms and lifestage in influencing the responses 

14 to O3. Co-exposure to other pollutants has also been considered.

5.2.9.1. Gene -Environment Interactions 

15 The significant inter-individual variation in responses to O3 infers that genetic background is 

16 an important determinant of susceptibility to O3 (Cho and Kleeberger, 2007, 195616; Kleeberger et 

17 al., 1997, 095736). Strains of mice which are prone or resistant to O3-induced effects have been used 

18 to systematically identify candidate susceptibility genes. Genome wide linkage analyses (also known 

19 as positional cloning) demonstrated quantitative trait loci for O3-induced lung inflammation and 

20 hyperpermeability on chromosome 17 (Kleeberger et al., 1997, 095736) and chromosome 4 

21 (Kleeberger et al., 2000, 014895), respectively, using these recombinant inbred strains of mice. More 

22 specifically these studies found that Tnf, whose protein product is the inflammatory cytokine TNF-α, 

23 and Tlr4, whose protein product is TLR4, were candidate susceptibility genes (Kleeberger et al., 

24 1997, 095736; Kleeberger et al., 2000, 014895). Other investigations in inbred mouse strains found 

25 that differences in expression of certain proteins, such as CCSP (Broeckaert et al., 2003, 055490) and 

26 MARCO (Dahl et al., 2007, 196986), are responsible for phenotypic characteristics, such as 

27 epithelial permeability and scavenging of oxidized lipids, respectively, which confer sensitivity to 

28 O3. 

29 Genetic polymorphisms have received increasing attention as modulators of O3-mediated 

30 effects. Functionally relevant polymorphisms in candidate susceptibility genes have been studied at 

31 the individual and population level in humans and also in animal models. Genes whose protein 
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1 products are involved in antioxidant defense/oxidative stress and xenobiotic metabolism, such as 

2 glutathione-S-transferase M1 (GSTM1) and NADPH:quinone oxidoreductase 1 (NQO1), have also 

3 been major focuses of these efforts. This is because oxidative stress resulting from O3 exposure is 

4 thought to contribute to the pathogenesis of asthma and because xenobiotic metabolism detoxifies 

5 secondary oxidation products formed by O3 which contribute to oxidative stress (Islam et al., 2008, 

6 097348). TNF-α is of interest since it is linked to a candidate O3 susceptibility gene and since it plays 

7 a key role in initiating airways inflammation (Li et al., 2006, 090972). Polymorphisms of genes 

8 coding for GST M1, NQO1 and TNF-α have been associated with altered susceptibility to O3-

9 mediated effects (Bergamaschi et al., 2001, 052670; Corradi et al., 2002, 035448; Li et al., 2006, 

10 090972; Romieu et al., 2004, 056796; Yang et al., 2005, 077211). Additional studies have focused on 

11 functional variants in other genes involved in antioxidant defense such as catalase (CAT), 

12 myeloperoxidase, heme oxygenase (HMOX-1) and manganese superoxide dismutase (MnSOD) 

13 (Islam et al., 2008, 097348; Wenten et al., 2009, 597084). These studies are discussed below. 

14 GSTM1 is a phase II antioxidant enzyme which is transcriptionally regulated by NF-E2-

15 related factor 2-antioxidant response element (Nrf2-ARE) pathway. A large proportion (40-50%) of 

16 the general public (across ethnic populations) has the GSTM1 null genotype, which has been linked 

17 to an increased risk of adverse health effects due to exposure to air pollutants (London, 2007, 

18 093279). A role for GSTs in metabolizing electrophiles such as 4-hydroxynonenal, which is a 

19 secondary oxidation product formed following O3 exposure, has been demonstrated (Awasthi et al., 

20 2004, 644649). A recent study found that the GSTM1 genotype modulated the time course of the 

21 neutrophilic inflammatory response following acute O3 exposure (0.4 ppm for 2 hours with 

22 intermittent exercise) in healthy adults (Alexis et al., 2009, 628542). In GSTM1-null and sufficient 

23 subjects, O3-induced sputum neutrophilia was similar at 4 hours. However, neutrophilia resolved by 

24 24 hours in sufficient subjects but not in GSTM1-null subjects. It is not known whether this effect 

25 was due to the persistence of pro-inflammatory stimuli, impaired production of downregulators or 

26 impaired neutrophil apoptosis and clearance. In addition, O3 exposure increased the expression of the 

27 surface marker CD14 in airway neutrophils of GSTM-1 null subjects compared with sufficient 

28 subjects. Furthermore, numbers of airway macrophages were decreased at 4 and 24 hours following 

29 O3 exposure in GSTM1-sufficient subjects (Alexis et al., 2009, 628542). Airways macrophages in 

30 GSTM1 null subjects were greater in number and found to have greater oxidative burst and 

31 phagocytic capability than those of sufficient subjects. Airways macrophages and dendritic cells 

32 from GSTM1 null subjects exposed to O3 expressed higher levels of the surface marker HLA-DR, 

33 suggesting activation of the innate immune system (Alexis et al., 2009, 628542). These differences 

34 in inflammatory responses between the GSTM1 null and sufficient subjects may provide biological 

35 plausibility for the differences in O3-mediated effects reported in controlled human exposure studies 

36 (Bergamaschi et al., 2001, 052670; Corradi et al., 2002, 035448). It should also be noted that 

37 GSTM1 genotype did not affect the acute pulmonary function (spirometric) response to O3 which 

38 provides additional evidence for separate mechanisms underlying ozone’s effects on pulmonary 
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1 function and inflammation in adults (Alexis et al., 2009, 628542). However, GSTM1 null asthmatic 

2 children were previously found to be more sensitive to the effects of O3 on pulmonary function than 

3 GSTM1 sufficient asthmatic children (Romieu et al., 2004, 056796). 

4 NQO1 catalyzes the 2-electron reduction by NADPH of quinones to hydroquinones. 

5 Depending on the substrate, it is capable of both protective detoxification reactions and redox 

6 cycling reactions resulting in the generation of reactive oxygen species. A recent study using NQO1-

7 null mice demonstrated that NQO1 contributes to O3-induced oxidative stress, AHR and 

8 inflammation in mice (Voynow et al., 2009, 194311). These experimental results may provide 

9 biological plausibility for the increased biomarkers of oxidative stress and increased pulmonary 

10 function decrements observed in O3-exposed individuals bearing both the wild-type NQO1 gene and 

11 the null GSTM1 gene (Bergamaschi et al., 2001, 052670; Corradi et al., 2002, 035448). 

12 Two studies reported relationships between TNF promoter variants and O3-induced effects in 

13 humans. In one study, O3-induced change in lung function was significantly lower in adult subjects 

14 with TNF promoter variants -308A/A and -308G/A compared with adult subjects with the variant -

15 308G/G (Yang et al., 2005, 077211). This response was modulated by a specific polymorphism of 

16 LTA (Yang et al., 2005, 077211), a previously identified candidate susceptibility gene whose protein 

17 product is lymphotoxin-α (Kleeberger et al., 1997, 095736). In the second study, an association 

18 between the TNF promoter variant -308G/G and decreased risk of asthma and lifetime wheezing in 

19 children was found (Li et al., 2006, 090972). The protective effect on wheezing was modulated by 

20 ambient O3 levels and by GSTM1 and GSTP1 polymorphisms. The authors suggested that the 

21 TNF-308 G/G genotype may have a protective role in the development of childhood asthma (Li et 

22 al., 2006, 090972). 

23 Similarly, a promoter variant of the gene HMOX-1, consisting of a smaller number of (GT)n 

24 repeats, was associated with a reduced risk for new-onset asthma in non-Hispanic white children 

25 (Islam et al., 2008, 097348). The number of (GT)n repeats in this promoter has been shown to be 

26 inversely related to the inducibility of HMOX-1. A modulatory effect of O3 was demonstrated since 

27 the beneficial effects of this polymorphism were seen only in children living in low O3 communities 

28 (Islam et al., 2008, 097348). This study also identified an association between a polymorphism of the 

29 CAT gene and increased risk of new-onset asthma in Hispanic children; however no modulation by 

30 O3 was seen (Islam et al., 2008, 097348). No association was observed in this study between a 

31 MnSOD polymorphism and asthma (Islam et al., 2008, 097348). 

32 Studies to date indicate that some variability in individual responsiveness to O3 may be 

33 accounted for by functional genetic polymorphisms. Further, the effects of gene-environment 

34 interactions may be different in children and adults.

5.2.9.2. Pre exis ting Dis eas es and Conditions

35 Several preexisting diseases and conditions have been described which modulate the response 

36 to O3 exposure. Atopy and asthma are important factors in humans. For example, asthmatics were 
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1 more susceptible to O3-mediated inflammation (Balmes et al., 1997, 086092; Basha et al., 1994, 

2 075950; Scannell et al., 1996, 080755), while not exhibiting any increase in responsiveness as 

3 measured by spirometry. Ozone exposure resulted in eosinophilia of the nasal and lower airways in 

4 individuals with preexisting allergic disease (Peden et al., 1995, 076189; Vagaggini et al., 2002, 

5 035191) and increased bronchial reactivity to challenge with inhaled specific antigen in mild allergic 

6 asthmatics (Jorres et al., 1996, 078122; Kehrl et al., 1999, 022101; Molfino et al., 1991, 042379). 

7 Increased bronchial reactivity was also reported in individuals with allergic rhinitis (Jorres et al., 

8 1996, 078122). It has been proposed that sensitivity is conferred by the presence of greater numbers 

9 of resident airway inflammatory cells in disease states such as asthma (Mudway and Kelly, 2000, 

10 010452). 

11 In addition, smoking and COPD status are important determinants since responsiveness to O3, 

12 as measured by spirometry, is decreased in individuals with these conditions (U.S. EPA, 2006, 

13 088089). Furthermore, obesity may alter susceptibility. In a reanalysis of the data of Hazucha (2003, 

14 048168), increasing body mass index in young women was associated with increased O3

15 responsiveness (Bennett et al., 2007, 418827). In animal models, diet-induced obesity augmented 

16 inflammation and injury, as measured by BAL markers, as well as innate AHR, in mice exposed 

17 acutely to O3 (Johnston et al., 2008, 597625). In contrast, the inflammatory response following sub18 acute exposure to O3 was dampened by obesity in a different mouse model (Shore et al., 2009, 

19 201551). Finally, nutritional status may impact the response to O3. Many investigations have focused 

20 on antioxidant deficiency as a modulator of O3-mediated effects (see above). Although results of 

21 these studies are mixed, studies in humans demonstrate that supplementation with ascorbate and 

22 alpha-tocopherol was protective against O3-induced pulmonary function deficits in healthy adults 

23 who were ascorbate-deficient (Samet et al., 2001, 019034) and in asthmatic children living in 

24 Mexico City (Romieu et al., 2002, 034711; Sienra-Monge et al., 2004, 196422). Furthermore, 

25 supplementation with ascorbate, alpha-tocopherol and beta-carotene was found to be protective in 

26 Mexico City streetworkers (Romieu et al., 1998, 086756). 

5.2.9.3. Life s ta ge : Pos tna tal developme nt

27 An interesting set of studies conducted over the last 10 years in the infant rhesus monkey has 

28 identified numerous O3-mediated perturbations in the developing lung and immune system (Plopper 

29 et al., 2007, 596412). 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 remodeling of 

31 the epithelial-mesenchymal trophic unit during postnatal development of the tracheobronchial 

32 airway wall. Rhesus monkeys were used in these studies because the branching pattern and 

33 distribution of airways in this model are more similar to humans than are those of rodents to humans. 

34 In addition, a model of allergic airways disease, which exhibits the main features of human asthma, 

35 had already been established in the adult rhesus monkey. Studies in infant monkeys were designed to 

36 determine whether repeated exposure to O3 altered postnatal growth and development, and if so, 
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1 whether such effects were reversible. In addition, exposure to O3 was evaluated for its potential to 

2 increase the development of allergic airways disease. Infant rhesus monkeys (30 days old) were 

3 exposed to cyclic episodic O3 over a 5-month period. This involved biweekly cycles of alternating 

4 O3 (5 consecutive days of 0.5 ppm O3, 8 h/day) and filtered air (9 consecutive days). Some animals 

5 were sensitized to house dust mite allergen (HDMA) and then exposed to HDMA aerosol for 2 h/day 

6 on days 3-5 of either filtered air or O3 exposure.

7 Key findings are numerous. First, baseline airway resistance and AHR in the infant monkeys 

8 were dramatically increased by combined exposure to both HDMA and O3 (Joad et al., 2006, 

9 596390; Schelegle et al., 2003, 053778). Secondly, O3 exposure alone led to a large increase in BAL 

10 eosinophils (Schelegle et al., 2003, 053778) while HDMA exposure alone led to a large increase of 

11 eosinophils in airways tissue (Joad et al., 2006, 596390; Schelegle et al., 2003, 053778). Thirdly, the 

12 growth pattern of distal airways was significantly changed by exposure to O3 alone and in 

13 combination with HDMA. More specifically, longer and narrower airways resulted and the number 

14 of conducting airway generations between the trachea and the gas exchange area was decreased 

15 (Fanucchi et al., 2006, 096491). This latter effect was not ameliorated by a recovery period of 

16 6 months. Fourthly, exposure to both HDMA and O3 altered the abundance and distribution of 

17 CD25+ lymphocytes in the airways (Miller et al., 2009, 596406). Lastly, several effects were seen at 

18 the level of the epithelial mesenchymal trophic unit in response to O3. These include altered 

19 organization of the airways epithelium (Schelegle et al., 2003, 053778), increased abundance of 

20 mucous goblet cells (Schelegle et al., 2003, 053778), disruption of the basement membrane zone 

21 (Evans et al., 2004, 596379), reduced innervation (Larson et al., 2004, 057062), increased 

22 neuroendocrine-like cells (Joad et al., 2006, 596390), and altered orientation and abundance of 

23 smooth muscle bundles (Plopper et al., 2007, 596412; Tran et al., 2004, 628626). Six months of 

24 recovery in filtered air led to reversal of some but not all of these effects (Evans et al., 2004, 596379; 

25 Kajekar et al., 2007, 567661; Plopper et al., 2007, 596412). The authors concluded that cyclic 

26 challenge of infant rhesus monkeys to allergen and O3 during the postnatal period compromised 

27 airway growth and development and resulted in changes which favor allergic airways responses 

28 (Plopper et al., 2007, 596412). 

29 Nasal mucous membranes are also a target of O3-mediated effects. The infant rhesus monkey 

30 was used as a model since its nasal airways are similar to those of children (Carey et al., 2007, 

31 195752). Lesions in airways epithelium in the developing nasal passages of immature monkeys were 

32 determined following both acute (5 consecutive days of 0.5 ppm 8 h/day) and cyclic episodic (as 

33 described above) O3 exposure. Similar effects were observed in response to acute and episodic O3. 

34 Histological analysis demonstrated necrotizing rhinitis in the nasal mucosa lining of the main nasal 

35 chamber and focal regions of epithelial exfoliation, especially in the anterior maxilloturbinate. An 

36 anterior to posterior decrease was observed in the severity of these lesions. Morphometric analysis 

37 demonstrated a 65% reduction in the mean thickness of the nasal epithelium in the anterior 

38 maxilloturbinate and loss of volume density of airway cilia, epithelial cytoplasm and nuclei. The 
March 2011 5-60 DRAFT – DO NOT CITE OR QUOTE 

1 authors reported that the O3-induced nasal lesions observed in this study are similar to those reported 

2 for adult monkeys. However, unlike the adult monkeys, no epithelial hyperplasia or metaplasia was 

3 observed in the young monkeys suggesting that persistent necrotizing rhinitis may be the long term 

4 sequelae in the absence of protective adaptations. 

5 Effects of O3 on early postnatal airways development has also been studied in rats. A recent 

6 study demonstrated that O3 exposure during critical postnatal periods resulted in increased SP nerve 

7 fiber density in lung smooth muscle (Hunter et al., 2010, 382064). The authors proposed that O3 may 

8 lead to enhanced responsiveness of airway sensory nerves. Another study found increases in 

9 immediate-early gene responses in airways epithelium of rats exposed postnatally to O3 (Johnston et 

10 al., 2006, 097439). Further, neonatal mice exhibit strain-specific differential susceptibility to O3

11 (Vancza et al., 2009, 596419). 

5.2.9.4. Lifes tage : Aging

12 On the other side of the lifestage spectrum is aging. The spirometric response to O3 is lost in 

13 humans as they age (Drechsler-Parks, 1995, 076085; Hazucha et al., 2003, 048168). In mice, 

14 physiological responses to O3 were also attenuated with age (Hamade et al., 2010, 666324). 

15 Mechanisms accounting for this effect have not been well-studied but could include altered number 

16 and sensitivity of receptors or altered signaling pathways involved in neural reflexes. 

5.2.9.5. Ada pta tio n

17 The decrease in pulmonary function and increase in bronchoconstriction, airways 

18 inflammation and bronchial reactivity observed on the first and second days of consecutive daily 

19 exposure in response to O3 were not seen after 4 or 5 days (see above). Several mechanisms have 

20 been postulated. First, the upregulation of antioxidant defenses (or conversely, a decrease in critical 

21 O3-reactive substrates) may protect against O3-mediated adverse effects. Increases in antioxidant 

22 content of the BAL have been demonstrated by Devlin (1997, 083577), Tepper (1989, 041991), and 

23 others. Second, IL-6 was demonstrated to be an important mediator of adaptation (McKinney et al., 

24 1998, 086751). Third, a protective role for increases in mucus producing cells and mucus 

25 concentrations in the airways has also been proposed (Devlin et al., 1997, 083577). Fourth, epithelial 

26 hyperplasia or metaplasia may decrease susceptibility to subsequent O3 challenge (Carey et al., 2007, 

27 195752; Harkema et al., 1987, 040816; Harkema et al., 1987, 041496). These morphologic changes 

28 have been observed in nasal and lower airways. Although there is some evidence to support these 

29 possibilities, there is no consensus on mechanisms underlying adaptation. Recent studies 

30 demonstrating that O3 activates TRP receptors suggest that modulation of TRP receptor number or 

31 sensitivity by repeated O3 exposures may also contribute to adaptation. 
March 2011 5-61 DRAFT – DO NOT CITE OR QUOTE 

5.2.9.6. Co-Exposures with Pa rtic ula te Ma tte r 

1 Numerous studies have investigated the effects of co-exposure to O3 and PM because of the 

2 prevalence of these pollutants in ambient air. Results are highly variable and depend on whether 

3 exposures are simultaneous or sequential, the type of PM employed and the endpoint examined. In 

4 humans, simultaneous exposure to O3 (0.12 ppm for 2 h at rest) and CAPs resulted in a diminished 

5 systemic IL-6 response compared with exposure to CAPs alone (Urch et al., 2010, 387113). 

6 Exposure to O3 alone did not alter blood IL-6 levels (Urch et al., 2010, 387113). The authors 

7 provided evidence that O3 mediated a switch to shallow breathing which may have accounted for this 

8 effect (Urch et al., 2010, 387113). Further, simultaneous exposure to CAPs and O3, but not exposure 

9 to either alone, resulted in increased diastolic blood pressure in human subjects (Fakhri et al., 2009, 

10 191914). In some strains of mice, pre-exposure to O3 (0.5 ppm for 2 hours) modulated the effects of 

11 carbon black PM on heart rate, HRV and breathing patterns (Hamade and Tankersley, 2009, 596386). 

12 Another recent study in mice demonstrated that treatment with carbon nanotubes followed 12 hours 

13 later by O3 exposure (0.5 ppm for 3 hours) resulted in a dampening of some of the pulmonary effects 

14 of carbon nanotubes measured as markers of inflammation and injury in the BAL (Han et al., 2008, 

15 596387). The authors suggest that this may represent “cross-tolerance.” Harkema et al. (2005, 

16 078340) found that epithelial and inflammatory responses in the airways of rats were enhanced by 

17 co-exposure to O3 and LPS (used as a model of biogenic PM) or to O3 and OVA (used as a model of 

18 an aeroallergen). Furthermore, one recent study demonstrated maternal-fetal effects of PM exposure 

19 on O3 responses. In this study, maternal exposure to PM resulted in augmented lung mediators of 

20 inflammation, airway epithelial mucous metaplasia and enhanced O3-mediated AHR in young mice 

21 (Auten et al., 2009, 200760). Overall, these findings are hard to interpret but demonstrate the 

22 complexity of interactions between PM and O3 exposures. 

5.2.9.7. Summary

23 Collectively, these older and more recent studies provide evidence for mechanisms which may 

24 underlie the variability in responsiveness seen among individuals. Certain functional genetic 

25 polymorphisms, pre-existing conditions and diseases, lifestages and co-exposures contribute to 

26 enhanced susceptibility to O3. Adaptation may also be important, but it is incompletely understood, 

27 both in terms of the pathways involved and the resulting consequences. 

5.2.10. Overall Summary

28 Key events in the toxicity pathway of O3 have been identified in humans and animal models.

29 They include the formation of secondary oxidation products in the lung, activation of neural reflexes, 

30 pulmonary injury and inflammation and increased bronchial reactivity. In addition, evidence is 

31 accumulating that immune system modulation may lead to impaired host defense and the 
March 2011 5-62 DRAFT – DO NOT CITE OR QUOTE 

1 exacerbation and/or induction of asthma and allergic responses (Figure 5-6). Systemic inflammation 

2 and vascular oxidative/nitrosative stress may be critical to the extrapulmonary effects of O3. 

Formation of secondary oxidation products

Ozone + Respiratory Tract 

Activation of 

neural reflexes

↓Inspiratory

Capacity

Mild 

bronchoconstriction

Decrements in pulmonary function

Inflammation 

and injury

Increased

bronchial 

reactivity

(AHR)

↑Epithelial 

permeability 

Airways 

neutrophilia

Impaired host defense/ 

respiratory tract infections

Allergic priming 

& sensitization

Exacerbation/induction of 

asthma and allergic responses

Decreased 

pathogen clearance 

Repair or 

remodeling

Immune system

modulation

Figure 5-6. Schematic depicting key events in ozone's toxicity pathway. Solid arrows denote 

pathways for which there is greater certainty. Broken arrows represent pathways of 

emerging interest.

5.2.11. Gaps in Knowledge

3 Despite a vast body of knowledge regarding the effects of O3 exposure, the current 

4 understanding of mechanisms underlying important health effects in humans is incomplete. 

5 Additional research will be useful to elucidate the biologic pathways by which exposure to O3: 

6  Primes the immune system, including promotion of adaptive immunity and activation of 

7 innate immunity 

8  Alters early postnatal development of the lung and immune system

9  Affects the cardiovascular system
March 2011 5-62 DRAFT – DO NOT CITE OR QUOTE 

References

A list of all references considered for inclusion in the dosimetry section of this chapter can be found at 

http://hero.epa.gov/index.cfm?action=search.do&submit=Search&group_id=409

A list of all references considered for inclusion in the mode of action section of this chapter can be found at 

http://hero.epa.gov/index.cfm?action=search.do&submit=Search&group_id=392

Ahmad, S.; Ahmad, A.; McConville, G.; Schneider, B. K.; Allen, C. B.; Manzer, R.; Mason, R. J.; White, C. W. (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 196429

Aibo, D. I.; Birmingham, N. P.; Lewandowski, R.; Maddox, J. F.; Roth, R. A.; Ganey, P. E.; Wagner, J. G.; Harkema, J. R. 

(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 378559

Alexis, N. E.; Lay, J. C.; Hazucha, M.; Harris, B.; Hernandez, M. L.; Bromberg, P. A.; Kehrl, H.; Diaz-Sanchez, D.; Kim, 

C.; Devlin, R. B.; Peden, D. B. (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 

628538

Alexis, N. E.; Zhou, H.; Lay, J. C.; Harris, B.; Hernandez, M. L.; Lu, T. S.; Bromberg, P. A.; Diaz-Sanchez, D.; Devlin, R. 

B.; Kleeberger, S. R.; Peden, D. B. (2009). The glutathione-S-transferase Mu 1 null genotype modulates ozoneinduced airway inflammation in human subjects. J Allergy Clin Immunol, 124: 1222-1228. 

http://dx.doi.org/10.1016/j.jaci.2009.07.036 628542

Alexis, N.; Soukup, J.; Nierkens, S.; Becker, S. (2001). Association between airway hyperreactivity and bronchial 

macrophage dysfunction in individuals with mild asthma. Am J Physiol Lung Cell Mol Physiol, 280: L369-L375. 

190013

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. 013072

Alfaro, M. F.; Putney, L.; Tarkington, B. K.; Hatch, G. E.; Hyde, D. M.; Schelegle, E. S. (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. 053551

Alfaro, M. F.; Walby, W. F.; Adams, W. C.; Schelegle, E. S. (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-

33. http://dx.doi.org/10.1080/01902140701364367 196567

Aris, R. M.; Christian, D.; Hearne, P. Q.; Kerr, K.; Finkbeiner, W. E.; Balmes, J. R. (1993). Ozone-induced airway 

inflammation in human subjects as determined by airway lavage and biopsy. Am J Respir Crit Care Med, 148: 

1363-1372. 038275

Aris, R. M.; Tager, I.; Christian, D.; Kelly, T.; Balmes, J. R. (1995). Methacholine responsiveness is not associated with 

O3-induced decreases in FEV1. Chest, 107: 621-628. 075945

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 042285

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. 077430

Asplund, P. T.; Ben-Jebria, A.; Rigas, M. L.; Ultman, J. S. (1996). Longitudinal distribution of ozone absorption in the 

lung: effect of continuous inhalation exposure. Arch Environ Occup Health, 51: 431-438. 082505

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments such as the Integrated Science Assessments (ISAs) and the Integrated Risk Information System (IRIS).
March 2011 5-63 DRAFT – DO NOT CITE OR QUOTE 

Auten, R. L.; Potts, E. N.; Mason, S. N.; Fischer, B.; Huang, Y.; Foster, W. M. (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 200760

Awasthi, Y. C.; Yang, Y.; Tiwari, N. K.; 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 644649

Ballinger, C. A.; Cueto, R.; Squadrito, G.; Coffin, J. F.; Velsor, L. W.; Pryor, W. A.; Postlethwait, E. M. (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 076649

Balmes, J. R.; Aris, R. M.; Chen, L. L.; Scannell, C.; Tager, I. B.; Finkbeiner, W.; Christian, D.; Kelly, T.; Hearne, P. Q.;

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. 

086092

Balmes, J. R.; Chen, L. L.; Scannell, C.; Tager, I.; Christian, D.; Hearne, P. Q.; Kelly, T.; Aris, R. M. (1996). Ozoneinduced decrements in FEV1 and FVC do not correlate with measures of inflammation. Am J Respir Crit Care 

Med, 153: 904-909. 080830

Bang, S.; Kim, K. Y.; Yoo, S.; Kim, Y. G.; Hwang, S. W. (2007). Transient receptor potential A1 mediates acetaldehydeevoked pain sensation. Eur J Neurosci, 26: 2516-2523. http://dx.doi.org/10.1111/j.1460-9568.2007.05882.x 628545

Basha, M. A.; Gross, K. B.; Gwizdala, C. J.; Haidar, A. H.; 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. 

075950

Bates, M. L.; Brenza, T. M.; Ben-Jebria, A.; Bascom, R.; Ultman, J. S. (2009). Longitudinal distribution of ozone 

absorption in the lung: Comparison of cigarette smokers and nonsmokers. Toxicol Appl Pharmacol, 236: 270-275. 

195727

Beckett, W. S.; McDonnell, W. F.; Horstman, D. H.; House, D. E. (1985). Role of the parasympathetic nervous system in 

acute lung response to ozone. J Appl Physiol, 59: 1879-1885. 039758

Belvisi, M. G.; Stretton, C. D.; Verleden, G. M.; Ledingham, S. J.; Yacoub, M. H.; Barnes, P. J. (1992). Inhibition of 

cholinergic neurotransmission in human airways by opioids. J Appl Physiol, 72: 1096-1100. 644681

Bennett, W. D.; Hazucha, M. J.; Folinsbee, L. J.; Bromberg, P. A.; Kissling, G. E.; London, S. J. (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 418827

Bergamaschi, E.; De Palma, G.; Mozzoni, P.; Vanni, S.; Vettori, M. V.; 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. 052670

Bhalla, D. K.; Gupta, S. K. (2000). Lung injury, inflammation, and inflammatory stimuli in rats exposed to ozone. J 

Toxicol Environ Health, 59: 211-228. 015036

Blomberg, A.; Mudway, I. S.; Nordenhall, C.; Hedenstrom, H.; Kelly, F. J.; Frew, A. J.; Holgate, S. T.; Sandstrom, T. 

(1999). Ozone-induced lung function decrements do not correlate with early airway inflammatory or antioxidant 

responses. Eur Respir J, 13: 1418-1428. 001267

Bosson, J.; Blomberg, A.; Pourazar, J.; Mudway, I. S.; Frew, A. J.; Kelly, F. J.; 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 399331

Bosson, J.; Stenfors, N.; Bucht, A.; Helleday, R.; Pourazar, J.; Holgate, S. T.; Kelly, F. J.; Sandstrom, T.; Wilson, S.; Frew, 

A. J.; Blomberg, A. (2003). Ozone-induced bronchial epithelial cytokine expression differs between healthy and 

asthmatic subjects. Clin Exp Allergy, 33: 777-782. 051687

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. 055490

Brook, R. D.; Brook, J. R.; Urch, B.; Vincent, R.; Rajagopalan, S.; Silverman, F. (2002). Inhalation of fine particulate air 

pollution and ozone causes acute arterial vasoconstriction in healthy adults. Circulation, 105: 1534-1536. 024987
March 2011 5-64 DRAFT – DO NOT CITE OR QUOTE 

Brook, R. D.; Urch, B.; Dvonch, J. T.; Bard, R. L.; Speck, M.; Keeler, G.; Morishita, M.; Marsik, F. J.; Kamal, A. S.; 

Kaciroti, N.; Harkema, J.; Corey, P.; Silverman, F.; Gold, D. R.; Wellenius, G.; Mittleman, M. A.; Rajagopalan, S.; 

Brook, J. R. (2009). Insights into the mechanisms and mediators of the effects of air pollution exposure on blood 

pressure and vascular function in healthy humans. Hypertension, 54: 659-U420. 

http://dx.doi.org/10.1161/hypertensionaha.109.130237 195611

Bush, M. L.; Asplund, P. T.; Miles, K. A.; Ben-Jebria, A.; Ultman, J. S. (1996). Longitudinal distribution of O3 absorption 

in the lung: gender differences and intersubject variability. J Appl Physiol, 81: 1651-1657. 080763

Bush, M. L.; Zhang, W.; Ben-Jebria, A.; Ultman, J. S. (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. 016665

Caceres, A. I.; Brackmann, M.; Elia, M. D.; Bessac, B. F.; del Camino, D.; D'Amours, M.; Witek, J. S.; Fanger, C. M.; 

Chong, J. A.; Hayward, N. J.; Homer, R. J.; Cohn, L.; Huang, X.; Moran, M. M.; Jordt, S. E. (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 628549

Carey, S. A.; Minard, K. R.; Trease, L. L.; Wagner, J. G.; Garcia, G. J.; Ballinger, C. A.; Kimbell, J. S.; Plopper, C. G.; 

Corley, R. A.; Postlethwait, E. M.; Harkema, J. R.; Einstein, D. R. (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 195752

Chang, L.-Y.; Huang, Y.; Stockstill, B. L.; Graham, J. A.; Grose, E. C.; Menache, M. G.; Miller, F. J.; Costa, D. L.; Crapo, J. 

D. (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 042387

Chen, C.; Arjomandi, M.; Balmes, J.; Tager, I.; Holland N (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 145956

Chen, C.; Arjomandi, M.; Qin, H.; Balmes, J.; Tager, I.; Holland, N. (2006). 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 196504

Cho, H. Y.; Hotchkiss, J. A.; Harkema, J. R. (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. 011985

Cho, H. Y.; Kleeberger, S. R. (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 195616

Cho, H.-Y.; Zhang, L.-Y.; Kleeberger, S. R. (2001). Ozone-induced lung inflammation and hyperreactivity are mediated via 

tumor necrosis factor-"alpha" receptors. Am J Physiol, 280: L537-L546. 016160

Christian, D. L.; Chen, L. L.; Scannell, C. H.; Ferrando, R. E.; Welch, B. S.; Balmes, J. R. (1998). Ozone-induced 

inflammation is attenuated with multiday exposure. Am J Respir Crit Care Med, 158: 532-537. 029925

Chuang, G. C.; Yang, Z.; Westbrook, D. G.; Pompilius, M.; Ballinger, C. A.; White, R. C.; Krzywanski, D. M.; Postlethwait, 

E. M.; Ballinger, S. W. (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 197202

Cohen-Hubal, E. A.; Kimbell, J. S.; Fedkiw, P. S. (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. 043785

Cole, M. P.; Freeman, B. A. (2009). Promotion of cardiovascular disease by exposure to the air pollutant ozone. Am J 

Physiol Lung Cell Mol Physiol, 297: L209-L216. 597507

Coleridge, H. M.; Coleridge, J. C. G.; Ginzel, K. H.; Baker, D. G.; Banzett, R. B.; Morrison, M. A. (1976). Stimulation of 

`irritant' receptors and afferent C-fibers in the lungs by prostaglandins. Nature, 264: 451-453. 038612

Coleridge, J. C. G.; Coleridge, H. M.; Schelegle, E. S.; Green, J. F. (1993). Acute inhalation of ozone stimulates bronchial 

C-fibers and rapidly adapting receptors in dogs. J Appl Physiol, 74: 2345-2352. 038695
March 2011 5-65 DRAFT – DO NOT CITE OR QUOTE 

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. 035448

Costa, D. L.; Schafrank, S. N.; Wehner, R. W.; 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 040273

Cross, C. E.; Motchnik, P. A.; Bruener, B. A.; Jones, D. A.; Kaur, H.; Ames, B. N.; 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 

625299

Cueto, R.; Squadrito, G. L.; Bermudez, E.; Pryor, W. A. (1992). Identification of heptanal and nonanal in bronchoalveolar 

lavage from rats exposed to low levels of ozone. Biochem Biophys Res Commun, 188: 129-134. 

http://dx.doi.org/10.1016/0006-291X(92)92359-6 042770

Dahl, A. R. (1990). Dose concepts for inhaled vapors and gases. Toxicol Appl Pharmacol, 103: 185-197. 094536

Dahl, M.; Bauer, A. K.; Arredouani, M.; Soininen, R.; Tryggvason, K.; Kleeberger, S. R.; 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 196986

Delaunois, A.; Segura, P.; Montano, L. M.; Vargas, M. H.; Ansay, M.; Gustin, P. (1998). Comparison of ozone-induced 

effects on lung mechanics and hemodynamics in the rabbit. Toxicol Appl Pharmacol, 150: 58-67. 015779

Devlin, R. B.; Folinsbee, L. J.; Biscardi, F.; Hatch, G.; Becker, S.; Madden, M. C.; Robbins, M.; Koren, H. S. (1997). 

Inflammation and cell damage induced by repeated exposure of humans to ozone. Inhal Toxicol, 9: 211-235. 

083577

Devlin, R. B.; McDonnell, W. F.; Mann, R.; Becker, S.; House, D. E.; Schreinemachers, D.; Koren, H. S. (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. 040359

Dimeo, M. J.; Glenn, M. G.; Holtzman, M. J.; Sheller, J. R.; Nadel, J. A.; Boushey, H. A. (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. 039662

Dormans, J. A. M. A.; Van Bree, L.; Boere, A. J. F.; Marra, M.; Rombout, P. J. A. (1999). Interspecies differences in time 

course of pulmonary toxicity following repeated exposure to ozone. Inhal Toxicol, 11: 309-329. 040766

Dostert, C.; Petrilli, V.; Van Bruggen, R.; Steele, C.; Mossman, B. T.; Tschopp, J. (2008). Innate Immune Activation 

Through Nalp3 Inflammasome Sensing of Asbestos and Silica. Science, 320: 674-677. 155753

Drechsler-Parks, D. M. (1995). The dose-response relationship in older men exposed to ozone. Exp Gerontol, 30: 65-75. 

076085

Duan, X.; Buckpitt, A. R.; Pinkerton, K. E.; Ji, C.; Plopper, C. G. (1996). Ozone-induced alterations in glutathione in lung 

subcompartments of rats and monkeys. Am J Respir Cell Mol Biol, 14: 70-75. 080791

Duan, X.; Buckpitt, A. R.; Plopper, C. G. (1993). Variation in antioxidant enzyme activities in anatomic subcompartments 

within rat and rhesus monkey lung. Toxicol Appl Pharmacol, 123: 73-82. 086326

Emmons, K.; Foster, W. M. (1991). Smoking cessation and acute airway response to ozone. Arch Environ Occup Health, 

46: 288-295. 042430

Enami, S.; Hoffmann, M. R.; Colussi, A. J. (2008). Acidity enhances the formation of a persistent ozonide at aqueous 

ascorbate/ozone gas interfaces. PNAS, 105: 7365-7369. 195833

Enami, S.; Hoffmann, M. R.; Colussi, A. J. (2008). Ozonolysis of uric acid at the air/water interface. J Phys Chem B, 112: 

4153–4156. http://dx.doi.org/10.1021/jp712010k 195834

Enami, S.; Hoffmann, M. R.; Colussi, A. J. (2009). 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 195835

Enami, S.; Hoffmann, M. R.; Colussi, A. J. (2009). Ozone oxidizes glutathione to a sulfonic acid. Chem Res Toxicol, 22: 

35-40. http://dx.doi.org/10.1021/tx800298j10.1021/tx800298j 197791
March 2011 5-66 DRAFT – DO NOT CITE OR QUOTE 

Enami, S.; Hoffmann, M. R.; Colussi, A. J. (2009). 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 

195621

Evans, M. J.; Fanucchi, M. V.; Baker, G. L.; Van Winkle, L. S.; Pantle, L. M.; Nishio, S. J.; Schelegle, E. S.; Gershwin, L. 

J.; Miller, L. A.; Hyde, D. M.; Plopper, C. G. (2004). The remodelled tracheal basement membrane zone of infant 

rhesus monkeys after 6 months of recovery. Clin Exp Allergy, 34: 1131-1136. http://dx.doi.org/10.1111/j.1365-

2222.2004.02004.x CEA2004 596379

Fabbri, L. M.; Aizawa, H.; O'Byrne, P. M.; Bethel, R. A.; Walters, E. H.; Holtzman, M. J.; Nadel, J. A. (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 040276

Fakhri, A. A.; Ilic, L. M.; Wellenius, G. A.; Urch, B.; Silverman, F.; Gold, D. R.; Mittleman, M. A. (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 191914

Fanucchi, M. V.; Plopper, C. G.; Evans, M. J.; Hyde, D. M.; Van Winkle, L. S.; Gershwin, L. J.; Schelegle, E. S. (2006). 

Cyclic exposure to ozone alters distal airway development in infant rhesus monkeys. Am J Physiol Lung Cell Mol 

Physiol, 291: L644-L650. http://dx.doi.org/10.1152/ajplung.00027.2006 096491

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 596381

Folinsbee, L. J.; Bedi, J. F.; Horvath, S. M. (1980). Respiratory responses in humans repeatedly exposed to low 

concentrations of ozone. Am Rev Respir Dis, 121: 431-439. 038880

Foster, W. M.; Freed, A. N. (1999). Regional clearance of solute from peripheral airway epithelia: recovery after sublobar 

exposure to ozone. J Appl Physiol, 86: 641-646. 001202

Foster, W. M.; Stetkiewicz, P. T. (1996). Regional clearance of solute from the respiratory epithelia: 18--20 h postexposure 

to ozone. J Appl Physiol, 81: 1143-1149. 079920

Frampton, M. W.; Morrow, P. E.; Torres, A.; Cox, C.; Voter, K. Z.; Utell, M. J.; Gibb, F. R.; Speers, D. M. (1997). Ozone 

responsiveness in smokers and nonsmokers. Am J Respir Crit Care Med, 155: 116-121. 082692

Frampton, M. W.; Morrow, P. E.; Torres, A.; Voter, K. Z.; Whitin, J. C.; Cox, C.; Speers, D. M.; Tsai, Y.; Utell, M. J. 

(1997). 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. 086111

Frampton, M. W.; Pryor, W. A.; Cueto, R.; Cox, C.; Morrow, P. E.; Utell, M. J. (1999). Ozone exposure increases aldehydes 

in epithelial lining fluid in human lung. Am J Respir Crit Care Med, 159: 1134-1137. 040757

Frank, R.; Liu, M. C.; Spannhake, E. W.; Mlynarek, S.; Macri, K.; Weinmann, G. G. (2001). Repetitive ozone exposure of 

young adults: evidence of persistent small airway dysfunction. Am J Respir Crit Care Med, 164: 1253-1260. 

093491

Freed, A. N.; Chou, C. L.; Fuller, S. D.; Croxton, T. L. (1996). Ozone-induced vagal reflex modulates airways reactivity in 

rabbits. Respir Physiol Neurobiol, 105: 95-102. 080798

Freed, A. N.; Cueto, R.; Pryor, W. A. (1999). Antioxidant transport modulates peripheral airway reactivity and 

inflammation during ozone exposure. J Appl Physiol, 87: 1595-1603. 011829

Fujita, M.; Sasayama, S.; Ohno, A.; Nakajima, H.; Asanoi, H. (1987). Importance of angina for development of collateral 

circulation. Heart, 57: 139-143. 004280

Gao, X.; Raghavamenon, A. C.; D'Auvergne, O.; Uppu, R. M. (2009). 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 200764

Garantziotis, S.; Li, Z.; Potts, E. N.; Kimata, K.; Zhuo, L.; Morgan, D. L.; Savani, R. C.; Noble, P. W.; Foster, W. M.; 

Schwartz, D. A.; Hollingsworth, J. W. (2009). Hyaluronan mediates ozone-induced airway hyperresponsiveness in 

mice. J Biol Chem, 284: 11309-11317. http://dx.doi.org/10.1074/jbc.M802400200 597603
March 2011 5-67 DRAFT – DO NOT CITE OR QUOTE 

Garantziotis, S.; Li, Z.; Potts, E. N.; Lindsey, J. Y.; Stober, V. P.; Polosukhin, V. V.; Blackwell, T. S.; Schwartz, D. A.; 

Foster, W. M.; Hollingsworth, J. W. (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 624947

Gerrity, T. R.; Biscardi, F.; Strong, A.; Garlington, A. R.; Brown, J. S.; Bromberg, P. A. (1995). Bronchoscopic 

determination of ozone uptake in humans. J Appl Physiol, 79: 852-860. 042785

Gerrity, T. R.; McDonnell, W. F.; House, D. E. (1994). The relationship between delivered ozone dose and functional 

responses in humans. Toxicol Appl Pharmacol, 124: 275-283. 041314

Gerrity, T. R.; Weaver, R. A.; Berntsen, J.; House, D. E.; O'Neil, J. J. (1988). Extrathoracic and intrathoracic removal of O3 

in tidal-breathing humans. J Appl Physiol, 65: 393-400. 040899

Giamalva, D.; Church, D. F.; Pryor, W. A. (1985). A comparison of the rates of ozonation of biological antioxidants and 

oleate and linoleate esters. Biochem Biophys Res Commun, 133: 773-779. 595129

Gilmour, M. I.; Park, P.; Doerfler, D.; Selgrade, M. K. (1993). Factors that influence the suppression of pulmonary 

antibacterial defenses in mice exposed to ozone. Exp Lung Res, 19: 299-314. 

http://dx.doi.org/10.3109/01902149309064348 039620

Gong, H. Jr; Wong, R.; Sarma, R. J.; Linn, W. S.; Sullivan, E. D.; Shamoo, D. A.; Anderson, K. R.; Prasad, S. B. (1998). 

Cardiovascular effects of ozone exposure in human volunteers. Am J Respir Crit Care Med, 158: 538-546. 029938

Graham, D. E.; Koren, H. S. (1990). Biomarkers of inflammation in ozone-exposed humans: comparison of the nasal and 

bronchoalveolar lavage. Am J Respir Crit Care Med, 142: 152-156. 042299

Graham, J. A.; Menzel, D. B.; Mole, M. L.; Miller, F. J.; Gardner, D. E. (1985). Influence of ozone on pentobarbital 

pharmacokinetics in mice. Toxicol Lett, 24: 163-170. http://dx.doi.org/10.1016/0378-4274(85)90053-0 040289

Gross, E. A.; Starr, T. B.; Randall, H. W.; Morgan, K. T. (1987). Morphometric analysis of the primate nasal cavity 

[Abstract]. Toxicologist, 7: 193. 625447

Gross, E. A.; Swenberg, J. A.; Fields, S.; Popp, J. A. (1982). Comparative morphometry of the nasal cavity in rats and 

mice. J Anat, 135: 83-88. 040121

Gunnison, A. F.; Hatch, G. E. (1999). O3-induced inflammation in prepregnant, pregnant, and lactating rats correlates with 

O3 dose estimated by 18O. Am J Physiol, 276: L332-L340. 087204

Gunnison, A. F.; Hatch, G. E.; Crissman, K.; Bowers, A. (1996). Comparative sensitivity of lactating and virgin female rats 

to ozone-induced pulmonary inflammation. Inhal Toxicol, 8: 607-623. 080803

Hackney, J. D.; Linn, W. S.; Mohler, J. G.; Collier, C. R. (1977). Adaptation to short-term respiratory effects of ozone in 

men exposed repeatedly. J Appl Physiol, 43: 82-85. 038282

Hamade, A. K.; Misra, V.; Rabold, R.; Tankersley, C. G. (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 666324

Hamade, A. K.; Tankersley, C. G. (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 596386

Hamilton, R. F.; Li, L.; Eschenbacher, W. L.; 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. 086157

Han, S. G.; Andrews, R.; Gairola, C. G.; Bhalla, D. K. (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 596387

Haque, R.; Umstead, T. M.; Freeman, W. M.; Floros, J.; Phelps, D. S. (2009). The impact of surfactant protein-A on ozoneinduced changes in the mouse bronchoalveolar lavage proteome. Proteome Science, 7: 12. 

http://dx.doi.org/10.1186/1477-5956-7-12 200767

Haque, R.; Umstead, T. M.; Ponnuru, P.; Guo, X.; Hawgood, S.; Phelps, D. S.; Floros, J. (2007). Role of surfactant proteinA (SP-A) in lung injury in response toacute ozone exposure of SP-A deficient mice. Toxicol Appl Pharmacol, 220: 

72-82. http://dx.doi.org/10.1016/j.taap.2006.12.017 597606
March 2011 5-68 DRAFT – DO NOT CITE OR QUOTE 

Harkema, J. R.; Hotchkiss, J. A.; Barr, E. B.; Bennett, C. B.; Gallup, M.; Lee, J. K.; Basbaum, C. (1999). Long-lasting 

effects of chronic ozone exposure on rat nasal epithelium. Am J Respir Cell Mol Biol, 20: 517-529. 001209

Harkema, J. R.; Plopper, C. G.; Hyde, D. M.; StGeorge, J. A.; Dungworth, D. L. (1987). Effects of an ambient level of 

ozone on primate nasal epithelial mucosubstances: quantitative histochemistry. Am J Pathol, 127: 90-96. 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1899606/. 040816

Harkema, J. R.; Plopper, C. G.; Hyde, D. M.; StGeorge, J. A.; Wilson, D. W.; Dungworth, D. L. (1987). 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. 041496

Harkema, J. R.; Wagner, J. G. (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 078340

Hatch, G. E. (1992). Comparative biochemistry of airway lining fluid. In RA Parent (Ed.), Comparative biology of the 

normal lung: v. I, treatise on pulmonary toxicology (pp. 617-632). Boca Raton, FL: CRC Press, Inc. 043901

Hatch, G. E.; Slade, R.; Harris, L. P.; McDonnell, W. F.; Devlin, R. B.; Koren, H. S.; Costa, D. L.; 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. 038953

Hatch, G. E.; Slade, R.; Stead, A. G.; Graham, J. A. (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 040472

Hatch, G. E.; Wiester, M. J.; Overton, J. H. 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 (pp. 553-560). Nijmegend, the Netherlands: Elsevier Science Publishers B.V. 041799

Hazbun, M. E.; Hamilton, R.; Holian, A.; Eschenbacher, W. L. (1993). Ozone-induced increases in substance P and 8-epiprostaglandin F2"alpha" in the airways of human subjects. Am J Respir Cell Mol Biol, 9: 568-572. 043914

Hazucha, M. J.; Bates, D. V.; Bromberg, P. A. (1989). Mechanism of action of ozone on the human lung. J Appl Physiol, 

67: 1535-1541. 041909

Hazucha, M. J.; Folinsbee, L. J.; Bromberg, P. A. (2003). Distribution and reproducibility of spirometric response to ozone 

by gender and age. J Appl Physiol, 95: 1917-1925. 048168

Hazucha, M. J.; Madden, M.; Pape, G.; Becker, S.; Devlin, R.; Koren, H. S.; Kehrl, H.; Bromberg, P. A. (1996). Effects of 

cyclo-oxygenase inhibition on ozone-induced respiratory inflammation and lung function changes. Eur J Appl 

Physiol, 73: 17-27. 043923

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 625266

Hollingsworth, J. W.; Cook, D. N.; Brass, D. M.; Walker, J. K. L.; Morgan, D. L.; Foster, W. M.; Schwartz, D. A. (2004). 

The role of Toll-like receptor 4 in environmental airway injury in mice. Am J Respir Crit Care Med, 170: 126-132. 

097816

Hollingsworth, J. W.; Free, M. E.; Li, Z.; Andrews, L. N.; Nakano, H.; Cook, D. N. (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. 635786

Hollingsworth, J. W.; Maruoka, S.; Li, Z.; Potts, E. N.; Brass, D. M.; Garantziotis, S.; Fong, A.; Foster, W. M.; Schwartz, 

D. A. (2007). Ambient ozone primes pulmonary innate immunity in mice. J Immunol, 179: 4367-4375. 

http://www.jimmunol.org/cgi/content/abstract/179/7/4367. 597609

Holtzman, M. J.; Cunningham, J. H.; Sheller, J. R.; Irsigler, G. B.; Nadel, J. A.; Boushey, H. A. (1979). Effect of ozone on 

bronchial reactivity in atopic and nonatopic subjects. Am Rev Respir Dis, 120: 1059-1067. 039220

Holtzman, M. J.; Fabbri, L. M.; O'Byrne, P. M.; Gold, B. D.; Aizawa, H.; Walters, E. H.; Alpert, S. E.; Nadel, J. A. (1983). 

Importance of airway inflammation for hyperresponsiveness induced by ozone. Am Rev Respir Dis, 127: 686-690. 

039745

Holz, O.; Jorres, R. A.; 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. 

058731
March 2011 5-69 DRAFT – DO NOT CITE OR QUOTE 

Horstman, D. H.; Ball, B. A.; Brown, J.; Gerrity, T.; Folinsbee, L. J. (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. 075834

Horvath, S. M.; Gliner, J. A.; Folinsbee, L. J. (1981). Adaptation to ozone: duration of effect. Am Rev Respir Dis, 123: 

496-499. 039221

Hotchkiss, J. A.; Harkema, J. R.; Henderson, R. F. (1991). Effect of cumulative ozone exposure on ozone-induced nasal 

epithelial hyperplasia and secretory metaplasia in rats. Exp Lung Res, 15: 589-600. 042441

Hu, P. C.; Miller, F. J.; Daniels, M. J.; 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 039418

Hu, S. C.; Ben-Jebria, A.; Ultman, J. S. (1992). Longitudinal distribution of ozone absorption in the lung: quiet respiration 

in healthy subjects. J Appl Physiol, 73: 1655-1667. 042794

Hu, S.-C.; Ben-Jebria, A.; Ultman, J. S. (1994). Longitudinal distribution of ozone absorption in the lung: effects of 

respiratory flow. J Appl Physiol, 77: 574-583. 041323

Hunter, D. D.; Wu, Z.; Dey, R. D. (2010). Sensory neural responses to ozone exposure during early postnatal development 

in rat airways. Am J Respir Cell Mol Biol, xx: xx-xx. http://dx.doi.org/10.1165/rcmb.2009-0191OC 382064

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. 041805

Ignatenko, A. V.; Cherenkevich, S. N. (1985). [Reactivity of amino-acids and proteins in reactions with ozone]. Kinet 

Catal, 26: 1332-1335. 625265

Islam, T.; McConnell, R.; Gauderman, W. J.; Avol, E.; Peters, J. M.; Gilliland, F. D. (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 097348

Iwasaki, T.; Takahashi, M.; Saito, H.; Arito, H. (1998). Adaptation of extrapulmonary responses to ozone exposure in 

conscious rats. Ind Health, 36: 57-60. 086165

Jakab, G. J.; Spannhake, E. W.; Canning, B. J.; Kleeberger, S. R.; Gilmour, M. I. (1995). The effects of ozone on immune 

function. Environ Health Perspect, 2: 77-89. 039548

Jiang, D.; Liang, J.; Fan, J.; Yu, S.; Chen, S.; Luo, Y.; Prestwich, G. D.; Mascarenhas, M. M.; Garg, H. G.; Quinn, D. A.; 

Homer, R. J.; Goldstein, D. R.; Bucala, R.; Lee, P. J.; Medzhitov, R.; Noble, P. W. (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 

628556

Joad, J. P.; Kott, K. S.; Bric, J. M. (1996). The local C-fiber contribution to ozone-induced effects on the isolated guinea 

pig lung. Toxicol Appl Pharmacol, 141: 561-567. 082711

Joad, J. P.; Kott, K. S.; Bric, J. M.; Peake, J. L.; Plopper, C. G.; Schelegle, E. S.; Gershwin, L. J.; Pinkerton, K. E. (2006). 

Structural and functional localization of airway effects from episodic exposure of infant monkeys to allergen and/or 

ozone. Toxicol Appl Pharmacol, 214: 237-243. http://dx.doi.org/10.1016/j.taap.2005.12.012 596390

Johansson, E.; Wesselkamper, S. C.; Shertzer, H. G.; Leikauf, G. D.; Dalton, T. P.; 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 644476

Johnston, C.; Holm, B.; Gelein, R.; Finkelstein, J. (2006). Postnatal lung development: Immediate-early gene responses 

post ozone and LPS exposure. Inhal Toxicol, 18: 875-883. http://dx.doi.org/10.1080/08958370600822466 097439

Johnston, R. A.; Mizgerd, J. P.; Shore, S. A. (2005). 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 596393

Johnston, R. A.; Schwartzman, I. N.; Flynt, L.; Shore, S. A. (2005). 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 

596394
March 2011 5-70 DRAFT – DO NOT CITE OR QUOTE 

Johnston, R. A.; Theman, T. A.; Lu, F. L.; Terry, R. D.; Williams, E. S.; Shore, S. A. (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 597625

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. 078122

Kabel, J. R.; Ben-Jebria, A.; Ultman, J. S. (1994). Longitudinal distribution of ozone absorption in the lung: comparison of 

nasal and oral quiet breathing. J Appl Physiol, 77: 2584-2592. 095597

Kafoury, R. M.; Pryor, W. A.; Squadrito, G. L.; Salgo, M. G.; Zou, X.; Friedman, M. (1998). Lipid ozonation products 

activate phospholipases A2, C, and D. Toxicol Appl Pharmacol, 150: 338-349. 016913

Kajekar, R.; Pieczarka, E. M.; Smiley-Jewell, S. M.; Schelegle, E. S.; Fanucchi, M. V.; Plopper, C. G. (2007). Early 

postnatal exposure to allergen and ozone leads to hyperinnervation of the pulmonary epithelium. Respir Physiol 

Neurobiol, 155: 55-63. http://dx.doi.org/10.1016/j.resp.2006.03.002 567661

Kanofsky, J. R.; Sima, P. D. (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. 075973

Kari, F.; Hatch, G.; Slade, R.; Crissman, K.; Simeonova, P. P.; 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. 

086171

Kehrl, H. R.; Peden, D. B.; Ball, B. A.; Folinsbee, L. J.; Horstman, D. H. (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. 022101

Kehrl, H. R.; Vincent, L. M.; Kowalsky, R. J.; Horstman, D. H.; O'Neil, J. J.; McCartney, W. H.; Bromberg, P. A. (1987). 

Ozone exposure increases respiratory epithelial permeability in humans. Am Rev Respir Dis, 135: 1124-1128. 

040824

Kermani, S.; Ben-Jebria, A.; Ultman, J. S. (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 195643

Kleeberger, S. R.; Levitt, R. C.; Zhang, L.-Y.; Longphre, M.; Harkema, J.; Jedlicka, A.; Eleff, S. M.; DiSilvestre, D.; 

Holroyd, K. J. (1997). Linkage analysis of susceptibility to ozone-induced lung inflammation in inbred mice. Nat 

Genet, 17: 475-478. 095736

Kleeberger, S. R.; Reddy, S.; Zhang, L.-Y.; Jedlicka, A. E. (2000). Genetic susceptibility to ozone-induced lung 

hyperpermeability: role of toll-like receptor 4. Am J Respir Cell Mol Biol, 22: 620-627. 014895

Kleeberger, S. R.; Seiden, J. E.; Levitt, R. C.; Zhang, L.-Y. (1993). Mast cells modulate acute ozone-induced inflammation 

of the murine lung. Am J Respir Crit Care Med, 148: 1284-1291. 044203

Kodavanti, U. P.; Costa, D. L.; Dreher, K. L.; Crissman, K.; Hatch, G. E. (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 077440

Kodavanti, U. P.; Thomas, R.; Ledbetter, A. D.; Schladweiler, M. C.; Shannahan, J. H.; Wallenborn, J. G.; Lund, A. K.; 

Campen, M. J.; Butler, E. O.; Gottipolu, R. R.; Nyska, A.; Richards, J. E.; Andrews, D.; Jaskot, R. H.; McKee, J.; 

Kotha, S. R.; Patel, R. B.; Parianandi, N. L. (In Press). Vascular and cardiac impairments in rats Inhaling ozone and 

diesel exhaust particles. Environ Health Perspect. http://dx.doi.org/10.1289/ehp.1002386 666323

Koenig, J. Q.; Covert, D. S.; Marshall, S. G.; Van Belle, G.; Pierson, W. E. (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. 041521

Kreit, J. W.; Gross, K. B.; Moore, T. B.; Lorenzen, T. J.; D'Arcy, J.; Eschenbacher, W. L. (1989). Ozone-induced changes in 

pulmonary function and bronchial responsiveness in asthmatics. J Appl Physiol, 66: 217-222. 041817

Krishna, M. T.; 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. http://ajrccm.atsjournals.org/cgi/content/full/156/3/943. 084262
March 2011 5-71 DRAFT – DO NOT CITE OR QUOTE 

Larsen, S. T.; Matsubara, S.; McConville, G.; Poulsen, S. S.; Gelfand, E. W. (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 628560

Larson, S. D.; Schelegle, E. S.; Walby, W. F.; Gershwin, L. J.; Fanuccihi, M. V.; Evans, M. J.; Joad, J. P.; Tarkington, B. K.; 

Hyde, D. M.; Plopper, C. G. (2004). Postnatal remodeling of the neural components of the epithelial-mesenchymal 

trophic unit in the proximal airways of infant rhesus monkeys exposed to ozone and allergen. Toxicol Appl 

Pharmacol, 194: 211-220. 057062

Last, J. A.; Gohil, K.; Mathrani, V. C.; Kenyon, N. J. (2005). Systemic responses to inhaled ozone in mice: cachexia and 

down-regulation of liver xenobiotic metabolizing genes. Toxicol Appl Pharmacol, 208: 117-126. 

http://dx.doi.org/10.1016/j.taap.2005.02.001 596400

Lay, J. C.; Alexis, N. E.; Kleeberger, S. R.; Roubey, R. A.; Harris, B. D.; Bromberg, P. A.; Hazucha, M. J.; Devlin, R. B.; 

Peden, D. B. (2007). Ozone enhances markers of innate immunity and antigen presentation on airway monocytes in 

healthy individuals. J Allergy Clin Immunol, 120: 719-22. http://dx.doi.org/10.1016/j.jaci.2007.05.005 196610

Li, Y.-F.; Gauderman, W. J.; Avol, E.; Dubeau, L.; Gilliland, F. D. (2006). Associations of tumor necrosis factor G-308A 

with childhood asthma and wheezing. Am J Respir Crit Care Med, 173: 970-976. 

http://dx.doi.org/10.1164/rccm.200508-1256OC 090972

Li, Z.; Potts, E. N.; Piantadosi, C. A.; Foster, W. M.; Hollingsworth, J. W. (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 670282

Linn, W. S.; Buckley, R. D.; Spier, C. E.; Blessey, R. L.; Jones, M. P.; Fischer, D. A.; Hackney, J. D. (1978). Health effects 

of ozone exposure in asthmatics. Am Rev Respir Dis, 117: 835-843. 038874

Linn, W. S.; Medway, D. A.; Anzar, U. T.; Valencia, L. M.; Spier, C. E.; Tsao FS-D; Fischer, D. A.; Hackney, J. D. (1982). 

Persistence of adaptation to ozone in volunteers exposed repeatedly for six weeks. Am Rev Respir Dis, 125: 491-

495. 039646

London, S. J. (2007). Gene-air pollution interactions in asthma. Proc Am Thorac Soc, 4: 217-220. 093279

Long, N. C.; Suh, J.; Morrow, J. D.; Schiestl, R. H.; Krishna Murthy, G. G.; Brain, J. D.; Frei, B. (2001). Ozone causes lipid 

peroxidation but little antioxidant depletion in exercising and nonexercising hamsters. J Appl Physiol, 91: 1694-

1700. 057301

Longphre, M.; Zhang, L.-Y.; Harkema, J. R.; Kleeberger, S. R. (1999). Ozone-induced pulmonary inflammation and 

epithelial proliferation are partially mediated by PAF. J Appl Physiol, 86: 341-349. 001199

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. 050626

McBride, J. T. (1992). Architecture of the tracheobronchial tree. In RA Parent (Ed.), Comparative biology of the normal 

lung (pp. 49-61). Boca Raton, FL: CRC Press. 078532

McKinney, W. J.; Jaskot, R. H.; Richards, J. H.; Costa, D. L.; Dreher, K. L. (1998). Cytokine mediation of ozone-induced 

pulmonary adaptation. Am J Respir Cell Mol Biol, 18: 696-705. 086751

Mikerov, A. N.; Gan, X.; Umstead, T. M.; Miller, L.; Chinchilli, V. M.; Phelps, D. S.; Floros, J. (2008). 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 597493

Mikerov, A. N.; Haque, R.; Gan, X.; Guo, X.; Phelps, D. S.; Floros, J. (2008). 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 201537

Mikerov, A. N.; Umstead, T. M.; Gan, X.; Huang, W.; Guo, X.; Wang, G.; Phelps, D. S.; Floros, J. (2008). 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 596405

Miller, F. J.; Overton, J. H. Jr; Jaskot, R. H.; Menzel, D. B. (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 040307
March 2011 5-72 DRAFT – DO NOT CITE OR QUOTE 

Miller, F. J.; Overton, J. H.; Gerrity, T. R.; Graham, R. C. (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 

Inc. 041545

Miller, L. A.; Gerriets, J. E.; Tyler, N. K.; Abel, K.; Schelegle, E. S.; Plopper, C. G.; Hyde, D. M. (2009). Ozone and 

allergen exposure during postnatal development alters the frequency and airway distribution of CD25+ cells in 

infant rhesus monkeys. Toxicol Appl Pharmacol, 236: 39-48. http://dx.doi.org/10.1016/j.taap.2008.12.031 596406

Molfino, N. A.; Wright, S. C.; Katz, I.; Tarlo, S.; Silverman, F.; McClean, P. A.; Szalai, J. P.; Raizenne, M.; Slutsky, A. S.; 

Zamel, N. (1991). Effect of low concentrations of ozone on inhaled allergen responses in asthmatic subjects. 

Lancet, 338: 199-203. 042379

Mudway, I. S.; Behndig, A. F.; Helleday, R.; Pourazar, J.; Frew, A. J.; Kelly, F. J.; Blomberg, A. (2006). Vitamin 

supplementation does not protect against symptoms in ozone-responsive subjects. Free Radic Biol Med, 40: 1702-

1712. 196536

Mudway, I. S.; Blomberg, A.; Frew, A. J.; Holgate, S. T.; Sandstrom, T.; Kelly, F. J. (1999). Antioxidant consumption and 

repletion kinetics in nasal lavage fluid following exposure of healthy human volunteers to ozone. Eur Respir J, 13: 

1429-1438. 001270

Mudway, I. S.; Housley, D.; Eccles, R.; Richards, R. J.; Datta, A. K.; Tetley, T. D.; Kelly, F. J. (1996). Differential depletion 

of human respiratory tract antioxidants in response to ozone challenge. Free Radic Res, 25: 499-513. 080730

Mudway, I. S.; Kelly, F. J. (1998). Modeling the interactions of ozone with pulmonary epithelial lining fluid antioxidants. 

Toxicol Appl Pharmacol, 148: 91-100. 000273

Mudway, I. S.; Kelly, F. J. (2000). Ozone and the lung: a sensitive issue. Mol Aspects Med, 21: 1-48. 010452

Mudway, I. S.; Kelly, F. J. (2004). 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 399328

Mudway, I. S.; Krishna, M. T.; Frew, A. J.; MacLeod, D.; Sandstrom, T.; Holgate, S. T.; Kelly, F. J. (1999). Compromised 

concentrations of ascorbate in fluid lining the respiratory tract in human subjects after exposure to ozone. Occup 

Environ Med, 56: 473-481. 011833

Mudway, I. S.; Stenfors, N.; Blomberg, A.; Helleday, R.; Dunster, C.; Marklund, S. L.; Frew, A. J.; Sandstrom, T.; Kelly, F. 

J. (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. 025327

Murphy, R. C.; Johnson, K. M. (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 197792

Nodelman, V.; Ultman, J. S. (1999). Longitudinal distribution of chlorine absorption in human airways: A comparison to 

ozone absorption. J Appl Physiol, 87: 2073-2080. http://jap.physiology.org/cgi/content/full/87/6/2073. 015112

Noviski, N.; Brewer, J. P.; Skornik, W. A.; Galli, S. J.; Drazen, J. M.; Martin, T. R. (1999). Mast cell activation is not 

required for induction of airway hyperresponsiveness by ozone in mice. J Appl Physiol, 86: 202-210. 001198

O'Byrne, P. M.; Walters, E. H.; Aizawa, H.; Fabbri, L. M.; Holtzman, M. J.; Nadel, J. A. (1984). Indomethacin inhibits the 

airway hyperresponsiveness but not the neutrophil influx induced by ozone in dogs. Am Rev Respir Dis, 130: 220-

224. 040066

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. 041366

Oslund, K. L.; Hyde, D. M.; Putney, L. F.; Alfaro, M. F.; Walby, W. F.; Tyler, N. K.; Schelegle, E. S. (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 195654

Oslund, K. L.; Hyde, D. M.; Putney, L. F.; Alfaro, M. F.; Walby, W. F.; Tyler, N. K.; Schelegle, E. S. (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 201539
March 2011 5-73 DRAFT – DO NOT CITE OR QUOTE 

Overton, J. H.; Graham, R. C.; Menache, M. G.; Mercer, R. R.; Miller, F. J. (1996). Influence of tracheobronchial region 

expansion and volume on reactive gas uptake and interspecies dose extrapolations. Inhal Toxicol, 8: 723-745. 

080733

Park, J. W.; Taube, C.; Swasey, C.; Kodama, T.; Joetham, A.; Balhorn, A.; Takeda, K.; Miyahara, N.; Allen, C. B.; 

Dakhama, A.; Kim, S. H.; Dinarello, C. A.; Gelfand, E. W. (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 644864

Passannante, A. N.; Hazucha, M. J.; Bromberg, P. A.; Seal, E.; Folinsbee, L.; Koch, G. (1998). Nociceptive mechanisms 

modulate ozone-induced human lung function decrements. J Appl Physiol, 85: 1863-1870. 030114

Pearson, A. C.; Bhalla, D. K. (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. 082686

Peden, D. B.; Setzer, R. W. Jr; Devlin, R. B. (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. 076189

Perepu, R. S.; Garcia, C.; Dostal, D.; Sethi, R. (2010). Enhanced death signaling in ozone-exposed ischemic-reperfused 

hearts. Mol Cell Biochem, 336: 55-64. http://dx.doi.org/10.1007/s11010-009-0265-4 385020

Perez-Gil, J. (2008). Structure of pulmonary surfactant membranes and films: The role of proteins and lipid-protein 

interactions. Biochim Biophys Acta, 1778: 1676-1695. http://dx.doi.org/10.1016/j.bbamem.2008.05.003 198890

Pichavant, M.; Goya, S.; Meyer, E. H.; Johnston, R. A.; Kim, H. Y.; Matangkasombut, P.; Zhu, M.; Iwakura, Y.; Savage, P. 

B.; DeKruyff, R. H.; Shore, S. A.; Umetsu, D. T. (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 596409

Picher, M.; Burch, L. H.; Boucher, R. C. (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 644780

Plopper, C. G.; Hatch, G. E.; Wong, V.; Duan, X.; Weir, A. J.; Tarkington, B. K.; Devlin, R. B.; Becker, S.; Buckpitt, A. R. 

(1998). Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone 

dose and glutathione depletion in rhesus monkeys. Am J Respir Cell Mol Biol, 19: 387-399. 087203

Plopper, C. G.; Mango, G. W.; Hatch, G. E.; Wong, V. J.; Toskala, E.; Reynolds, S. D.; Tarkington, B. K.; Stripp, B. R. 

(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 

596410

Plopper, C. G.; Smiley-Jewell, S. M.; Miller, L. A.; Fanucchi, M. V.; Evans, M. J.; Buckpitt, A. R.; Avdalovic, M.; 

Gershwin, L. J.; Joad, J. P.; Kajekar, R.; Larson, S.; Pinkerton, K. E.; Van Winkle, L. S.; Schelegle, E. S.; Pieczarka, 

E. M.; Wu, R.; Hyde, D. M. (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 

596412

Postlethwait, E. M.; Cueto, R.; Velsor, L. W.; Pryor, W. A. (1998). O3-induced formation of bioactive lipids: Estimated 

surface concentrations and lining layer effects. Am J Physiol, 274: L1006-L1016. 086754

Postlethwait, E. M.; Joad, J. P.; Hyde, D. M.; Schelegle, E. S.; Bric, J. M.; Weir, A. J.; Putney, L. F.; Wong, V. J.; Velsor, L. 

W.; Plopper, C. G. (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. 003000

Postlethwait, E. M.; Langford, S. D.; Bidani, A. (1994). Determinants of inhaled ozone absorption in isolated rat lungs. 

Toxicol Appl Pharmacol, 125: 77-89. 044219

Postlethwait, E. M.; Ultman, J. S. (2001). Airspace surface chemistry mediates O3-induced lung injury. Hum Ecol Risk 

Assess, 7: 1145-1159. http://dx.doi.org/10.1080/20018091094907 196805

Pryor, W. A. (1976). Free radical reactions in biology: Initiation of lipid autoxidation by ozone and nitrogen dioxide. 

Environ Health Perspect, 16: 180-181. 038940
March 2011 5-74 DRAFT – DO NOT CITE OR QUOTE 

Pryor, W. A. (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 042725

Pryor, W. A. (1994). Mechanisms of radical formation from reactions of ozone with target molecules in the lung. Free 

Radic Biol Med, 17: 451-465. 075987

Pryor, W. A.; Bermudez, E.; Cueto, R.; Squadrito, G. L. (1996). Detection of aldehydes in bronchoalveolar lavage of rats 

exposed to ozone. Toxicol Sci, 34: 148-156. 082697

Pryor, W. A.; Das, B.; Church, D. F. (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. 042482

Pryor, W. A.; Giamalva, D. H.; Church, D. F. (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 . 595130

Pulfer, M. K.; Murphy, R. C. (2004). Formation of biologically active oxysterols during ozonolysis of cholesterol present in 

lung surfactant. J Biol Chem, 279: 26331-26338. 076673

Pulfer, M. K.; Taube, C.; Gelfand, E.; Murphy, R. C. (2005). Ozone exposure in vivo and formation of biologically active 

oxysterols in the lung. J Pharmacol Exp Ther, 312: 256-264. 076663

Rashba-Step, J.; Tatoyan, A.; Duncan, R.; Ann, D.; Pushpa-Rehka, T. R.; 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 628562

Reeser, W. H.; Lee, G. M.; Taylor, A.; Wang, L.; Arnold, S. F.; Ultman, J. S.; 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 195718

Rigas, M. L.; Ben-Jebria, A.; Ultman, J. S. (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. 083602

Rigas, M. L.; Catlin, S. N.; Ben-Jebria, A.; Ultman, J. S. (2000). Ozone uptake in the intact human respiratory tract: 

Relationship between inhaled dose and actual dose. J Appl Physiol, 88: 2015-2022. 010454

Romieu, I.; Meneses, F.; Ramirez, M.; Ruiz, S.; Padilla, R. P.; Sienra, J. J.; Gerber, M.; Grievink, L.; Dekker, R.; Walda, I.; 

Brunekreef, B. (1998). Antioxidant supplementation and respiratory functions among workers exposed to high 

levels of ozone. Am J Respir Crit Care Med, 158: 226-232. 086756

Romieu, I.; Sienra-Monge, J. J.; Ramirez-Aguilar, M.; Moreno-Macias, H.; Reyes-Ruiz, N. I.; Estela del Rio-Navarro, B.; 

Hernandez-Avila, M.; London, S. J. (2004). 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. 

056796

Romieu, I.; Sienra-Monge, J. J.; Ramirez-Aguilar, M.; Tellez-Rojo, M. M.; Moreno-Macias, H.; Reyes-Ruiz, N. I.; Del 

Rio-Navarro, B. E.; Ruiz-Navarro, M. X.; 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. 034711

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. 001264

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 041704

Samet, J. M.; Hatch, G. E.; Horstman, D.; Steck-Scott, S.; Arab, L.; Bromberg, P. A.; Levine, M.; McDonnell, W. F.; 

Devlin, R. B. (2001). Effect of antioxidant supplementation on ozone-induced lung injury in human subjects. Am J 

Respir Crit Care Med, 164: 819-825. 019034

Santiago, L. Y.; Hann, M. C.; Ben-Jebria, A.; Ultman, J. S. (2001). Ozone absorption in the human nose during 

unidirectional airflow. J Appl Physiol, 91: 725-732. http://jap.physiology.org/cgi/content/abstract/91/2/725. 019841

Sarangapani, R.; Gentry, P. R.; Covington, T. R.; Teeguarden, J. G.; Clewell HJ 3rd (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. http://dx.doi.org/10.1080/713857276 054581
March 2011 5-75 DRAFT – DO NOT CITE OR QUOTE 

Sathishkumar, K.; Gao, X.; Raghavamenon, A. C.; Parinandi, N.; Pryor, W. A.; Uppu, R. M. (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 201549

Sathishkumar, K.; Murthy, S. N.; Uppu, R. M. (2007). 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 197785

Sathishkumar, K.; Xi, X.; Martin, R.; Uppu, R. M. (2007). Cholesterol secoaldehyde, an ozonation product of cholesterol, 

induces amyloid aggregation and apoptosis in murine GT1-7 hypothalamic neurons. J Alzheimers Dis, 11: 261-274. 

097758

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. 

http://www.journalarchive.jst.go.jp/english/jnlabstract_en.php?cdjournal=tjem1920&cdvol=130&noissue=2&startp

age=117. 039738

Sawyer, K.; Brown, J.; HazuchaM; Bennett, W. D. (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 195142

Scannell, C.; Chen, L.; Aris, R. M.; Tager, I.; Christian, D.; Ferrando, R.; Welch, B.; Kelly, T.; Balmes, J. R. (1996). 

Greater ozone-induced inflammatory responses in subjects with asthma. Am J Respir Crit Care Med, 154: 24-29. 

080755

Schelegle, E. S.; Adams, W. C.; Siefkin, A. D. (1987). Indomethacin pretreatment reduces ozone-induced pulmonary 

function decrements in human subjects. Am Rev Respir Dis, 136: 1350-1354. 041706

Schelegle, E. S.; Carl, M. L.; Coleridge, H. M.; Coleridge, J. C. G.; Green, J. F. (1993). Contribution of vagal afferents to

respiratory reflexes evoked by acute inhalation of ozone in dogs. J Appl Physiol, 74: 2338-2344. 039203

Schelegle, E. S.; Miller, L. A.; Gershwin, L. J.; Fanucchi, M. V.; Van Winkle, L. S.; Gerriets, J. E.; Walby, W. F.; Mitchell, 

V.; Tarkington, B. K.; Wong, V. J.; Baker, G. L.; Pantle, L. M.; Joad, J. P.; Pinkerton, K. E.; Wu, R.; Evans, M. J.; 

Hyde, D. M.; Plopper, C. G. (2003). Repeated episodes of ozone inhalation amplifies the effects of allergen 

sensitization and inhalation on airway immune and structural development in Rhesus monkeys. Toxicol Appl 

Pharmacol, 191: 74-85. 053778

Schelegle, E. S.; Siefkin, A. D.; McDonald, R. J. (1991). Time course of ozone-induced neutrophilia in normal humans. Am 

J Respir Crit Care Med, 143: 1353-1358. 042491

Schelegle, E. S.; Walby, W. F.; Adams, W. C. (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 195841

Schroter, R. C.; Sudlow, M. F. (1969). Flow patterns in models of the human bronchial airways. Respir Physiol Neurobiol, 

7: 341-355. 071359

Seltzer, J.; Bigby, B. G.; Stulbarg, M.; Holtzman, M. J.; Nadel, J. A.; Ueki, I. F.; Leikauf, G. D.; Goetzl, E. J.; Boushey, H. 

A. (1986). O3-induced change in bronchial reactivity to methacholine and airway inflammation in humans. J Appl 

Physiol, 60: 1321-1326. 040383

Servais, S.; Boussouar, A.; Molnar, A.; Douki, T.; Pequignot, J. M.; 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 195667

Shore, S. A.; Lang, J. E.; Kasahara, D. I.; Lu, F. L.; Verbout, N. G.; Si, H.; Williams, E. S.; Terry, R. D.; Lee, A.; Johnston, 

R. A. (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 201551

Shore, S. A.; Schwartzman, I. N.; Le Blanc, B.; Krishna Murthy, G. G.; Doerschuk, C. M. (2001). Tumor necrosis factor 

receptor 2 contributes to ozone-induced airway hyperresponsiveness in mice. Am J Respir Crit Care Med, 164: 602-

607. 018993

Sienra-Monge, J. J.; Ramirez-Aguilar, M.; Moreno-Macias, H.; Reyes-Ruiz, N. I.; Del Rio-Navarro, B. E.; Ruiz-Navarro, 

M. X.; Hatch, G.; Crissman, K.; Slade, R.; Devlin, R. B.; 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 196422
March 2011 5-76 DRAFT – DO NOT CITE OR QUOTE 

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. 042865

Slade, R.; Highfill, J. W.; Hatch, G. E. (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. 059465

Slade, R.; Watkinson, W. P.; Hatch, G. E. (1997). Mouse strain differences in ozone dosimetry and body temperature 

changes. Am J Physiol, 272: L73-L77. 082708

Stenfors, N.; Bosson, J.; Helleday, R.; Behndig, A. F.; Pourazar, J.; Tornqvist, H.; Kelly, F. J.; Frew, A. J.; Sandstrom, T.; 

Mudway, I. S.; Blomber, A. (2010). Ozone exposure enhances mast-cell inflammation in asthmatic airways despite 

inhaled corticosteroid therapy. Inhal Toxicol, 22: 133-139. http://dx.doi.org/10.3109/08958370903005736 386512

Stenfors, N.; Pourazar, J.; Blomberg, A.; Krishna, M. T.; Mudway, I.; Helleday, R.; Kelly, F. J.; Frew, A. J.; Sandstrom, T. 

(2002). Effect of ozone on bronchial mucosal inflammation in asthmatic and healthy subjects. Respir Med, 96: 352-

358. 030473

Sterner-Kock, A.; Kock, M.; Braun, R.; Hyde, D. M. (2000). Ozone-induced epithelial injury in the ferret is similar to 

nonhuman primates. Am J Respir Crit Care Med, 162: 1152-1156. 013033

Sun, J.; Koto, H.; Chung, K. F. (1997). Interaction of ozone and allergen challenges on bronchial responsiveness and 

inflammation in sensitised guinea pigs. Int Arch Allergy Immunol, 112: 191-195. 082724

Tarrant, J. M. (2010). Blood cytokines as biomarkers of in vivo toxicity in preclinical safety assessment: Considerations for 

their use. Toxicol Sci, 117: 4-16. 644810

Taylor, A. B.; Borhan, A.; Ultman, J. S. (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 195717

Taylor, A. B.; Lee, G. M.; Nellore, K.; Ben-Jebria, A.; Ultman, J. S. (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 

195731

Taylor-Clark, T. E.; McAlexander, M. A.; Nassenstein, C.; Sheardown, S. A.; Wilson, S.; Thornton, J.; Carr, M. J.; Undem, 

B. J. (2008). Relative contributions of TRPA1 and TRPV1 channels in the activation of vagal bronchopulmonary Cfibres by the endogenous autacoid 4-oxononenal. J Physiol, 586: 3447-3459. 

http://dx.doi.org/10.1113/jphysiol.2008.153585 628565

Taylor-Clark, T. E.; Undem, B. J. (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 377143

Tepper, J. S.; Costa, D. L.; Fitzgerald, S.; Doerfler, D. L.; Bromberg, P. A. (1993). Role of tachykinins in ozone-induced 

acute lung injury in guinea pigs. J Appl Physiol, 75: 1404-1411. 

http://jap.physiology.org/cgi/content/abstract/75/3/1404. 628570

Tepper, J. S.; Costa, D. L.; Lehmann, J. R.; Weber, M. F.; Hatch, G. E. (1989). Unattenuated structural and biochemical 

alterations in the rat lung during functional adaptation to ozone. Am J Respir Crit Care Med, 140: 493-501. 041991

Thomson, E.; Kumarathasan, P.; Goegan, P.; Aubin, R. A.; Vincent, R. (2005). Differential regulation of the lung endothelin 

system by urban particulate matter and ozone. Toxicol Sci, 88: 103-113. 087554

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. 

http://ebm.rsmjournals.com/cgi/content/abstract/231/6/979. 097483

Tran, M. U.; Weir, A. J.; Fanucchi, M. V.; Rodriguez, A. E.; Pantle, L. M.; Smiley-Jewell, S. M.; Van Winkle, L. S.; Evans, 

M. J.; Miller, L. A.; Schelegle, E. S.; Gershwin, L. J.; Hyde, D. M.; Plopper, C. G. (2004). Smooth muscle 

hypertrophy in distal airways of sensitized infant rhesus monkeys exposed to house dust mite allergen. Clin Exp 

Allergy, 34: 1627-1633. http://dx.doi.org/10.1111/j.1365-2222.2004.02057.x 628626

Trevisani, M.; Siemens, J.; Materazzi, S.; Bautista, D. M.; Nassini, R.; Campi, B.; Imamachi, N.; Andrè, E.; Patacchini, R.; 

Cottrell, G. S.; Gatti, R.; Basbaum, A. I.; Bunnett, N. W.; 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 628590

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 195842
March 2011 5-77 DRAFT – DO NOT CITE OR QUOTE 

U.S. EPA (1996). Air quality criteria for ozone and related photochemical oxidants (Report No. EPA/600/P-93/004AF). 

Research Triangle Park, NC: U.S. Environmental Protection Agency, Office of Research and Development. (NTIS 

No. PB94173127) 017831

U.S. EPA (2006). Air quality criteria for ozone and related photochemical oxidants (Report No. 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. 088089

Ultman, J. S.; Ben-Jebria, A.; Arnold, S. F. (2004). Uptake distribution of ozone in human lungs: intersubject variability in 

physiologic response. University Park, PA: Health Effects Institute. http://pubs.healtheffects.org/view.php?id=70. 

057197

Ultman, J. S.; Ben-Jebria, A.; Hu, S.-C. (1994). Noninvasive determination of respiratory ozone absorption: The bolusresponse method (Report No. research report no. 69). Cambridge, MA: Health Effects Institute. 041746

Uppu, R. M.; Cueto, R.; Squadrito, G. L.; Pryor, W. A. (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. 076212

Urch, B.; Speck, M.; Corey, P.; Wasserstein, D.; Manno, M.; Lukic, K. Z.; Brook, J. R.; Liu, L.; Coull, B.; Schwartz, J.; 

Gold, D. R.; Silverman, F. (2010). Concentrated ambient fine particles and not ozone induce a systemic interleukin6 response in humans. Inhal Toxicol, 22: 210-218. http://dx.doi.org/10.3109/08958370903173666 387113

Vagaggini, B.; Taccola, M.; Clanchetti, S.; Carnevali, S.; Bartoli, M. L.; Bacci, E.; Dente, F. L.; Di Franco, A.; Giannini, 

D.; Paggiaro, P. L. (2002). Ozone exposure increases eosinophilic airway response induced by previous allergen 

challenge. Am J Respir Crit Care Med, 166: 1073-1077. 035191

Van Bree, L.; Dormans, J. A. M. A.; Koren, H. S.; Devlin, R. B.; Rombout, P. J. A. (2002). Attenuation and recovery of 

pulmonary injury in rats following short-term, repeated daily exposure to ozone. Inhal Toxicol, 14: 883-900. 

035452

Vancza, E. M.; 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 

596419

Vasu, V. T.; Oommen, S.; Lim, Y.; Valacchi, G.; Hobson, B.; Eiserich, J. P.; Leonard, S. W.; Traber, M. G.; Cross, C. E.; 

Gohil, K. (2010). Modulation of ozone-sensitive genes in alpha-tocopherol transfer protein null mice. Inhal Toxicol, 

22: 1-16. 201561

Verhein, K. C.; Hazari, M. S.; Moulton, B. C.; Jacoby, I. W.; Jacoby, D. B.; Fryer, A. D. (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 670295

Vesely, K. R.; Schelegle, E. S.; Stovall, M. Y.; Harkema, J. R.; Green, J. F.; Hyde, D. M. (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. 051045

Vincent, R.; Vu, D.; Hatch, G.; Poon, R.; Dreher, K.; Guenette, J.; Bjarnason, S.; Potvin, M.; Norwood, J.; McMullen, E. 

(1996). Sensitivity of lungs of aging Fischer 344 rats to ozone: assessment by bronchoalveolar lavage. Am J 

Physiol, 271: L555-L565. 080778

Vivier, E.; Raulet, D. H.; Moretta, A.; Caligiuri, M. A.; Zitvogel, L.; Lanier, L. L.; Yokoyama, W. M.; 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 676697

Voynow, J. A.; Fischer, B. M.; Zheng, S.; Potts, E. N.; Grover, A. R.; Jaiswal, A. K.; Ghio, A. J.; Foster, W. M. (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. 194311

Wagner, J. G.; Harkema, J. R.; Jiang, Q.; Illek, B.; Ames, B. N.; Peden, D. B. (2009). Gamma-tocopherol attenuates ozoneinduced exacerbation of allergic rhinosinusitis in rats. Toxicol Pathol, 37: 481-491. 

http://dx.doi.org/10.1177/0192623309335630 201574

Wagner, J. G.; Hotchkiss, J. A.; Harkema, J. R. (2002). Enhancement of nasal inflammatory and epithelial responses after 

ozone and allergen coexposure in brown Norway rats. Toxicol Sci, 67: 284-294. 

http://toxsci.oxfordjournals.org/cgi/reprint/67/2/284.pdf. 026079
March 2011 5-78 DRAFT – DO NOT CITE OR QUOTE 

Wagner, J. G.; Jiang, Q.; Harkema, J. R.; Illek, B.; Patel, D. D.; Ames, B. N.; Peden, D. B. (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 596420

Watkinson, W. P.; Campen, M. J.; Nolan, J. P.; Costa, D. L. (2001). Cardiovascular and systemic responses to inhaled 

pollutants in rodents: effects of ozone and particulate matter. Environ Health Perspect, 109: 539-546. 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1240578/. 016245

Watkinson, W. P.; Campen, M. J.; Wichers, L. B.; Nolan, J. P.; Costa, D. L. (2003). Cardiac and thermoregulatory responses 

to inhaled pollutants in healthy and compromised rodents: modulation via interaction with environmental factors. 

Environ Res, 92: 35-47. 050547

Weibel, E. R. (1980). Design and structure of the human lung. In Fishman, A. P (Ed.), Assessment of pulmonary function 

New York, NY: McGraw-Hill. 079848

Weinmann, G. G.; Liu, M. C.; Proud, D.; Weidenbach-Gerbase, M.; Hubbard, W.; Frank, R. (1995). Ozone exposure in 

humans: Inflammatory, small and peripheral airway responses. Am J Respir Crit Care Med, 152: 1175-1182. 

http://ajrccm.atsjournals.org/cgi/content/abstract/152/4/1175. 077206

Weinmann, G. G.; Weidenbach-Gerbase, M.; Foster, W. M.; Zacur, H.; Frank, R. (1995). Evidence for ozone-induced smallairway dysfunction: lack of menstrual-cycle and gender effects. Am J Respir Crit Care Med, 152: 988-996. 038645

Welch, R. W.; Wang, Y.; Crossman A Jr; Park, J. B.; Kirk, K. L.; 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 644675

Wenten, M.; Gauderman, W. J.; Berhane, K.; Lin, P. C.; Peters, J.; Gilliland, F. D. (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 

597084

Wiester, M. J.; Stevens, M. A.; Menache, M. G.; McKee, J. L. Jr; Gerrity, T. R. (1996). Ozone uptake in healthy adult males 

during quiet breathing. Toxicol Sci, 29: 102-109. 041280

Wiester, M. J.; Tepper, J. S.; Winsett, D. W.; Crissman, K. M.; Richards, J. H.; Costa, D. L. (1996). Adaptation to ozone in 

rats and its association with ascorbic acid in the lung. Toxicol Sci, 31: 56-64. 080829

Williams, A. S.; Nath, P.; Leung, S. Y.; Khorasani, N.; McKenzie, A. N. J.; Adcock, I. M.; Chung, K. F. (2008). 

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 597546

Williams, A. S.; Eynott, P. R.; Leung, S. Y.; Nath, P.; Jupp, R.; De Sanctis, G. T.; Resnick, R.; Adcock, I. M.; Chung, K. F.

(2009). 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 628605

Williams, A. S.; Issa, R.; Durham, A.; Leung, S. Y.; Kapoun, A.; Medicherla, S.; Higgins, L. S.; Adcock, I. M.; Chung, K. 

F. (2008). 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 628607

Williams, A. S.; Issa, R.; Leung, S. Y.; Nath, P.; Ferguson, G. D.; Bennett, B. L.; Adcock, I. M.; Chung, K. F. (2007). 

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 628609

Williams, A. S.; Leung, S. Y.; Nath, P.; Khorasani, N. M.; Bhavsar, P.; Issa, R.; Mitchell, J. A.; Adcock, I. M.; Chung, K. F. 

(2007). 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 597545

Wu, Z. X.; Barker, J. S.; Batchelor, T. P.; Dey, R. D. (2008). 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 597548

Wu, Z. X.; Satterfield, B. E.; Dey, R. D. (2003). Substance P released from intrinsic airway neurons contributes to ozoneenhanced airway hyperresponsiveness in ferret trachea. J Appl Physiol, 95: 742-750. 

http://jap.physiology.org.jproxy.lib.ecu.edu/cgi/content/abstract/95/2/742. 628616
March 2011 5-79 DRAFT – DO NOT CITE OR QUOTE 

Yang, I. A.; Holz, O.; Jorres, R. A.; Magnussen, H.; Barton, S. J.; Rodriguez, S.; Cakebread, J. A.; Holloway, J. W.; 

Holgate, S. T. (2005). Association of tumor necrosis factor-"alpha" polymorphisms and ozone-induced change in 

lung function. Am J Respir Crit Care Med, 171: 171-176. 077211

Yokoyama, E.; Frank, R. (1972). Respiratory uptake of ozone in dogs. Arch Environ Occup Health, 25: 132-138. 039756

Yoon, H. K.; Cho, H. Y.; Kleeberger, S. R. (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 596422

Yost, B. L.; Gleich, G. J.; Jacoby, D. B.; Fryer, A. D. (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 597549