March 20 11 DRAFT – DO NOT CITE OR QUOTE 5-1 

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

### 1 chemical and/or its reaction products are absorbed and retained at target sites. The measure ment of 

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

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

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

### 5 O 3 might range from the concentration of gas in the air (units of ppm or mg/m 3 ), to the quantity of 

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

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

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

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

### 10 O zone is a highly reactive though poorly water soluble gas. The latter feature is believed to be 

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

### 12 chemically r eactive has suggested to some that its effective dose at the target sites exists in the form 

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

### 14 when O 3 interacts with components of the extracellular lining fluid ( ELF) such as lipids and 

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

### 16 exists in the deep lung where the ELF thickness narrows allowing O 3 to react directly with cells 

### 17 protruding from the ELF and surfa ce macrophages. Ozone uptake relates directly to these ELF 

### 18 substrate reactions and is termed “ reactive absorption. ” Thus the uptake of O 3 is related to both the 

### 19 concentration of O 3 as well as the availability of substrates within the ELF. 

### 20 Two types of meas urement have been used to arrive at the O 3 dose to target sites during 

### 21 breathing: ( 1) measurement of removal of O 3 from the air stream (termed “ uptake ”); and ( 2) 

### 22 measurement of chemical reactions or the product of those reactions with tissues or with 

### 23 biomo lecules known to be present in tissues (termed “ reactions ” ). The results of the above 

### 24

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 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 2 measurements have been incorporated into mathematical models for the purpose of explaining, 

### 1 predicting, and extrapolating O 3 dose in different exposure scenarios of inter est. 

### 2 This chapter is intended as an update of the past O 3 AQCD s (U.S. EPA, 1996, 017831 ; 

### 3 U.S. EPA, 2006, 088089 ) and restates the basic concepts derived from O 3 dosimetry literature 

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

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

### 6 O 3 and factors that affect the ability to extrapolate between species (e.g. , experimental animal to 

### 7 human). As there have been few O 3 dosimetry studies since the last AQCD, the reader is referred to 

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

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

### 10 as well as studies published since 2005, in the areas of reactions, uptake, and models for O 3 

### 11 dosimetry , is discussed in the following sections

. 

### 12 

## 5.1.2. Ozone Reactions and Reaction Products

5.1.2.1. Summary o f Findings fro m 2006 Ozone AQCD
Ozone dose can be examined by the chemical reactions or the products of these reactions that 

### 13 result from O 3 exposure. Since O 3 is chemically reactive with a wide spectrum of biomolecules, it is 

### 14 not feasible to delineate it s many reaction products. Measurements of reaction formation have 

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

### 16 of O 3 - derived oxygen to biomolecules through the use of oxygen

- 18 labeling. In vitro exp osure of 

### 17 ELF shows that O 3 disappearance from the gas phase depends on the characteristics of the ELF 

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

. 

### 19 To gain access to the underlying cellular compartments, O 3 must dissolve at the air -liquid 

### 20 interface of the airway surface and travel through the ELF layer. The ELF is comprised of the airw ay 

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

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

### 23 There is a progressive decrease in ELF thickness an d increase in interfacial surface with progression 

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

### 25 conducting airways (Figure 5

- 1). Some cells, such as macrophages, may protrude into the gas phase, 

### 26 allowing for direct contact between O 3 and cell membranes. The progressive thinning in the ELF 

### 27 decreases the distance O 3 must travel to reach the cellular tissue layer. A computational fluid 

### 28 dynamics (CFD) model was able to predict experimentally measured O 3 uptake, bu t only with nasal 

### 29 mucus layer thickness considered (Cohen

- Hubal et al., 1996, 043785 ) , reaffirming the importance of 

### 30
t he resistance imparted by the ELF layer in dose and lesion patterns in the nasal passage. 

### 31 Taking into account the high reactivity and low water solubility of O 3 , calculations suggest 

### 32 that O 3 will not penetrate ELF layers greater than 0.1 μm without being transformed to other more 

### 33 long

- lived reactive species, thus initiating a reaction cascade (Pryor, 1992, 042725 ) . It follows that 

### 34March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 3 the ELF should be considered an importa nt target for O 3 toxicity in the airways. Experimental 

### 1 support for this concept comes from several studies which measure the total oxygen

- addition product 

### 2 of O 3 reactions in the airways through the use of oxygen

- 18 labeled O 3 . High concentrations of O 3 

### 3

rea ction products are found in the nasal lavage cells, bronchoalveolar lavage (BAL) , mucus, 

### 4 surfactant, and cells, and in the epithelial cells of the lower airways, providing evidence that O 3 

### 5 reacts at the air

- liquid interface. Model calculations of the nasal cavity based on diffusion equations 

### 6 and reaction rates of O 3 with model substrates predict an O 3 penetration distance (0.5 μm) less than 

### 7 the thickness of the mucus layer (10 μm) (Santiago et al., 2001, 019841 ) . Thus, O 3 may cause injury 

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

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

### 10

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 b lood 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 t he blood.March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 4 Ozone may interact with many of the components that make up the ELF including 

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

### 2 (Perez

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

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

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

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

### 6 reduction reactions. 

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

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

### 9 chemical reactions such as the Criegee ozonolysis mechanism (Figure 5

- 2) (Pryor et al., 1991, 

### 10 042482 ) . Lipid ozonation products, such as the aldehydes hexanal, heptanal, and nonanal, have been 

### 11 recovered after O 3 exposure in human bronchial alveolar lavage fluid (BALF), rat BALF, isolated rat 

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

### 13 al., 1996, 082697 )

. It was estimated that 88% of the O 3 that does not com e in contact with 

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

### 15 ELF (Uppu et al., 1995, 0 76212 ) . Nonanal has been suggested as a relatively specific biomarker for 

### 16 O 3 exposure since the monounsaturated fatty acid parent compound, oleic acid, does not undergo 

### 17 autoxidation (Pryor et al., 1996, 082697 ) . A dducts of the aldehyde 4

- hydroxynonenal were found in 

### 18 human alveolar macrophages after O 3 exposure (Hamilton et al., 1998, 086157 )

. Polyunsaturated 

### 19 fatty acid (PUFA) reactions are limited by the availability of O 3 since lipids are so abundant in the 

### 20 ELF. Yields of O 3 - induced aldehydes were increased by the decrease in other substrates such as 

### 21 ascorbic acid (AH 2 ) (Postlethwait et al., 1998, 086754 ) . PUFA reactions may not generate sufficient 

### 22 bioactive materials to account for acute cell injury, however only mo dest amounts of products may 

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

### 24 1998, 086754 )

. 

### 25March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 5 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. Cholesterol is the most abundant neutral lipid in human ELF. Reaction of cholesterol with O 3 

### 1 results in biologically active cholesterol products such as the oxysterols, β

- epoxide and 6 -oxo- 3,5

- 

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

### 3 076663 ) . Product yields will depend on ozonolysis conditions, however cholesterol oz onolysis 

### 4 products were formed in similar abundance to phospholipid

- derived ozonolysis products in rat ELF 

### 5 (Pulfer and Murphy, 2004, 076673 )

. 

### 6 Antioxidant substances appe ar to be an important cellular defense against O 3 . The level and 

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

### 8 altered by O 3 exposure. Endogenous antioxidants such as urate (UA), ascorbate (AH 2 ), an d reduced 

### 9 glutathione (GSH) display high intrinsic reactivities toward O 3 , but do not possess equal O 3 

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

### 11 tested, followed by AH 2 (Mudway and Kelly, 1998, 000273 ) . In human BALF samples, the mean 

### 12 consumption of AH 2 was greater than UA (Mudway et al., 1996, 080730 ) . GSH was consistently less 

### 13 reactive than UA or AH 2 (Kanofsky and Sima, 1995, 075973 ; Mudway and Kelly, 1998, 000273 ; 

### 14 Mudway et al., 1996, 080730 ) . In a red cell based system, AH 2 augmented the in vitro uptake o f O 3 

### 15 by six fold as computed by the mass balance across the exposure chamber (Ballinger et al., 2005, 

### 16 076649 ) . In addition, O 3 - induced cell membrane oxidation required interactions with AH 2 and GSH, 

### 17 but not UA or the vitamin E analog Trolox, however UA could block reactions of O 3 with AH 2 

### 18 (Ballinger et al., 2005, 076649 ) . The consumption of antioxidants by O 3 was linear with time and 

### 19 positi vely correlated with initial substrate concentration and chamber O 3 concentration (Mudway and 

### 20 Kelly, 1998, 000273 ; Mudway et al., 1996, 080730 ) . However, estimated in vitro O 3 uptake was not 

### 21 proportional to the production of O 3 - derived aldehydes from red cell membrane exposure (Ballinger 

### 22 et al., 2005, 076649 ) . Studies with rats exposed to O 3 show consumption of ascorbate that correlates 

### 23March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 6 with O 3 exposure (Gunnison and Hatch, 1 999, 087204 ; Gunnison et al., 1996, 080803 ; Vincent et al., 

### 1 1996, 080778 ). 

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

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

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

### 5 methionine, tyrosine, and tryptophan). Ozone reaction with S

- containing biomolecules has been 

### 6 shown to fol low the following order: thiosulfate > ascorbate > cysteine ≈ methionine > glutathione 

### 7 (Kanofsky and Sima, 1995, 075973 ) . Rate constants for the reaction of amino acids with O 3 vary 

### 8 between investigations due to differing reac tion conditions and assumptions; however aliphatic 

### 9 amino acids consistently are very slow to react with O 3 (e.g. , alanine: 25

- 100 m oles/L/sec ) (Hoigné 

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

### 11 075973 ; Pryor et al., 1984, 595130 ) . Uppu et al. (1995, 076212 ) predicted that 12% of inhaled O 3 

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

### 13 PUFAs. 

### 14 ELF exists as a complex mixture, thus it is important to look a t O 3 reactivity in substrate 

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

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

. 

### 17 However, O 3 reactions with AH 2 predominated over the reaction with lipids, when exposed to 

### 18 substrate solution mixtures (Postlethwait et al., 1998, 086754 ) . It was suggested that O 3 may react 

### 19 with other substrates once AH 2 concentrations within the reaction plane fall sufficiently. 

### 20 Additionally, once AH 2 was consumed, the absorption efficiency diminished, allowing inhaled O 3 to 

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

### 22 concluded O 3 is more reac tive with AH 2 and UA than with the weakly reacting GSH (or cysteine or 

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

### 24 and Sima, 1995, 075973 ). 

### 25 

## 5.1.2.2. Recent Publications Further experiments on the reaction kinetics between O 3 and the antioxidants pres ent in the 

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

### 27 195643 ) evaluated the interfacial exposure of aqueous solutions of UA, AH 2 , and GSH (50

- 200 μM) 

### 28 with O 3 (1 -5 ppm). Similar to the results of Mudway and Kelly (1998, 000273 ) , this stu dy found the 

### 29 hierarchy in reactivity between O 3 and these antioxidants to be UA>AH 2 >>GSH. UA and AH 2 

### 30 shared a 1:1 stoichiometry with O 3 , whereas 2.5 moles of GSH were consumed per mole of O 3 . 

### 31 Using these stoichiometries, reaction rate constants were derive d (5.8 × 10 4 m oles/L/sec , 

### 32 5.5× 10 4 m oles/L/sec , and 57.5/ M 0.75 /s ec for the reaction of O 3 with UA, AH 2 , and GSH, 

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

### 34 (1998, 000273 ) . Other studies reported reactive rate constants that are two to three orders of 

### 35 magnitude larger, however these studies used higher concentrations of O 3 and antioxidants under less 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 7 physiologically relevant experimental condi tions (Giamalva et al., 1985, 595129 ; Kanofsky and 

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

. 

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

### 3 initial air

- liquid interface interactions of O 3 with ELF component s (e.g. , antioxidants and proteins) in 

### 4 ~1 millisecond (Enami et al., 2008, 195834 ; Enami et al., 2008, 195833 ; Enami et al., 2009, 197791 ; 

### 5 Enami et al., 2009, 195835 ; Enami et al., 2009, 195621 )

. Solutions of aqueous UA, AH 2 , GSH, 

### 6

α - tocopherol ( α - TOH), and protein cysteines (CyS) were sprayed as microdroplets in O 3 (g)/N 2 

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

### 8 studies demonstrated different reactivity toward AH 2 , UA, and GSH by O 3 in the gas phase 

### 9 compared to the liquid phase thus supporting the relevance of reactions between gas phase O 3 and 

### 10 ELF. 

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

### 12 195643 ) , the hierarchy of reactivity of these ELF components with O 3 ( g) was determined to be AH 2 

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

### 14 UA, AH 2 , and GSH with O 3 (g) as investigated by Enami et al. versus O 3 (aq) as described previously. 

### 15 UA was more reactive than AH 2 towar d O 3 (aq), but in reactions with O 3 (g), these antioxidants have 

### 16 equivalent reactivity (Enami et al., 2008, 195834 ) . As O 3 is a kinetically slow one

- electron acceptor 

### 17 but very reactive O

- atom donor, products of the interaction of O 3 with UA, AH 2 , GSH, CyS, and α 

### 18 TOH result from addition of n O- atoms ( n = 1

- 4). These products included epoxides (e.g. , U-O - ), 

### 19 peroxides (e.g. U -O 2 - ), and ozonides (e.g. , U-O 3 - ). For instance, GSH was oxidized to sulfonates 

### 20 (GS O 3 - /GS O 3 2- ) , not glutathione disulfide (GSSG) by O 3 (g) (Enami et a l., 2009, 197791 ) . However, 

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

### 22 in O 3 exposed ELF where as GSSG is. This is also supported by the fact that O 3 is much less reactive 

### 23 with GSH than other antioxidants, such that < 3% of O 3 will be scavenged by GSH when in 

### 24 equimolar amounts with AH 2 (Enami et al., 2009, 197791 )

. 

### 25 Ozonolysis pro duct yields and formation were affected by pH. Acidified conditions (pH ≈ 

### 26 3- 4), such as those that may result from acidic particulate exposure or pathological conditions like 

### 27 asthma ( pH ≈ 6), decreased the scavenging ability of UA and GSH for O 3 ; such that at low pH, the 

### 28 scavenging of O 3 must be taken over by other antioxidants, such as AH 2 (Enami et al., 2008, 195834 ; 

### 29 Enami et al., 2009, 197791 ) . Also, under acidic conditions (pH ≈ 5), the ozonolysis products of AH 2 

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

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

### 32 acidic co

- pollutant exposure will increase the toxicity of O 3 by preventing some antioxidant 

### 33 reactions and shifting the reaction products to more persistent compounds. 

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

### 35 barrier and target for inhaled O 3 . The thickness of the lining fluid and mucus layer is an important 

### 36 determinant of the dose of O 3 to the tissues. The antioxidant substances present in the ELF appear in 

### 37 most cases to limit interaction of O 3 with underlying tissues and to prevent penetration of O 3 deeper 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 8 into the lung . However, new findings indicate that in some cases, the antioxidants might themselves 

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

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

### 3 emphasize the importance of gaseous O 3 acting at the air

- liquid interface in initiating a reaction 

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

### 5 gaseous and aqueous O 3 reactivity toward airway antioxidants. In addition to the highly reactive O 3 , 

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

### 7 injury (Section 5.2). 

### 8 

## 5.1.3. Ozone Uptake

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

Past A QCD s (U.S. EPA, 1996, 017831 ; U.S. EPA, 2006, 088089 ) provide information on the 

### 9 majority of literature relevant to understanding the state of the science in O 3 dosimetry. One method 

### 10 of addressing the question of O 3 dosimetry is to measure the amount of O 3 removed from the air 

### 11 stream during breathing (termed “ uptake ” ). The percentage of the O 3 in the air stream that is 

### 12 removed is termed “ uptake efficiency. ” Uptake studies have utilized both bolus and continuous O 3 

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

### 14 of O 3 uptake between upper and lower respiratory tract regions. A number of studies that have 

### 15 measured the fractional O 3 uptake efficiency of the human respira tory tract (F RT ), upper airways 

### 16 (F UA ), and lower respiratory tract (F LRT ) are presented in Table 5-1 as a review. 

### 17

Table 5
- 1. Human respiratory tract uptake efficiency data

Reference Mouth/Nose a Inspiratory Flow (mL/s) V T (mL) f B (bpm) b F RT F UA F LRT CONTI NUOUS 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 0.80 d 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 0.46March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 9 

### Target Sites for Ozone Dose A primary uptake site of O 3 delivery to the lungs is believed to be the centriacinar region 

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

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

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

### 4 were confirmed as the site receiving the greatest O 3 dose ( 18 O mass/lung weight) in resting O 3 

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

. 

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

### 7 was dependent on the delivered O 3 dose to these tissues. However, 18 O label was detected to a lesser 

### 8 extent in other regions of the tracheobronchial air way tree, showing that O 3 is delivered to these 

### 9 compartments as well, resulting in a smaller dose. Models predict that the net O 3 dose ( O 3 flux to air



### 10 liquid interface) gradually decreases distally from the trachea toward the end of the tracheobronchial 

### 11

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

. 

### 12 However, the tissue dose ( O 3 flux to liquid

- tissue interface) is low in the trachea, increases to a 

### 13 maximum in the terminal bronchioles and the CAR, and then rapidly decreases dis tally into the 

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

### 15 reactions occurring between ELF constituents and O 3 after the 16th generation, representing the 

### 16 CAR region (Miller et al., 1985, 040307 )

. 

### 17 

### Nasopharyngeal Removal and Dose of O zone In both animals and humans, about 50% of the absorbed O 3 is removed in the head (nose, 

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

### 19 041799 ; Hu et al., 1992, 042794 ) . The nasopharyngeal region provides a defense against O 3 entering 

### 20 the lungs by removing half of the inhaled O 3 from the airstream. The limiting factors in nasal O 3 

### 21 uptake are simultaneous diffusion and chemical reaction of O 3 in the nasal ELF layer (Santiago et al., 

### 22 2001, 019841 ) . The lining layer in the nose is thicker than in the lungs, but, like for the lungs, 

### 23 Ultman et al. (1994, 041746 ) Mouthpiece 250 500 e 15 0.30 Mouthpiece 250 500 15 0.47 Ultman et al. (20 04, 057197 ) M 490 450 e 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; m outhpiece = exposure by mouthpiece; F RT = total RT uptake; F UA = upper airway uptake; F LRT = lower RT uptake

. b f B is either measured or is computed from flows and V T . 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 F UA from Santiago et al. (20 01, 019841 ) represents nasal absorption (F nose ). e V T is computed from flow and f B .March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 10 mathema tical estimates predict that O 3 penetrates less than the thickness of the ELF layer leaving the 

### 1 reaction products as likely the agents damaging the nasal tissue. The percentage of O 3 taken up is 

### 2 inversely related to flow rate and weakly related to inlet O 3 concentration. It was hypothesized that 

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

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

### 5 Uptake efficiencies have been calculated for various segme nts of the upper airways 

### 6 (Table 5- 1). Gerrity et al. (1995, 042785 ) reported unidirectional uptake efficiencies of O 3 inhaled 

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

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

### 9 35.5%), and essentially zero between the carina and the bronchus intermedius (totaling 32.5%). 

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

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

### 12 main bifurcation carina, and the bronchus intermedius, respectively. The lower effici encies seen in 

### 13 Gerrity et al. (1995, 042785 ) may have resulted from the mouthpiece scrubbing O 3 from the breath 

### 14 during inhalation. Overall, the nasopharyngeal regio n removes half of the inhaled O 3 by reactions in 

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

### 16 concentration. 

### 17 

### Pulmonary Ozone Uptake and Dose
O 3 uptake in rats is approximately 54% efficient (Hatch et al., 1989, 041799 ) , while in h umans 

### 18 at rest it ranges from 80

- 95% efficient (Hu et al., 1992, 042794 ). Approximately 43% of inhaled O 3 

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

### 20 number of variables, including tidal volume (V T ), breathing frequency (f B ), exposure time, minute 

### 21 volume, and O 3 concentration. Simulations from the Overton et al. (1996, 080733 ) single-path 

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

### 23 uptake by the upper airways was not considered, pred icted that fractional uptake and PAR O 3 dose 

### 24 increased with V T . Likewise, experimental studies found that O 3 uptake is positively correlated with 

### 25 changes in V T (Gerrity et al., 1988, 040899 ; Ultman et al., 2004, 057197 ) . Also, O 3 exposure leads to 

### 26 a reflex mediated increase in f B and reduction in V T , hypothesized to be protective by decreasing the 

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

### 28 volume, a decrease in V T will resu lt in an increase in f B . Nasal flow rate (Santiago et al., 2001, 

### 29 019841 ) and f B are inversely related to O 3 uptake, such tha t an increase in f B will decrease uptake 

### 30 efficiency (Figure 5

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

### 31 1996, 041280 ) . Modeling also predicted a decrease in fractional uptake with increased f B , but an 

### 32 increase in PAR dose with in creased f B (Overton et al., 1996, 080733 ) . Similarly, increased f B (80 - 

### 33 160 bpm) and rapid shallow breathing in ra ts resulted in a decrease in midlevel tracheal 18 O content 

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

### 35 dependence may be a result of frequency

- induced alterations in contact time that affects the first

- 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 11 order absorption rate for O 3 (Postlethwait et al., 1994, 044219 ). Also, an association with O 3 uptake 

### 1 efficiency was found with minute volume and exposure time. 

### 2

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

### 3 O 3 is absorbed in the upper airways and shifts uptake to the central and lower airways and 

### 4 respiratory airspaces (Hu et al., 1994, 041323 ; Nodelman and Ultman, 19 99, 015112 ; Ultman et al., 

### 5 1994, 041746 ) . Hu et al. (1994, 041323 ; Ultman et al., 1994, 041746 ) found that O 3 absorption 

### 6 increases relative to the respiratory tract penetration volume (Vp) of a bolus O 3 dose (Figure 5

- 4)

. 

### 7 Ozone uptake efficiency and Vp are not affected by bolus O 3 concentration (Gerrity et al., 1988, 

### 8 040899 ; Hu et al., 1992, 042794 ; Kabel et al., 1994, 095597 ) , indicating that O 3 uptake is a linear 

### 9 absorption process, where the diffusion and chemical reaction rates of O 3 are proportional to the O 3 

### 10 concentration. This relationship was not true for nasal cavity uptake, which was proportional to O 3 

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

### 12 uptake efficiency had a weak but significant negative dependence on O 3 concentration (Rigas et al., 

### 13 2000, 010454 ) . Overall O 3 inhalation uptake is over 80% efficient, but the exact efficiency that 

### 14 determines how much O 3 is available at longitudinally distributed compartments in the lung is 

### 15

s ensitive to changes in V T , f B , and exposure time. Increased f B will shift the O 3 uptake from the upper 

### 16 airways to the central airways and respiratory airspaces. 

### 17March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 12 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 (V p 

### ) in a representative subject. ﷒ Each point represents the ozone uptake of a bolus inspired through a mouthpiece by the subject. The volumes, V UA 

### and V D 

### , are the volume of the upper airways and anatomical dead space, respectively, and V p 

### 50% is the V p 

### at which 50% of the inspired bolus was absorbed. In 47 healthy subjects, Ultman et al. (2004, 057197 ) found that V p 

### 50% was well correlated wit h V D 

### and better correlated with the volume of the conducting airways, i.e., V D 

### minus V UA 

### . 

### Mode of Breathing
Ozone uptake and distribution is highly sensitive to the mode of breathing. Variability in 

### 1 conducting airways volume had a weaker influence on O 3 ab sorption during nasal breathing 

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

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

### 4 that O 3 uptake fraction is greater during nasal breathing than during oral breathing at each Vp (Kabel 

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

### 6 oral breathing results in deeper penetration of O 3 into the lung wi th a higher absorbed fraction in the 

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

### 8 results were obtained from O 3 uptake studies in dogs (Yokoya ma and Frank, 1972, 039756 ); 

### 9 however earlier human studies suggest that oral or oronasal breathing results in a higher O 3 uptake 

### 10 efficiency than nasal breath ing (Gerrity et al., 1988, 040899 ; Wiester et al., 1996, 041280 ). These 

### 11 human studies measured total respiratory tract absorption after continuous O 3 exposure using a 

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

### 13 

### Interindividual Variability Similarly exposed individuals vary in the amount of actual dose received with intersubject 

### 14 differences in fractional O 3 uptake by the respiratory tract accounting for the majority of total 

### 15March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 13 variation in O 3 uptake efficiency (Bush et al., 1996, 080763 ; Rigas et a l., 2000, 010454 ; Santiago et 

### 1 al., 2001, 019841 ) . Interindividual variability accounted for between 10

- 50% of the absolute 

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

. 

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

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

### 5 in O 3 uptake is the result of substituting a dose surrogate, such as exposure concentration or inhaled 

### 6 dose, for the actual O 3 dose delivered to the tissues. 

### 7 Va riability in local dose may be attributed to differences in the pulmonary physiology. Since 

### 8 the conducting airways (CA) remove the majority of inhaled O 3 before it reaches the gas exchange 

### 9 region, the volume and surface area of the upper airways will influence O 3 uptake. Models predict 

### 10 that fractional O 3 uptake and PAR dose (flux of O 3 to the PAR surfaces divided by exposure 

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

### 12 contrary, alveolar expansion had minimal effect on uptake efficiency as little O 3 reaches the 

### 13 peripheral lung (Bush et al., 2001, 016665 ; Overton et al., 1996, 080733 ) . Ozone uptake is vi rtually 

### 14 complete by the time O 3 rea ches the alveolar spaces of the lung (Postlethwait et al., 1994, 044219 ). 

### 15 Experimental studies have found that differences in CA volumes may account for 7 5% of the 

### 16 variation in absorp tion between subjects (Ultman et al., 2004, 057197 ) . In support of this concept, 

### 17 regression analysis show ed that O 3 absorption is positively correlated with anatomical dead space 

### 18 (V D ) and CA volume (i.e., V D minus V UA ), but not total lung capacity (TLC), forced vital capacity 

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

### 20 Postlethwait et al., 1994, 044219 ; Ultman et a l., 2004, 057197 )

. Variability in V D is correlated more 

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

### 22 in individual bronchial cross

- sectional area, indicating that changes in cross

- sectional area available 

### 23 for gas diffusion are related to overall O 3 retention (Reeser et al., 2005, 195718 ; Ultman et al., 2004, 

### 24 057197 ) . These studies provide support to the pulmonary physiology, especially the CA volume and 

### 25 surface area, playing a key role in variability of O 3 uptake between individuals. 

### 26 When absorption data between genders is normalized to Vp/V D , then the variability attributed 

### 27 to gen der differences is no longer distinguishable (Bush et al., 1996, 080763 ) . A physiologically 

### 28 based pharmacokinetic (PBPK) model simu lating O 3 uptake indicates that regional extraction of O 3 

### 29 is relatively insensitive to age, but extraction per unit surface area is two - to eight fold higher in 

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

### 31

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

### 32 054581 )

. 

### 33 

### Correlation of Dose and Response Two studies have investigated the correlation of O 3 uptake with the pulmonary function 

### 34 responses to O 3 exposure (Gerrity et al., 1994, 041314 ; Reeser et al., 2005, 195718 ) . These studies 

### 35 found that the large subj ect

- to

- subject variability in %ΔFEV 1 response to O 3 does not appear to have 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

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

### 1 %ΔFEV 1 and fractional absorption of O 3 using the bolus method. Contrary to previous findings, the 

### 2 percent change in dead space volume of the respiratory tract (%ΔV D ) did not correlate with O 3 

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

### 4 041314 ) found that intersubject variability in FEV 1 and airway resistance was not related to 

### 5 differences in the O 3 dose delivered to the lower airways, whereas minute ventilation was predictive 

### 6 of FEV 1 decrement. No study has yet demonstrated that subjects show a consistent pattern of O 3 

### 7 retention when re - exposed over weeks of time, as has been shown to be the case for the FEV 1 

### 8 response, or that within

- subject variation in FEV 1 response is related to fluctuations in O 3 uptake. 

### 9 On the contrary, cellular injury and inflammation have been found to correlat e with the site



### 10 specific O 3 dose. Contained within the CAR, the respiratory bronchioles were confirmed as the site 

### 11 receiving the greatest O 3 dose ( 18 O mass/lung weight) and sustained the greatest cellular injury in O 3 

### 12 (0.4 and 1.0 ppm) exposed resting rhes us monkeys (Plopper et al., 1998, 087203 ) . The respiratory 

### 13 bronchioles, having the highest concentration of local O 3 dose, were also the site of significant GSH 

### 14 reduction. 

### 15 

### Co
- Pollutant and Sequential Ozone Exposure
Previous continuous O 3 exposure (0.12 or 0.36 ppm) decreased bolus O 3 uptake, possibly due 

### 16 to depletion of compounds able to react with O 3 (Asplund et al., 1996, 082505 ; Rigas et al., 1997, 

### 17 083602 ) . Conversely, O 3 (0.36 ppm) bolus uptake was increased with prior NO 2 (0.36 or 0.72 ppm) 

### 18 or SO 2 (0.36 ppm) exposure (Rigas et al., 1997, 083602 ) . It was hypothesized that this increased 

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

### 20 oxidant-i nduced airway inflammation. 

### 21 

### Physical Activity Exercise increases the overall exposure of the lung due in most part to the increased volume of 

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

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

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

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

### 26 higher dose of O 3 in the lower airway . In addition to increasing the quantity of O 3 in the lung, 

### 27 exercis e also has been shown to lead to a switch to oronasal breathing. By increasing flow to what is 

### 28 common in moderate exercise, the upper airways absorbed a smaller fraction of the O 3 (~0.50 at 

### 29 quiet breathing to 0.10 at exercise); however, the trachea and mor e distal conducting airways 

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

### 31 and 0.25 absorbed in the respiratory zone) (Hu et al., 1994, 041323 ). The same shift in the O 3 dose 

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

### 33 (Nodelman and Ultman, 1999, 015112 )

. 

### 34March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 15 

## 5.1.3.2. Recent Publications Few new studies have investigated the uptake of O 3 in the respiratory tract since the end of the 

### 1 last O 3 assessment (U.S. EPA, 2006, 088089 ) . The studies that have been conducted agree with the 

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

### 3 Past studies have shown that O 3 - induced epithelial damage to the lung occurs with a 

### 4 reproducible pattern of severity between daughter branches of individual bifurcations that is 

### 5 dependent on the O 3 concentration

- time prof ile of the inhaled gas. A 3

- dimensional computational 

### 6 fluid dynamics model was created to investigate the dose

- response relationship leading to the 

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

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

### 9 and a sharp carinal ridge. Various flow scenarios w ere simulated using Reynolds numbers (Re) 

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

### 11 both lung size and minute ventilation, such that the range in Re from 100

- 500 would encompass 

### 12 generations 1

- 5, 3 -7, and 6

- 10 for an adult during quiet breathing, light exertion, and heavy exercise, 

### 13 respectively, whereas the same Re range corresponds to generations 0

- 4, 1

- 6, and 4

- 8 for a 4

- year 

### 14 old child. Consistent with early physical models of Schroter and Sudlow (1969, 071359 ) , the model 

### 15 predicted that during inspiration, the velocity and O 3 concentration distribution were axisymmetric 

### 16 throughout the parent branch, but skewed towards the inner wall within the daughter bra nches. 

### 17 During expiration, the model predicted that the velocity and O 3 concentration distribution was 

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

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

### 20 expiration on the parent branch wall downstream of the branching region. 

### 21 Past studies investigating nasal uptake of O 3 have shown that the nose partially protects the 

### 22 rest of the respiratory tract from damage fr om inspired O 3 (Gerrity et al., 1988, 040899 ; Santiago et 

### 23 al., 2001, 01984 1 ) . Sawyer et al. (2007, 195142 ) further investigated nasal uptake of O 3 in healthy 

### 24 adults during exercise. Fractional O 3 uptake, ac oustic rhinometry (AR), and nasal NO measurements 

### 25 were taken on ten adults (8 W, 2 M) exposed to 0.2 ppm O 3 before and after moderate exercise at 

### 26 two flow rates (10 and 20 L/min). The percent nasal uptake of O 3 was ~50% greater at 10 L/min 

### 27 compared to 20 L/min both pre - and postexercise. However, the inhaled O 3 delivery rate to the lung 

### 28 (i.e., flow rate X [ O 3 ppm] X nasal O 3 penetration) was 1.6

- fold greater at the higher flow than at the 

### 29 lower flow (2.5 compared to 0.9 ppm ·L/min). Exercise did not affect O 3 uptake at either flow rate, 

### 30 but did significantly increase nasal volume (Vn) and AR measurements of nasal cross

- sectional area 

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

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

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

### 34 outliers and gender segregation. 

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

### 36 affect the fractional uptake of a bolus dose of O 3 in apparently healthy smokers with limited smoking 

### 37March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 16 history (Bates et al., 2009, 195727 ) . Despite similar internal O 3 dose distribution, the smokers 

### 1 exhibited greater pulmonary responses to O 3 bolus exposures, measured as FEV 1 decrements and 

### 2 increases in the normalized slope of the alveolar p lateau (S N ). This is contrary to previous studies 

### 3 conducted in smokers with a greater smoking history that found decreased O 3 induced decrements in 

### 4 FEV 1 in smokers (Emmons and Foster, 1991, 042430 ; Frampton et al., 1997, 082692 )

. 

### 5 Recent studies have reiterated the importance of intersubject variation in O 3 uptake. The 

### 6 intersubject variability in nasal O 3 uptake determined by Sawyer et al. (2007, 195142 ) ranged from 

### 7 26.8 to 65.4% (pre - and postexercise). A second study investigating the use of the CO 2 expirogram to 

### 8 quantify pulmonary responses to O 3 found that intersubject variability accounted for 50% of the 

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

. 

### 10 In summary, O 3 uptake efficiency is sensitive to a number of factors. As discussed before, the 

### 11 characteristics of the ELF layer is a key determinant in the dose of O 3 that reaches the tissue layer. 

### 12 Fractional absorption will decrease with increased flow and increase proportional to V T . Decreased 

### 13 uptake efficiency due to increased f B and oronasal breathing, as occurs during exercise, will shift the 

### 14 O 3 dose distribution deeper and lead to a greater dose to the lower respira tory tract. Individual total 

### 15 airway O 3 uptake efficiency is also sensitive to large changes in O 3 concentration, exposure time, and 

### 16 V E . Major sources of variability in absorption of O 3 include O 3 concentration, exposure time, 

### 17 breathing frequency, minute vo lume, and tidal volume, but the interindividual variation is the 

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

### 19 that the large differences in biological response between subjects (FEV 1 , BAL cell inflamm atory 

### 20 response, etc.) are explainable by the differences in O 3 uptake. Recent studies have provided 

### 21 evidence for hot spots of O 3 flux around bifurcations in the airways. 

### 22 

## 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 Find ings fro m 2006 Ozone AQCD
The 2006 O 3 AQCD (U.S. EPA, 2006, 088089 ) discu ssed the suitability of animal models for 

### 23 comparison with human O 3 exposure and concluded that the acute and chronic functional responses 

### 24 of laboratory animals to O 3 appear qualitatively homologous to human responses. Thus, animal 

### 25 studies can provide impor tant data in determining cause

- effect relationships between exposure and 

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

### 27 comparing quantitative dose

- response relationships in animal models to humans due t o obvious 

### 28 interspecies differences. 

### 29 Physiological and anatomical differences exist between experimental species. Primates are 

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

### 31 breathers with a monopodial bran ching lung structure. In addition, rodents have fewer terminal 

### 32 bronchioles, the major site of O 3 uptake, compared to humans or canines (McBride, 1992, 078532 )

. 

### 33March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

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

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

### 2 structure between primates and rodents could affect the site and amount of O 3 upt ake. Also, because 

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

### 4 exposure leading to lowered O 3 dose and toxicity (Iwasaki et al., 1998, 086165 ; Slade et al., 1997, 

### 5 082708 ; Watkinson et al., 2003, 050547 ) . In addition to lowering the O 3 dose to the lungs, this 

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

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

### 8 rate which may lead to: (1) decreased cardiac outp ut (CO), (2) lowered blood pressure (BP), and (3) 

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

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

### 11 from animals to humans. 

### 12 Sensitivity to heath effects from O 3 varies between and within species, a s well as between 

### 13 endpoints. Rodents appear to have a slightly higher tachypneic response to O 3 and are less sensitive 

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

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

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

### 17 the most responsive to O 3 - induced inflammatory cell and protein influx. Rabbits were the least 

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

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

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

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

### 22 O 3 (0.1-1.0 ppm for 60 min utes ) exposure (Arsalane et al., 1995, 077430 )

. 

### 23 Humans and animal models are similar in the patt ern of regional O 3 dose, but absolute values 

### 24 differ. Hatch et al. (1994, 038953 ) reported that exercising humans exposed to oxygen - 18 labeled O 3 

### 25 (0.4 ppm) accumulated 4

- 5 times higher concentrations of O 3 reaction product in BAL cells, 

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

### 27

necessa ry to expose resting rats to 2 ppm O 3 to achieve the same BAL accumulation of 18 O reaction 

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

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

### 30 cellular effects of the O 3 exposure observed such that these responses to 2.0 ppm O 3 were similar to 

### 31 those of the 0.4 ppm O 3 in exercising humans. 

### 32 As O 3 absorption and activity relies on ELF antioxidant substances as described in Sectio n 5.2, 

### 33 variability in antioxidant concentrations and metabolism between species may affect dose and O 3 - 

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

### 35 GSH peroxidase, and lower vitamin E levels in the lung c ompared to rats (Ichinose et al., 1988, 

### 36 041805 ; Sagai et al., 1987, 041704 ). Nasal lavage fluid analysis shows that humans have a higher 

### 37 proportion of their nasal antioxidants as UA and low levels of AH 2 whereas mice, r ats, or guinea pigs 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 18 have high levels of AH 2 and undetectable levels of UA (Figure 5

- 5a). GSH is not detected in the 

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

### 2 have a higher antioxidant to protein rati o in nasal lavage and BAL fluid than humans (Hatch, 1992, 

### 3 043901 ) . The BALF profile differs from the nasal lavage (Figure 5

- 5b). Humans ha ve a higher 

### 4 proportion of GSH and less AH 2 making up their BALF content compared to the guinea pigs and rats 

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

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

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

. 

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

### 9 species and lung regions did not appear to be the primary factor in O 3 ind uced tissue injury. 

### 10 However, a close association between site

- specific O 3 dose, the degree of epithelial injury, and 

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

. 

### 12March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 19 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 permis sion 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 There have been few new publications ex amining interspecies differences in dosimetry and 

### 1 response to O 3 since the last AQCD. These studies do not overtly change the conclusions discussed 

### 2 above from the previous document (U.S. EPA, 2006, 088089 )

. 

### 3March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 20 A quantitative comparison of O 3 transport in the airways of rats, dogs, and humans was 

### 1 conducted using a three

- compartmen t airways model, based on upper and lower airway casts and 

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

### 3 designed as cylindrical tubes with constant volume and one

- dimensional gas movement and no 

### 4 airway branching patterns. It used data for solubility of O 3 as well as measured nasopharyngeal 

### 5 removal rates of O 3 published previously. This model exami ned how interspecies anatomical and 

### 6 physiological differences affect intra

- airway O 3 concentrations and the amount of gas absorbed (10% 

### 7 O 3 exposure). Peak, real

- time, and mean O 3 concentrations were higher in the upper and lower 

### 8 airways of humans compared to rats and dogs, but lowest in the alveoli of humans. The amount of O 3 

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

### 10 1.1 × 10 -7 in rats and 1.46 × 10 -7 in dogs). The intra

- airway concentration decreased distall y in all 

### 11 species. Sensitivity analysis demonstrated that V T , f B , and upper and lower airways surface area had 

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

### 13 reactions in the ELF or consider gas diffusion as a driving force for O 3 transport. Also, the model 

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

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

### 16 To further understand the genetic basis for age

- dependent differential response to O 3 , adult 

### 17 (15 wee ks old) and neonatal (15

- 16 days old) mice from 8 genetically diverse strains were examined 

### 18 for O 3 - induced (0.8 ppm for 5 h ours ) pulmonary injury and lung inflammation (Vancza et al., 2009, 

### 19 596419 ) . Ozone exposure increased polymorphonuclear leukocytes (PMN) influx in all strains of 

### 20 mice tested, but significantly in only some sensitive strains, suggesting a ge netic background effect. 

### 21 This strain difference was not due to differences in delivered dose of O 3 to the lung, evidenced by 

### 22
18 O lung enrichment. The sensitivity of strains for O 3 - induced increases in BAL protein and PMNs 

### 23 was different for different strain s of rats suggesting that genetic factors contribute to heightened 

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

### 25 of O 3 than corresponding strain neonates. Thus, it appeared that the infant mice showed a two - to 

### 26 three fold higher response than the adults when expressed relative to the accumulated O 3 reaction 

### 27 product in their lungs. The apparent decrease in delivered O 3 dose in neonates could be a result of a 

### 28 more rapid loss of body temperature in infant ra ts incident to maternal separation and chamber air 

### 29 flow. 

### 30 The three- dimensional detail of the nasal passages of immature Rhesus mac a que mo n keys 

### 31 were analyzed for developing p re dictive dosimetry models and exposure -dose- response relationships 

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

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

### 34 adultho od (comparing to (Gross et al., 1982, 040121 ; Gross et al., 1987, 625447 )) . Ozone exposures 

### 35 (0.5 ppm, 8 h/day under acute [5 day s] and episodic conditions [ 5 replicates of the acute paradigm 

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

### 37 most sensitive cell types in the nasal cavity, showing 50

- 80% decreases in epithelial thickness and 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 21 epithelial cell volume. The character and locati on of nasal lesions resulting from O 3 exposure a re 

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

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

### 3 develop persistent necrotizing rhinitis following episodic longer

- term exposures. 

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

### 5 extrapolate to human O 3 exposures. Rats required 4

- 5 times higher exposure to O 3 to achieve 

### 6

co mparable increases in BAL protein and PMNs to exercising humans. New studies have shown that 

### 7 varied O 3 response in different mouse strains was not due to differences in delivered dose of O 3 to 

### 8 the lung but more likely genetic sensitivity, and that infant m ice show greater toxicity relative to the 

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

### 10 comparison between species, the acute and chronic functional responses of laboratory animals to O 3 

### 11 appear qualitatively h omologous to those of the human making them a useful tool in determining 

### 12 mechanistic and cause

- effect relationships with O 3 exposure. 

### 13 

# 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
As described in the previous section, O 3 is a highly reactive oxidant gas w ith low water 

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

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

### 16 proteins, and carbohydrates and results i n their oxidative modification. Because of its chemical 

### 17 reactivity, inhaled O 3 directly targets components residing on the airways and alveolar surfaces, 

### 18 including ELF and surface macrophages. Although the O 3 molecule is consumed and may not reach 

### 19 the apic al plasma membrane of airways and alveolar epithelium, secondary oxidation products 

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

### 21 mast cells and eosinophils. Thus, O 3 effects are mediated by componen ts of ELF and by the multiple 

### 22 cell types found in the respiratory tract. 

### 23 Three distinct short

- term responses have been well

- characterized in humans challenged with 

### 24 O 3 : decreased pulmonary function, airways inflammation, and increased bronchial reactivity. In 

### 25 addition, evidence has been accumulating that O 3 exposure exacerbates, and possibly causes, asthma 

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

### 27 including inflammation and injury, have also been describe d. Animal studies have demonstrated a 

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

### 29 cardiovascular and other organ effects occur following short - and long

- term exposures of animals to 

### 30 O 3 . Mechanisms res ponsible for these effects are incompletely understood. 

### 31 This section of the ISA highlights findings of studies published since the last O 3 AQCD 

### 32 (U.S. EPA, 2006, 088089 ) which provide insight into the biological pathways underlying the effects 

### 33March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 22 of O 3 . Older studies which represent the current state of the science are also discus sed. Studies 

### 1 conducted at more environmentally

- relevant concentrations of O 3 are of greater interest, since 

### 2 mechanisms responsible for effects at low O 3 concentrations may not be identical to those occurring 

### 3 at high O 3 concentrations. In fact, some evidenc e suggests a concentration

- dependent hierarchy of 

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

### 5 modes of action responsible for the pulmonary and extrapulmonary effects of O 3 exposure. 

### 6 

## 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

Since O 3 does not diffuse far into the aqueous layer of the ELF without reacting, it is not likely 

### 7 to directly impact the underlying cells of the respiratory tract (Pryor, 1992, 042725 )

. This does not 

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

### 9 surface macrophages. The secondary oxidation products formed in the ELF following O 3 exposure 

### 10

are primarily responsible for ozone’s effects at the molecular, cellular and tissue level . T he amount 

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

### 12 reaction and injury due to O 3 exposure, as will be discussed below. 

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

### 14 reactions. Free radicals are generated during O 3 - mediated oxidation reactions (Pryor, 1994, 075987 )

. 

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

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

### 17 presence of the lipid

- soluble free radical scavenger alpha

- tocopherol (Fu jita et al., 1987, 004280 ; 

### 18 Pryor, 1976, 038940 ; Pryor, 1994, 075987 ) . Although O 3 can react with all hydrocarbons, its 

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

### 20 are one preferred target of O 3 . Following reaction of O 3 with unsaturated fatty acids in the ELF, 

### 21

measur able amounts of aldehydes were found in human bronchoalveolar lavage (BAL) fluid 

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

### 23 lipids is an important mechanism underlying O 3 -induced injury (Mudway and Kelly, 2000, 010452 ; 

### 24 Pryor, 1976, 038940 ) . This could occur by free

- radical reactions initiated by O 3 in the ELF or by 

### 25 direct effects of O 3 on membranes of cells, like surface macrophages, which extend be yond the ELF. 

### 26 Markers of lipid peroxidation have been demonstrated in lung tissue and BAL fluid following O 3 

### 27 exposure and are enhanced in alpha

- tocopherol deficient animals (Mudway and Kelly, 2000, 

### 28 010452 ) . Ozone

- mediated lipid peroxidation leads to the rapid formation of eicosanoids, another 

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

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

### 31 2004, 076673 ) , indicating that O 3 reacts with the cholesterol found in surfactant. In addition, O 3 

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

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

### 34 and Kelly, 2000, 01045 2 )

. 

### 35March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 23 The ELF contains numerous antioxidants including alpha

- tocopherol, albumin, ascorbate, 

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

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

### 3 concentrations in the surface liqui d of human conducting airways and are known to be preferred 

### 4 targets of O 3 . These antioxidants are thought to be the first line of defense against inhaled O 3 , 

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

### 6 010452 ) . In vitro studies have demonstrated consumption of water

- soluble antioxidants and the 

### 7 formation of oxidation products by O 3 as well as a reactive hierarchy with O 3 (Cross et al., 1992, 

### 8 625299 ; Mudway and Kelly, 1998, 000273 ) . When examined as a single antioxidant in solution, 

### 9 urate exhibited the greatest reactivity, followed b y ascorbate and glutathione ( Mudway and Kelly, 

### 10 1998, 000273 ) . Results using mixtures have demonstrated greater complexity (Mudway and Kelly, 

### 11 2000, 010452 )

. 

### 12 Although ELF con stituents such as antioxidants may protect against the deleterious effects of 

### 13 O 3 , there is some evidence that antioxidants may paradoxically facilitate O 3 - mediated damage. This 

### 14 apparent contradiction should be viewed in terms of the concentration

- dependent role of the ELF 

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

### 16 between O 3 and the low molecular weight antioxidants ascorbate and glutathione generated 

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

### 18 2005, 076649 ) . Rea ctions between O 3 and these antioxidant species exhibited a biphasic 

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

### 20 concentrations of antioxidant. In this way, endogenous reactants led to the formation of se condary 

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

### 22 Aqueous phase reactions between O 3 and urate or bovine serum albumin did not result in membrane 

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

### 24 protected against lipi d and protein oxidation resulting from the reaction of O 3 and ascorbate 

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

### 26 some antioxidants was opposed by quenching reactions involving other antioxidants. 

### 27 Local scavenging of inhaled O 3 by antioxidants in specific respiratory regions has been 

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

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

### 30 0.2 ppm O 3 for 2 h ours indicating that urate is the predominant antioxidant with respect to O 3 

### 31 reactivity in the nasal cavity (Mudway et al., 1999, 001270 ) . In addition, depletion of urat e during O 3 

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

### 33 1999, 001270 ) . Efforts to identify the predominant antioxidant(s) in other respiratory tract regions 

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

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

### 36 human subjects to 0.2 ppm O 3 (Blomberg et al., 1999, 001267 )

. 

### 37March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 24 Since exposure to O 3 often leads to airway inflammation characterized by neutrophilia and 

### 1 since neutrophil

- derived oxidants often scavenge ELF antioxidants, concentrations of ELF 

### 2 antioxidants were examined during ai rways neutrophilia which generally occurs 4

- 6 h ours 

### 3 postexposure to O 3 (Gunnison and Hatch, 1999, 087204 ; Long et al. , 2001, 057301 ; Mudway et al., 

### 4 1999, 011833 ) . In humans exposed to 0.2 ppm O 3 for 2 h ours , urate, glutathione and alpha



### 5 tocopherol levels remained unchanged in BAL fluid 6 hours postexposure while ascorbate was 

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

### 7 study involving the same protocol reported a loss of ascorbate from bronchial wash fluid and BA L 

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

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

### 10 urate levels in response to O 3 (Mudway et al., 2001, 025327 ) . Further, O 3 exposu re (0.8 ppm , 

### 11 4 hours ) in female rats resulted in a 50% decrease in BAL fluid ascorbate immediately postexposure 

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

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

### 14 hamsters exposed to 3 ppm O 3 for 6 h ours found no depletion of ascorbate, glutathione or alpha



### 15 tocopherol in B AL fluid (Long et al., 2001, 057301 ) . Instead an increase in BAL fluid urate and a 

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

. 

### 17 Although it is known that ELF antioxidants are v ariably distributed among regions of the 

### 18 respiratory tract, mechanisms underlying this variability are not well

- understood. It is thought that 

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

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

### 21 source appears to be the plasma (Peden et al., 1995, 076189 ) . Reple tion of urate in nasal lavage fluid 

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

. 

### 23 When these subjects were exposed to O 3 , nasal lavage urate was significantly decreased while 

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

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

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

. 

### 27 Regulation of ascorbate, gluta thione and alpha

- tocopherol concentrations within the ELF is less clear 

### 28 than that of urate (Mudway et al., 2006, 1 96536 ) . In a sequential nasal lavage study in humans, 

### 29 wash

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

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

### 31 in BALF glutathione and decreases in BALF and plasma ascorbate levels several hours following O 3 

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

### 33 025327 ) . Furthermore, high levels of dehydroascorbate, the oxidized form of ascorbate, have been 

### 34 reported in human ELF (Mudway et a l., 2006, 196536 ) . Other investigators have demonstrated 

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

### 36 export of reduced ascorbate (Welch et al., 1995, 644675 )

. 

### 37March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

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

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

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

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

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

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

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

### 7 suggestion that the amount and composition of ELF antioxidants, the capac ity to replenish 

### 8 antioxidants in the ELF or the balance between beneficial and injurious interactions between 

### 9 antioxidants and O 3 may contribute to O 3 sensitivity which varies between individuals and species 

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

. 

### 11 The complexity of these interactions was demonstrated by a study in which O 3 exposure resulted in 

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

### 13 and healthy controls, despite differences in ELF antioxidant concentrations prior to O 3 exposure 

### 14 (Mudway et al., 2001, 025327 ) . Further, the O 3 - induced increase in oxidized glutathione and 

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

### 16 (Mudway et al., 2001, 025327 ) . W hile the authors concluded that basal ascorbate and oxidized 

### 17 glutathione concentrations were not predictive of responsiveness to O 3 , they also suggested that the 

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

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

### 20 antioxidant capacity may play a role in modulating responses to O 3 . 

### 21 Several studies in animals evaluated the relationships between 18 O- labeled O 3 dose markers , 

### 22 injury markers an d ascorbate concentrations following O 3 exposure. In female rats exposed to 

### 23 0.8 ppm O 3 for 4 h ours , BAL indicators of injury and inflammation (protein and neutrophil number) 

### 24 and 18 O reaction product were increased inversely with the reduction in ascorbate (Gunn ison and 

### 25 Hatch, 1999, 087204 )

. In another study, aging rats (9 and 24 mo nths old) were shown to have 49% 

### 26 and 64% lower ascorbat e in lung tissue, respectively , than 2

- mo nth- old rats (Vincent et al., 1996, 

### 27 080778 )

. However, aging

- induced ascorbate lo ss did not increase the accumulation of 18 O reaction 

### 28 products following O 3 exposure (0.4 -0.8 ppm , 2

- 6 h ours)

. Pregnancy and lactation also caused lower 

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

### 30 of 18 O reaction products following O 3 exposure (Gunnison and Hatch, 1999, 087204 )

. Kari et al. 

### 31 (1997, 086171 ) observed that a 3

- week caloric restriction (75%) in rats abrogated the toxicity of O 3 

### 32 (2 ppm , 2 h ours ), measured as BAL fluid increases in protein, fibronectin and neutrophils, which 

### 33 was seen in normally fed rats. Accompanying this resistance to O 3 toxicity, was a 30% higher basal 

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

### 35 above normal and re duction (30%) in the accumulation of 18 O reaction product in the lungs. These 

### 36 investigations demonstrated an inverse relationship between ascorbate levels and O 3 dose and 

### 37 provide evidence for ascorbate playing a protective role following O 3 exposure in these studies. 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 26 Many investigations have focused on antioxidant deficiency and supplementation as 

### 1 modulators of O 3 - mediated effects. Ascorbate deficiency has been shown to increase the effects of 

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

### 3 Slade et al., 1989, 059465 ) . Supplementation with ascorbate and alpha

- tocopherol was protective in 

### 4 healthy adults who were on an ascorbate

- deficient diet and exposed to 0.4 ppm O 3 for 2 hours while 

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

### 6 reduction in forced expiratory volume in one second (FEV 1 ) following O 3 exposure (Samet et al., 

### 7 2001, 019034 ) . However the inflammatory response (influx of neutrophils and levels of IL

- 6) 

### 8 measured in BAL fluid 1 h our after O 3 exposure was not different between supplemented and non



### 9 supplemented subjects (Samet et al., 2001, 019034 ) . Supplementation with ascorbate and alpha



### 10 tocopherol also protected against pulmonary function decrements and nas al inflammatory responses 

### 11 which were associated with high levels of ambient O 3 in asthmatic children living in Mexico City 

### 12 (Romieu et al., 2002, 034711 ; Sienra

- Monge et al., 2004, 196422 ) . Similarly, supplementation with 

### 13

a scorbate, alpha

- tocopherol and beta

- carotene improved pulmonary function in Mexico City 

### 14 streetworkers (Romieu et al., 1998, 086756 ) . However, ascorbate and alpha

- tocopherol 

### 15 supplementation failed to ameliorate the pulmonary function decrements or airways neutrophilia 

### 16 observed in humans exposed to 0.2 ppm O 3 for 2 h ours (Mudway et al., 2006, 196536 ) . It was 

### 17 suggested that supplementation may be ineffective in the absen ce of antioxidant deficiency (Mudway 

### 18 et al., 2006, 196536 ) . Furthermore, protective effects of supplementation w ith alpha

- tocopherol 

### 19 alone have not been observed in humans (Mudway and Kelly, 2000, 010452 )

. 

### 20 Recent studies in animals demonstrated protection against O 3 - induced effects using gamma



### 21 tocopherol supplementation in models of aller gic rhinosin usitis (Wagner et al., 2009, 201574 ) and 

### 22 lower airway all ergic inflammation (Wagner et al., 2007, 596420 ) . Previous studies demonstrated 

### 23 that supplementati on with alpha- tocopherol was ineffective in these models (Wagner et al., 2007, 

### 24 596420 ) . Other investigators found that alpha

- tocopherol deficiency led to an increase in liver lipid 

### 25 peroxidation (Sato et al., 1980, 039738 ) and a drop in liver alpha

- tocopherol levels following O 3 

### 26 exposure (Vasu et al., 2010, 201561 ) . A recent study used alpha

- tocopherol transfer protein null mice 

### 27 as a model of alpha

- tocopherol deficiency and demonstrated an altered adaptive response of the lung 

### 28 genome to O 3 exposure (Vasu et al., 2010, 201561 ) . Take n together, these studies provide evidence 

### 29 that the tocopherol system modulates O 3 - induced responses. 

### 30 Other antioxidants have been shown to confer resistance to O 3 - induced injury. In a recent 

### 31 study, lung hyperpermeability in response to O 3 was unexpectedly reduced in mice deficient in the 

### 32 glutamate

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

### 33 2010, 644476 ) . Since the lungs of these mice exhibited 70% glutathione depletion, protection against 

### 34 O 3 - induced injury was unexpected (Johansson et al., 2010, 644476 ) . However it was found that 

### 35 several other antioxidant defenses, including metallothionein , were upregulated in response to O 3 to 

### 36 a greater degree in the glutathione

- deficient mice compared with sufficient mice (Johansson et al., 

### 37 2010, 644476 ) . The authors suggested that resistance to O 3 - induced lung injury was due to 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 27 compensatory augmentation of antioxidant defenses (Johansson et al., 2010, 644476 ) . A ntioxidant 

### 1 effects have also been attributed to Clara cell secretory pr otein (CCSP) and surfactant protein A 

### 2 (SP

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

### 3 exposed to O 3 (0.2 or 1 ppm for 8 h ours) by an unknown mechanism (Plopper et al., 2006, 596410 )

. 

### 4 SP

- A protected against O 3 - induced airways inflammation and injury, possibly by acting as a 

### 5

sacr ificial substrate (Haque et al., 2007, 597606 ). 

### 6 A role for plasma antioxidants in modulating O 3 - induced respiratory effects has also been 

### 7 suggested (Aibo et al., 2010, 378559 )

. In this study, pretreatment of rats with a high dose of 

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

### 9 in to the lung following 6 h exposure to 0. 25 and 0.5 ppm O 3 (Aibo et al., 2010, 378559 ) . These 

### 10 effects were not observed in response to O 3 alone

. Although not measured in this study, glutathione 

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

### 12

source o f plasma glutathione, acetaminophen treatment may have lowered plasma glutathione level s 

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

### 14 interdependence between respiratory tract, plasma and liver responses to O 3 , poss ibly related to 

### 15 glutathione status. 

### 16 An other important consideration is the non

- uniformity of the injury response to O 3 throughout 

### 17 the respiratory tract

. Several mechanisms have been proposed to explain this phenomenon

. First, 

### 18 dosimetry may be a key determi nant since the sites receiving the largest dose might be expected to 

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

### 20 003000 ) . An important coro ll ary is that the uneven distribution of mucus in the respiratory tract 

### 21 airways may influence the capacity of O 3 to reach the aqueous layer (Mudway and Kelly, 2000, 

### 22 010452 ) . Secondly, the non

- homogeneous formation of cytotoxic products in the ELF may account 

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

### 24 the respiratory tract, being greater in the u pper airways and less more distally. Further the 

### 25 composition of the ELF varies along the respiratory tract. Thus, t he a mount of protective 

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

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

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

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

### 30 the formatio n of oxidized surfactant lipids is likely restricted to th e alveolar region and respiratory 

### 31 bronchioles where surfactant is found

. Thus, region

- specific formation of particular oxidation 

### 32 products may dicta te patterns of epithelial injury in the respiratory tract. 

### 33 The relationship between site

- specific O 3 dose, epithelial injury and glutathione concentration 

### 34 was inve stigated by Plopper (1998, 087203 )

. Adult rhesus monkeys were exposed for 2 h ours to 0.4 

### 35 and 1.0 ppm O 3 , whi ch was labeled with 18 O, and tissues were analyzed immediately postexposure 

### 36 for dose, epithelial injury and glutathione levels

. Results indicated that exposure to 1 ppm O 3 

### 37 resulted in the greatest epithelial injury in the respiratory bronchioles although i njury was observed 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 28 at all of the airway sites but not in the lung parenchyma

. Exposure to 0.4 ppm O 3 resulted in 

### 1 epithelial injury only in the respiratory bronchioles

. Local O 3 dose was found to be highly variable 

### 2 among the different sites with the greates t levels found in the respiratory bronchioles and lowest 

### 3 levels found in the parenchyma following 1 ppm O 3 . Glutathione levels varied in the different 

### 4 airways sites in monkeys exposed to filtered air

. Exposure to 1 ppm O 3 decreased glutathione levels 

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

### 6 exposure dose of O 3 (uptake of 18 O) and the degree of initial epithelial injury at a particular site in 

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

### 8 a protective role during O 3 exposure. 

### 9 While the formation of secondary oxidation products is the key event leading to O 3 - mediated 

### 10 effects, scavenging and/or metabolism of those products is likely to be an importan t determinant of 

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

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

### 13 recent study demonstrated increased gene expression o f MARCO in the lungs of an O 3 - resistant C3H 

### 14 mouse strain (HeJ) but not in an O 3 - sensitive, genetically nearly identical strain (OuJ) (Dahl et al., 

### 15 2007, 196986 ) . Upregulation of MARCO occurred in mice exposed to 0.3 ppm O 3 for 24

- 48 h ours ; 

### 16 inhalation exposure for 6 h ours at this concentration was insufficient for this response. Animals 

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

### 18 neutrophils, protein and isoprostanes, following exposure to 0.3 ppm O 3 (Dahl et al., 2007, 196986 )

. 

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

### 20 2007, 196986 ) . Scavenging of oxidized lipids may li mit O 3 - induced injury since ozonized 

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

### 22 076663 ) stimulate apoptosis and cytotoxicity (Gao et al., 2009, 200764 ; Sathishkumar et al., 2007, 

### 23 097758 ; Sathishkumar et al., 2007, 197785 ; Sathishkumar et al., 2009, 201549 ) in vitro. While these 

### 24 studies have focused on the alveolar compartment (alveolar macrophages, surfactant lipids), 

### 25 comparable pathways have yet to be elucidated in the conducting airways. A second mechanism 

### 26 likely to impact O 3 - mediated effects is the metabolism of secondary oxidation products catalyzed by 

### 27 antioxidant enzymes such as glutathione peroxidase, glutathione S

- transferases (GST) and 

### 28 NADPH:quinone oxidoreductase 1 (NQO1). Evidence for the i mportance of GST and NQO1 in 

### 29 modulating the effects of O 3 is presented below (see Gene

- Environment Interactions). 

### 30 Secondary oxidation products formed as a result of O 3 exposure initiate numerous responses at 

### 31 the cellular, tissue and whole organ level of t he respiratory system. These responses include the 

### 32 activation of neural reflexes, injury and inflammation, hyperpermeability, increased bronchial 

### 33 reactivity and altered host defenses, as will be discussed below. In addition, the enhancement of 

### 34 asthma and a llergic responses demonstrated as a result of O 3 exposure is likely due to secondary 

### 35 oxidation products. Exposure to O 3 also results in effects on other organ systems such as the 

### 36 cardiovascular, hepatic and central nervous systems. Mechanisms underlying th ese extrapulmonary 

### 37 responses are not well understood. It is unlikely that lipid ozonides and other secondary oxidation 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 29 products, which are bioactive and cytotoxic in the respiratory system, gain access to the vascular 

### 1 space (Chuang et al., 2009, 197202 )

. However O 3 exposure may result in systemic oxidative stress, 

### 2 as suggested by studies in humans reporting an association between O 3 exposure and both levels of 

### 3 plasma 8

- isoprostanes and the presence of peripheral blood lymphocyte micronuclei (Chen et al., 

### 4 2006, 196504 ; Chen et al., 2007, 145956 ). 

### 5 

## 5.2.2.1. Summary The initial key event in ozone’s toxicity pathway is the formation of secondary oxidation 

### 6 products in the respiratory tract. Pathways for the removal of those products are also of great 

### 7 importance. Due to the highly reactive nature of O 3 , direct reactions most likely involve components 

### 8 of the ELF and/or plasma membranes of surface macrophages which extend beyond the ELF. 

### 9 Reaction products likely mediate ozone’s effec ts on respiratory tract epithelium. 

### 10 

## 5.2.3. Ac tiva tio n o f Ne ural Re fle xe s

Acute O 3 exposure results in reversible effects on lung function parameters through activation 

### 11 of neural reflexes. The involvement of bronchial C

- fibers, a type of nociceptive sensory nerve, has 

### 12 been demonstrated in dogs (Coleridge et al., 1993, 038695 ; Schelegle et al., 1993, 039203 ) and the 

### 13 involvement of nociceptive sensory nerves has been demonstrated in humans (Passannante et al., 

### 14 1998, 030114 ) . Furthermore there is evidence that substance P (SP), a tachykinin which is known to 

### 15 be released from C

- fibers, plays a role in O 3 - mediated effects (Hazbun et al., 1993, 043914 ; Krishna 

### 16 et al., 1997, 084262 )

. 

### 17 The response to O 3 in humans is characterized by substernal discomfort, especially on deep 

### 18 inspiration, accompanied by involuntary truncation of inspiration (Hazucha et al., 1989, 041909 )

. 

### 19 This leads to decreased inspiratory capacity and to decreased forced vital capacity (FVC) and forced 

### 20 expiratory volume in one second (FEV 1 ), as measured by spirometry, and is accompanied by a 

### 21 decreased tidal volume and increased respirat ory frequency in human subjects during exercise 

### 22 (Hazuch a et al., 1989, 041909 ) . For example, these pulmonary function responses have been noted 

### 23 immediately after a 1

- h exposure to 0.3 ppm O 3 , resolving by 6 h ours after exposure (Schelegl e et 

### 24 al., 1991, 042491 ) , and during and immediately after a 4

- h exposure to 0.2 ppm O 3 in exercising 

### 25 humans (Aris et al., 1993, 038275 ; Balmes et al., 1996, 080830 ) . Although spirometric changes 

### 26 began to return to baseline shortly after exposure, small residual spirometric decrements were 

### 27 reported at 24 h ours postexposure (Hazucha et al., 1996, 043923 ) . Spirometric changes in FEV 1 and 

### 28 FVC were not due to changes in respiratory muscle strength (Hazucha et al., 1989, 0 41909 )

. 

### 29 Changes in FVC or symptoms were not modified by treatment with bronchodilators such as atropine 

### 30 (Beckett et al., 1985, 039758 ) . Thus, parasympathetic involv ement in the O 3 - mediated decreases in 

### 31 lung volume was not significant (Mudway and Ke lly, 2000, 010452 ) . However, the loss of vital 

### 32 capacity was reversible with intravenous administration of the rapid

- acting opioid agonist, 

### 33 sufentanyl, indicating an involvement of opioid receptor

- containing nerve fibers and/or more central 

### 34March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 30 neurons (Passannante et al., 1998, 030114 ) . The effect s of sufentanyl may be attributed to blocking 

### 1 C- fiber stimulation by O 3 since activation of opioid receptors downregulates C

- fiber function 

### 2 (Belvisi et al., 1992, 644681 ) . There is some evidence that eicosanoids play a role in the neural 

### 3 reflex since cyclooxygenase inhibition with indomethacin (Alexis et al., 2000, 013072 ; Schelegle et 

### 4 al., 1987, 041706 ) or ibuprofen, which also blocks some lipoxygenase activity (Hazucha et al., 1996, 

### 5 043923 ) , before exposure to O 3 significantly blunted the spirometric responses. In the latter study, 

### 6 ibuprofen treatment resulted in measurable decreases in BAL levels of PGE2 and TXB2 at 1 h our 

### 7 postexposure (Hazucha et al., 1996, 043923 ) . Although an earlier study demonstrated that PGE2 

### 8 stimulated bronchial C

- fibers (Coleridge et al., 1976, 038612 ; Coleridge et al., 1993, 038695 ) and 

### 9 suggested that PGE2 mediated O 3 - induced decreases in pulmonary function, no correlation was 

### 10 ob served between the degree of ibuprofen

- induced inhibition of BAL PGE2 levels and blunting of 

### 11 the spirometric response to O 3 (Hazucha et al., 1996, 043923 ) . Nonetheless, recent studies continue 

### 12 to provide evidence that arachidonic acid metabolites, as well as oxidative stress, contribute to 

### 13 human responsiveness to O 3 (Alfaro et al., 2007, 196567 ). 

### 14 A delay in onset of O 3 - induced pulmonary function responses has been noted in numerous 

### 15 studies. Recently the delay was characterized in terms of changes in breathing frequency (Schelegle 

### 16 et al., 2007, 195841 ) . In humans exposed to O 3 , no change in breathing frequency was observed until 

### 17 a certain cumulative inhaled dose of O 3 had been reached. Subsequently, the magnitude of the 

### 18 change in breathing frequency was correlated with the inhaled do se rate (Schelegle et al., 2007, 

### 19 195841 ) . These investigators proposed that initial reactions of O 3 with ELF resulted i n a time



### 20 dependent depletion of ELF antioxidants, and that activation of neural reflexes occurred only after 

### 21 the antioxidant defenses were overwhelmed (Schelegle et al., 2007, 195841 )

. 

### 22 There is a large range of pulmonary function responses to O 3 among healthy young adults 

### 23 (Balmes et al., 1996, 080830 ; Hazucha et al., 2003, 048168 ) . Since individual responses are 

### 24 relatively consistent across time, it is thought that responsiveness reflects an intrinsic characteristic 

### 25 of the subject (Mudway and Kelly, 2000, 010452 ) . Older adults are generally not responsive to O 3 

### 26 (Hazucha et al., 2003, 048168 ) , while obese young women may be more responsive than lean young 

### 27

women (Bennett et al., 2007, 418827 ) . The lack of spirometric responsiveness is not attributable to 

### 28 the presence of endogenous endorphins, which could potentially block C

- fiber stimulation by O 3 , as 

### 29 demonstrated in a study involving intravenous admini stration of naloxone immediately following the 

### 30 O 3 exposure to weak responders (Passannante et al., 1998, 030114 ) . Currently, the mechanis ms 

### 31 underlying the inter

- individual variation in responsiveness to O 3 are not known. It has been proposed 

### 32 that some of the variation in response may be genetically determined (Yang et al., 2005, 077211 )

. 

### 33 More discussion on this topic is found below ( S ection 5.2. 9 .1). 

### 34 In addition to the spirometric changes, mild airways obstruction occurs as a result of O 3 

### 35 exposure. This pulmonary function decrement is generally measured as specific airway resistance 

### 36 ( sRaw ) which is the product of airway resistance and thoracic gas volume . Increased sRaw occurred 

### 37 fairly rapid ly in exercis ing humans, peaking at 1

- 6 h ours and resolving by 24 h ours following O 3 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 31 exposure (Aris et al., 1993, 038275 ; Balmes et al., 1996, 080830 ; Hazucha et al., 1996, 043923 ; 

### 1 Schelegle et al., 1991, 042491 )

. Small but statistically significant increases in sRaw during O 3 

### 2 exposure (0.2 ppm for 4 h ours with intermittent exercise) and immediately following O 3 exposure 

### 3 (0.3 ppm for 1 h our , moderate exercise or 0.4 ppm for 2 h ours , intermittent exercise) were observed 

### 4 in several studies (Aris et al., 1993, 038275 ; Balmes et al., 1996, 080830 ; Hazucha et al., 1996, 

### 5 043923 ; Schelegl e et al., 1991, 042491 ) . These changes in sRaw correlated with changes in 

### 6 inflammatory and injury endpoints measured 18 h ours postexposure, as will be discussed below, but 

### 7 not with the time course or degree of spirometric change measured during exposure (Aris et al., 

### 8 1993, 038275 ; Balmes et al., 1996, 080830 ; Schelegle et al., 1991, 042491 )

. In addition, a small but 

### 9 persistent increase in airways resistance associated with narrowing of small peripheral airways 

### 10 (measured as changes in isoV FEF 25

- 75 ) was demonstrated in O 3 - exposed humans (0.35 ppm for 

### 11 130 min utes with intermittent exercise) (Weinmann et al., 1995, 077206 ; Weinmann et al., 1995, 

### 12 038645 ) . A similar study (0.4 ppm O 3 for 2 h ours with intermittent exercise) found decreases in 

### 13 FEF 25

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

### 14 dysfunction (Kreit et al., 1989, 041817 ) . In separate studies, a statistically significant increase in 

### 15 residual volume (Hazucha et al., 1989, 041909 ) and a statistically significant decrease in FEF 25

- 75 

### 16 (Horstman et al., 1995, 075834 ) were observed following O 3 exposure. 

### 17 Mechanisms underlying the rapid increase in airways resistance following O 3 exposure are 

### 18 incompletely understood. However p retreatment with atropine was found to decrease baseli ne sRaw 

### 19 and prev ent O 3 - induced increases in sRaw (Beckett et al., 1985, 039758 ), indicating the involvement 

### 20 of muscarinic cholinergic receptors of the parasympathetic nervous system. Interestingly, atropine 

### 21 pretreatment partially blocked the decrease in FEV 1 , but had no effect on the decrease in FVC, 

### 22 breathing rate, tidal volume or respiratory symptoms (Beckett et al., 1985, 039758 ) . Thus pulmonary 

### 23 function decrements measured as FEV 1 may reflect both restrictive and obstructive type changes in 

### 24 airways responses. Using a beta

- adrenergic agonist, it was shown that smooth muscle contraction, 

### 25 not inc reased airways mucus section, was responsible for O 3 - induced increases in airways resistance 

### 26 (Beckett et al., 1985, 039758 ) . Furthermore, tachykinins may contribute t o O 3 - mediated increases in 

### 27 airways resistance. Bronchopulmonary C fibers mediate local axon responses by releasing 

### 28 tachykinins such as SP. Tachykinins bind to neurokinin (NK) receptors resulting in responses such as 

### 29 bronchoconstriction. In one study in whi ch bronchial biopsies were performed and studied by 

### 30 immunohistochemistry, SP was substantially diminished in submucosal sensory nerves 6 h ours 

### 31 following O 3 exposure (0.2 ppm O 3 for 2 h ours with exercise) (Krishna et al., 1997, 084262 ) . A 

### 32 statistically significant correlation was obse rved between loss of SP immunoreactivity from neurons 

### 33 in the bronchial mucosa and changes in FEV 1 measured 1 h our postexposure (Krishna et al., 1997, 

### 34 084262 ) . Another study found that SP was increased in lavage fluid of human subjects immediately 

### 35 after O 3 challenge (0.25 ppm O 3 for 1 hour with heavy exercise) (Hazbun et al., 1993, 043914 ) These 

### 36 results provide evidence that the increased airways resistance obser ved following O 3 exposure is due 

### 37March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 32 to vagally

- mediated responses and possibly by local axon reflex responses through 

### 1 bronchopulmonary C fiber

- mediated release of SP. 

### 2 In responsive individuals, a striking degree of response attenuation occurs following repea ted 

### 3 daily exposures to O 3 . This phenomena has been reported for both lung function and symptoms such 

### 4 as upper airway irritation, nonproductive cough and substernal discomfort and pain upon deep 

### 5 inspiration (Folinsbee et al., 1980, 038880 ; Hackney et al., 1977, 038282 ; Horvath et al., 1981, 

### 6 039221 ) . Repeated daily exposures also led to an attenuation of the sRaw response in exercising 

### 7 humans (Christian et al., 1998, 029925 ). It is well

- established that a young O 3 responder will no 

### 8 longer be responsive on the fourth or fifth day of consecutive daily O 3 exposure (0.4 ppm O 3 for 

### 9 4 hours or 0.2 ppm O 3 for 4 hours ) and that after developing this tolerance it takes up to 7

- 10 d ays of 

### 10

non - exposure in order for the subject to regain O 3 responsiveness (Christian et al., 1998, 029925 ; 

### 11 Devlin et al., 1997, 083577 ; Folinsbee et al., 1980, 038880 ; Hackney et al., 1977, 038282 ; Horvath 

### 12 et al., 1981, 039221 ; Linn et al., 1982, 039646 ) . One group reported persistent small airway 

### 13 dysfunction despite attenuation of the FEV 1 response on the third day of consecutive O 3 exposure 

### 14 (Frank et al., 2001, 093491 ) . Studies in animals also indicate an attenuation of the physiologic 

### 15 response as measured by breathing patterns and tidal volume following five consecutive days of O 3 

### 16 exposure (Tepper et al., 1989, 041991 ) . The mechanisms underlying this attenuation in humans and 

### 17 animal models are n ot well understood (Devlin et al., 1997, 083577 ), although some studies have 

### 18 implicated an alteration in lung antioxidant capacity, enhanced mucus production or factors related 

### 19 to epithelial hyperplasia following O 3 exposure (Devlin et al., 1997, 083577 ) . These potential 

### 20 mechanisms and others are discussed below. Adaptation of O 3 - induced bradycardic responses, which 

### 21 also result from activation of neural reflexes, have also been reported in animal studies (Hamade and 

### 22 Tankersley, 2009, 596386 ; Watkinson et al., 2001, 016245 )

. 

### 23 Finally, the degree of acute decrease of vital capacity in young, healthy adults does not 

### 24 correlate (positively) with the degree of neutrophilic inflammation observed in their airways or the 

### 25 degree of airways obstruction elicited by O 3 exposure (Aris et al., 1993, 038275 ; Aris et al., 1995, 

### 26 075945 ; Balmes et al., 1996, 080830 ; Schelegle et al., 1991, 042491 ) , implying that different 

### 27 mechanisms are at work. Further evidence is provided by the finding that pretreatment with 

### 28 ibuprofen attenuated O 3 - induced lung function changes and increases in BAL fluid PGE2 levels, but 

### 29 had no effect on neutrophili a (Hazucha et al., 1996, 043923 )

. 

### 30 

## 5.2.3.1. Ne w Ce llula r a n d Mole c ula r Ins igh ts
Recent studies in animals provide new information regarding the effects of O 3 on reflex 

### 31 responses mediated by bronchopulmonary C

- fibers, a type of nociceptive sensory nerve. Ozone 

### 32 exposure in mice was found to selectively activate a subset of receptors on bronchopulmonary C



### 33 fibers which are TRPA1 ion channels (Taylor- Clark and Undem, 2010, 377143 ) . TRPA1 ion 

### 34 channels are members of the TRP family of ion channels, w hich are known to mediate the responses 

### 35 of sensory neurons to inflammatory mediators (Caceres et al., 2009, 628549 ) . In addition to TRPA1 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 33 ion channels possibly playing a key role in O 3 - induced decrements in pulmonary fun ction, they may 

### 1 mediate allergic asthma (Caceres et al., 2009, 628549 ) . Activation of TRPA1 ion channels following 

### 2 O 3 exposure is likely due to the formation of secondary products such as aldehydes and 

### 3 prostaglandins (Taylor- Clark and Undem, 2010, 377143 ) . Ozonation of unsaturated fatty acids in the 

### 4 ELF results in the generation of aldehydes (Frampton et al., 1999, 040757 ) . For example 

### 5 4- hydroxynonenal and 4

- oxononenal are derived from the peroxidation of omega

- 6 unsaturated fatty 

### 6 acids (Taylor

- Clark et al., 2008, 628565 ; Trevisani et al., 2007, 628590 ) . 4

- oxononenal is a stronger 

### 7 electrophile than 4

- hydroxynonenal and exhibits greater potency towards the TRPA1 channels 

### 8 (Taylor

- Clark et al., 2008, 628565 ) . TRPA1 channels can be activated by aldehydes and other 

### 9 electrophiles through covalent modification of cysteine and lysine residues (Trevisani et al., 2007, 

### 10 628590 ) . In addition, PGE2 is known to sensitize TRPA1 channels (Bang et al., 2007, 628545 ) and, 

### 11 as noted pr eviously, PGE2 (or other products of cyclooxygenase

- catalyzed reactions) has been 

### 12 proposed to mediate the pulmonary function changes observed following O 3 exposure in humans. 

### 13 In addition to stimulating central nervous system reflexes, bronchopulmonary C f ibers mediate 

### 14 local axon responses by releasing neuropeptides such as SP, neurokinin (NK) A and calcitonin gene



### 15 related peptide (CGRP). Oslund et al. (2008, 195654 ) demonstrated that NK

- 1 receptor blockade had 

### 16 no effect on O 3 - stimulated physiologic responses such as tidal volume and breathing frequency in 

### 17 rats over the 8

- h exposure period. However, SP and NK receptors contributed to vagally

- mediated 

### 18 bronchoconstriction in guinea pigs 3 days after a single exposure to O 3 (2 ppm for 4 h ours ) (Verhein 

### 19 et al., 2011, 670295 )

. 

### 20 

## 5.2.3.2. Summary A key event in ozone’s toxici ty pathway is the activation of neural reflexes which leads to 

### 21 decrements in pulmonary function. Evidence is accumulating that secondary oxidation products are 

### 22 responsible for this effect. Eicosanoids have been implicated in humans while eicosanoids and 

### 23 al dehydes are effective in animal models. Different receptors on bronchial C

- fibers have been shown 

### 24 to mediate separate effects of O 3 on pulmonary function. Nociceptor sensory nerves are involved in 

### 25 the involuntary truncation of respiration which results in decreases in FVC, FEV 1 , tidal volume and 

### 26 an increase in respiratory frequency and pain upon deep inspiration. Opioids block these responses 

### 27 while atropine does not. New evidence in an animal model suggests that TRPA1 receptors on 

### 28 bronchial C

- fibers mediate this pathway. Ozone exposure also results in activation of vagal sensory 

### 29 nerves and a mild increase in airways obstruction measured as increased sRaw . Atropine and beta



### 30 adrenergic agonists blocked this response in one study indicating that the airway obst ruction was due 

### 31 to bronchoconstriction. Other studies in humans implicated SP release from bronchial C

- fibers 

### 32 resulting in airway narrowing due to either neurogenic edema or bronchoconstriction. New evidence 

### 33 in an animal model suggests that the SP

- NK recep tor pathway caused bronchoconstriction following 

### 34 O 3 exposure. Considerable inter

- individual variability exists in O 3 responsiveness measured by 

### 35 decrements in pulmonary function. Further, attenuation of these pulmonary function decrements 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 34 occurs following O 3 exposure for several consecutive days. Mechanisms responsible for these effects 

### 1 are not known but may be related to inherent differences in neural sensitivity. 

### 2 

## 5.2.4. Re s pirato ry Tra c t Inju ry a nd Infla mma tio n
As described above, O 3 reacts with components of th e ELF resulting in the generation of 

### 3 secondary oxidation products. Higher concentrations of these products may directly injure 

### 4 respiratory tract epithelium. Lower concentrations may initiate cellular responses including cytokine 

### 5 generation, adhesion molecu le expression and modification of tight junctions leading to 

### 6 inflammation and increased permeability across airways epithelium (Dahl et al., 2007, 196986 ; 

### 7 Mudway and Kelly, 2000, 010452 ) . Subsequent epithelial remodeling may also occur (Mudway and 

### 8 Kelly, 2000, 010452 )

. 

### 9 Injury and inflammation have been observed in many d ifferent regions of the respiratory tract 

### 10 following O 3 exposure (Plopper et al., 1998, 087203 ; Postlethwait et al., 2000, 003000 ) . The nasal 

### 11 airways, conducting airways and distal airways (i.e. respiratory bronchioles or centriacinar region 

### 12 depending on the species) have all been identified as sites of O 3 - mediated injury and inflammat ion 

### 13 (Mudway and Kelly, 2000, 010452 ) . One study found greater injury in conducting airways 

### 14 downstream of bifurcations where local doses of O 3 were higher (Postlethwait et al., 2000, 003000 )

. 

### 15 Although the extent of O 3 - induced injury is variable along the respiratory tract, common features of 

### 16 the injury response have been noted (Mudway and Kelly, 200 0, 010452 ) . In the conducting airways, 

### 17 necrosis of ciliated cells and degranulation of secretory cells has been observed and in the alveolar 

### 18 region, necrosis of Type 1 pneumocytes occurs. 

### 19 Further, O 3 - induced injury and inflammation responses are variable between species

. For 

### 20 example , Dormans et al. (1999, 040766 ) found that rats, mice, and guinea pigs all exhibited 

### 21 O 3 - induced (0.2 - 0.4 ppm for 3

- 56 day s) inflammation ; however, guinea pigs were the most 

### 22 sensitive with respect to alveolar macrophage elicitation and pulmonary cell densit y in the 

### 23 centriacinar region. Mice were the most sensitive to bronchiolar epithelial hypertrophy and 

### 24 biochemical changes (e.g. lactate dehydrogenase, glutathione reductase, glucose -6-phosphate 

### 25 dehydrogenase activity), and had the slowest recovery from O 3 e xposure. All species displayed 

### 26 increased collagen in the ductal septa and large lamellar bodies in Type II pneumocytes at the longest 

### 27 exposure and highest concentration, whereas this response occurred in the rat and guinea pig at 

### 28 lower O 3 levels (0.2 ppm ) as well. Since no dose metric was measured, it is possible that some of 

### 29 these differences may be attributable to disparate total inhaled dose or local organ dose. Overall, the 

### 30 authors rated mice as most susceptible, followed by guinea pigs, then rats (Dormans et al., 1999, 

### 31 040766 ) . Rats were also less sensitive to epithelial necrosis and inflammatory responses from O 3 

### 32 (1.0 ppm for 8 h) than monkeys and ferrets, which manifested a similar response (Sterner

- Kock et 

### 33 al., 2000, 013033 ) . These data suggest that ferrets may be a good animal m odel for O 3 -induced 

### 34 airway effects due to the similarities in pulmonary structure between primates and ferrets. 

### 35March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 35 While injury and inflammation often accompany each other, in many cases epithelial injury 

### 1 precedes observable inflammatory effects and in other cases inflammation leads to injury of the 

### 2 surrounding cells and tissues. In addition to being species

- dependent, the acute injury response is 

### 3 focal, site

- specific and dependent on exposure parameters and the time that elapsed since exposure 

### 4 (Postlethwait et al., 2000, 003000 ) . The presence of shed epithelial cells in the BAL, increases in 

### 5 levels of BAL lactate dehydrogenase and protein and increased epithelial permeability have been 

### 6 observed and are indicative of epithelial injury. In addition, histologic analysis has demonstrated 

### 7 damage to tight junctions between epithelial cells, suggesting an increase in epithelial permeability. 

### 8 Several studies have measured epithelial permeability as the flux of the small solute 99mTc



### 9 DTPA which was introduced into the air spaces in different regions of the respiratory tract. An early 

### 10 study demonstrated i ncreased pulmonary epithelial permeability , measured as the clearance of 

### 11 99mTc

- DTPA, in humans exposed for 2 h to 0.4 ppm O 3 while exercising moderately (Ke hrl et al., 

### 12 1987, 040824 ) . Another study found that increased epithelial permeability occurred at 1

- 3 h ours and 

### 13 18

- 20 h ours postexposure and did not resolve for several days (Foster and Stetkiewicz, 1996, 

### 14 079920 ) . Increased bronchial permeability was also observed in dogs immediately after and 18 h ours 

### 15 postexposure to 0.2 ppm O 3 for 6 h ours (Fr eed et al., 1996, 080798 ) . Increased epithelial 

### 16 permeability has been proposed to play a role in allergic sensitization (Matsumura, 1970, 050626 ) , in 

### 17 activation of neural reflexes and in stimulation of smooth muscle receptors (Dimeo et al., 1981, 

### 18 039662 ) . Studies in animals have also demonstrated increased vascular perm eability, as measured by 

### 19 BAL protein and albumin (Costa et al., 1985, 040273 ; Hu et al., 1982, 039418 )

. 

### 20 An important hallmark of a cute O 3 exposure in humans and animals is neutrophilic airways 

### 21 inflammation. Although neutrophil influx into nasal airways has been demonstrated in human 

### 22 subjects (Graham and Koren, 1990, 042299 ) , most studies of neutrophil influx have focused on the 

### 23 lower airways (Aris et al., 1993, 038275 ; Hazucha et al., 1996, 043923 ) . The time course of this 

### 24 respo nse and its resolution is slower than that of the decrements in pulmonary function (Hazucha et 

### 25 al., 1996, 043923 ) . In general, airways neutrophilia is observable within 1

- 2 h ours , peaks at 

### 26 4-6 hours and is returning to baseline levels at 24 h following exposure to O 3 in exercising humans 

### 27 involving 0.4 ppm for 2 h ours (Devlin et al., 1991, 040359 ) or 0.3 ppm for 1 h our (Schelegle et al., 

### 28 1991, 042491 ) . Since the influx and persistence of neutrophils in airways following O 3 exposure 

### 29 correlates with the temporal profile of epithelial injury (Hu et al., 1982, 039418 ) , neutrophils are 

### 30 likely to be injurious. However , neutrophils can contribute to the repair of O 3 - injured epithelium by 

### 31 removing necrotic epithelial cells (Mudway and Kelly, 2000, 010452 ; Vesely et al., 1999, 051045 )

. 

### 32 The degree of airways inflammation due to O 3 is thought to have more important long

- term 

### 33 consequences th an the more quickly resolving changes in pulmonary function since airways 

### 34 inflammation is often accompanied by tissue injury (Balmes et al., 1996, 080830 )

. 

### 35 The influx of inflammatory cells in the airways of human subjects has been assessed by 

### 36 bronchoscopy and by morphometric measurements in bronchial mucosal biopsies. Bronchoscopy can 

### 37 be used to sample fl uid from all of the airways and from the lung parenchyma distal to the wedged 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 36 bronchoscope tip or just the more proximal portion. While many studies have documented a distal 

### 1 lung inflammatory response using conventional (pooled) BAL, a lesser number of stu dies have 

### 2 documented inflammation in the proximal airways using “proximal” BAL sampling (i.e. , liquid 

### 3 aspirated from a 20

- 30 mL initial lavage aliquot after wedging the bronchoscope or from the left 

### 4 main bronchus transiently isolated by inflation of proxim al and distal balloons) or by using bronchial 

### 5 mucosal biopsy (Aris et al., 1993, 038275 ; Schelegle et al., 1991, 042491 ) . Airways neutrophilia was 

### 6 observed at 1 and 6 h ours postexposure in proximal airways BAL but only at 6 h ours postexposure 

### 7 in BAL from all of the airways combined (Schelegle et al., 1991, 042491 ) . This result demonstrated 

### 8 that measurements made in proximal airways BAL better reflected the earliest phase of airways 

### 9 inflammation than measurements made in conventional multi

- aliquot (pooled) BAL. 

### 10 Inter

- individual variability in the neutrophilic response has been noted (Devlin et al., 1991, 

### 11 040359 ; Holz et al., 1999, 058731 ; Schelegle et al., 1991, 042491 ) . One study demonstrated a 

### 12 three fold difference in airways neutrophilia, measured as percent of total cells in proximal BAL, 

### 13 among human subjects exposed to 0.3 ppm O 3 for 1 h our while exercising (Schelegle et al., 1991, 

### 14 042491 ) , while a 20 -fol d difference was demonstrated in BAL neutrophils following exposure to 

### 15 0.08-0.10 ppm O 3 for 6.6 h ours while exercising (Devlin et al., 1991, 040359 ) . Reproducibility of 

### 16 intra- individual responses to 0.25 ppm O 3 , measured as sputum neutrophilia, was demonstrated by 

### 17 Holz (1999, 058731 ) . Few studies have examined the dose

- responsiveness of airways neutrophilia in 

### 18 O 3 - exposed humans (Devlin et al., 19 91, 040359 ; Holz et al., 1999, 058731 ) . No dose



### 19 responsiven ess was observed in healthy human subjects exposed for 1 h our to 0.125 and 0.25 ppm 

### 20 O 3 and a statistically significant increase in sputum neutrophilia was observed only at the higher 

### 21 dose (Holz et al., 1999, 058731 ) . However, dose

- dependent and statistically significant increases in 

### 22 BAL neutrophils and the inflammatory mediator IL

- 6 were reported following exposure to 0.08 an d 

### 23 0.1 ppm O 3 for 6.6 hours in exercising humans (Devlin et al., 1991, 040359 ) . Additi onal evidence is 

### 24 provided by a meta

- analysis of the O 3 dose- inflammatory response in controlled human exposure 

### 25 studies involving exposure to 0.08

- 0.6 ppm O 3 for 60

- 396 min utes (Mudway and Kelly , 2004, 

### 26 399328 ) . Results demonstrated a linear relationship between inhaled O 3 dose (determined as the 

### 27 product of concentration, ventilation and time) and B AL neutrophils at 0

- 6 h ours and 18

- 24 h ours 

### 28 following O 3 exposure (Mudway and Kelly, 2004, 399328 )

. 

### 29 O zone exposure results in alteration s in other airways inflammatory cells besides neutrophils. 

### 30 Numbers of lymphocytes and total cells in BAL fluid were decreased early after O 3 exposure, 

### 31 preceding the neutrophil influx (Blomberg et al., 1999, 001267 ; Krishna et al., 1997, 084262 ; 

### 32 Mudway and Kelly, 2000, 010452 ) . The decrease in total cells was thought to reflect decreases in 

### 33 airway macrophages, although it was not clear whether the cells were necrotic or whether membrane 

### 34 adhesive properties were altered making them more difficult to obtain by lavage (Blomberg et al., 

### 35 1999, 001267 ; Frampton et al., 1997, 086111 ; Mudway and Kelly, 2000, 010452 ; Mudway et al., 

### 36 1999, 011833 ; Pearson and Bhalla, 1997, 082686 ) . Recent studies have demonstrated increases in 

### 37 numbers of sputum monocytes and dendritic

- like cells (Alexis et al., 2010, 628538 ) (discussed 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 37 further in section 5.2.7). Increases in submucosal m ast cells were observed 1.5 hours after a 2 -h 

### 1 exposure of healthy human subjects to 0.2 ppm O 3 (Blomberg et al., 1999, 001267 ) and increases in 

### 2 BAL mast cell number were observed 18 h ours after O 3 exposure (Frampton et al., 1997, 086111 )

. 

### 3 Mast cells may play an important role in mediating neutrophil influx since they are an important 

### 4 source of several pro

- inf lammatory cytokines and since their influx precedes that of the neutrophils 

### 5 (Blomberg et al., 1999, 001267 ; Stenfors et al., 2002, 030473 ) . Further, a study using mast cell



### 6 deficient mice demonstrated decreased neutrophili c inflammation in response to O 3 compared with 

### 7 wild type mice (Kleeberger et al., 1993, 044203 ). The mechanisms involved in clearing O 3 - provoked 

### 8 inflammatio n remain to be clarified. 

### 9 T he cellular and molecular signals involved in injury and inflammatory responses following 

### 10 O 3 exposure have been extensively evaluated (U.S. EPA, 2006, 088089 ). Eicosanoids are one class 

### 11 of secondary oxidation products which may be formed rapidly following O 3 exposure and which 

### 12 may mediate injury and i nflammation. Eicosanoids are metabolites of arachidonic acid, a 20

- carbon 

### 13 polyunsaturated fatty acid, which is released from membrane phospholipids by phospholipase 

### 14 A2

- mediated catalysis. Activation of phospholipase A2 occurs by several cell signaling path ways 

### 15 and may be triggered by O 3 - mediated lipid peroxidation of cellular membranes (Rashba- Step et al., 

### 16 1997, 628562 )

. Additionally, cellular phospholipases A2, C and D may be activated by lipid 

### 17 ozonation products (Kafoury et al., 1998, 016913 ) . While the c onversion of arachidonic acid to 

### 18 prostaglandins, leukotrienes and other eicosanoid products is generally catalyzed by 

### 19 cyclooxygenases and lipoxygenases, non

- enzymatic reactions also occur during oxidativ e stress 

### 20 leading to the generation of a wide variety of eicosanoids and reactive oxygen species. Further, t he 

### 21 release of arachidonic acid from phospholipids is accompanied by the formation of 

### 22 lysophospholipids which are precursors for platelet activating f actors. Thus, formation of 

### 23 eicosanoids, reactive oxygen species and platelet activating factors accompanies O 3 - mediated lipid 

### 24 peroxidation. 

### 25 Additional cell signaling mediators are generated subsequent to O 3 exposure. Secondary 

### 26 reaction products may stimul ate airway macrophages to produce cytokines such as IL

- 1, IL

- 6 and 

### 27 TNF

- α which in turn activate IL

- 8 production by epithelial cells. Ozone exposure is also known to 

### 28 upregulate the vascular endothelial adhesion molecules P

- selectin and ICAM

- 1 (Blomberg et al., 

### 29 1999, 001267 ; Krishna et al., 1997, 084262 ) and to increase the expression of pro -inflammatory 

### 30 mediators GM

- CSF, Gro

- α and IL -8 (Mudway and Kelly, 2000, 010452 ) . In addition, lung epithelial 

### 31 cells may release ATP in response to O 3 exposure (Ahmad et al., 2005, 196429 )

. ATP and its 

### 32 metabolites (catalyzed by ecto

- enzymes) can bind to cellular purinergic receptors resulting in 

### 33 activation of cell signaling path ways (Picher et al., 2004, 644780 ) . One such metabolite, adenine, is 

### 34 capable of undergoing oxidation leading to the formation of urate which, if present in high 

### 35 concentrations, could activate inflammasomes and result in caspase 1 activation and the maturation 

### 36 and secretion of IL

- 1beta and IL

- 18 (Dostert et al., 2008, 155753 )

. 

### 37March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 38 Many studies have focused on cell signaling pathways leading to airways neutrophilia in 

### 1 humans or animal models which generally peaks 4

- 6 h ours after exposure. Although IL

- 8 has b een 

### 2 proposed to play a role in neutrophil chemotaxis, measurements of IL

- 8 in lavage fluid from humans 

### 3 exposed to O 3 show increases that are too late to account for this effect (Mudway and Kelly, 2000, 

### 4 010452 ) . However, the profiles of PGE2 and IL

- 6 responses suggest that they may play a role in 

### 5 neutrophil chemotaxis. A study in mice demonstrated that PAF may be important in this response 

### 6 (Longphre et al., 1999, 001199 ) , while macrophage inflammatory protein

- 2 (MIP

- 2) and ICAM

- 1 

### 7 have also been implicated in a rat model (Bhalla and Gupta, 2000, 015036 ) . Other studies have 

### 8 inve stigated mechanisms involved in earlier or later phases of neutrophilic airways inflammation and 

### 9 inflammation occurring in the nasal airways and more distal lung (U.S. EPA, 2006, 088089 )

. 

### 10 One set of studies in humans focused on the earliest phase of airways inflammation (1

- 2 h ours 

### 11 following exposure). Exercising subjects were exposed to 0.2 ppm O 3 for 2 h ours and bronchial 

### 12 biopsy tissues were obtained 1.5 and 6 h ours after exposure (Blomberg et al., 1999, 001267 ; Bosson 

### 13 et al., 2003, 051687 ; Bosson et al., 2009, 399331 ; Stenfors et al., 2002, 030473 ) . Results 

### 14 demonstrated upregulation of vascular endothelial adhesion molecules P

- selectin and ICAM

- 1 at 

### 15 both 1.5 and 6 hours (Blomberg et al., 1999, 001267 ; Stenfors et al., 2002, 030473 ) . Submucosal 

### 16 mast cell numbers were increased at 1.5 h ours in the biopsy samples without an accompanying 

### 17 increase in neutrophil number (Blomberg e t al., 1999, 001267 ) . Pronounced neutrophil infiltration 

### 18 was observed at 6 h in the bronchial mucosa ( Stenfors et al., 2002, 030473 ) . Surprisingly, 

### 19 suppression of the NFkB and AP

- 1 pathways at 1.5 h ours and a lack of increased IL

- 8 at 1.5 or 

### 20 6 hours in bronch ial epithelium was observed (Bosson et al., 2009, 399331 ) . The authors suggested 

### 21 that vascular endothelial adhesion molecules, rather than redox sensitive transcription factors, are 

### 22 key to early neutrophil recruitment in response to O 3 . 

### 23 Multi

- day exposure to O 3 has been found to dampen the inflammatory respons e, but not the 

### 24 injury response, compared with a single day exposure (Christian et al., 1998, 029925 ; Devlin et al., 

### 25 1997, 083577 ) . In human subjects exposed for 4 h ours to 0.2 ppm O 3 during moderate exercise, 

### 26 decreased numbers of BAL neutrophils were observed after 4 d ays of consecutive exposure 

### 27

com pared with responses after 1 d ay (Christian et al., 1998, 029925 ) . Results indicated an 

### 28 attenuation of the inflammatory response in both proximal airways and distal lung. However 

### 29 repeated exposure did not result in attenuation of the injury markers lactate dehydrogenase and 

### 30 protein in the BAL. Similar results were found in a study of humans undergoing heavy intermittent 

### 31 exercise who were e xposed for 2 h ours to 0.4 ppm O 3 for 5 consecutive days (Devlin et al., 1997, 

### 32 083577 ) . In this latter study, partial recov ery of the inflammatory response was noted 10 days 

### 33 following the exposure (Devlin et al., 1997, 083577 ) . In an animal stud y conducted in parallel (Van 

### 34 Bree et al., 2002, 035452 ) , full susceptibility to O 3 challenge following exposure to O 3 for 5 

### 35 consecutive days required 15

- 20 d ays recovery. Further, no attenuation of cellular proliferation in 

### 36 terminal bronchioles was observed during the 5 consecutive days of O 3 exposure. In a separate study 

### 37 in rats involving repeated O 3 exposures, a lack of attenuation of the injury marker lavagable protein, 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 39 the persistence of macrophages in the centriacinar region, and histological evidence of progressive 

### 1 tissue injury was demonstrated (Tepper et al., 1989, 041991 ) . Thus, the inflammatory response 

### 2 resembled that of the pulmonary function response which was attenuated with repeated short

- term 

### 3 O 3 exposure in both human sub jects and animals (Christian et al., 1998, 029925 ; Hackney et al., 

### 4 1977, 038282 ; Horvath et al., 1981, 039221 ) . Findings that injury, measured by BAL markers or by 

### 5 histopathology, persisted in the absence of inflammation or pulmonary function decrements suggests 

### 6 that, despite adaptation, repeated exposure to O 3 may have serious long

- term consequen ces such as 

### 7 airway remodeling. The mechanisms involved in clearing O 3 - provoked inflammation remain to be 

### 8 clarified. 

### 9 Increases in markers of inflammation and of injury occurred to a comparable degree in 

### 10 subjects with mild (least sensitive) and more remarkab le (more sensitive) spirometric responses to 

### 11 O 3 (Balmes et al., 1996, 080830 ) . Two other studies using similar exposure protocols found that 

### 12 acute spirometric changes were not positively correlated with cellular and biochemical indicators of 

### 13 inflammation (Aris et al., 1993, 038275 ; Sc helegle et al., 1991, 042491 ) . However inflammation was 

### 14 correlated with changes in sRaw (Balmes et al. , 1996, 080830 ) . In another study, pretreatment with 

### 15 ibuprofen had no effect on neutrophilia although it blunted the spirometric response (Hazucha et al., 

### 16 1996, 043923 ) . Taken together, results from these studies indicate different mechanisms underlying 

### 17 the spirometric and inflammatory responses to O 3 . 

### 18 In contrast, a common mechanism underlying both inflammation and impaired pulmonary 

### 19 function was suggested by (Krishna et al., 1997, 084262 ) . This study, conducted in exercising 

### 20 humans exposed to 0.2 ppm O 3 for 2 h ours , demonstrated a correlation between loss of SP 

### 21 immunoreactivity from neurons in the bronchial mucosa and numbers of neutrophils and epithelial 

### 22 cells (shed epithelial cells are an index of injury) in the BAL 6 h postexposure. Furthermore, the loss 

### 23 of SP immunoreactivity was correlated with the observed changes in FEV 1 . SP is a neuropeptide 

### 24 released by sensory nerves which mediates neurogenic edema and bronchoconstriction (Krishna et 

### 25 al., 1997, 084262 ) . Further, another study found that SP was increased in lavage fluid of human 

### 26 subjects immediately after O 3 challenge (Hazbun et al., 1993, 043914 ) . Taken together, these 

### 27 findings suggest O 3 - mediated stimulation of sensory nerves leading to activation of central and local 

### 28 axon reflexes as a common effector pathway leading to impaired pulmonary function and 

### 29 inflammation. 

### 30 Chronic exposure to O 3 has been studied in animal models. In the nasal airways, exposure to 

### 31 O 3 for days or weeks results in mucous cell metaplasia of nasal transitional epithelium (Harkema et 

### 32 al., 1999, 001209 ; Hotchkiss et al., 1991, 042441 ) . This remodeling effect was characterized by 

### 33 neutrophilic infiltration, a loss of sensitive nasal epithelial cells, the proliferation of resistant 

### 34 epithelial cells and mucin gene ov erexpression (Cho et al., 1999, 011985 ) . Bronchiolitis, bronchiolar 

### 35 metaplasia of alveolar ducts, proliferation of Type 2 pneumocytes and fibrosis were reported 

### 36 following chronic O 3 exposure (Mudway and Kelly, 2000, 010452 ) . It was suggested that remodeling 

### 37 of respiratory epithelium may lead to enhanced resistance or tolerance to O 3 (Mudway and Kelly, 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 40 2000, 010452 ) . Deposition of collagen in the airways and sustained lung function decrements 

### 1 especially in small airways have also been demonstrated as a response to chronic O 3 exposure 

### 2 (Chang et al., 199 2, 042387 ; Mudway and Kelly, 2000, 010452 )

. 

### 3 

## 5.2.4.1. New Cellular a n d Mole c ula r Ins igh ts Recent investigations in animal models have elucidated additional mechanisms involved in 

### 4 O 3 - induced inflammation and injury. In one study, tachykinins working through NK

- 1 and CGRP 

### 5 receptors were found to contribute to airways epithel ial injury, but not to neutrophil influx, in O 3 - 

### 6 exposed rats (Oslund et al., 2008, 195654 ; Oslund et al., 2009, 201539 ) . Key roles for CXCR2, a 

### 7 receptor for the cytokines KC and MIP

- 2, and for IL

- 6 in O 3 - mediated neutrophil influx were 

### 8 demonstrated in mice (Johnston et al., 2005, 596393 ; Johnston et al., 2005, 596394 ) . Activation of 

### 9 JNK and p3 8 pathways and cathepsin

- S were also found to be important in this response (Williams 

### 10 et al., 2007, 628609 ; Williams e t al., 2008, 628607 ; Williams et al., 2009, 628605 ) . Furt hermore, 

### 11 matrix metalloproteinase

- 9 (MMP

- 9) protected against O 3 - induced airways inflammation and injury 

### 12 in mice (Yoon et al., 2007, 596422 )

. 

### 13 Williams et al. (2007, 597545 ) found that the toll

- like receptor (TLR) adaptor protein MyD88 

### 14 was important in mediating O 3 - induced neutrophilia in mice exposed to 3 ppm O 3 for 3 h ours , with 

### 15 TLR4 and TLR2 contributing to the speed of the response. Moreover, MyD88, TLR2 and TLR4 

### 16 contributed to inflammatory gene expression in this model and O 3 upregulated MyD88, TLR4 and 

### 17 TLR4 gene expression. These results complement those of Hollingsworth et al. (2004, 097816 ) who 

### 18 demonstrated airways neutrophilia following acute (2 ppm O 3 for 3 h ours ) and subchronic O 3 

### 19 exposure (0.3 ppm for 3 d ays ) in a mouse model (Hollingsworth et al., 2004, 097816 ) . In this study, 

### 20 airways neutrophilia was not dependent on TLR4 (Hollingsworth et al., 2004, 097816 ) . Ozone 

### 21 effects on lung hyperpermeability, which is often correlated with neutrophil influx, were prev iously 

### 22 found to require a functioning TLR4 (Kleeberger et al., 2000, 014895 )

. 

### 23 Other studies focused on the role of hyaluronan in mediating a later phase (24 h ours ) of 

### 24 O 3 - induced inflammation in mice (Garantziotis et al., 2009, 597603 ; Garantziotis et al., 2010, 

### 25 624947 ) . Hyaluronan is an extracellular matrix component which is normally found in the ELF as a 

### 26 large polymer. Exposure to 2 ppm O 3 for 3 h ours resulted in elevated levels of soluble low mole cular 

### 27 weight hyaluronan in the BAL fluid 24 h ours postexposure (Garantziotis et al., 2009, 597603 ; 

### 28 Garantziotis et al., 2010, 624947 )

. Ozone may have caused the depolymerization of hyaluronan to 

### 29 soluble fragments which are known to be endogenous ligands of the CD44 receptor and TLR4 in the 

### 30 macrophage (Jiang et al., 2005, 628556 ) . Binding of hyaluronan fragments to the CD44 receptor 

### 31 activates hyaluronan clearance, while binding to TLR4 results in signaling through MyD88 to 

### 32 produce chemokines that stimulate the influx of inflammatory cells (Jiang et al., 2005, 628556 )

. 

### 33 Activation of NFkB occurred in both airway epithelia and alveolar macrophages 24 h ours 

### 34 postexposure to O 3 . Increases in BAL pro

- inflammatory factors KC, IL

- 1beta, MCP

- 1, tumor 

### 35 necrosis factor

- α (TNF

- α ) and IL

- 6 observed 24 h ours following O 3 exposure were found to be 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 41 partially dependent on TLR4 while increases in BAL inflammatory cells, which consisted mainly of 

### 1 macrophages, were dependent on CD44. BAL inflammatory cells number and injury mark ers 

### 2 following O 3 exposure were similar in wild

- type and TLR4

- deficient animals. 

### 3 

## 5.2.4.2. Summary Injury and inflammation are key events in ozone’s toxicity pathway. Secondary oxidation 

### 4 products have been implicated in a number of these processes. Although there may be inter

- species 

### 5 differences with respect to specific mediators, mechanisms involved in the acute responses to O 3 

### 6 include epithelial injury and airways neutrophilia. Longer

- term exposures may result in mucus cell 

### 7 metaplasia of nasal epithelium or airways remodeling and fibrosis. Work from several laboratories in 

### 8 humans and animal models suggest that O 3 triggers the release of tachykinins such as SP from 

### 9 airway sensory nerves which could contribute to downstream effects including injury and 

### 10 inflammation. N ew investigations show that O 3 exposure leads to the generation of hyaluronan 

### 11 fragments which activate TLR4 and CD44

- dependent signaling pathways in macrophages and result 

### 12 in a greater turnover of macrophage populations in the lung. Activation of these pat hways occurs 

### 13 later than the acute neutrophilic response suggesting that they may contribute to longer

- term effects 

### 14 of O 3 . The mechanisms involved in clearing O 3 - provoked inflammation remain to be clarified. 

### 15 Similar to the pulmonary function responses discu ssed in the previous section, considerable 

### 16 inter

- individual variability exists in O 3 responsiveness as measured by airways neutrophilia. Further, 

### 17 attenuation of the inflammatory response occurs following O 3 exposure for several consecutive days. 

### 18 However ev idence suggests that injury may continue despite the dampening of the inflammatory 

### 19 response during repeated exposures. Mechanisms responsible for inter

- individual variability and 

### 20 response attenuation, or the lack thereof, are not known. It should be noted that inflammation, as 

### 21 measured by airways neutrophilia, is not correlated with decrements in pulmonary function as 

### 22 measured by spirometry. Consequently, spirometric measures are not a good surrogate for the degree 

### 23 of inflammation in any given individual fo llowing O 3 exposure. Furthermore, airways neutrophilia 

### 24 may not be a good indicator of O 3 - mediated lung injury. 

### 25 

## 5.2.5. Inc re a s e d Bron c hial Rea c tivity
In addition to causing mild airways obstruction as discussed above, acute O 3 exposure results 

### 26 in reversible increa ses in bronchial reactivity by mechanisms which are not well understood. These 

### 27 effects may be more significant in human subjects with already compromised airways 

### 28 (S ection 5.2.6 ). Bronchial reactivity is generally determined in terms of a response to a chal lenge 

### 29 agent. Non

- specific bronchial reactivity in humans is assessed by measuring the effect of inhaling 

### 30 increasing concentrations of a bronchoconstrictive drug on lung mechanics ( sRaw or FEV 1 ). 

### 31 Methacholine is most commonly employed but histamine and othe r agents are also used. Specific 

### 32 bronchial reactivity is assessed by measuring effects in response to an inhaled allergen in individuals 

### 33March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 42 (or animals) already sensitized to that allergen. An increase in sRaw in response to non

- specific or 

### 1 specific challenge agents indicates airways hyperresponsiveness (AHR). 

### 2 O zone may sensitize bronchial smooth muscle to stimulation through a direct effect on smooth 

### 3 muscle or through effects on the sensory nerves in the epithelium or on the motor nerves innervating 

### 4 the smoot h muscle (Holtzman et al., 1979, 039220 ; O'Byrne et al., 1983, 041366 ; O'Byrne et al., 

### 5 1984, 040066 ) . One possibility may be related to O 3 - mediated increases in epithelial permeability, 

### 6

w hich would improve access of agonist to smooth muscle receptors (Holtzman et al., 1979, 039220 )

. 

### 7 Neurally

- mediated effects have b een demonstrated in several studies. In one, p retreatment with 

### 8 atropine was found to block O 3 - induced AHR, suggesting the involvement of cholinergic 

### 9 postgan glionic pathways (Holtzman et al., 1979, 039220 ) . Other studies in animals demonstrated 

### 10 that O 3 - induced AHR involved vagally

- mediated responses (Freed et al., 1996, 080798 ) and local 

### 11 axon reflex responses through bronchopulmonary C fiber

- mediated release of SP (Joad et al., 1996, 

### 12 082711 ) . Further, pretreatment with capsai cin to deplete nerve fibers of SP blocked O 3 - mediated 

### 13 bronchial reactivity measured as AHR (Tepper et al., 1993, 628570 ). 

### 14 Some evidence suggested the involvement of arachidonic acid metabolites (Fabbri et al., 1985, 

### 15 040276 ; Seltzer et al., 1986, 040383 )

. An early study found AHR in exe rcising humans immediately 

### 16 post exposure to 0.6 ppm O 3 for 2 h ours , which was associated with increases in BAL neutrophils and 

### 17 cycloo xygenase products (Seltzer et al., 19 86, 040383 ) . Other investigators found that ibuprofen 

### 18 pretreatment had no effect on AHR following exposure to 0.4 ppm O 3 for 2 h ours , although 

### 19 spirometric re spo nses were blunted (Hazucha et al., 1996, 043923 )

. This study indicated that the 

### 20 arachidonic a cid metabolites whose generation was blocked by ibuprofen, an inhibitor of 

### 21 cyclooxygenase and some lipoxygenase activity, (i.e. prostaglandins, thromboxanes and some 

### 22 leukotrienes) did not play a role in AHR. Experiments in dogs demonstrated a close correla tion 

### 23 between O 3 - induced AHR and airway neutrophilic inflammation measured in tissue biopsies 

### 24 (Holtzman et al., 1983, 039745 ) . Furthermore , the increased AHR observed in dogs following O 3 

### 25 exposure was inhibited by neutrophil depletion (O'Byrne et al., 1983, 041366 ) and by pre

- treatment 

### 26 with inhibitors of arachidonic acid metabolism. In one of these studies, indomethacin pre

- treatment 

### 27 did not prevent airway neutrophilia in response to O 3 providing evidence that the subset of 

### 28 arachidonic acid metabolites whose generation was inhibitable by the cyclooxygenase inhibitor 

### 29 indomethacin (i.e. , prostaglandins and thromboxanes) was not responsible for neutrophil influx 

### 30 (O'Byrne et al., 1984, 040066 ) . Taken together, these findings suggest that arachidonic acid 

### 31 metabolites, but probably not prostaglandins or thromboxanes, may be involved in the AHR res ponse 

### 32 following O 3 exposure in dogs. 

### 33 Later it was recognized that increased bronchial reactivity can be both a rapidly occurring and 

### 34 persistent response to O 3 (Foster and Freed, 1999, 001202 ) . Secondary oxidation products of O 3 and 

### 35 tachykinins have been proposed as early mediators of the response and inflammation

- derived 

### 36 products have been proposed as mediators of the later response (Foster and Freed, 1999, 001202 )

. 

### 37 Some studies have suggested an involvement of IL

- 1 (Park et al., 2004, 644864 ) and TNF

- α (Cho et 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 43 al., 2001, 016160 ; Shore et al., 2001, 018993 ) . Furthermore, multiday exposure to O 3 has been found 

### 1 to dampen the AHR respon se compared with a single day exposure (Dimeo et al., 1981, 039662 ). 

### 2 

## 5.2.5.1. Ne w Ce llula r a n d Mol e c ula r Ins igh ts
Recent studies in animal models provide new evidence for mechanisms underlying increased 

### 3 bronchial reactivity. In guinea pigs, AHR was found to be mediated by different pathways at 1 and 

### 4 3 days postexposure to a single dose of O 3 (2 ppm fo r 4 h ours ) (Verhein et al., 2011, 670295 ; Yost et 

### 5 al., 2005, 597549 ) . At 1 day, airway hyperreactivity was due to activation of airway pa rasympathetic 

### 6 nerves rather than to a direct effect on smooth muscle (Yost et al., 2005, 597549 ) . This effect 

### 7 occurred as a result of O 3 - stimulated release of major basic protein from eosinophils (Yost et al., 

### 8 2005, 597549 ) . Major basic protein is known to block inhibitory M2 muscarinic receptors which 

### 9 normally dampen acetylcholine release from parasympathetic nerves (Yost et al., 2005, 597549 ) . The 

### 10 resulting increase in acetylcholine release caused an increase in smooth muscle contraction 

### 11 following O 3 exposure (Yost et al., 2005, 597549 ) . Eosinophils played a different role 3 days 

### 12 postexposure to O 3 in guinea pigs (Yost et al., 2005, 597549 )

. Ozone

- mediated influx of eosinophils 

### 13 in to lung airways resulted in a different population present 3 days postexposure compared to those 

### 14 present at one day (Yost et al., 2005, 597549 ) . At this point, eosinophil

- derived major basic protein 

### 15 increased smooth muscle responsiveness to acetylcholine which also contributed to AHR (Yost et al., 

### 16 2005, 597549 ) . However, the major effect of eosinophils was to protect against vagal hyperreactivity 

### 17 (Yost et al., 2005, 597549 ) . The authors suggested that these beneficial effects were due to the 

### 18 production of nerve growth factor (Yost et al., 2005, 597549 ) . Further work by these investigators 

### 19 demonstr ated a key role for IL

- 1beta in mediating AHR three days postexposure to O 3 (Verhein et 

### 20 al., 2011, 670295 ) . In this study, IL

- 1beta increased nerve growth factor and SP which acted through 

### 21 the NK1 receptor to cause vagally

- mediated bronchoconstriction (Verhein et al., 2011, 670295 ) . The 

### 22 mechanism by which SP caused acetylcholine release from parasympathetic nerves following O 3 

### 23 exposure was not determined (Verhein et al., 2011, 670295 ) . Two studies by other investigators 

### 24 demonstrated that SP released from airways nociceptive neuron s contributed to O 3 - induced AHR 

### 25 measured 3 h ours postexposure to 2 ppm O 3 in ferrets ( Wu et al., 2003, 628616 ; Wu et al., 2008, 

### 26 597548 ) . These authors further suggested that SP expression in airway neurons was upregulated by 

### 27 IL

- 1 which was released in response to O 3 (Wu et al., 2008, 597548 ) . Taken together, the above 

### 28 study results indicate that mechanisms involved in O 3 - mediated AHR can vary over time 

### 29 postexposure and that eosinophils and SP can play a role. 

### 30 Evidence for cytokine and chemokine involvement in the AHR response to O 3 has recently 

### 31 been described. Williams et al. (2008, 597546 ) demonstrated that the Th2 cytokine IL

- 13 contributed 

### 32 to AHR, as well as to airways neutrophilia, following exposure of mice to 3 ppm O 3 for 3 h ours

. 

### 33 Other studi es in mice have demonstrated a key role for CXCR2, the chemokine receptor for the 

### 34 neutrophil chemokines KC and MIP

- 2, but not for IL

- 6 in O 3 - mediated AHR (Johnston et al., 20 05, 

### 35 596394 ; Johnston et al., 2005, 596393 ) . In contrast, CXCR2 and IL

- 6 were both required for 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 44 neutrophil influx in this model (Johnston et al., 2005, 596393 ; Johnston et al., 2005, 596394 ) , as 

### 1 discussed above. 

### 2 Other studies have focused on the role of TLR4. Hollingsworth measured AHR, as well as 

### 3 airways neutrophilia, in mice 6 and 24 h ours following acute (2 ppm O 3 for 3 h ours ) and subchronic 

### 4 (0.3 ppm for 3 d ays ) exposure to O 3 (Hollingsworth et al., 2004, 097816 ) . TLR4 is a key component 

### 5 of the innate immune system and is responsible for the immediate inflammatory response seen 

### 6 following challenge with endotoxin and other pathogen

- associ ated substances. In the 2004 study, a 

### 7 functioning TLR4 was required for the full AHR response following O 3 exposure but not for airways 

### 8 neutrophilia (Hollingsworth et al., 2004, 097816 ) . These findings are complemented by an older 

### 9 study demonstrating that O 3 effects on lung hyperpermeability required a functioning TLR4 

### 10 (Kleeberger et al., 20 00, 014895 ) . Williams et al. (2007, 597545 ) found that TLR2, TLR4 and the 

### 11 TLR adaptor protein MyD88 contributed to AHR in mice exposed to 3 ppm O 3 for 3 h ours . Ozone 

### 12 was also found to upregulate MyD88, TLR4 and TLR4 gene expression in this model (Williams et 

### 13 al., 2007, 597545 )

. 

### 14 A newly recognized mechanistic basis for O 3 - induced AHR is provided by studies focusing on 

### 15 the role of hyaluronan following O 3 exposure in mice (Garantziotis et al., 2010, 624947 ) 

### 16 (Garantziotis et al., 2009, 597603 ) . Briefly, TLR4 and CD44 were found to mediate AHR in response 

### 17 to O 3 and hyaluronan. Hyaluronan is an extracellular matrix component which is normally found in 

### 18 the ELF as a large polymer. Exposure to 2 ppm O 3 for 3 h ours resulted in enhanced AHR and 

### 19 elevated levels of soluble low molecular weight hyaluronan in the BAL fluid 24 h ours postexposure 

### 20 (Garantziotis et al., 2009, 597603 ; Garantziotis et al ., 2010, 624947 ) . Ozone may have caused the 

### 21 depolymerization of hyaluronan to soluble fragments which are known to be endogenous ligands of 

### 22 the CD44 recepto r and TLR4 in the macrophage (Jiang et al., 2005, 628556 ) . In the two recent 

### 23 studies, O 3 - induced AHR was attenuated in CD44 and TLR4

- deficient mice (Garantziotis et al., 

### 24 2009, 597603 ; Garantziotis et al., 2010, 624947 ) . Hyaluronan fragment

- mediated stimulation of 

### 25 AHR was found to require functioning CD44 recep tor and TLR4 (Garantziotis et al., 2009, 597603 ; 

### 26 Garantziotis et al., 2010, 624947 ) . In contrast, high

- molecular

- weight hyaluronan blocked AHR in 

### 27 response to O 3 (Garantziotis et al., 2009 , 597603 ) . In another study high

- molecular -weight 

### 28 hyaluronan enhanced repair of epithelial injury (Jiang et al., 2005, 628556 ) . These studies provide a 

### 29 link between innate immunity and the development of AHR following O 3 exposure, and indicate a 

### 30 role for TLR4 in increasing bronchial smooth muscle reactivity. 

### 31 

## 5.2.5.2. Summary Increased bronchial reactivity is a key event in the toxicity pathway of O 3 . It can be bo th a 

### 32 rapidly occurring and persistent response, although adaptation can also occur during multi -day 

### 33 exposures. Both direct effects on smooth muscle and neurally

- mediated effects on smooth muscle 

### 34 have been proposed to contribute to AHR following O 3 exposure . Currently, more evidence has 

### 35 accumulated for the latter mechanism. In humans exposed to O 3 , atropine was found to block the 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 45 early AHR response indicating the involvement of cholinergic postganglionic pathways. Inhibition 

### 1 of arachidonic acid metabolism wa s ineffective in blocking this response in humans while mixed 

### 2 results were found in animal models. Studies in O 3 - exposed animals have demonstrated a role for SP 

### 3 release from bronchial C fibers in mediating neurally

- mediated effects on smooth muscle. Later 

### 4 phases of increased bronchial reactivity may involve the induction of IL

- 1beta which in turn 

### 5 upregulates SP production. In guinea pigs, eosinophil

- derived major basic protein contributed to the 

### 6 stimulation of cholinergic postganglionic pathways. A novel ro le for hyaluronan in mediating the 

### 7 later phase effects of O 3 has recently been demonstrated. High molecular weight polymers of 

### 8 hyaluronan normally found in the ELF were degraded following O 3 exposure in mice. The resulting 

### 9 hyaluronan fragments stimulated A HR in a TLR4 - and CD44 receptor

- dependent manner. Previous 

### 10 work has shown that O 3 - mediated increases in lung permeability required a functioning TLR4 

### 11 suggesting a possible relationship between increased epithelial permeability and AHR in this model. 

### 12 Other cytokines and chemokines have been implicated in the AHR response to O 3 in animals models. 

### 13 

## 5.2.6. Exa ce rba tio n a n d Ind u ction o f As thma a nd Allergic Res po n s e s
In individuals with asthma, there is increased responsiveness to bronchoconstrictor challenge. 

### 14 This resu lts from a combination of structural and physiological factors including increased inner



### 15 wall thickness, smooth muscle responsiveness and mucus secretion. Although inflammation is likely 

### 16 to contribute, its relationship to AHR is not clear (U.S. EPA, 2006, 088089 ) . However, some 

### 17 asthmatics have higher baseline levels of neutroph ils, lymphocytes, eosinophils and mast cells in 

### 18 bronchial washes and bronchial biopsy tissue (Stenfors et al., 2002, 030473 ) . Evidence is 

### 19 accumulating that O 3 exposure exacerbates asthmatic and allergic responses in sensitive individuals. 

### 20 Further , some studies suggest that O 3 exposure leads to the development of asthmatic and allergic 

### 21 responses. 

### 22 In order to determine whether asthmatics exhibit greater sensitivity to O 3 , several older studies 

### 23 compared pulmonary function responses in asthmatic and non

- asthmatic subjects following O 3 

### 24 exposure. While the majority focused on measurements of FEV 1 and FVC and found no differences 

### 25 between the two groups (Holz et al., 1999, 058731 ; Koenig et al., 1987, 041521 ; Linn et al., 1978, 

### 26 038874 ; Mudway et al., 2001, 025327 ; Scannell et al., 1996, 080755 ; Stenfors et al., 2002, 030473 ) , 

### 27 there were notable exceptions. In one study, greater airways obstruction in asthmatics compared with 

### 28

non - asthmatic subjects was observed immediately following a 2

- h exposure to 0.4 ppm O 3 with 

### 29 intermittent exercise (Kreit et al., 1989, 041817 ) . These changes were measured as statistically 

### 30 significant greater decreases in FEV 1 and FEF 25

- 75 in the absence of a bronchoconstrictor challenge 

### 31 (Kreit et al., 1989, 041817 ) . These results suggest that this group of asthmatics responded to 

### 32 O 3 - exposure with a greater degree of vagally

- mediated bronchoconstriction compared with the non 

### 33 asthmatics. A second study demon strated a statistically significant greater decrease in FEV 1 and 

### 34 FEV 1 /FVC in asthmatics compared with non

- asthmatics exposed to 0.12 ppm O 3 for 7.6 h ours with 

### 35 light exercise (Horstman et al., 1 995, 075834 ) . These responses were accompanied by wheezing and 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 46 inhaler use in the asthmatics ( Horstman et al., 1995, 075834 ) . Aerosol bolus dispersion 

### 1 measurements demonstrated a statistically significant greater change in asthmatics compared with 

### 2

non - asthmatics which was suggestive of O 3 - induced small airway dysfunction (Horstman et al., 

### 3 1995, 075834 ) . Furthermore, a statistically significant correlation was observed between the degree 

### 4 of baseline airway status and the FEV 1 response to O 3 in the asthmatic subjects (Horstman et al., 

### 5 1995, 075834 ) . A third study found similar decreases in FVC and FEV 1 in both asthmatics and non



### 6 asthmatics exposed to 0.4 ppm O 3 for 2 h ours with mild exerc ise (Alexis et al., 2000, 013072 ). 

### 7 However , a statistically significant decrease in FEF 75 , a me asure of small airway function, was 

### 8 observed in asthmatics but not in non

- asthmatics (Alexis et al., 2000, 013072 ) . Taken together these 

### 9 latter studies indicate that while the magnitude of restrictive type spirometric decline was similar in 

### 10 asthmatics and non

- asthmatics, that obstructive type changes (i.e. bronchoconstriction) were greater 

### 11 in asthma tics. Further, asthmatics exhibited greater sensitivity to O 3 in terms of small airways 

### 12 function. 

### 13 Since asthma exacerbations occur in response to allergens and/or other triggers, some studies 

### 14 have focused on O 3 - induced changes in AHR following a bronchoco nstrictor challenge. No 

### 15 difference in sensitivity to methacholine bronchoprovocation was observed between asthmatics and 

### 16

non - asthmatics exposed to 0.4 ppm O 3 for 2 h ours with moderate exercise (Kreit et al., 1989, 

### 17 041817 ) . However, increas ed bronchial reactivity to inhaled allergens was demonstrated in mild 

### 18 allergic asthmatics ( Jorres et al., 1996, 078122 ; Kehrl et al., 1999, 022101 ; Mol fino et al., 1991, 

### 19 042379 ) and in a llergen

- sensitized guinea pigs following O 3 exposure (Sun et al., 1997, 082724 )

. 

### 20 Similar , but modest, responses were reported for individuals with allergic rhinitis (Jorres et al., 1996, 

### 21 078122 )

. Further, t he contractile response of isolated airways was increased by pre- exposure to O 3 in 

### 22 human subjects sensitized and chal lenged with allergen (Roux et al., 1999, 001264 )

. 

### 23 In terms of airways neutrophilia, larger responses were observed in asthmatics compared to 

### 24

non - asthmatics subjects exposed to O 3 in some (Balmes et al., 1997, 086092 ; Basha e t al., 1994, 

### 25 075950 ; Scannell et al., 1996, 080755 ) but not all (Mudway et al., 2001, 025327 ) of the older 

### 26 studies. Further, statistically significantly increases in myeloperoxidase levels (an indicator of 

### 27 neutrophil activation) in bronchial washes was observed in mild asthmatics compared with non



### 28 asthmatics, despite no dif ference in O 3 - stimulated neutrophil influx between the 2 groups following 

### 29 exposure to 0.2 ppm O 3 for 2 h ours with mild exercise (Stenfors et al., 2002, 030473 )

. 

### 30 Eosinophils and associated proteins are thought to affect muscari ni c cholinergic receptor s 

### 31 which are involved in vagally

- mediated bronchocons triction (Mudway and Kelly, 2000, 010452 )

. 

### 32 Studies described in Section 5.2.5.1 which demonstrated a key role of eosinophils in O 3 - mediated 

### 33 AHR may not be entirely relevant to humans given the large numbers of eosinophils normally 

### 34 present in guinea pig airways compared with humans (Yost et al., 2005, 597549 ) . However airways 

### 35 eosinophilia often occurs in human allergic airways dis ease, suggesting greater plausibility of this 

### 36 mechanism in allergic asthmatics. Furthermore, O 3 exposure sometimes often results in airways 

### 37 eosinophilia in allergic subjects or animal models. For example, e osinophilia of the nasal and other 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 47 airways was o bs erved in individuals with pre existing allergic dise ase following O 3 inhalation (Peden 

### 1 et al., 1995, 076189 ; Vagaggini et al., 2002, 035191 ) . Further, O 3 exposure increased allergic 

### 2 responses, such as eosinophilia and augmented intraepithelial mucosubstances, i n the nasal airways 

### 3 of ovalbumin (OVA)

- sensitized rats (Wagner et al., 2002, 026079 )

. In contrast, Stenfors (2002, 

### 4 030473 ) found no stimulation of eosinophil influx measured in bronchial washes and BAL fluid of 

### 5 mild asthmatics following exposure to a lower concentration of O 3 . 

### 6 The role of mast cells in O 3 - mediated asthma exacerbations has been investigated. Mast c ells 

### 7 are thought to play a key role in O 3 -induced airways inflammation, since airways neutrophilia was 

### 8 decreased in mast cell

- deficient mice exposed to O 3 (Kleeberger et al., 1993, 044203 ) . However, 

### 9 another study found that mast cells were not involved in the develo pment of increased bronchial 

### 10 reactivity in O 3 - exposed mice (Noviski et al., 1999, 001198 ) . Nonetheless, mast cells release a wide 

### 11 variety of important inflammatory mediators which may lead to asthma exacerbations (S tenfors et 

### 12 al., 2002, 030473 ) . A large increase in m ast ce ll number in bronchial submucosa was observed in 

### 13

non - asthmatics and a significant decrease in mast cell number in bronchial epithelium was observed 

### 14 in mild asthmatics 6 h ours following exposure to 0.2 ppm O 3 for 2 h ours during mild exercise 

### 15 (Stenfors et al., 2002, 030473 ) . While these results point to an O 3 - mediated flux in bronchial mast 

### 16 cell populations which differed between the non

- asthmatics and mild asthmatics, interpretation of 

### 17 these findings is difficult. Furthermore, mast cell number did not change in airway lavages in either 

### 18 group in response to O 3 (Stenfors et al., 2002, 030473 ) 

### 19 Cytokine profiles in the airways have been investigated as an indicator of O 3 sensitivity. 

### 20 Differences in epithelial cytokine expression were observed in bronchial biopsy samples in non



### 21 asthmatic and asthmatic subjec ts both at baseline and 6 h postexposure to 0.2 ppm O 3 for 2 h ours 

### 22 (Bosson et al., 2003, 051687 ) . The asthmatic subjects had a higher baseline expression of IL

- 4 and 

### 23 IL

- 5 compared to non

- asthmatics. In addition, expression of IL

- 5, IL

- 8, GM

- CSF, and ENA

- 78 in 

### 24 asthmatics was increased significantly following O 3 exposure compared to non

- asthmatics (Bosson 

### 25 et al., 2003, 051687 ) . Some of these (IL

- 4, IL

- 5 and GM

- CSF) are T helper type 2 (Th2)

- related 

### 26 cytokines or neutrophil chemoattractants, and play a role in IgE production, airway eosinophilia and 

### 27 suppression of Th1

- cytokine production (Bosson et al., 2003, 051687 ) . These findings suggest a link 

### 28 between adaptive immunity an d enhanced sensitivity of asthmatics to O 3 . 

### 29 

## 5.2.6.1. New Cellular a n d Mole c ula r Ins igh ts Since asthmatics may have enhanced sensitivity to O 3 , one recent study investigated whether 

### 30 O 3 exposure exacerbated asthmatic responses in persistent asthmatics. These subject s had been 

### 31 therapeutically treated with inhaled corticosteroids for several months prior to the study. Exposure of 

### 32 these subjects to 0.2 ppm O 3 for 2 h ours with mild exercise resulted in decrements in FEV 1 and FVC 

### 33 and an increase in sRaw immediately postex posure (Stenfors et al., 2010, 386512 ) . In addition, large 

### 34 increase s in neutrophil number and myeloperoxidase levels (an indicator of neutrophil activation) in 

### 35 airway lavages and in mast cell number in bronchial submucosa tissue obtained by biopsy was 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 48 obs erved 18 h ours postexposure (Stenfors et al., 2010, 386512 ). No change in bronchial wash or 

### 1 BAL fluid mast cell number or eosinophil number in any compartment was observed. (Stenfors et 

### 2 al., 2010, 386512 ) . These results suggest that some standard treatme nts for asthma may not protect 

### 3 against the effects of environmental O 3 and that this group may be particularly prone to asthma 

### 4 exacerbations given that airways neutrophilia occurred despite the inhaled corticosteroid treatment 

### 5 (Stenfors et al., 2010, 386512 ). 

### 6 Other recent studies in humans and animals provide evidence that O 3 causes the development 

### 7 of allergic responses and/or asthma. Several of these suggested that O 3 - mediated activation of innate 

### 8 immunity contributes to O 3 - induced, as well as to O 3 - ampli fied, allergic responses. In one study 

### 9 involving human subjects, the authors hypothesized that O 3 exposure would result in recruitment of 

### 10 activated innate immune cells to the airways. Healthy individuals were exposed to 0.08 ppm O 3 for 

### 11 6.6 h ours with inter mittent exercise and airways inflammation was characterized in induced sputum 

### 12 18 h postexposure (Ale xis et al., 2010, 628538 )

. Previous studies demonstrated that induced sputum 

### 13 contains liquid and cellular constituents of the lining fluid from central conducting airways (Alexis 

### 14 et al., 2001, 190013 ) and also identified the se airways as a site of preferential O 3 absorption du ring 

### 15 exercise (Hu et al., 1 994, 041323 ). Ozone exposure resulted in s putum neutrophilia and increased 

### 16 numbers of airways monocytes and dendritic

- like cells (Alexis et al., 2010, 628538 )

. In addition, 

### 17 increased expression of cell surface markers characteristic of innate immunity and antigen 

### 18 presentation (i.e. CD

- 14 and HLA

- DR) was demonstrated on airways monocytes (Alexis et al., 2010, 

### 19 628538 ) . Enhanced antigen presentation contributes t o exaggerated T cell responses and promotes 

### 20 Th2 inflammation and an allergic phenotype (Lay et al., 2007, 196610 ) . U pregulation of pro



### 21 inflammatory cytoki nes in sputum was also demonstrated in O 3 - exposed subjects (Alexis et al., 

### 22 2010, 628538 ) . One of these cytokin es, IL

- 12p70, correlated with numbers of dendritic

- like cells in 

### 23 the sputum, and is an indicator of dendritic cell activation (Alexis et al., 2010, 628538 )

. These 

### 24 authors have previously reported that O 3 activates monocytes and macrophages (Lay et al., 2007, 

### 25 196610 ) which could play a role in exacerbating existing asthma by activa ting allergen

- specific 

### 26 memory T

- cells. The current study confirms these findings and extends them by suggesting a 

### 27 potential mechanism whereby O 3 -a ctivated dendritic cells could stimulate n aïve T

- cells to promote 

### 28 the development of asthma (Alexis et al., 2010, 628538 ) . A companion study by these same 

### 29 investigators (described in detail in S ection 5.2.9 .1) provides evidence of dendritic cell activation, 

### 30 measured as increased expression of HLA

- DR, in a subset of the human subjects (GSTM1 null) 

### 31 exposed to 0.4 ppm O 3 for 2 h ours with intermittent exercise (Alexis et al., 2009, 628542 ) 

### 32 Another recent study demonstrated O 3 - mediated activation of the innate immune system and 

### 33 lin ked it to the development of non

- specific AHR in a mouse model (Pichava nt et al., 2008, 596409 )

. 

### 34 Repeated exposure to 1 ppm O 3 for 3 h ours induced non

- specific AHR measured 24 h ours following 

### 35 the last exposure (Pichavant et al., 2008, 596409 ) . This response was found to require NKT cells, 

### 36 which are effector lymphocytes of innate immunity, as well as IL

- 17 and airways neutrophilia 

### 37 (Pichavant et al., 2008, 596409 ) . Since gl ycolipids such as galactosyl ceramide are ligands for the 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 49 invariant CD1 receptor on NKT cells and serve as endogenous activators of NKT cells, a role for 

### 1 O 3 - oxidized lipids in activating NKT cells was proposed (Pichavant et al., 2008, 596409 ) . The 

### 2 authors contrasted this innate immunity pathway with that of allergen

- provoked specific AHR which 

### 3 involves adaptive immunity, the cytokines IL

- 4, IL

- 13, IL

- 17, and airways eosinophilia (Pichavant 

### 4 et al., 2008, 596409 ) . Interestingly, NKT cells were required for both the specific AHR provoked by 

### 5 allergen and the non

- specific AHR provoked by O 3 (Pichavant et al., 2008, 596409 ) . Different 

### 6 cytokine profiles of the NKT cells from all ergen and O 3 - exposed mice in mediating was proposed to 

### 7 account for the different pathways (Pichavant et al., 2008, 596409 ) . More recently, NKT cells have 

### 8 been found to function in both innate and adaptive immunity (Vivier et al., 2011, 676697 ). 

### 9 Priming of the innate immune system by O 3 was reported by Hollingsworth et al., (2007, 

### 10 597609 ) . In this study, exposure of mice to 2 ppm O 3 for 3 h ours led to non specific AHR at 24 and 

### 11 48 h ours postexposure, an effect which subsided by 72 h ours (Hollingsworth et al., 2007, 597609 )

. 

### 12 However in mice treated with aerosolized endotoxin immediately following O 3 exposure, AHR was 

### 13 greatly enhanced at 48 and 72 h ours postexposure (Hollingsworth et al., 2007, 597609 )

. Ozone pre

### 14

e xposure was found to reduce the number of inflammatory cells, to increase cytokine production and 

### 15 total protein in the BAL fluid and to increase systemic IL

- 6 following exposure to endotoxin 

### 16 (Hollingsworth et al., 2007, 597609 ) . Furthermore, O 3 stimulated the apoptosis of alveolar 

### 17 macrophages 24 h ours postexposure, an effect which was greatl y enhanced by endotoxin treatment. 

### 18 Apoptosis of blood monocytes was also observed in response to the combined exposures 

### 19 (Hollingsworth et al., 2007, 597609 )

. Ozone pre

- exposure enhanced the response of lung 

### 20 macrophages to endotoxin (Hollingsworth et al., 2007, 597609 ) . Taken together, these findings 

### 21 demonstrated that O 3 exposure increased innate immune responsiveness to endotoxin. The authors 

### 22 proposed that this effect was mediated by TLR4

- dependent pathways since O 3 increased surface 

### 23 expression of TLR4 on macrophages (Hollin gsworth et al., 2007, 597609 ) . More recently, these 

### 24 authors demonstrated that hyaluronan contributed to the O 3 - primed response to endotoxin (Li et al., 

### 25 2010, 670282 ) . In this study, exposure of mice to 1 ppm O 3 for 3 h resulted in enhanced responses to 

### 26 endo toxin, which was mimicked by intratracheal instillation of hyaluronan fragments (Li et al., 2010, 

### 27 67028 2 ) . Hyaluronan, like O 3 , was also found to induce TLR4 receptor peripheralization in the 

### 28 membrane (Hollingsworth et al., 2007, 597609 ; Li et al., 2010, 670282 ) , an effect which is 

### 29 associated with enh anced responses to endotoxin. This study and previous ones by the same 

### 30 investigators showed elevation of BAL hyaluronan in response to O 3 exposure (Garantziotis et al., 

### 31 2009, 597603 ; Garantziotis et al., 2010, 624947 ; Li et al., 2010, 670282 ) , providing evidence that 

### 32 ozone’s effects on innate immunity are at least in part mediated by hyaluronan fragments. The 

### 33 authors note that excessive TLR4 signaling can lead to lung injury and suggest that O 3 may be 

### 34

re sponsible for an exaggerated innate immune response which may underlie lung injury and 

### 35 decreased host defense (Li et al., 2010, 670282 ) (Section 5.2. 7.1 ). 

### 36 An interaction between allergen and O 3 in the induction of nonspecific AHR was shown in 

### 37 another animal study (Lar sen et al., 2010, 628560 ) . Mice were sensitized with the aerosolized 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 50 allergen OVA on 10 consecutive days followed by exposure to O 3 (0.1-0.5 ppm for 3 h ours ) (Larsen 

### 1 et al., 2010, 628560 ) . While allergen sensitization alone did not alter airway responsiveness to a 

### 2 nonspecific challenge, O 3 exposure of sensitized mice resulted in nonspecific AHR at 6 and 24 h ours 

### 3 postexposure (Larsen et al. , 2010, 628560 ) . The effects of O 3 on AHR were independent of airways 

### 4 eosinophilia and neutrophilia (Larsen et al., 2010, 628560 ) . However, OVA pretreatment led to 

### 5 goblet cell metaplasia which was enhanced by O 3 exposure (Larsen et al., 2010, 628560 ) . It should 

### 6 be noted that OVA sensitization using only aerosolized antigen in this study is less common than the 

### 7 usual procedure for OVA sensitization achieved by one or more initial systemic injections of OVA 

### 8 and adjuvant followed by repeated inhalation exposure to OVA. 

### 9 Furthermore, O 3 was foun d to act as an adjuvant for allergic sensitization (Hollingsworth et 

### 10 al., 2010, 635786 ) . In this study in mice, oropharyngeal aspiration of OVA on day 0 and day 6 failed 

### 11 to lead to allergic sensitization unless mice were first exposed to 1 ppm O 3 for 2 h ours 

### 12 (Hollingsworth et al., 2010, 635786 ) . The O 3 - mediated response involved Th2 (IL

- 4, IL

- 5 and IL

- 9) 

### 13 and Th17 cytokines (IL

- 17) and was dependent on a functioning TLR4 (Hollingsworth et al., 2010, 

### 14 635786 ) . Ozone exposure also activated OVA

- bearing dendritic cells in the thoracic lymph nodes, as 

### 15 measured by the presence of the CD86 surface marker, which suggests naïve T

- cell stimulation and 

### 16 the involvement of Th2 pathways (Hollingsworth et al., 2010, 635786 ) . Thus ozone’s adjuvant 

### 17 effects may be due to activation of both innate and adaptive immunity. 

### 18 Results of recent studies demonstrate participation of innate immune pathways and also 

### 19 suggest involvement of adaptive immune pathways in both the induction and the exacerbation of 

### 20 allergic responses and AHR by O 3 . 

### 21 

## 5.2.6.2. Summary Collectively these older and mor e recent studies provide insight into ozone’s ability to provoke 

### 22 asthma exacerbations in humans. Greater airways inflammation and/or greater bronchial reactivity 

### 23 have been demonstrated in asthmatics compared to non

- asthmatics. This pre

- existing inflammatio n 

### 24 and altered baseline bronchial reactivity may contribute to the enhanced bronchoconstriction seen in 

### 25 asthmatics exposed to O 3 . Furthermore , inflammation may contribute to O 3 - mediated AHR. Animal 

### 26 studies have demonstrated a role for eosinophil

- derived pro teins in mediating these effects. Since 

### 27 airways eosinophilia occurs in both allergic humans and allergic animal models, this pathway may 

### 28 underlie the exacerbation of allergic asthma by O 3 . In addition, differences have been noted in 

### 29 epithelial cytokine exp ression in bronchial biopsy samples of healthy and asthmatic subjects. A Th2 

### 30 phenotype, indicative of adaptive immune system activation and enhanced allergic responses, was 

### 31 observed before O 3 exposure and was increased by O 3 exposure in asthmatics. Since e osinophilia is a 

### 32 hallmark of a Th2 phenotype, these findings support links between allergic asthma, sensitivity to O 3 

### 33 and adaptive immunity. Studies in humans and animal models also provide evidence for activation of 

### 34 innate immunity by O 3 . In humans, O 3 ex posure resulted in increased numbers of airways monocytes 

### 35 and dendritic

- like cells. Altered expression of cell surface markers characteristic of innate immunity 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 51 and antigen presentation was observed on monocytes and macrophages. Recruitment of these 

### 1 activa ted immune cells could lead to activation of allergen

- specific memory T

- cells in allergic 

### 2 individuals and result in the exacerbation of existing asthma in response to an allergen trigger. In 

### 3 animal studies, O 3 exposure primed the innate immune system and led to increased endotoxin



### 4 induced AHR by a mechanism involving hyaluronan and TLR4. The exaggerated immune response 

### 5 to O 3 + endotoxin could lead to a more pronounced lung injury response to a bacterial trigger. 

### 6 Enhanced bronchial reactivity, airways eosinophilia, Th2 phenotype, recruitment of activated innate 

### 7 immune cells, and enhanced responsiveness to endotoxin all provide biological plausibility for 

### 8 epidemiologic evidence of asthma exacerbations associated with exposure to O 3 . Thus, the influx of 

### 9 immuno modulatory cells and the activation of innate and adaptive immunity lead to the exacerbation 

### 10 of asthma and allergic responses which is emerging as a key event in the toxicity pathway of O 3 . 

### 11 Recent studies in humans and animal models also provide evidence that O 3 exposure causes 

### 12 induction of AHR and allergic responses. Both activation of innate immunity and promotion of 

### 13 adaptive immunity have been implicated. In humans, O 3 exposure resulted in increased numbers of 

### 14 dendritic

- like cells and levels of a cytoki ne associated with dendritic cell activation in the sputum, 

### 15 suggesting the presence of a population of activated dendritic cells which could stimulate naïve 

### 16 T- cells to promote the development of asthma. Evidence for activated dendritic cells was also found 

### 17 in GSTM 1 null human subjects ( S ection 5.2. 9 .1) and in allergen

- sensitized animals exposed to O 3 . In 

### 18 the latter study, O 3 acted as an adjuvant for allergic sensitization and the development of AHR by a 

### 19 mechanism involving TLR4. In a different animal model, O 3 - induced AHR required the presence of 

### 20 NKT cells and IL

- 17, both of which indicate innate immune system activation. Ozone -induced 

### 21 goblet cell metaplasia has also been demonstrated. These findings suggest that O 3 may be capable of 

### 22 causing new onset asthma and allergic responses in humans. Thus, promotion of adaptive immunity 

### 23 and activation of innate immunity leads to the induction of AHR and allergic responses which is 

### 24 emerging as a key event in the toxicity pathway of O 3 . 

### 25 

## 5.2.7. Impaire d Hos t De fen s e

O 3 impacts host defense by a variety of different mechanisms . Animal models have 

### 26 demonstrated decreased mucociliary particle clearance and effects on alveolar macrophages, 

### 27 including inhibited phagocytosis and production of reactive oxygen intermediates, and altered 

### 28 chemotaxis and adhesion in response to O 3 exposure (U.S. EPA, 2006, 088089 )

. Ozone has been 

### 29 shown to target SP

- A, resulting in a decrease in its function (U.S. EPA, 2006, 088089 ) . In addition, 

### 30 reduced clearance of bacterial pathogens and enhanced susceptibility to bacterial lung infections 

### 31 were observed in rodents exposed acutely to O 3 (Gilmour et al., 1993, 039620 ; U.S. EPA, 2006, 

### 32 088089 ) . Further, O 3 - induced alterations in immune function have been demonstrated (Jakab et al., 

### 33 1995, 039548 ; U.S. EPA, 20 06, 088089 )

. 

### 34March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 52 

## 5.2.7.1. New Cellular and Molec ular Ins igh ts As described above, priming of the innate immune system by O 3 was reported by 

### 1 Hollingsworth et al. (2007, 597609 ) . Besides effects on AHR, exposure of mice to 2 ppm O 3 for 

### 2 3 hours reduced inflammatory cell influx in the airways in response to endotoxin (Hollingsworth et 

### 3 al., 2007, 597609 ) . In addition, O 3 exposure stimulated the apoptosis of alveolar macrophages 

### 4 24 hours postexposure, an effect which was enhanced by a subsequent treatment with endotoxin 

### 5 (Hollingsworth et al., 2007, 597609 ) . Apoptosis of circulating blood monocytes wa s also observed in 

### 6 response to O 3 and endotoxin (Hollingsworth et al., 2007, 597609 ) . The authors attributed these 

### 7 effects to the increased surface expression of TLR4 and increased signaling in macrophages 

### 8 observed in the study (Hollingsworth et al., 2007, 597609 ) . It was proposed that the resulting 

### 9 decrease in airways inflammatory cells could account for O 3 - mediated de creased clearance of 

### 10 bacterial pathogens observed in numerous animal models (Hollingsworth et al., 2007, 597609 ) . A 

### 11 more recent study by these investigators provided evidence that hyaluronan mediates O 3 - priming of 

### 12 innate immunity and suggested that exaggerated innate immune responses may underlie lung injury 

### 13 and decreased host defense (Li et al., 2010, 670282 )

. 

### 14 Recent studies also demonstrated SP

- A oxidation by O 3 . SP

- A is an important innate immune 

### 15 protein which plays a number of roles in host defense including acting as opsonin for the recognition 

### 16 of some pathogens (Haque et al., 2009, 200767 ) . These investigations demonstrated that O 3 - 

### 17 mediated carbonylation of SP

- A was associated with impaired macrophage phagocytosis in vitro 

### 18 (Mikerov et al., 2008, 596405 ) . Furthermore, O 3 exposure i n mice was found to increase 

### 19 susceptibility to pneumonia infection in mice through an impairment of SP

- A dependent 

### 20 phagocytosis (Mikerov et al., 2008, 201537 ; Mikerov et al., 2008, 597493 )

. 

### 21 Another recent study demonstrated impaired antigen

- specific immunity following subacute O 3 

### 22 exposure (0.6 ppm, 10 h/day for 15 days) in mice (Feng et al., 2006, 596381 ) . Specifically, O 3 

### 23 exposure altered the lymphocyte subset and cytokine profile and impacted thymocyte early 

### 24 development lea ding to immune dysfunction. 

### 25 

## 5.2.7.2. Summary Collectively these older and more recent studies in animal models provide several mechanisms 

### 26 by which O 3 exposure could enhance susceptibility to lung infections. Both decreased mucociliary 

### 27 particle clearance and decrea sed numbers and function of alveolar macrophage have been 

### 28 implicated. Recent studies suggest that O 3 - mediated oxidation of SP

- A oxidation and priming of the 

### 29 innate immune system may contribute to decreased pathogen clearance. Immune dysfunction outside 

### 30 of the lung has also been demonstrated. Thus, immune system modulation resulting in impaired host 

### 31 defense is emerging as a key event in ozone’s toxicity pathway. 

### 32March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 53 

## 5.2.8. Extrapulmonary Effects
Extrapulmonary effects of O 3 have been noted for decades (U.S. EPA, 2006, 088089 ) . One 

### 1 such effect is hypothermia, which in rodents occurs subsequ ent to the activation of neural reflexes 

### 2 and involves the parasympathetic nervous system (Watkinson et al., 2001, 016245 ) . Other 

### 3 mechanisms are likely to be involved in extrapulmonary effects. It has been proposed that lipid 

### 4 oxidation products resulting from reaction of O 3 with lipids in the ELF are responsible for systemic 

### 5 effects, however it is not known whether they gain access to the vascular space (Chuang et al. , 2009, 

### 6 197202 ) . Alternatively, extrapulmonary release of diffusible mediators may initiate or propagate 

### 7 inflammatory responses in the vascular or in system ic compartments (Col e and Freeman, 2009, 

### 8 597507 ) . For example, one such mediator, the cytokine IL

- 6, is known to have pleitropic effects such 

### 9 as inducing the acute phase respon se, activating the hypothalamus

- pituitary

- adrenal axis and altering 

### 10 serum cholesterol levels (Tarrant, 2010, 644810 )

. 

### 11 

## 5.2.8.1. Ca rdiova s c ula r Effe c ts
Effects of O 3 on the cardiovascular system hav e been demonstrated in both humans and animal 

### 12 models (U.S. EPA, 2006, 08808 9 )

. Several mechanisms have been proposed to account for these 

### 13 responses (Perepu et al., 2010, 385020 ) . First, O 3 may impair alveolar

- arteri al oxygen transfer and 

### 14 reduce the supply of arterial oxygen to the myocardium. This may have a greater impact in 

### 15 individuals with compromised cardiopulmonary systems. Gong et al. (1998, 029938 ) provided 

### 16 evidence of a small decrease in arterial oxygen saturation in human subjects exposed to O 3 . In 

### 17 addition, Delaunois et al. (1998, 015779 ) demonstrated pulmonary vasoconstriction in O 3 -exposed 

### 18 rabbits. Although of interest, the contribution of this pathway to O 3 - induced cardiovascular effects 

### 19 remains uncertain. Se condly, O 3 may trigger neural reflexes which stimulate the autonomic nervous 

### 20 system and alter electrophysiologic responses of the heart. For example, bradycardia, altered HRV 

### 21 and arrhythmia have been demonstrated in animals exposed to O 3 (Arito et al., 1990, 042285 ; 

### 22 Hamade and Tankersley, 2009, 596386 ; Watkinson et al., 2001, 016245 ) . Third, O 3 - induced 

### 23 p ulmonary inflammation may lead to inflammatory or injury responses in the cardiovascular system 

### 24 (Cole and Freeman, 2009, 597507 )

. 

### 25 Some recent studies have suggested that O 3 ma y alter the systemic vasculature. In resting 

### 26 humans, exposure to fine particulate matter (PM) + O 3 resulted in arterial vasoconstriction and 

### 27 increased diastolic blood pressure during the 2

- h exposure (Brook et al., 2002, 024987 ) . However, a 

### 28 recent study in humans observed no changes in HRV or blood pressure in healthy nonsmokers 

### 29 exposed at rest to 0.12 ppm O 3 for 2 h ou rs (Brook et al., 2009, 195611 ) . Thus, the previously 

### 30 observed effects of the combined O 3 and PM exposure may have been attributable to PM alone. A 

### 31 role for O 3 in modulating endothelin, a potent vasoconstrictor, has also been proposed. Studies in 

### 32 animals found that O 3 - induced endothelin system genes in the lung and increased circulating levels 

### 33 of endothelin (Thomson et al., 2005, 087554 ; Thomson et al., 2006, 097483 )

. 

### 34March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 54 

### Recent Cellular and Molecular Insights
Ozone

- induced perturbations of the cardiovascular system were recently investigated in young 

### 1 mice and monkeys (Chuang et al., 2009, 197202 ) . Young mice exposed to 0.5 ppm O 3 for 5 day s 

### 2 demonstrated increased heart rate and blood pressure. Decreases in endothelial

- dependent 

### 3 vasorelaxation and NO homeostasis were observed in arterial tissue. Oxidative and nitrosative stress 

### 4 were demonstrated in lung and aortic tissue following O 3 exposur e. Mitochondrial DNA was 

### 5 damaged in lung and aortas from young mice and infant rhesus monkeys similarly exposed to O 3 . 

### 6 Since altered NO homeostasis, mitochondrial DNA damage and oxidative stress are known to 

### 7 contribute to the development of atherosclerosis , young Apo E null mice were exposed 

### 8 subchronically to O 3 (0.5 ppm O 3 for 5 d ays /week over 8 week s ). Ozone exposure augmented the 

### 9 aortic lesion areas compared with controls exposed to filtered air, suggesting an acceleration of 

### 10 atherogenesis. This is the f irst study to definitively identify the systemic vasculature as a target of 

### 11 O 3 - induced effects. 

### 12 A second recent study in animals demonstrated effects on the heart due to chronic O 3 exposure 

### 13 (Perepu et al., 2010, 385020 ) . Rats were exposed to 0.8 ppm O 3 for 28 and 56 d ays and isolated 

### 14 hearts were subjected to ischemia

- reperfusion injury. Ozone exposure enhanced the sensitivity to 

### 15 injury in this model, as demonstrated by decreased cardiac function compared with control rats that 

### 16 were exposed to filtered air. Further, markers of lipid peroxidation and inflammation were greater in 

### 17 the hearts of O 3 - exposed rats. This study is the first to definitivel y identify the heart as a target for 

### 18 O 3 - induced effects. 

### 19 Further evidence for O 3 - induced effects in the systemic vasculature and heart is provided by a 

### 20 recent chronic study in rats (Kodavanti et al., In Press, 666323 ) . Episodic exposure to 0.4 ppm O 3 for 

### 21 16 weeks (5 h /d ay for 1 d ay /w ee k) resulted in increased aortic levels of mRNA for biomarkers of 

### 22 oxidative stress, thrombosis, vasoconstriction and prot eolysis. Ozone exposure also increased lectin



### 23 like oxidized

- low density lipoprotein receptor

- 1(LOX

- 1) mRNA and protein levels in the aorta. 

### 24 Depletion of cardiac mitochondrial phospholipid fatty acids was also observed. Taken together, these 

### 25 results suggest a role for circulating oxidized lipids in mediating the effects of O 3 . 

### 26 

## 5.2.8.2. Hepa tic Effe c ts Changes in hepatic gene expression have also been noted following O 3 exposure. Specifically, 

### 27 downregulation of gene families related to lipid, fatty acid and carbohydr ate metabolism was 

### 28 demonstrated in the liver of O 3 - exposed mice (Last et al., 2005, 596400 ) . In addition, transcription of 

### 29 enzymes involved in xenobiotic metabolism was decreased. Impairment of hepatic drug metabolism 

### 30 was suggested in an older study which found that mice exposed to O 3 had prolonged pentobarbital 

### 31 sleeping time (Graham et al., 1985, 040289 ) . Further evidence of hepatic effects is provided by a 

### 32 recent study in which exposure to 0.25 and 0.5 ppm O 3 for 6 h ours resulted in exacerbation of drug



### 33 induced liver injury in mice pre

- exposed to acetaminophen. This included a greater increase in 

### 34March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 55 hepatic neutrophil accumulation and greater alteration in gene expression profiles in mice exposed to 

### 1 O 3 and acetaminoph en compared with either exposure alone (Aibo et al., 2010, 378559 )

. 

### 2 

## 5.2.8.3. Summary Collectively, these older and more recent studies in animal models provide evidence for 

### 3 extrapulmonary effects of O 3 . Although it was suggested that these effects are directly mediated by 

### 4 secondary oxidation products formed in the lung as a result of O 3 exposure, there is no evidence that 

### 5 these species enter the circulation. Alternatively, extrapulmonary effects may be due to activation of 

### 6 neural reflexes or to release of diffusible mediators which may initiate or propagate inflammatory 

### 7 responses in the vascular or systemic compartments. Recent studies suggest that oxidative/nitrosative 

### 8 stress contributes to O 3 - induced cardiovascular effects. Thus, systemic inflammation and vascular 

### 9 oxidative/nitrosative stress are emerging as key events in the toxic ity pathway of O 3 . 

### 10 

## 5.2.9. Fa c to rs Affe c tin g Res po n s e s to Ozone
Responses to O 3 are variable within the population and the basis for this variability is n ot clear 

### 11 (Mudway and Kelly, 2000, 010452 ) . Research has focused on the role of gen e- environme nt 

### 12 interactions, pre existing conditions, adaptive mechanisms and lifestage in influencing the responses 

### 13 to O 3 . Co

- exposure to other pollutants has also been considered. 

### 14 

## 5.2.9.1. Gene -E nvironment I nteractions The significant inter

- individual variation in responses to O 3 infers that genetic background is 

### 15 an important determinant of susceptibility to O 3 (Cho and Kleeberger, 2007, 195616 ; Kleeberger et 

### 16 al., 1997, 095736 ) . Strains of mice which are prone or resistant to O 3 - induced effects have been used 

### 17 to systematically identify cand idate susceptibility genes. Genome wide linkage analyses (also known 

### 18 as positional cloning) demonstrated quantitative trait loci for O 3 - induced lung inflammation and 

### 19 hyperpermeability on chromosome 17 (Kleeberger et al., 1997, 095736 ) and chromosome 4 

### 20 (Kleeberger et al., 2000, 014895 ) , respectively, using these recombinant inbred strains of mice. More 

### 21 specifically these studies found that Tnf, whose protein product is the inflammatory cytokine TNF

- α , 

### 22 and Tlr4, whose protein product is TLR4, were candidate susceptibility genes (Kleeberger et al., 

### 23 1997, 095736 ; Kleeberger et al., 2000, 014895 ) . Other investigations in inbred mouse strains found 

### 24 that differences in expression of certain proteins, such as CCSP (Broeckaert et al., 2003, 055490 ) and 

### 25 MARCO (Dahl et al., 2007, 196986 ) , are responsible for phenotypic characteristics, such as 

### 26 epithelial permeability and scavenging of oxidize d lipids, respectively, which confer sensitivity to 

### 27 O 3 . 

### 28 Genetic polymorphisms have received increasing attention as modulators of O 3 - mediated 

### 29 effects. Functionally relevant polymorphisms in candidate susceptibility genes have been studied at 

### 30 the individual and population level in humans and also in animal models. Genes whose protein 

### 31March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 56 products are involved in antioxidant defense/oxidative stress and xenobiotic metabolism, such as 

### 1 glutathione -S- transferase M1 (GSTM1) and NADPH:quinone oxidoreductase 1 (NQO1), have also 

### 2 been major focuses of these efforts. This is because oxidative stress resulting from O 3 exposure is 

### 3 thought to contribute to the pathogenesis of asthma and because xenobiotic metabolism detoxifies 

### 4 secondary oxidation products formed by O 3 which contribute to oxidative stress (Islam et al., 2008, 

### 5 097348 ) . TNF

- α is of interest since it is linked to a candidate O 3 susceptibility gene and since it plays 

### 6 a key role in initiating airways inflammation (Li et al., 2006, 090972 ) . Polymorphisms of genes 

### 7 coding for GST M1, NQO1 and TNF

- α have been associated with altered susceptibility to O 3 - 

### 8 mediated effects (Bergamaschi et al., 2001, 052670 ; Corradi et al., 2002, 035448 ; Li et al., 2006, 

### 9 090972 ; Romieu et al., 2004, 056796 ; Yang et al., 2005, 077211 ) . Additional studies have focused on 

### 10 functional variants in other genes involved in antioxidant defense such as catalase (CAT), 

### 11 myeloperoxidase, heme oxygenase (HMOX

- 1) and manganese superoxide dismutase (MnSOD) 

### 12 (Islam et al., 2008, 097348 ; Wenten et al., 2009, 597084 ) . These studies are discussed below. 

### 13 GST M1 is a phase II antioxidant enzyme which is transcriptionally regulated by NF

- E2

- 

### 14 related factor 2

- antioxidant response element (Nrf2

- ARE) pathway. A large proportion (40

- 50%) of 

### 15 the general public (across ethnic populations) has the GSTM1 null genotype, which has been linked 

### 16 to an increased risk of adverse health effects due to exposure to air pollutants (London, 2007, 

### 17 093279 ) . A role for GSTs in me tabolizing electrophiles such as 4

- hydroxynonenal, which is a 

### 18 secondary oxidation product formed following O 3 exposure, has been demonstrated (Awasthi et al., 

### 19 2004, 644649 ) . A recent study found that the GSTM1 genotype modulated the time course of the 

### 20 neutrophilic inflammatory response following acute O 3 exposure (0.4 ppm for 2 h ours with 

### 21 intermittent exercise) in healthy adults (Alexis et al., 2009, 628542 ) . In GSTM1

- null and sufficient 

### 22 subjects, O 3 - induced sputum neutrophilia was similar at 4 h ours . However, neutrophilia resolved by 

### 23 24 hours in sufficient subjec ts but not in GSTM1

- null subjects. It is not known whether this effect 

### 24 was due to the persistence of pro

- inflammatory stimuli, impaired production of downregulators or 

### 25 impaired neutrophil apoptosis and clearance. In addition, O 3 exposure increased the expression of the 

### 26 surface marker CD14 in airway neutrophils of GSTM

- 1 null subjects compared with sufficient 

### 27 subjects. Furthermore, numbers of airway macrophages were decreased at 4 and 24 h ours following 

### 28 O 3 exposure in GSTM 1- sufficient subjects (Alexis et al., 2009, 628542 ) . Airways macrophages in 

### 29 GSTM 1 null subjects were greater in number and found to have greater oxidative burst and 

### 30 phagocytic capability than those of sufficient subjects. Airways macrophages and dendritic cells 

### 31 from GST M1 null subjects exposed to O 3 expressed higher levels of the surface marker HLA

- DR, 

### 32 suggesting activation of the innate immune system (Alexis et al., 2009, 628542 ) . These differences 

### 33 in inflammatory responses between the GSTM1 null a nd sufficient subjects may provide biological 

### 34 plausibility for the differences in O 3 - mediated effects reported in controlled human exposure studies 

### 35 (Bergamaschi et al., 20 01, 052670 ; Corradi et al., 2002, 035448 ) . It should also be noted that 

### 36 GSTM1 genotype did not affect the acute pulmonary function (spirometric) response to O 3 which 

### 37 provides additional evidence for separate mechanisms underlying ozone’s effects on pulmonary 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 57 function and inflammation in adults (Alexis et al., 2009, 628542 ) . However , GSTM 1 null asthmatic 

### 1 children were previously found to be more sensitive to the effects of O 3 on pulmonary function than 

### 2 GSTM 1 sufficient asthmatic children (Romieu et al., 2004, 056796 ). 

### 3 NQO1 catalyzes the 2

- electron reducti on by NADPH of quinones to hydroquinones. 

### 4 Depending on the substrate, it is capable of both protective detoxification reactions and redox 

### 5 cycling reactions resulting in the generation of reactive oxygen species. A recent study using NQO1

- 

### 6 null mice demonstr ated that NQO1 contributes to O 3 - induced oxidative stress, AHR and 

### 7 inflammation in mice (Voynow et al., 2009, 194311 ) . These experimental results may provide 

### 8 biological plausibility for the increased biomarkers of oxidative stress and increased pulmonary 

### 9 function decrements observed in O 3 - exposed individuals bearing both the wild -type NQO1 gene and 

### 10 the null GSTM1 gene (Bergamaschi et al., 2001, 052670 ; Cor radi et al., 2002, 035448 )

. 

### 11 Two studies reported relationships between TNF promoter variants and O 3 - induced effects in 

### 12 humans. In one study, O 3 - induced chan ge in lung function was significantly lower in adult subjects 

### 13 with TNF promoter variants

- 308A/A and

- 308G/A compared with adult subjects with the variant

- 

### 14 308G/G (Yang et al., 2005, 077211 ) . This response was modulated by a specific polymorphism of 

### 15 LTA (Yang et al., 2005, 077211 ) , a previously identified candidate susceptibility gene whose protein 

### 16 product is lymphotoxin

- α (Kleeberger et al., 1997, 095736 ) . In the second study, an association 

### 17 between the TNF promoter v ariant

- 308G/G and decreased risk of asthma and lifetime wheezing in 

### 18 children was found (Li et al., 2006, 090972 ) . The protective effect on wheezing was modulated by 

### 19 ambient O 3 levels and by GSTM1 and GSTP1 polymorphisms. The authors suggested that the 

### 20 TNF

- 308 G/G genotype may have a protective role in the development of childhood asthma (Li et 

### 21 al., 2006, 090972 ). 

### 22 Similarly, a promoter variant of the gene HMOX

- 1, consisting of a smaller number of (GT)n 

### 23 repeats, was associated with a reduced risk for new

- onset asthma in non

- Hispanic white children 

### 24 (Islam et al., 2008, 097348 ) . The number of (GT)n repeats in this promoter has been shown to be 

### 25 inversely related to the inducibility of HMOX

- 1. A modulatory effect of O 3 was demonstrated since 

### 26 the beneficial effects of this polymorphism were seen only in children living i n low O 3 communities 

### 27 (Islam et al., 2008, 097348 ) . This study also identified an association between a polymorphism of the 

### 28 CAT gene and increased risk of new

- on set asthma in Hispanic children; however no modulation by 

### 29 O 3 was seen (Islam et al., 2008, 097348 ) . No association was observed in this study between a 

### 30 MnSOD polymorphism and asthma (Islam et al., 2008, 097348 ). 

### 31 Studies to date indicate that some variability in individual responsiveness to O 3 may be 

### 32 accounted for by functional genetic polymorphisms. Further, the effects of gene

- environment 

### 33 int eractions may be different in children and adults. 

### 34 

## 5.2.9.2. Pre exis ting D is eas es and C onditions Several preexisting diseases and conditions have been described which modulate the response 

### 35 to O 3 exposure. Atopy and asthma are important factors in humans. For example , asthmatics were 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 58 more susceptible to O 3 - mediated inflammation (Balmes et al., 1997, 086092 ; Basha et al., 1994, 

### 1 075950 ; Scannell et al., 1996, 080755 ) , while not exhibiting any increase in responsiveness as 

### 2 measured by spirometry. Ozone exposure resulted in eosinophilia of the nasal and lower airways in 

### 3 individuals with preexisting allergic disease (Peden et al., 1995, 076189 ; Vagaggini et al., 2002, 

### 4 035191 ) and increased bronchial reactivity to challenge with inhaled s pecific antigen in mild allergic 

### 5 asthmatics (Jorres et al., 1996, 078122 ; Kehrl et al., 1999, 022101 ; Molfino et al., 1991, 042379 )

. 

### 6 Increased bronchial reactiv ity was also reported in individuals with allergic rhinitis (Jorres et al., 

### 7 1996, 078122 ). It has been proposed that s ensitivity is c onferred b y the presence of greater numbers 

### 8 of resident airway inflammatory cells in disease states such as asthma (Mudway and Kelly, 2000, 

### 9 010452 ). 

### 10 In addition, smoking and COPD status are important determinants since responsiveness to O 3 , 

### 11

as measure d by spirometry, is decreased in individuals with these conditions (U.S. EPA, 2006, 

### 12 088089 ) . Furthermore, obesity may alter susceptibility. In a reanalysis of the data of Hazucha (2003, 

### 13 048168 ) , increasing body mass index in young women was assoc iated with incr eased O 3 

### 14 responsiveness (Bennett et al., 2007, 418827 ) . In animal models, diet

- induced obesity augmented 

### 15 inflammation and injury, as measured by BAL markers, as well as innate AHR, in mice exposed 

### 16 acutely to O 3 (Johnston et al., 2008, 597625 ) . In contrast, the inflammatory response following sub



### 17 acute exposure to O 3 was dampened by obesity in a different mouse model (Shore et al., 2009, 

### 18 20155 1 ). Finally, nutritional status may impact the response to O 3 . Many investigations have focused 

### 19 on antioxidant deficiency as a modulator of O 3 - mediated effects (see above). Although results of 

### 20 these studies are mixed, studies in humans demonstrate that s upplementation with ascorbate and 

### 21 alpha- tocopherol was protective against O 3 - induced pulmonary function deficits in healthy adults 

### 22 who were ascorbate

- deficient (Samet et al., 2001, 019034 ) and in asthmatic children living in 

### 23 Mexico City (Romieu e t al., 2002, 034711 ; Sienra

- Monge et al., 2004, 196422 ) . F urthermore, 

### 24 supplementation with ascorbate, alpha

- tocopherol and beta

- carotene was found to be protective in 

### 25 Mexico City streetworkers (Romieu et al., 1 998, 086756 )

. 

### 26 

## 5.2.9.3. Life s ta ge : Pos tna tal developme nt An interesting set of studies conducted over the last 10 y ears in the infant rhesus monkey has 

### 27 identified num erous O 3 - mediated perturbations in the developing lung and immune system (Plopper 

### 28 et al., 2007, 596412 ) . These investigations were prompted b y the dramatic rise in the incidence of 

### 29 childhood asthma and focused on the possible role of O 3 and allergens in promoting remodeling of 

### 30 the epithelial

- mesenchymal trophic unit during postnatal development of the tracheobronchial 

### 31 airway wall. Rhesus monkey s were used in these studies because the branching pattern and 

### 32 distribution of airways in this model are more similar to humans than are those of rodents to humans. 

### 33 In addition, a model of allergic airways disease, which exhibits the main features of human asthma, 

### 34 had already been established in the adult rhesus monkey. Studies in infant monkeys were designed to 

### 35 determine whether repeated exposure to O 3 altered postnatal growth and development, and if so, 

### 36March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 59 whether such effects were reversible. In addition, e xposure to O 3 was evaluated for its potential to 

### 1 increase the development of allergic airways disease. Infant rhesus monkeys (30 days old) were 

### 2 exposed to cyclic episodic O 3 over a 5

- month period. This involved biweekly cycles of alternating 

### 3 O 3 (5 consecutive days of 0.5 ppm O 3 , 8 h/d ay ) and filtered air (9 consecutive days). Some animals 

### 4 were sensitized to house dust mite allergen (HDMA) and then exposed to HDMA aerosol for 2 h/day 

### 5 on days 3

- 5 of either filtered air or O 3 exposure. 

### 6 Key findings are numerou s. First, baseline airway resistance and AHR in the infant monkeys 

### 7 were dramatically increased by combined exposure to both HDMA and O 3 (Joad et al., 2006, 

### 8 596390 ; Schelegle et al., 2003, 053778 ) . Secondly, O 3 exposure alone led to a large increase in BAL 

### 9 eosinophils (Schelegle et al., 2003, 053778 ) while HDMA exposure alone led to a large increase of 

### 10 eosinophils in airways tissue (Joad et al., 2006, 596390 ; Schelegle et al., 2003, 053778 ) . Thirdly, th e 

### 11 growth pattern of distal airways was significantly changed by exposure to O 3 alone and in 

### 12 combination with HDMA. More specifically, longer and narrower airways resulted and the number 

### 13 of conducting airway generations between the trachea and the gas excha nge area was decreased 

### 14 (Fanucchi et al., 2006, 096491 ) . This latter effect was not ameliorated by a recovery period of 

### 15 6 mon ths . Fourthly, exposure to both HDMA and O 3 altered the abundance and distribution of 

### 16 CD25+ lymphocytes in the airways (Miller et al., 2009, 596406 ) . Lastly, several effects were seen at 

### 17 the level of the epithelial mesenchymal trophic unit in response to O 3 . These include altered 

### 18 organization of the airways epithelium (Schelegle et al., 2003, 053778 ) , increased abundance of 

### 19 mucous goblet cells (Schelegle et al., 2003, 053778 ) , disruption of the basement membrane zone 

### 20 (Evans et al., 2004 , 596379 ) , reduced innervation (Larson et al., 2004, 057062 ) , increased 

### 21 neuroendocrine

- like cells (Joad et al., 2006, 596390 ) , and altered o rientation and abundance of 

### 22 smooth muscle bundles (Pl opper et al., 2007, 596412 ; Tran et al., 2004, 628626 ) . S ix months of 

### 23 recovery in filtered air led to reversal of some but not all of these effects (Evans et al., 2004, 596379 ; 

### 24 Kajekar et al., 2007, 567661 ; Plopper et al., 2007, 596412 ) . The authors concluded that cyclic 

### 25 challenge of infant rhesus m onkeys to allergen and O 3 during the postnatal period compromised 

### 26 airway growth and development and resulted in changes which favor allergic airways responses 

### 27 (Plopper et al., 2007, 596412 )

. 

### 28 Nasal mucous membranes are also a target of O 3 - mediated effects. The infant rhesus monkey 

### 29 was used as a model since its nasal airways are similar to those of children (Carey et al., 2007, 

### 30 195752 ) . Lesions in airways epithelium in the developing nasal passages of immature monkeys were 

### 31 determined following both acute (5 consecutive days of 0.5 ppm 8 h/d ay ) and cyclic episodic (as 

### 32 described above) O 3 exposure. Similar effects were observed in response to acute and episodic O 3 . 

### 33 Histological analysis demonstrated necrotizing rhinitis in the nasal mucosa lining of the main nasal 

### 34 chamber and focal regions of epithelial exfoliation, especially in the anterior maxilloturbinate. An 

### 35 anterior to posterior decrease was observed in the severity of these lesions. Morphometric analysis 

### 36 demonstrated a 65% reduction in the mean thickness of the nasal epithelium in the anterior 

### 37 maxilloturbinate and loss of v olume density of airway cilia, epithelial cytoplasm and nuclei. The 

### 38March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 60 authors reported that the O 3 - induced nasal lesions observed in this study are similar to those reported 

### 1 for adult monkeys. However, unlike the adult monkeys, no epithelial hyperplasia or m etaplasia was 

### 2 observed in the young monkeys suggesting that persistent necrotizing rhinitis may be the long term 

### 3 sequelae in the absence of protective adaptations. 

### 4 Effects of O 3 on early postnatal airways development has also been studied in rats. A recen t 

### 5 study demonstrated that O 3 exposure during critical postnatal periods resulted in increased SP nerve 

### 6 fiber density in lung smooth muscle (Hunter et al., 2010, 382064 ) . The authors proposed that O 3 may 

### 7 lead to enhanced responsiveness of airway sensory nerves. Another study found increases in 

### 8 immediate

- early gene responses in airways epithelium of rats exposed postnatally to O 3 (Johnston et 

### 9 al., 2006, 097439 ) . Further, neonatal mice exhibit strain

- specific differential susceptibility to O 3 

### 10 (Vancza et al., 2009, 596419 )

. 

### 11 

## 5.2.9.4. Lifes tage : Aging On the other side of the lifestage spectrum is aging. The spirometri c response to O 3 is lost in 

### 12 humans as they age (Drechsler

- Parks, 1995, 076085 ; Hazucha et al., 2003, 048168 ) . In mice, 

### 13 physiological responses to O 3 were also attenuated with age (Hamade et al., 2010, 666324 )

. 

### 14 Mechanisms accounting for this effect have not been well

- studied but could include altered number 

### 15 and sensitivity of receptors or altered signaling pathways involved in neural reflexes

. 

### 16 

## 5.2.9.5. Ada pta tio n The decrease in pulmonary function and increase in bronchoconstriction, airways 

### 17 inflammation and bronchia l reactivity observed on the first and second days of consecutive daily 

### 18 exposure in response to O 3 were not seen after 4 or 5 days (see above). Several mechanisms have 

### 19 been postulated. First, the upregulation of antioxidant defenses (or conversely, a decre ase in critical 

### 20 O 3 - reactive substrates) may protect against O 3 - mediated adverse effects. Increases in antioxidant 

### 21 content of the BAL have been demonstrated by Devlin (1997, 083577 ) , Tepper (1989, 041991 ) , and 

### 22 others. Second, IL

- 6 was demonstrat ed to be an important mediator of adaptation (McKinney et al., 

### 23 1998, 086751 ) . Third, a protective role for increases in mucus producing cells and mucus 

### 24 concentra tions in the airways has also been proposed (Devlin et al., 1997, 083577 ) . Fourth, epithelial 

### 25 hyperplasia or metaplasia ma y decrease susceptibility to subsequent O 3 challenge (Carey et al., 2007, 

### 26 195752 ; Harke ma et al., 1987, 040816 ; Harkema et al., 1987, 041496 ) . The se morphologic changes 

### 27 have been observed in nasal and lower airways. Although there is some evidence to support these 

### 28 possibilities, there is no consensus on mechanisms underlying adaptation. Recent studies 

### 29 demonstrating that O 3 activates TRP receptors su ggest that modulation of TRP receptor number or 

### 30 sensitivity by repeated O 3 exposures may also contribute to adaptation. 

### 31March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 61 

## 5.2.9.6. Co

- Exposures w ith Pa rtic ula te Ma tte r
Numerous studies have investigated the effects of co

- exposure to O 3 and PM because of the 

### 1 prevale nce of these pollutants in ambient air. Results are highly variable and depend on whether 

### 2 exposures are simultaneous or sequential, the type of PM employed and the endpoint examined. In 

### 3 humans, simultaneous exposure to O 3 (0.12 ppm for 2 h at rest) and CAP s resulted in a diminished 

### 4 systemic IL

- 6 response compared with exposure to CAPs alone (Urch et al., 2010, 387113 )

. 

### 5 Exposure to O 3 alone did not alter blood IL

- 6 levels (Urch et al., 2010, 387113 ) . The authors 

### 6 provided evidence that O 3 mediated a switch to shallow breathing which may have accounted for this 

### 7 effect (Urch et al., 2010, 387113 ) . Further, simultaneous exposure to CAPs and O 3 , but not exposure 

### 8 to either alone, resulted in increased diastolic blood pressure in human subjects (Fakhri et al., 2009, 

### 9 191914 ) . In some strains of mice, pre

- exposure to O 3 (0.5 ppm for 2 h ours ) modulate d the effects of 

### 10 carbon black PM on heart rate, HRV and breathing patterns (Hamade and Tankersley, 2009, 596386 )

. 

### 11 Another recent study in mice demonstrated that treatment with carbon nanotubes followed 12 h ours 

### 12 later by O 3 exposure (0.5 ppm for 3 h ours ) resulted in a dampening of some of the pulmonary effects 

### 13 of carbon nanotubes meas ured as markers of inflammation and injury in the BAL (Han et al., 2008, 

### 14 596387 ) . The authors suggest that this may represent “ cross -tolerance. ” Harkema et al. (2005, 

### 15 078340 ) found that epithe lial and inflammatory responses in the airways of rats were enhanced by 

### 16

co - exposure to O 3 and LPS (used as a model of biogenic PM) or to O 3 and OVA (used as a model of 

### 17 an aeroallergen). Furthermore, o ne recent study demonstrated maternal

- fetal effects of P M exposure 

### 18 on O 3 responses. In this study, maternal exposure to PM resulted in augmented lung mediators of 

### 19 inflammation, airway epithelial mucous metaplasia and enhanced O 3 - mediated AHR in young mice 

### 20 (Auten et al., 2009, 200760 ). Overall, these findings are hard to interpret but demonstrate the 

### 21 complexity of interactions between PM and O 3 exposures. 

### 22 

## 5.2.9.7. Summary Collectively, these older and more recent studies provide evidence for mechanisms which may 

### 23 underlie the variability in responsiveness seen among individuals. Certain functional genetic 

### 24 polymorphisms, pre

- existing conditions and diseases, lifestages and co

- exposures contribute to 

### 25 enhanced susceptibility to O 3 . Adaptation may also be important, but it is inc ompletely understood, 

### 26 both in terms of the pathways involved and the resulting consequences. 

### 27 

## 5.2.10. Overall Summary
Key events in the toxicity pathway of O 3 have been identified in humans and animal models. 

### 28 They include the formation of secondary oxidation products in the lung, activation of neural reflexes, 

### 29 pulmonary injury and inflammation and increased bronc hial reactivity. In addition, evidence is 

### 30 accumulating that immune system modulation may lead to impaired host defense and the 

### 31March 20 11 DRAFT – DO NOT CITE OR QUOTE 5

- 62 exacerbation and/or induction of asthma and allergic responses (Figure 5

- 6). Systemic inflammation 

### 1 and vascular oxidative/nitrosa tive stress may be critical to the extrapulmonary effects of O 3 . 

### 2



### 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 inte rest.


## 5.2.11. Gaps in Knowledge
Despite a vast body of knowledge regarding the effects of O 3 exposure, the current 

### 3 understanding of mechanisms underlying important health effects in humans is incomplete. 

### 4 Additional research will be useful to elucidate the biologic pathways by which exposure to O 3 : 

### 5

 Primes the immune system, including promotion of adaptive immunity and activation of 

### 6 innate immunity 

### 7

 Alter s early postnatal development of the lung and immune system 

### 8

 Affects the cardiovascular system 

### 9March 2011 5- 62 DRAFT – DO NOT CITE OR QUOTE 

# References

A lis t 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&gro up_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=searc h.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

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- Sanchez, D.; Kim, C.; Devlin, R. B.; Peden, D. B. (2010). Low

- level ozone exposure induces a irways inflammation and modifies cell surface phenotypes in healthy humans. Inhal Toxicol, 22: 593

- 600. http://dx.doi.org/10.3109/08958371003596587 628538 Al exis, 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 ozone

induced airway inflammati on 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.; Becke r, 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 breat hing 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

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- 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

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