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## 5 DOSIMETRY AND MODE O F ACTION

5.1 Introduction

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

### 1 the dosimetry of O 3 and to discuss factors which influence it. The second is to describe 

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

### 3
7. This chapter is not intended to be a comprehensive overview, but rather, it updates the 

### 4 basic concepts de r ived from O 3 literature presented in previous documents ( U.S. EPA, 

### 5 2006b
, 1996a ) and introduces the recent relevant literature. 

### 6
In Section 5.2, particular attention is given to dosimetric factors influencing individual 

### 7 risk of developing effects from O 3 exposure. As there have been few O 3 dosimetry 

### 8 studies publishe d since the last AQCD, the reader is referred to previous documents ( U.S. 

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

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

### 11 2005, in the areas of uptake, reactions, and models for O 3 dosimetry, is discussed. 

### 12
Section 5.3 highlights findings of studies published since the 2006 O 3 AQCD, which 

### 13 provide insight into the biological pathways by which O 3 exer ts its actions. Since 

### 14 common mechanisms lead to health effects from both short

- and long

- term exposure to 

### 15 O 3 , these pathways are discussed in Chapter 5 rather than in later chapters. The relevant 

### 16 sections of Chapters 6 and 7 are indicated. Older studies wh ich represent the current state 

### 17 of the science are also discussed. Studies conducted at more environmentally

- relevant 

### 18 concentrations of O 3 are of greater interest, since mechanisms responsible for effects at 

### 19 low O 3 concentrations may not be identical to th ose occurring at high O 3 concentrations. 

### 20 The topics of dosimetry and mode of action are bridged by reactions of O 3 with 

### 21 components of the extracellular lining fluid (ELF), which play a role in both O 3 uptake 

### 22 and biological responses (Figure 5

- 1). 

### 23

In addit ion, this chapter discusses interindividual variability in responses, and issues 

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

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

### 26 mecha nistic considerations. 

### 27Draft

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### Figure 5

- 1 Schematic of the O 3 

### exposure and response pathway. O 3 

### concentrations can be reported as the exposure concentration, inhaled dose, the net dose, or the local tissue dose. The net dose refers to the total absorption of O 3 

### and is the sum of all the tissue compartmental doses. Chapter 5 discusses the concepts of dose and modes of action that result in the health effects discussed in Chapters 6 and 7.



## 5.2 Human and Animal Ozone Dosimetry

5.2.1 Introduction

Dosimetry refers to the meas urement 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. Dose refers 

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

### 3 surrogates of dose of reactive gases, such as O 3 , can range in refinement from their 

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

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

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

### 7 expression of the dose of O 3 might range from the quantity of gas inhaled as the product 

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

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

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

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

### 12 epithelium. 

### 13
Ozone is a highly reactive, though poorly water soluble, gas at physiologic al temperature. 

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

### 15 lower respiratory tract (LRT). Figure 5

- 2 presents the basic structure of the human 

### 16 respiratory tract (RT). The lung can be divided into three m ajor regions: the extrathoracic 

### 17 O 3 exposure Inhaled O 3 dose Net O 3 dose

Tissue O 3 dose and product formation Modes of Action Health EffectsDraft

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- 3 September 2011 (ET) region or upper respiratory tract (URT, from the nose/mouth to larynx); the 

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

### 2 pulmonary region (from the respiratory bronchioles t o the terminal alveolar sacs). The 

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

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

### 5 respiratory bronchioles. Respiratory bronchioles, the tran sition region between ciliated 

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

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

### 8 pigs, sheep, and pigs. The branching structure of th e ciliated bronchi and bronchioles also 

### 9 differs between species from being a rather symmetric and dichotomous branching 

### 10 network of airways in humans to a more monopodial branching network in other 

### 11 mammals. 

### 12

Source: Based on ICRP ( 1994 )


### Figure 5

- 2 Representation of r espiratory tract regions in humans. Structures are anterior nasal passages, ET 1 

### ; oral airway and posterior nasal passages, ET 2 

### ; bronchial airways, BB; bronchioles, bb; and alveolar interstitial, AI.Draft

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- 4 September 2011 Source: Panel (a) reprinted with permission from McGr aw
- Hill ( Weibel, 1980 )


### Figure 5

- 3 Structure of lower airways with progression from the large airways to the alveolus. (a) Illustrates basic airway anatomy. Structures are epithelial cells, EP; basement membrane, BM; smooth muscle cells, SM; and fibrocartilaginous coat, FC. (b) Illustrates the relative amounts of liquid, tissue, and blood with distal progression. In the b ronchi there is a thick surface lining over a relatively thick layer of tissues. With distal progress, the lining diminishes allowing increased access of compounds crossing the air

- liquid interface to the tissues and the blood. (c) Presents the factors act ing in the gas and liquid phases of O 3 

### transport. c. Transport Factors Gas Phase

Convection Diffusion Dispersion Liquid Phase

Solubility Diffusion Chemical Reaction Convection O 3 transportDraft

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- 5 September 2011 Figure 5

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

### 1 the TB region to the alveolus in the alveolar region. The fact that O 3 is so chemically 

### 2 reactive has suggested to s ome that its effective dose at the target sites exists in the form 

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

### 4 products are formed when O 3 interacts with components of the ELF such as lipids and 

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

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

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

### 8 exists in the LRT where the thinness of the ELF layer allows O 3 to react directly with 

### 9 cells protruding from the ELF (Figure 5

- 3b). Ozone uptake relates directly to these ELF 

### 10 substrate reactions and is termed “ reactive absorption. ” Thus, the uptake of O 3 is rela ted 

### 11 to both the concentration of O 3 as well as the availability of substrates within the ELF. 

### 12
Chemical reactions are not the only processes controlling the uptake of O 3 from the 

### 13 airstream into compartments of the RT (Figure 5

- 3c). Ozone uptake is affected by 

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

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

### 16 processes of gas transport, as well as the physical and chemical properties of the E LF and 

### 17 tissue layers. The role of these processes varies throughout the length of the RT and as O 3 

### 18 moves from the gas to liquid compartments of the RT. 

### 19
Two types of measurements have been used to arrive at the O 3 dose to target sites during 

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

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

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

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

### 24 extrapolating O 3 dose in different exposure scenarios. Few new studies have investigated 

### 25 the uptake of O 3 in the RT since the last O 3 assessment ( U.S. EPA, 2006b ) . T he studies 

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

### 27 change the dosimetry conclusions of the last document. 

### 28

5.2.2 Ozone Uptake

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

### 29 the state of the science in O 3 dosimetry. One method of quantifying O 3 dosimetry is to 

### 30 measure the amount of O 3 removed from the air stream during breathing (termed 

### 31

“ uptake ” ). The O 3 in the breath that is removed during the breathing period i s termed 

### 32

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

### 33 continuous O 3 breathing techniques as well as modeling to investigate uptake efficiency 

### 34Draft

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- 6 September 2011 and distribution of O 3 uptake between the upper and lower respiratory trac t. A number of 

### 1 the studies that have measured the fractional uptake of O 3 in the human RT (F RT ), URT 

### 2 (F URT ), and LRT (F LRT ) are presented in Table 5

- 1. 

### 3

Table 5

- 1 Human respiratory tract uptake efficiency data 4

5.2.2.1 Gas Transport Principles

Transport of O 3 in the gas phase is governed by bulk flow or convection, effective axial 

### 5 dispersion, and loss to airway walls (Figure 5

- 3c) ( Miller, 1995 ) . The relative importance 

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

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

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

### 9 the alveolar region of the lung. 

### 10 Reference Mouth/Nose a Inspiratory Flow (mL/s) V T (mL) f B (bpm) b F RT F URT F LRT CONTINUOUS EXPOSURE Gerrity et al. ( 1988 ) M 509 832 18 0.40 0.91 N 456 754 18 0.36 0.91 M/N 350 832 12 0.41 0.93 M/N 634 778 24 0.38 0.89 Gerrity et al. ( 1994 ) c M 1,360 1,650 25 0.81 0.37 0.43 M 1,360 1,239 35 0.78 0.41 0.36 Gerrity et al. ( 1995 ) Mouthpiece 330 825 12 0.91 0.27 0.95 Wiester et al. ( 1996c ) M 539 631 16 0.76 N 514 642 16 0.73 Santiago et al. ( 2001 ) N 50 0.80 d
N 250 0.33 Rigas et al. ( 2000 ) Face mask 480 1,100 27.6 0.86 BOLUS EXPOSURE Hu et al. ( 1992 ) Mouthpiece 250 0.96 0.46 Ultman et al. ( 1994 ) Mouthp iece 250 500 e 15 0.30 Mouthpiece 250 500 15 0.47 Ultman et al. ( 2004 ) M 490 450 e 32.7 0.87 M 517 574 27 0.91 Nodelman and Ultman ( 1999 ) Nasal Cannula 150 500 18 0.90 Nasal Cannula 1,000 500 120 0.45 Mouthpiece 150 500 18 0.80 0.95 Mouthpiece 1,000 500 120 0.25 0.90

a M = mouth exposure during spontaneous breathing; N = nasal exposure during spontaneous breathing; M/N = pooled data from mouth and nasal exposure; mouthpiece = exposure by mouthpiece; F RT = total RT uptake; F U RT = upper RT uptake; F LRT = lower RT uptake.

b f B is either measured or i s computed from flows and V T .
c Total RT uptake reported by Gerrity et al. ( 1988 ) and Gerrity et al. ( 1994 ) did not include the contribution from U RT uptake efficiency during expiration. The data include an expiratory U RT co ntribution, assuming it equals inspiratory U RT uptake efficiency.

d F U RT from Santiago et al. ( 2001 ) represents nasal absorption (F nose ).

e V T is computed from flow and f B .Draft

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- 7 September 2011 Gas transport in the TB region occurs by a combination of bulk flow and mixing 

### 1 ( Ultman, 1 985 ) . Mixing can occur by diffusion processes associated with the molecular 

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

### 3 complexity of the airway structure and surface affects the bulk airflow patterns so tha t not 

### 4 all nasal and lung surfaces receive the same O 3 exposure or dose ( Miller and Kimbell, 

### 5 1995 ) . The principal influence on mixing in the TB region comes from the axial velocity 

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

### 7 inspirat ory profile is sharper than the more flattened expiratory profile ( Schroter and 

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

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

### 10 fluctuations), and is influenced by Tayl or dispersion forces. In humans, turbulent flow 

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

### 12 species and flow rates. For example, airf l ow is nonturbulent in the rat nose at any 

### 13 physiologic flow rate but may be hi ghly turbulent in the human nose during exercise 

### 14 ( Miller, 1995 )

. 

### 15
Conversely, the principal mechanism of gas mixing in the lung periphery is molecular 

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



### 17 sectional area of the airways rapidly increases and linear velocities decrease, leading to a 

### 18 greater role of molecular diffusion of gases. Gas molecules clos e to the alveolocapillary 

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

### 20 the diffusion of O 3 into the ELF where chemical reactions occur drives alveolar gas 

### 21 uptake. 

### 22

5.2.2.2 Target Sites for Ozone Dose

A primary uptake site of O 3 delivery to the lung epithelium is believed to be the 

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

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

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

### 26 the CAR, the respiratory bronchioles were confirmed as the site receiving the greatest O 3 

### 27 dose ( 18 O mass/lung weight) in resting O 3 exposed rhesus monkeys, when not 

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

### 29 occurred in the vicinity of the respiratory bronchioles and was dependent on the deliver ed 

### 30 O 3 dose to these tissues (see also Section 5.4.1). However, 18 O label was detected to a 

### 31 lesser extent in other regions of the TB airway tree, showing that O 3 is delivered to these 

### 32 compartments as well, although in a smaller dose. Earlier models predicte d that the net 

### 33 O 3 dose (total absorption, O 3 flux to air

- liquid interface) gradually decreased with distal 

### 34 progression from the trachea to the end of the TB region and then rapidly decreased in the 

### 35Draft

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- 8 September 2011 alveolar region ( Miller et al., 1985 ) . However, the tissue O 3 dose (O 3 flux to liquid

- tissue 

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

### 2 and the CAR, and then rapidly decreased in the alveolar region. Despit e the exclusion of 

### 3 the URT and O 3 reactions with ELF constituents after the 16th generation, the model 

### 4 predicted experimental results showing that the CAR received the greatest O 3 tissue dose 

### 5 ( Miller et al., 1985 )

. 

### 6
Inhomogeneity in the RT structure may affect the dose delivered to this target site. 

### 7 Models have predicted that the farther the PAR is from the trachea, the less the O 3 tissue 

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

### 9 approximately a 50 to 300% greater PAR dose for the shortest path relativ e to the longest 

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

### 11 path distance and ventilatory unit size affected dose. The variation of O 3 dose among 

### 12 anatomically equivalent ventilatory units was predicted to vary as much as six

- fold, as a 

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

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

### 15 considered insignificant, local regions of t he RT may receive significantly higher than 

### 16 average doses and therefore be at greater risk of effects. 

### 17

5.2.2.3 Upper Respiratory Tract Ozone Removal and Dose

The URT provides a defense against O 3 entering the lungs by removing half of the 

### 18 inhaled O 3 from the airst ream. In both animals and humans, about 50% of the absorbed 

### 19 O 3 was removed in the head (nose, mouth, and pharynx), about 7% in the larynx/trachea, 

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

### 21 fraction of O 3 taken up was inversely related to flow rate and weakly related to inlet O 3 

### 22 concentration ( Yokoyama and Frank, 1972 ) . The limiting factors in nasal O 3 uptake were 

### 23 simultaneous diffusion and chemical reaction of O 3 in the nasal ELF layer ( Santiago et 

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

### 25 mathematical estimates predicted that O 3 penetrates less than the thickness of the ELF 

### 26 layer; reaction products are likely the agents damaging the nasal tissue and not O 3 itself. 

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

### 28 depleting substrates in the nasal ELF becoming the limitin g factor of the reaction 

### 29 ( Santiago et al., 2001 )

. 

### 30
Uptake efficiencies have been measured for various segments of the URT (Table 5

- 1). 

### 31 Gerrity et al. ( 1995 ) reported unidirectional uptake efficiencies of O 3 inhaled from a 

### 32 mouthpie ce; of 17.6% from the mouth to vocal cords, 9.5% from the vocal cords to the 

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

### 34Draft

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- 9 September 2011 carina (totaling 35.5%), and essentially zero between the carina and the bronchus 

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

### 2 ( 1992 ) that reported uptake efficiencies of 21, 36, 44, and 46% between the mouth and 

### 3 the vocal cords, the upper trachea, the main bifurcation carina, and the bronchus 

### 4 intermedius, respectively. The lower eff iciencies seen in Gerrity et al. ( 1995 ) may have 

### 5 resulted from the mouthpiece scrubbing O 3 from the breath during inhalation. 

### 6
Past studies investigating nasal uptake of O 3 have shown that the nose partially protects 

### 7 the LRT from damage from inspired O 3 ( Santiago et al., 2001 ; Gerrity et al., 1988 )

. 

### 8 Sawyer et al. ( 2007 ) further investigated nasal uptake of O 3 in healthy adults during 

### 9 exercise. Fractional O 3 uptake, acoustic rhinometry (AR), and nasal NO measurements 

### 10 were taken on ten adults (8 women, 2 men) exposed to 200 ppb O 3 before and after 

### 11 moderate exercise at two flow rates (10 and 20 L/min). The percent nasal uptake of O 3 

### 12 was ~50% greater at 10 L/min compared to 20 L/min both pre

- and post

- exercise. 

### 13 However, the inhaled O 3 deli vered dose to the LRT (i.e., flow rate × [O 3 ppm] × nasal O 3 

### 14 penetration) was 1.6

- fold greater at the higher flow than at the lower flow (2.5 compared 

### 15 to 0.9 ppm·L/min). Prior exercise did not affect O 3 uptake at either flow rate, but did 

### 16 significantly inc rease nasal volume (Vn) and AR measurements of nasal cross

- sectional 

### 17 area (minimum cross

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

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

### 19 corresponds to the poste rior edge of the nasal turbinates) (p ≤ 0.05). Conversely, exercise 

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

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

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

### 23 gender segregation. 

### 24

Overall, the URT removes half of the inhaled O 3 by reactions in the nasal ELF. The exact 

### 25 uptake efficiency will change due to variations in flow rate and inhale d concentration. 

### 26

5.2.2.4 Lower Respiratory Tract Ozone Uptake and Dose

Total O 3 uptake in the entire RT in rats and guinea pigs ranges from 40

- 54% efficient 

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

### 28 ranges from 80

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

### 29 absorbed in the LRT of both humans and animals. Models predicted t hat the net O 3 dose 

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

### 31 decreases in the alveolar region ( Miller et al., 1985 ) . However, these models predicte d 

### 32 low tissue O 3 dose in the trachea and large bronchi. As injury has been seen in these 

### 33 areas, net dose may be a better predictor of local toxic tissue dose. 

### 34Draft

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- 10 September 2011 Uptake efficiency depends on a number of variables, including O 3 exposure 

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

### 2 respiratory patterns are uniquely described by breathing frequency (f B ) and tidal volume 

### 3 (V T ); by minute volume (MV = f B x V T ) and f B ; or by MV and V T . Simulations from the 

### 4 Overton et al. ( 1996 ) single

- path anatomical respiratory tract model, where the upper and 

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

### 6 predicted that fractional uptake and PAR O 3 dose increased with V T when f B was held 

### 7 constant. Likewise, experimental studies found that O 3 uptake was positively correlated 

### 8 with changes in V T ( Ultman et al., 2004 ; Gerrity et al., 1988 ) . Also, O 3 exposure led to a 

### 9 reflex mediated increase in f B and reduction in V T , hypothesized to be protective by 

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

### 11 O 3 uptake was inversely proportional to flow rate ( Santiago et al., 2001 ) , so that an 

### 12 increase in MV will increase O 3 delivery to the lower airways. At a fixed MV, increasing 

### 13
V T (corresponding to decreasing f B ) drove O 3 deeper into the lungs a nd increased total 

### 14 respiratory uptake efficiency (Figure 5

- 4) ( Ultman et al., 2004 ; Wiester et al., 1996c ; 

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

### 16 increased f B when V T was held constant, but an increase in PAR dose with increased f B 

### 17 ( Overton et al., 1996 ) . Similarly, incre ased f B (80

- 160 bpm) and shallow breathing in 

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

### 19 bronchi ( Alfaro et al., 2004 ) . This dependence may be a res ult of frequency

- induced 

### 20 alterations in contact time that affects the first

- order absorption rate for O 3 ( Postlethwait 

### 21 et al., 1994 ) . Also, an association of O 3 uptake efficiency was found with MV and 

### 22 exposure time. 

### 23
Increasing flow leads to deeper penetration of O 3 into the lung, such that a smaller 

### 24 fraction of O 3 is absorbed in the URT and uptake shifts to the TB airways and respiratory 

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

### 26 ( 1994 ) and Ultman et al. ( 1994 ) found that O 3 absorption increased with volumetric 

### 27 penetration (Vp) of a bolus of O 3 into th e RT (Figure 5

- 5). Ozone uptake efficiency and 

### 28 Vp were not affected by bolus O 3 concentration ( Kabel et al., 1994 ; Hu et al., 1992 )
, 

### 29 indicating that O 3 uptake is a linear absorption process, where the diffusion and chemical 

### 30 reaction r ates of O 3 are proportional to the O 3 concentration. This relationship was also 

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

### 32 significant negative dependence of O 3 concentration on uptake efficiency in exercising 

### 33 indi viduals; however, only due to large changes in O 3 concentration suggesting that O 3 

### 34 uptake is likely still essentially linear with respect to O 3 concentration. This study also 

### 35 found that exposure time had a small but significant influence on uptake efficien cy; 

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

### 37 substrates from the ELF. 

### 38Draft

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- 11 September 2011 Source: Reprinted with permission of Health Effects Institute ( Ultman et al., 2004 )


### Figure 5

- 4 Total ozone uptake efficiency as a function of breathing frequency at a constant minute ventilation of 30 L/min. Subjects breathed 0.25 ppm O 3 

### oronasally via a breathing mask. The uptake efficiency was well correlated with breathing frequency (r =

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

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

### 1 reproducible pattern of severity between daughter branches of individual bifurcations that 

### 2 is dependent on the O 3 concentration

- time profile of the inhaled gas. A 3

- D 

### 3 computational fl uid dynamics model was created to investigate the dose

- response 

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

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

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

### 7 were simulated using Reynolds numbe rs (Re) ranging from 100 to 500. The Re that 

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

### 9 such that the range in Re from 100

- 500 would encompass generations 1

- 5, 3

- 7, and 6

- 10 

### 10 for an adult during quiet breathing, ligh t exertion, and heavy exercise, respectively, 

### 11 whereas the same Re range corresponds to generations 0

- 4, 1

- 6, and 4

- 8 for a 4

- year

- old 

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

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

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

### 15 daughter branches. During expiration, the model predicted that the velocity and O 3 

### 16 concentration distribution was slightly skewed towar ds the outer walls of the daughter 

### 17 branches. Hot spots of wall flux existed at the carina during inspiration and expiration 

### 18Draft

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- 12 September 2011 with Re >100. Additional hot spots were found during expiration on the parent branch 

### 1 wall downstream of the branching region. 

### 2

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


### Figure 5

- 5 Ozone uptake fraction as a function of volumetric penetration (V p 

### ) in a representative subject. Each point represents the O 3 

### 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 ) found that V p 

### 50% was well correlated with V D 

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

### minus V UA 

### .

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

### 3 that determines how much O 3 is available at longitudinally distributed compartments in 

### 4 the lung is sensitive to changes in V T , f B , and to a minor extent, exposure time. 

### 5 Decreased f B at a fixed penetration volume will shift the O 3 uptake from the upper 

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

### 7

5.2.2.5 Mode of Breathing

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

### 8 airways volume had a weaker influence on O 3 absorption during nasal breathing 

### 9 compared to oral breathing. This could be a result of O 3 scrubbing in the nasal 

### 10Draft

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- 13 September 2011 passageways that are bypassed by oral breathing. Studies by Ultman and colleag ues using 

### 1 bolus inhalation demonstrated that O 3 uptake fraction was greater during nasal breathing 

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

### 3 oral breathing at 150 mL/s and 0.45 during nasal breathing and 0.25 d uring oral breathing 

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

. 

### 5 Therefore, oral breathing results in deeper penetration of O 3 into the RT with a higher 

### 6 absorbed fraction in the URT, TB, and alveolar airways ( Nodelman and Ultman, 1999 )

. 

### 7 Similar results were obtained from O 3 uptake studies in dogs ( Yokoyama and Frank, 

### 8 1972 ) . Earlier human studies suggesedt that oral or oronasa l breathing results in a higher 

### 9 O 3 uptake efficiency than nasal breathing ( Wiester et al., 1996c ; Gerrity et al., 1988 ) ; 

### 10 however the difference observed between inspired O 3 taken up during oral versus nasal 

### 11 breathing may not be biologic ally significant. These human studies measured total RT 

### 12 absorption after continuous O 3 exposure using a pharyngeal sampling tube, which may 

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

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

### 15 distribution of O 3 deposited in the distal airways. 

### 16

5.2.2.6 Interindividual Variability in Dose

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

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

### 18 accounted for between 10

- 50% of the absolute variability in O 3 uptake measurements 

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

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

### 21 hypothesized that interindividual variation in O 3 induced response such as FEV 1 is the 

### 22 result of interindividual variation in delivered dose or regional O 3 uptake among exposed 

### 23 individuals. 

### 24
Recent studies have reiterated the importance of intersubject variation in O 3 uptake. The 

### 25 intersubject variability in nasal O 3 uptake determined by Sawyer et al. ( 2007 ) ranged 

### 26 from 26.8 to 65.4% (pre

- and post

- exercise). A second stu dy investigating the use of the 

### 27 CO 2 expirogram to quantify pulmonary responses to O 3 found that intersubject 

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

. 

### 29
Variability in local dose may be attributed to differences in the pulmonary ph ysiology, 

### 30 anatomy, and biochemistry. Since the TB airways remove the majority of inhaled O 3 

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

### 32 airways will influence O 3 uptake. Models predicted that fractional O 3 uptake and PAR 

### 33 dose (flux of O 3 to the PAR surfaces divided by exposure concentration) increase with 

### 34Draft

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- 14 September 2011 decreasing TB volume and decreasing TB region expansion. On the contrary, alveolar 

### 1 expansion had minimal effect on uptake efficiency as relatively little O 3 reaches the 

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

### 3 complete by the time O 3 reaches the alveolar spaces of the lung ( Postlethwait et al., 

### 4 199 4 ) . Experimental studies have found that differences in TB volumes may account for 

### 5 75% of the variation in absorption between subjects ( Ultman et al., 2004 ) . In support of 

### 6 this concept, regression analysis showed that O 3 absorption was positively correlated 

### 7 with anatomical dead space (V D ) and TB v olume (i.e., V D minus V URT ), but not total lung 

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

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

. 

### 10 Variability in V D was correlated more with the variability in the TB volume than the URT 

### 11 volume. Similarly, uptake was correlated with changes in individual bronchial cross



### 12 secti onal area, indicating that changes in cross

- sectional area available for gas diffusion 

### 13 are related to overall O 3 retention ( Reeser et al., 2005 ; Ultman et al., 2004 ) . These studies 

### 14 provide support to the pulmonary p hysiology, especially the TB volume and surface area, 

### 15 playing a key role in variability of O 3 uptake between individuals. 

### 16
When absorption data were normalized to Vp/V D , variability attributed to gender 

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

### 18 has been predicted. Overton and Graham ( 1989 ) predicted that the total qu antity of O 3 

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

### 20 that the LRT distribution of absorbed O 3 and the CAR O 3 tissue dose were not sensitive 

### 21 to age during quiet breathing. However, during heavy exercise or work O 3 uptake was 

### 22 dependent on age. A physiologically based pharmacokinetic model simulating O 3 uptake 

### 23 predicted that regional extraction of O 3 was relatively insensitive to age, but extraction 

### 24 per unit surface area was two

- to eightfold higher in infants compa red to adults, due to 

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

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

. 

### 27
Smoking history, with its known increase in mucus production, was not found to 

### 28 significantly affect the fractional uptake of a bolus dose of O 3 in apparently healthy 

### 29 smokers with limited smoking history ( Bates et al., 2009 ) . Despite similar internal O 3 

### 30 dose distribution, the smokers exhibited greater pulmonary responses to O 3 bolus 

### 31 exposures, measured as FEV 1 decrements and increases in the normalized slope of the 

### 32 alveolar plateau (S N ). This was contrary to previous studies conducted in smokers with a 

### 33 greater smoking history that found decreased O 3 induced decrements in FEV 1 in smokers 

### 34 during continuous O 3 exposure ( Frampton et al., 1997b ; Emmons and Foster, 1991 )

. 

### 35Draft

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- 15 September 2011 

### 5.2.2.7 Physical Activity

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

### 1 part, to the increased intake of air. As exercise increases from a low to moderate level, V T 

### 2 increases. This increase in V T is achieved by encroaching upon both the inspiratory and 

### 3 expirator y reserve volumes of the lung ( Dempsey et al., 1990 ) . After V T reaches about 

### 4 50% of the vital capacity, generally during heavy exercise, furth er increases in ventilation 

### 5 are achieved by increasing f B . Ventilatory demands of heavy exercise require airway flow 

### 6 rates that often exceed 10 times resting levels and V T that approach 5 times resting levels 

### 7 ( Dempsey et al., 2008 )

. 

### 8

This increase in V T and flow associated with exercise in humans shifts the O 3 dose 

### 9 further into the periphery of the RT causing a disproportionate in crease in distal lung 

### 10 dose. In addition to increasing the bulk transport of O 3 into the lung, exercise also leads 

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

### 12 lower

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

### 13 ventilatory parameters from normal respiration levels predicted a 10

- fold increase in total 

### 14 mass uptake of O 3 ( Miller et al., 1985 )

. This model also predicted that as exercise and 

### 15 ventilatory demand increased the maximum tissue dose moved distally into the RT 

### 16 (Figure 5

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

### 17 flow = 750

- 1
, 000 mL/s compared to 250 mL/s at rest), the URT absorbed a small er 

### 18 fraction of the O 3 (~0.50 at rest to 0.10 at exercise); however, the trachea and more distal 

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

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

### 21 TB with almost no O 3 reaching the alveolar zone at rest) ( Hu et al., 1994 ) . The same shift 

### 22 in the O 3 dose distribution more distally in the lung occurred in other studies mimicking 

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

### 24 sensitive to age only under exercise condi tions ( Overton and Graham, 1989 ) . The total 

### 25 quantity of O 3 absorbed per minute was predicted to increase with age during heavy work 

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

### 27 ventilation led to only a 1.6

- fold higher delivered dose rate of O 3 to the lung. Past models 

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

### 29 compartments and regions. Tissue and mucus lay er dose in the TB region increased ~1.4

- 

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

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

### 32 al., 1985 )

. 

### 33Draft

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- 16 September 2011 Source: Reprinted with permission

. ( Miller et al., 1985 )


### Figure 5

- 6 Modeled effect of exercise on tissue dose of the LRT. Curve 1: V T 

### = 500 mL; f B 

### = 15 breaths/min. Curve 2: V T 

### = 1 , 000 mL; f B 

### = 15 breaths/min. Curve 3: V T 

### = 1 , 750 mL; f B 

### = 20.3 breaths/min. Curve 4: V T 

### = 2 , 250 mL; f B 

### = 30 breaths/min. TB = tracheobronchial region; P = pulmonary region.

5.2.2.8 Summary

In summary, O 3 uptake is affected by com plex interactions between a number of factors 

### 1 including RT morphology, breathing route, frequency, and volume, physicochemical 

### 2 properties of the gas, physical processes of gas transport, as well as the physical and 

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

### 4 throughout the length of the RT and as O 3 moves from the gas into liquid compartments 

### 5 of the RT. The primary uptake site of O 3 delivery to the lung epithelium is believed to be 

### 6 the CAR, however inhomogeneity in t he RT structure may affect the dose delivered to 

### 7 this target site with larger path lengths leading to smaller locally delivered doses. Recent 

### 8 studies have provided evidence for hot spots of O 3 flux around bifurcations in airways. 

### 9 Experimental studies and m odels have suggested that the net O 3 dose gradually decreases 

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

### 11 the alveolar region. However, the tissue O 3 dose is low in the trachea, increases to a 

### 12Draft

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- 17 September 2011 maximum in the term inal bronchioles and the CAR, and then rapidly decreases distally 

### 1 into the alveolar region. 

### 2

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

### 3 decrease with increased flow and increase proportional to V T , so that at a fix ed MV, 

### 4 increasing V T (or decreasing f B ) drives O 3 deeper into the lungs and increases total 

### 5 respiratory uptake efficiency. Individual total airway O 3 uptake efficiency is also 

### 6 sensitive to large changes in O 3 concentration, exposure time, and MV. Major sou rces of 

### 7 variability in absorption of O 3 include O 3 concentration, exposure time, f B , MV, and V T , 

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

### 9 majority of this interindividual variability is due to differenc es in TB volume and surface 

### 10

area. 

### 11

An increase in V T and f B are both associated with increased physical activity. These 

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

### 13 of O 3 into the lung with a higher absorbed fract ion in the ET, TB, and alveolar airways. 

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

### 15 O 3 distally into the RT and into the alveolar region. 

### 16

5.2.3 Ozone Reactions and Reaction Products

Ozone dose can be examined by the c hemical reactions or the products of these reactions 

### 17 that result from O 3 exposure. Since O 3 is chemically reactive with a wide spectrum of 

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

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

### 20 appearance of plausible products, or the addition of O 3 - derived oxygen to biomolecules 

### 21 through the use of oxygen

- 18 labeling. In vitro exposure of ELF showed that O 3 

### 22 disappearance from the gas phase depends on the characteristics of the ELF substrates 

### 23 ( Postlethwait et al., 1998 ; Hu et al., 1994 )

. 

### 24

F or O 3 to gain access to the underlying cellular compartments, O 3 must dissolve at the 

### 25 air

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

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

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

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

### 29 1995 ) . There is a progressive decrease in ELF thickness and increase in interfacial 

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

. 

### 31 Some cells, such as macrophages, may protrude into the gas phase, a llowing for direct 

### 32 contact between O 3 and cell membranes. The progressive thinning of the ELF while 

### 33 moving further down the RT decreases the radial distance O 3 must travel to reach the 

### 34Draft

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- 18 September 2011 cellular tissue layer. A computational fluid dynamics model was able to predict 

### 1 experimentally measured O 3 uptake, but only with nasal mucus layer thickness 

### 2 considered ( Cohen

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

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

### 4
Taking into acc ount the high reactivity and low water solubility of O 3 , calculations 

### 5 suggest that O 3 will not penetrate ELF layers greater than 0.1 μm without being 

### 6 transformed to other more long

- lived reactive species, thus initiating a reaction cascade 

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

### 8 possibly allowing for direct interaction of O 3 with the underlying epithelial cells. O ne 

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

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

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

### 12 an important target for O 3 and the resulting secondary oxidation products should be 

### 13 considered key mediators of toxicity in the airways (role of reaction products in O 3 

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

### 15 on diffusion equations and reaction rates of O 3 with model substrates predict an O 3 

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

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

### 18 which measured the total oxygen

- addition product of O 3 reactions in the airways through 

### 19 th e use of oxygen

- 18 labeled O 3 . High concentrations of O 3 reaction products were found 

### 20 in the bronchoalveolar lavage (BAL) mucus and surfactant providing evidence that O 3 

### 21 reacts at the air

- liquid interface. Thus, O 3 may cause injury by direct reaction with 

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

### 23 underlying the lining fluid. The reaction cascade resulting from the interaction of O 3 with 

### 24 ELF substrates acts to carry the oxidative burden deeper into the tissu es. 

### 25
Ozone may interact with many of the components in the ELF including phospholipids, 

### 26 neutral lipids, free fatty acids, proteins, and low molecular weight antioxidants ( Perez



### 27 Gil, 2008 ; Uppu et al., 1995 ) . It was estimated that 88% of the O 3 that does not come in 

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

### 29 those contained within phospholipids or neutral lipids ( Uppu et al., 1995 ) . Ozon e reacts 

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

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

### 32 via chemical reactions such as the Criegee ozonolysis mechanism (Figu re 5

- 7) ( Pryor et 

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

### 34 nonanal, have been recovered after O 3 exposure in human BAL fluid (BALF), rat BALF, 

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

### 36 Pryor et al., 1996 ) . Nonanal has been suggested as a relatively specific biomarker for O 3 

### 37 exposure since the monounsaturated fatt y acid parent compound, oleic acid, does not 

### 38Draft

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- 19 September 2011 undergo autoxidation ( Pryor et al., 1996 ) . Adducts of the aldehyde 4

- hydroxynonenal 

### 1 were found in human alveolar macrophages after O 3 exposure ( Hamilton et al., 1998 )

. 

### 2 Polyunsaturated fatty acid (PUFA) reactions are limited by the availability of O 3 since 

### 3 lipids are so abundant in the ELF. Yields of O 3 - induced aldehydes were increased by the 

### 4 decrease in other substrates such as ascorbic acid (AH 2 ) ( Postlethwait et al., 1998 ) . Free 

### 5 radicals are also generated during O 3 - mediated oxidation react ions with PUFA ( Pryor, 

### 6 1994 ) . These reactions are reduced by the presence of the lipid

- solub le fre e radical 

### 7
scavenger α

- tocopherol (α

- TOH) ( Pryor, 1994 ; Fujita et al., 1987 ; Pryor, 1976 ) . PUFA 

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

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

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

. 

### 11

Source: U .S. EPA ( 2006b )


### Figure 5

- 7 Schematic overview of ozone interaction with PUFA in ELF and lung cells. It should be noted that not all secondary reaction products are shown.

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

### 12
O 3 results in biologically active cholesterol products such as the oxysterols, β

- epoxide 

### 13 and 6

- oxo

- 3,5

- diol ( Murphy and Johnson, 2008 ; Pulfer et al., 2005 ; Pulfer and Murphy, 

### 14 2004 ) . Product yields will depend on ozonolysis conditions, however cholesterol 

### 15 ozonolysis products were formed in similar abundance to phospholipid

- derived 

### 16 ozonolysis products in rat ELF ( Pulfer and Murphy, 2004 )

. 

### 17
The ELF also contains proteins present in blood plasma as well as proteins secreted by 

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

### 19 reactions as well as reactions of their constit uent amino acids (the most reactive of which 

### 20 are cysteine, histidine, methionine, tyrosine, and tryptophan). Ozone has been shown to 

### 21Draft

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- 20 September 2011 preferentially react with biomolecules in the following order: thiosulfate > ascorbate > 

### 1 cysteine ≈ methionine > glutathion e ( Kanofsky and Sima, 1995 ) . Rate constants for the 

### 2 reaction of amino acids with O 3 vary between investigations due to differing reaction 

### 3 conditions and assumptions; however al iphatic amino acids consistently were very slow 

### 4 to react with O 3 (e.g., alanine: 25

- 100 moles/L/sec) ( Kanofsky and Sima, 1995 ; 

### 5 Ignatenko and Cherenkevich, 1985 ; Pryor et a l., 1984 ; Hoigné and Bader, 1983 ) . Uppu et 

### 6 al. ( 1995 ) predicted that 12% of inhaled O 3 that does not react with antioxidants will 

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

### 8
Reactions of ozone with low molecular weight antioxidants have been extensively 

### 9 studied. The consumpti on of antioxidants such as uric acid (UA), ascorbate (AH 2 ), and 

### 10 reduced glutathione (GSH) by O 3 was linear with time and positively correlated with 

### 11 initial substrate con c entration and chamber O 3 concentration ( Mudway and Kelly, 1998 ; 

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

### 13 concentrations in the ELF of the human TB airways and displa y high intrinsic reactivities 

### 14 toward O 3 , but do not possess equal O 3 reactivity. In individual and in limited composite 

### 15 mixtures, UA was the most reactive antioxidant tested, followed by AH 2 ( Mudway and 

### 16 Kelly, 1998 ) . GSH was consistently less reactive than UA or AH 2 ( Mudway and Kelly, 

### 17 1998 ; Mudway et al., 1996 ; Kanofsky and Sima, 1995 ) . To quantify these reactions, 

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

### 19 UA, AH 2 , and GSH (50

- 200 μM) with O 3 (1

- 5 ppm). Similar to the results of Mudway 

### 20 and Kelly ( 1998 ) , this study found the hierarchy in reactivity between O 3 and these 

### 21 antioxidants to be UA≥AH 2 >>GSH. UA and AH 2 shared a 1:1 stoichiometry with O 3 , 

### 22 whereas 2.5 moles of GSH were consumed per mole of O 3 . Using these st oichiometries, 

### 23 reaction rate constants were derived (5.8×10 4 M - 1 sec - 1 , 5.5×10 4 M - 1 sec - 1 , and 57.5 M - 

### 24

0.75 /sec [20.9 M - 1 sec - 1 ] for the reaction of O 3 with UA, AH 2 , and GSH, respectively). 

### 25 These values are similar to those derived from data presented in Mu dway and Kelly 

### 26 ( 1998 ) . Other studies reported reactive rate constants that are two to three orders of 

### 27 magnitude larger, however these studies used higher concentrations of O 3 and 

### 28 antioxidants under less physiologically relevant experimental conditions ( Kanofsky and 

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

. 

### 30
A series of studies used new techniques to investigate the reaction products resulting 

### 31 from initial air

- liquid interface interactions of O 3 with ELF components (e.g., 

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

. 

### 33 Solutions of aqueous UA, AH 2 , GSH, α

- TOH, and protein cysteines (CyS) were sprayed 

### 34 as microdroplets in O 3 /N 2 mixtures at atmospheric pressure and analyzed by electrospray 

### 35 mass spectrometry. These recent studies demonstrated different reactivity toward AH 2 , 

### 36 UA, and GSH by O 3 when the large surface to volume ratio of microdroplets promote an 

### 37 interfacial reaction compared to previous studies using bulk liquid phase bioreactors, thus 

### 38Draft

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- 21 September 2011 supporting the relevance of reactions between gas phase O 3 and antioxidants found in the 

### 1
E LF. 

### 2
As was seen in previous studies ( Kermani et al., 2006 ; Kanofsky and Sima, 1995 ) , the 

### 3 hierarchy of reactivity of these ELF components with O 3 was determined to be AH 2 ≈ 

### 4 UA > CyS > GSH. There was some va riance between the reaction rates and product 

### 5 formation of UA, AH 2 , and GSH with O 3 as investigated by Enami et al. versus O 3 

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

### 7 reactive than AH 2 toward O 3 in previous studies, but in reactions with O 3 with 

### 8 microdroplets, these antioxidants had equivalent reactivity ( Enami et al., 2008b ) . As O 3 is 

### 9 a kinetically slow one

- electron acceptor but very reactive O

- atom donor, products of the 

### 10 interaction of O 3 with UA, AH 2 , GSH, CyS, and α

- TOH result from addition of n O



### 11 atoms ( n = 1

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

- O - ), peroxides (e.g. U

- O 2 - ), 

### 12 and ozonides (e.g., U

- O 3 - ). For instance, GSH was oxidized to sulfonates (GSO 3 - /GSO 3 2

- 

### 13 ), not glutathione disulfide (GSSG) by O 3 ( Enami et al., 2009b ) . Howev er, it is possible 

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

### 15 in O 3 exposed ELF whereas GSSG is. This is also supported by the fact that O 3 is much 

### 16 less reactive with GSH than other antioxidants, such that < 3 % of O 3 will be scavenged 

### 17 by GSH when in equimolar amounts with AH 2 ( Enami et al., 2009b )

. 

### 18
Ozonolysis product yields and for mation were affected by pH. Acidified conditions (pH ≈ 

### 19 3

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

### 20 conditions like asthma (pH ≈ 6), decreased the scavenging ability of UA and GSH for O 3 ; 

### 21 such that at low pH, the sca venging of O 3 must be taken over by other antioxidants, such 

### 22
as AH 2 ( Enami et al., 2009b
, 2008b ) . Also, under acidic conditions (pH ≈ 5), the 

### 23 ozonolysis products of AH 2 shifted from the innocuous dehydroascorbic acid to the more 

### 24 persistent products, AH 2 ozonide and threonic acid ( Enami et al., 2008a ) . It is possible 

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

### 26 of O 3 by preventing some antioxidant reactions and shifting the reaction products to more 

### 27 persistent compounds. 

### 28

In a red blood cell (RBC) based s ystem, AH 2 augmented the in vitro uptake of O 3 by six 

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

### 30 2005 ) . However, estimated in vitro O 3 uptake was not proportional to the production of 

### 31 O 3 - derived aldehyde s from exposing O 3 to RBC membranes ( Ballinger et al., 2005 ) . In 

### 32 addition, O 3 induced cell membrane oxidation which required interactions with AH 2 and 

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

### 34 between O 3 an d bovine serum albumin did not result in membrane oxidation ( Ballinger et 

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

### 36 protein oxidation resulting from the reaction of O 3 and AH 2 ( Ballinger et al. , 2005 ) . This 

### 37Draft

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- 22 September 2011 study provided evidence that antioxidants may paradoxically facilitate O 3 - mediated 

### 1 damage. This apparent contradiction should be viewed in terms of the concentration



### 2 dependent role of the ELF antioxidants. Reactions between O 3 and antioxid ant species 

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

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

### 5 led to the formation of secondary oxidation products which wer e injurious and also led to 

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

### 7 oxidation products mediated by some antioxidants was opposed by quenching reactions 

### 8 involving other antioxidants. 

### 9
Alterations in ELF composition ca n result in alterations in O 3 uptake. Bolus O 3 uptake in 

### 10 human subjects can be decreased by previous continuous O 3 exposure (120

- 360 ppb), 

### 11 possibly due to depletion of compounds able to react with O 3 ( Rigas et al., 1997 ; 

### 12 Asplund et al., 1996 ) . Conversely, O 3 (360 ppb) bolus uptake was increased with prior 

### 13 NO 2 (360

- 720 ppb) or SO 2 (360 ppb) exposure ( Rigas et al., 1997 ) . It was hypothesized 

### 14 that this increased fractional absorption of O 3 c ould be due to increased production of 

### 15 reactive substrates in the ELF due to oxidant

- induced airway inflammation. 

### 16

Besides AH 2 , GSH and UA, the ELF contains numerous antioxidant substances that 

### 17 appear to be an important cellular defense against O 3 includin g α

- TOH, albumin, 

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

### 19 1999 ) . The level and type of an tioxidant present in ELF varies between species, regions 

### 20 of the RT, and can be altered by O 3 exposure. Mechanisms underlying the regional 

### 21 variability are not well

- understood. It is thought that both plasma ultrafiltrate and locally 

### 22 secreted substances cont ribute to the antioxidant content of the ELF ( Mudway et al., 

### 23 2006 ; Freed et al., 1999 ) . In the case of UA, the major source appears to be the plasma 

### 24 ( Peden et al., 1995 ) . Repletion of UA in nasal lava ge fluid was demonstrated during 

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

### 26 were exposed to 200 ppb O 3 for 2 hours while exercising , nasal lavage fluid UA was 

### 27 significantly decreased while plasma UA levels were significantly increased ( Mudway et 

### 28 al., 1999a ) . The finding that UA, but not AH 2 or GSH, w as depleted in nasal lavage fluid 

### 29 indicated that UA was the predominant antioxidant with respect to O 3 reactivity in the 

### 30 nasal cavity ( Mudway et al., 1999a )

. In addition, con centrations of UA were increased by 

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

### 32 O 3 for 2 hours, which suggested that increased mucosal gland secretions were an 

### 33 important source ( Peden et al., 1995 ) . Using the O 3 - specific antioxidant capacity assay on 

### 34 human nasal lavage sampl es, Rutkowski et al. ( 2011 ) concluded that about 30% of the 

### 35 antioxi dant capacity of the nasal liquid lining layer was attributed to UA activity. This 

### 36 assay predicted that more than 50% of the subject

- to

- subject differences in antioxidant 

### 37 capacity were driven by differences in UA concentration. However, day

- to

- day within

- 

### 38Draft

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- 23 September 2011 s ubject variations in measured antioxidant capacity were not related to the corresponding 

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

### 2 predominant antioxidant(s) in other RT regions besides the nasal cavity have failed t o 

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

### 4
AH 2 was greater than UA ( Mudway et al., 1996 )

. 

### 5
Regulation of AH 2 , GSH and α

- TOH concentrations within the ELF is less clear than that 

### 6 of UA ( Mudway et al., 2006 ) . In a sequential nasal lavage study in humans, wash

- out of 

### 7
AH 2 and GSH occurred, indicating the a bsence of rapidly acting repletion mechanisms 

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

### 9 decreases in BALF and plasma AH 2 levels several hours following O 3 exposure (200 ppb 

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

### 11 1999b ) . Furthermore, high levels of dehydroascorbate, the oxidized form of AH 2 , have 

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

### 13 demonstrated cellular uptake of oxidized AH 2 by several cell types leading to 

### 14 intracellular reduction and export of reduced AH 2 ( Welch et al., 1995 ) . Studies with rats 

### 15 exposed to 0.4

- 1.1 ppm O 3 for 1

- 6 hours have shown consumption of AH 2 that correlates 

### 16 with O 3 exposure ( Gunnison and Hatch, 1999 ; Gunnison et al., 1996 ; Vincent et al., 

### 17 1996a )

. 

### 18
ELF exists as a complex mixture, thus it is important to look at O 3 reactivity in substrate 

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

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

### 21 Kelly, 1998 ) . However, O 3 reactions with AH 2 predominated over the reaction with 

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

### 23 suggested that O 3 may react with other s ubstrates once AH 2 concentrations within the 

### 24 reaction plane fall sufficiently. Additionally, once AH 2 was consumed, the absorption 

### 25 efficiency diminished, allowing inhaled O 3 to be distributed to more distal airways 

### 26 ( Postlethwait et al., 1998 ) . Multiple studies have concluded O 3 is more reactive with AH 2 

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

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

. 

### 29
In addition to reactions with components of the ELF, O 3 may react with plasma 

### 30 membranes of c ells which reside in the RT. Eicosanoids are an important class of 

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

### 32 Eicosanoids are metabolites of arachidonic acid, a 20

- carbon PUFA, which is released 

### 33 from membrane phospholipids by phospholipase A2

- mediated catalysis. Activation of 

### 34 phospholipase A2 occurs by several cell signaling pathways and may be triggered by O 3 - 

### 35 mediated lipid peroxidation of cellular membranes ( Rashba

- Step et al., 1997 )

. 

### 36 Additionally, cellular phospholipases A2, C and D may be activated by lipid ozonation 

### 37Draft

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- 24 September 2011 products ( Kafoury et al., 1998 ) . While the conversion of arac hidonic acid to 

### 1 prostaglandins, leukotrienes and other eicosanoid products is generally catalyzed by 

### 2 cyclooxygenases and lipoxygenases, non

- enzymatic reactions also occur during oxidative 

### 3 stress leading to the generation of a wide variety of eicosanoids an d reactive oxygen 

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

### 5 the formation of lysophospholipids which are precursors for platelet activating factors 

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

### 7 O 3 - mediated lipid peroxidation. 

### 8

5.2.3.1 Summary

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

### 9 barrier and target for inhaled O 3 (Figure 5

- 8). The thickness of the lining fluid and mucus 

### 10 layer is a n important determinant of the dose of O 3 to the tissues. The antioxidant 

### 11 substances present in the ELF appear in most cases to limit interaction of O 3 with 

### 12Draft

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- 25 September 2011 Contents of this figure not discussed in Section 5.2 will be discussed in Section 5.3. Clara cell secretory prote in, CCSP; Surfactant Protein

- A, SP

- A; Platelet activating factor, PAF.


### Figure 5

- 8 Details of the O 3 

### interaction with the airway ELF to form secondary oxidation products. Ozone will react with components of the ELF to produce reaction products that may lead to cellular injury and cell signaling as discussed in Section 5.3.

underlying tissues and to prevent penetration of O 3 deeper into the lung. The formation of 

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

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

### 3 antioxidant substrate pools as well as to remove secondary reaction products from tissu e 

### 4 interactions. Important differences exist in the reaction rates for O 3 and these ELF 

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

### 6 and AH 2 are more reactive with O 3 than GSH, proteins, or lipids. In addition to contri



### 7 buting to the driving force for O 3 uptake, formation of secondary oxidation products may 

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

### 9 indicate that the antioxidants might be participating in reactions w here the resulting 

### 10 secondary oxidation products might penetrate into the tissue layer and cause injury. 

### 11Draft

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- 26 September 2011 

## 5.3 Possible Pathways/Modes of Action

5.3.1 Introduction

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

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

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

### 3 pathways describe the processes by which perturbation of normal biological processes 

### 4 produce changes sufficient to lead to c ell injury and subsequent events such as adverse 

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

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

### 7 short

- term and long

- term exposures to O 3 . The extensive research carr ied out over 

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

### 9 mechanisms by which O 3 exerts its effects. This section will discuss some of the 

### 10 representative studies with particular emphasis on studies published since the 2006 O 3 

### 11 AQCD and on studies in humans which inform biological mechanisms underlying 

### 12 responses to O 3 . 

### 13

It is well

- appreciated that secondary oxidation products, which are formed as a result of 

### 14 O 3 exposure, initiate numerous responses at the cellular, tissue and whole organ level of 

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

### 16 initiation of inflammation, alteration of epithelial barrier function, sensitization of 

### 17 bronchial smooth muscle, modification of innate/adaptive im munity and airways 

### 18 remodeling, as will be discussed below. Exposure to O 3 also may result in effects on 

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

### 20 systems. It is unlikely that lipid ozonides and other secondar y oxidation products, which 

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

### 22 ( Chuang et al., 2009 ) . However the inhalation of O 3 may result in systemic ox idative 

### 23 stress. The following subsections describe the current understanding of potential 

### 24 pathways and modes of action responsible for the pulmonary and extrapulmonary effects 

### 25 of O 3 exposure. 

### 26

5.3.2 Activation of Neural Reflexes

Acute O 3 exposure results in reve rsible effects on lung function parameters through 

### 27 activation of neural reflexes. The involvement of bronchial C

- fibers, a type of nociceptive 

### 28 sensory nerve, has been demonstrated in dogs exposed through an endotracheal tube to 2

- 

### 29 3 ppm O 3 for 20

- 70 minutes ( Coleridge et al., 1993 ; Schelegle et al., 1993 ) . This vagal 

### 30Draft

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- 27 September 2011 afferent pathway was found to be r esponsible for O 3 - mediated rapid shallow breathing 

### 1 and other changes in respiratory mechanics in O 3 - exposed dogs ( Schelegle et al., 1993 )

. 

### 2 Ozone also triggers neural reflexes which stimulate the auto nomic nervous system and 

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

### 4 and arrhythmia have been demonstrated in rodents exposed to 0.1

- 0.6 ppm O 3 ( Hamade 

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

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

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

### 8 pathways originating in the respiratory tract may also be responsible for O 3 - mediated 

### 9 activation of nucleus tractus solit arius neurons which resulted in neuronal activation in 

### 10

stress

- responsive regions of the central nervous system (CNS) (rats, 0.5

- 2.0 ppm O 3 for 

### 11 1.5

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

. 

### 12
Recent studies in animals provide new information regarding the effects of O 3 on reflex 

### 13 responses mediated by bronchopulmonary C

- fibers. In ex vivo mouse lungs, O 3 exposure 

### 14 selectively activated a subset of C

- fiber receptors which are TRPA1 ion channels ( Taylor



### 15 Clark and Undem, 2010 ) . TRPA1 ion channels are mem bers of the TRP family of ion 

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

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

### 18 key role in O 3 - induced decrements in pulmonary function, they may mediate allergic 

### 19 asthma ( Caceres et al., 2009 ) . Activation of TRPA1 ion channels following O 3 exposure 

### 20 is likely initiated by secondary oxidation products such as alde hydes and prostaglandins 

### 21 ( Taylor

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

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

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

### 24
4

- hydroxynonenal and 4

- oxononenal ( Taylor

- Clark et al., 2008 ; Trevisani et al., 2007 ) . 4

- 

### 25 oxononenal is a stronger electrophile than 4

- hydroxynonenal and exhibits greater potency 

### 26 towards the TRPA1 channels ( Taylor

- Clark et al., 2008 )

. ( Trevisani et al., 2007 ) . In 

### 27 addition, PGE 2 is known to sens itize TRPA1 channels ( Bang et al., 2007 )

. 

### 28

In exercisi ng humans, the response to O 3 (500 ppb for 2 h) was characterized by 

### 29 substernal discomfort, especially on deep inspiration, accompanied by involuntary 

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

### 31 and to decreased forced vital capacity (FVC) and forced expiratory volume in on e second 

### 32 (FEV 1 ), as measured by spirometry. These changes, which occurred during O 3 exposure, 

### 33 were accompanied by decreased V T and increased respiratory frequency in human 

### 34 subjects. Spirometric changes in FEV 1 and FVC were not due to changes in respiratory 

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

### 36 O 3 - mediated decreases in lung volume was minimal ( Mudway and Kelly, 2000 )
, since 

### 37 changes in FVC or symptoms were not modified by treatment with bronchodilators such 

### 38Draft

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- 28 September 2011 as atropine in exercising human subjects exposed to 400 ppb O 3 for 0.5 hour ( Beckett et 

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

### 2 admi nistration of the rapid

- acting opioid agonist, sufentanyl, in exercising human 

### 3 subjects exposed to 420 ppb O 3 for 2 hours, which indicated the involvement of opioid 

### 4 receptor

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

. 

### 5 The effects of sufentanyl may be attributed to blocking C

- fiber stimulation by O 3 since 

### 6 activation of opioid receptors downregulated C

- fiber function ( Belvisi et al., 1992 ) . Thus, 

### 7 nociceptive sensory nerves, presumably bronchial C

- fibers, are responsible for O 3 - 

### 8 mediated responses in humans ( Passannante et al., 1998 ) . This v agal afferent pathway is 

### 9 responsible for pain

- related symptoms and inhibition of maximal inspiration in humans 

### 10 ( Hazucha et al., 1989 )

. 

### 11
There is some evidence that eicosanoids (see Section 5.3.3) play a role in the neural 

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

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

### 14 1996 ) , before exposure to O 3 significantly blunted the spirometric responses. These 

### 15 studies involved exposures of 1

- 2 hours to 350

- 400 ppb O 3 i n exercising human subjects. 

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

### 17 of PGE 2 and TXB 2 at 1

- hour postexposure ( Hazucha et al., 1996 ) . Although an earlier 

### 18 study demonstrated that PGE 2 stimulated bronchial C

- fibers ( Coleridge et al., 1993 ; 

### 19 Coleridge et al., 1976 ) and suggested that PGE 2 mediated O 3 - induced decreases in 

### 20 pulmonary function, no correlation was observed between the degree of ibuprofen



### 21 induced inhibition of BALF PGE 2 levels and blunting of the spirometric response to O 3 

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

### 23 Further, as noted above, PGE 2 may play a role in the neural reflex by sensitizing TRPA1 

### 24 channels. A recent study in exercising human subjects exposed for 1 hour to 350 ppb O 3 

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

### 26 contribute to human responsiveness to O 3 ( Alfaro et al., 2007 )

. 

### 27
In addition to the spirometric changes, mild airways obstruction occurred in exercising 

### 28 humans during O 3 exposure (500 ppb for 2 hours) ( Hazucha et al., 1989 ) . This pulmonary 

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

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

### 31 exercising human subjects exposed for 1

- 4 hours to 200

- 300 ppb O 3 , changes in sRaw 

### 32 correlated with changes in inflammatory and injury endpoints measured 18

- hours 

### 33 postexposure, but did not fo llow the same time course or change to the same degree as 

### 34 spirometric changes (i.e. FEV 1 , FVC) measured during exposure ( Balmes et al., 1996 ; 

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

### 36 airway r esistance associated with narrowing of small peripheral airways (measured as 

### 37 changes in isovolumetric FEF 25

- 75 ) was demonstrated in O 3 - exposed human subjects (350 

### 38Draft

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- 29 September 2011 ppb for 130 minutes with exercise) ( Weinmann et al., 1995a ; Weinmann et al., 1995b ) . A 

### 1 similar study (400 ppb O 3 for 2 hours in exercising human subjects) found decre ases in 

### 2
FEF 25

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

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

### 4 increase i n residual volume (500 ppb for 2 hours) ( Hazucha et al., 1989 ) and a 

### 5 statistically significant decrease in FEF 25

- 75 (160 ppb for 7.6 hours) ( Horstman et al., 

### 6 1995 ) were observed following O 3 exposure in exercising hu man subjects, providing 

### 7 further support for an O 3 - induced effect on small airways. 

### 8
Mechanisms underlying this rapid increase in airway resistance following O 3 exposure 

### 9 are incompletely understood. Pretreatment with atropine decreased baseline sRaw and 

### 10
prev ented O 3 - induced increases in sRaw in exercising human subjects (400 ppb for 0.5 

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

### 12 receptors of the parasympathetic nervous system. Interestingly, atropine pretreatment 

### 13 partially b locked the decrease in FEV 1 , but had no effect on the decrease in FVC, 

### 14 breathing rate, tidal volume or respiratory symptoms ( Beckett et al., 1985 ) . Using a β



### 15 adrenergic agonist, it was shown that smooth muscle contraction, not increased airway 

### 16 mucus secretion, w as responsible for O 3 - induced increases in airway resistance ( Beckett 

### 17 et al., 1985 ) . Thus, pulmonary function decrements measured as FEV 1 may reflect both 

### 18 restrictive (such as decreased inspiratory capacity) and obstructive (such as 

### 19 bronchoconstriction) type cha nges in airway responses. This is consistent with 

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

### 21
FEV 1 (r=

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

- 0.16, 

### 22 p=0.10) in exercising human subjects exposed for 2.5 hours to 120

- 400 ppb O 3 . 

### 23
Furthermore, tachykinins may contribute to O 3 - mediated increases in airway resistance. 

### 24 In addition to stimulating CNS reflexes, bronchopulmonary C

- fibers mediate local axon 

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

### 26 calcitonin gene

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

### 27
responses such as bronchoconstriction. Recent studies in animals demonstrated that NK

- 1 

### 28 receptor blockade had no effect on O 3 - stimulated physiologic responses such as V T and 

### 29
f B in rats over the 8 hour exposure to 1 ppm O 3 ( Oslund et al., 2008 ) . However, SP and 

### 30 NK receptors contributed to vagally

- mediated bronchoconstriction in guinea pigs 3 days 

### 31 af ter a single 4

- hour exposure to 2 ppm O 3 ( Verhein et al., 2011 ) . In one human study in 

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

### 33 substantially diminished in submucosal sensory nerves 6 hours following O 3 expos ure 

### 34 (200 ppb for 2 hours with exercise) ( Krishna et al., 1997 ) . A statistically significant 

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

### 36 bronchial mucosa and changes in FEV 1 measured 1

- hour po stexposure ( Krishna et al., 

### 37 1997 ) . Another study found that SP was increased in lavage fluid of human subjects 

### 38Draft

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- 30 September 2011 immediately after O 3 challenge (250 ppb for 1 hour with exercise) ( Hazbun et al., 1993 )

. 

### 1 These results provide evidence that the increased airway resistance observed following 

### 2 O 3 exposure is due to vagally

- mediated responses and possibly by local axon reflex 

### 3 responses through bronchopulmonary C

- fiber

- mediated release of SP. 

### 4

5.3.3 Initiation of inflammation

As described previously (5.2.3), O 3 reacts with components of the ELF and cellular 

### 5 membranes resulting in the generation of secondary oxidation products. Higher 

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

### 7 concentrations may initiate cellular resp onses including cytokine generation, adhesion 

### 8 molecule expression and modification of tight junctions leading to inflammation and 

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

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

### 11 5.3.7) ( Mudway and Kelly, 2000 )

. 

### 12
An important hallmark of acute O 3 exposure in humans and animals is neutrophilic 

### 13 airways in flammation. Although neutrophil influx into nasal airways has been 

### 14 demonstrated in exercising human subjects (400 ppb O 3 , 2 hours) ( Graham and Koren, 

### 15 1990 ) , most studies of neutrophil influx have focused on the lower airways ( Hazucha et 

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

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

### 18 human s ubjects exposed for 2 hours to 500 ppb O 3 ( Hazucha et al., 1996 ) . In several 

### 19 studies, ai rways neutrophilia was observable within 1

- 2 hours, peaked at 4

- 6 hours and 

### 20 was returning to baseline levels at 24 hours following exposure of 1

- 2 hours to 300

- 400 

### 21 ppb O 3 in exercising humans ( Devlin et al., 1991 ; Scheleg le et al., 1991 ) . Since the influx 

### 22 and persistence of neutrophils in airways following O 3 exposure correlated with the 

### 23 temporal profile of epithelial injury (guine a pigs, 0.26

- 1 ppm O 3, 72 hours) ( Hu et al., 

### 24 1982 ) , neutrophils were probably injurious. However, neutrophils have also been shown 

### 25 to contribute to repair of O 3 - injured epithelium in rats exposed for 8 hours to 1 ppm O 3, 

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

### 27 1999 ) . Nonetheless, the degree of airways inflammation due to O 3 is thought to have 

### 28 more important long

- te rm consequences than the more quickly resolving changes in 

### 29 pulmonary function since airways inflammation is often accompanied by tissue injury 

### 30 ( Balmes et al., 1996 )

. 

### 31
Ozone exposure results in alterations in other airways inflammatory cells besides 

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

### 33 some of these cells accounts for the later (i.e. 18

- 20 hours) phase of inflammation 

### 34Draft

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- 31 September 2011 following O 3 exposure. Numbers of lymphocytes and total cells in BALF were decreased 

### 1 early after O 3 exposure in exercising humans exposed for 2 hours to 200 ppb O 3 , which 

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

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

### 4 although it was not clear whether the cells were necrot ic or whether membrane adhesive 

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

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

### 7
Pearson an d Bhalla, 1997 ) . A recent study in exercising human subjects exposed for 6.6 

### 8 hours to 80 ppb O 3 demonstrated an increase in numbers of spu tum monocytes and 

### 9 dendritic

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

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

### 11 increase in submucosal mast cells was observed 1.5 hours after a 2 hour

- exposure to 200 

### 12 ppb O 3 ( Blomberg et al., 1999 ) and an increase in BAL mast cell number was observed 

### 13 18 hours after a 4

- hour exposure to 220 ppb O 3 exposu re in exercising human subjects 

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

### 15 influx since they are an important source of several pro

- inflammatory cyto kines and since 

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

### 17 to 200 ppb O 3 ( Stenfors et al., 2002 ; Blomberg et al., 1999 ) . Further, a study using mast 

### 18 cell

- deficient mice demonstrated decreased neutrophilic inflammation in response to O 3 

### 19 (1.75 ppm, 3 hours) compared with wild type mice ( Kleeberger et al., 1993 ) . Influx of 

### 20 these inflammatory cell types in the lung is indicative of O 3 - mediated activation of innate 

### 21 immunity as will be discussed in Section 5.3.6. 

### 22
Much is known about the cellular and molecular signals involved in inflammatory 

### 23 responses to O 3 exposure ( U.S. EPA, 20 06b ) . Eicosanoids are one class of secondary 

### 24 oxidation products which may be formed rapidly following O 3 exposure and which may 

### 25 mediate inflammation. In addition, secondary reaction products may stimulate 

### 26 macrophages to produce cytokines such as IL

- 1, I L

- 6 and TNF

- α which in turn activate 

### 27
IL

- 8 production by epithelial cells. Although IL

- 8 has been proposed to play a role in 

### 28 neutrophil chemotaxis, measurements of IL

- 8 in BALF from humans exposed to O 3 

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

. 

### 30 The time

- course profiles of PGE 2 and IL

- 6 responses suggest that they may pl ay a role in 

### 31 neutrophil chemotaxis in humans ( Mudway and Kelly, 2000 ) . However, pretreatment 

### 32 with ibuprofen at tenuated O 3 - induced increases in BALF PGE 2 levels, but had no effect 

### 33 on neutrophilia in exercising human subjects exposed for 2 hour to 400 ppb O 3 ( Hazucha 

### 34 et al., 1996 )

. 

### 35
One set of studies in humans focused on the earliest phase of airways inflammation (1

- 2 

### 36 hours following exposure). Exercising subjects were exposed to 200 ppb O 3 for 2 hour s 

### 37 and bronchial biopsy tissues were obtained 1.5 and 6 hours after exposure ( Bosson et al., 

### 38Draft

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- 32 September 2011 2009 ; Bosson et al., 2003 ; Stenfors et al., 2002 ; Blomberg et al., 1999 ) . Results 

### 1 demonstrat ed upregulation of vascular endothelial adhesion molecules P

- selectin and 

### 2 ICAM

- 1 at both 1.5 and 6 hours ( Stenfors et al., 2002 ; Blomberg et al., 1999 )

. 

### 3 Submucosal mast cell numbers were increased at 1.5 hours in the biopsy samples without 

### 4 an accompanying increase in neutrophil number ( Blomberg et al., 1999 ) . Pronounced 

### 5 neutrophil infiltration was observed at 6 hours in the bronchial mucosa ( Stenfors et al., 

### 6 2002 ) . Surprisingly, suppression of the NF

- κB and AP

- 1 pathways at 1.5 hours and a lack 

### 7 of increased IL

- 8 at 1.5 or 6 hours in bronchial epithelium was observed ( Bosson et al., 

### 8 2009 )

. The authors suggested that vascular endothelial adhesion molecules, rather than 

### 9 redox sensitive transcription factors, play key roles in early neutrophil recruitment in 

### 10 response to O 3 . 

### 11
Increases in markers of inflammation occurred to a comparable degree in exercising 

### 12 human subjects with mild (least sensitive) and more remarkable (more sensitive) 

### 13 spirometric responses to O 3 (200 ppb, 4 hours) ( Balmes et al., 1996 ) . Two other studies 

### 14 using similar protocols (200 ppb for 4 hours and 300 ppb for 1 hour) found tha t acute 

### 15 spirometric changes were not positively correlated with cellular and biochemical 

### 16 indicators of inflammation ( Aris et al., 1993 ; Schelegle et al., 1991 ) . However 

### 17 inflamm ation was correlated with changes in sRaw ( Balmes et al., 1996 ) . In another 

### 18 study, pretreatment with ibuprofen had no effect on neutrophilia although it blunted the 

### 19 spirometric response in exercising human subjects exposed for 2 hours to 400 ppb O 3 

### 20 ( Hazucha et al., 1996 ) . Taken together, results from these studies indicate different 

### 21 mechanisms un derlying the spirometric and inflammatory responses to O 3 . 

### 22
A common mechanism underlying both inflammation and impaired pulmonary function 

### 23 was suggested by Krishna et al. ( 1997 ) . This study, conducted in exercising humans 

### 24
expos ed to 200 ppb O 3 for 2 hours, demonstrated a correlation between loss of SP 

### 25 immunoreactivity from neurons in the bronchial mucosa and numbers of neutrophils and 

### 26 epithelial cells (shed epithelial cells are an index of injury) in the BALF 6

- hours 

### 27 postexposure. Furthermore, the loss of SP immunoreactivity was correlated with the 

### 28 observed changes in FEV 1 . Another study found that SP was increased in lavage fluid of 

### 29 exercising human subjects immediately after O 3 challenge (250 ppb, 1 hour) ( Hazbun et 

### 30 al., 1993 ) . SP is a neuropeptide released by sensory nerves which mediates neurogenic 

### 31 edema and bronchoconstriction ( Krishna et al., 1997 ) . Taken together, these findings 

### 32 suggest that O 3 - mediated stimulation of sensory nerves which leads to activation of 

### 33 central and local axon reflexes is s a common effector pathway leading to impaired 

### 34 pulmonary function and inflammation. 

### 35
Studies in animal models have confirmed many of these find ings and provided evidence 

### 36 for additional mechanisms involved in O 3 - induced inflammation. A study in mice (2 ppm 

### 37Draft

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- 33 September 2011 O 3 , 3 hours) demonstrated that PAF may be important in neutrophil chemotaxis 

### 1 ( Longphre et al., 1999 ) , while ICAM

- 1 and macrophage inf lammatory protein

- 2 (MIP

- 2), 

### 2 the rodent IL

- 8 homologue, have been implicated in a rat model (1 ppm O 3 , 3 hours) 

### 3 ( Bhalla and Gupta, 2000 ) . Key roles for CXCR2, a receptor for keratinocyte

- derived 

### 4 chemokine (KC) and MIP

- 2, and for IL

- 6 in O 3 - mediated neutrophil influx were 

### 5 demonstrated in mice (1 ppm O 3 , 3 hours) ( Johnston et al., 2005a ; Johnston et al., 

### 6 2005b ) . Activation of JNK and p38 pathways and cathepsin

- S were also found to be 

### 7 important in this response (3 ppm O 3 , 3 hours) ( Williams et al., 2009a ; Williams et al., 

### 8 2008b ; Williams et al., 2007a ) . Matrix metalloproteinase

- 9 (MMP

- 9) protected against 

### 9 O 3 - induced airways inflammation and i njury in mice (0.3 ppm O 3 , 6

- 72 hours) ( Yoon et 

### 10 al., 2007 ) . Interleukin

- 10 (IL

- 10) was also found to be protective since IL

- 10 deficient 

### 11 mice responded to O 3 exposure (0.3 ppm, 24

- 72 hours) with enhanced numbers of BAL 

### 12 neutrophils, enhanced NF

- κB activation and MIP

- 2 levels compared with IL

- 10 sufficient 

### 13 mice ( Backus et al., 2010 )

. 

### 14
In addition, lung epithelial cells may release ATP in response to O 3 exposure ( Ahmad et 

### 15 al., 2005 ) . ATP and its metabolites (catalyzed by ecto

- enzym es) can bind to cellular 

### 16 purinergic receptors resulting in activation of cell signaling pathways ( Picher et al., 

### 17
2004 ) . One such metabolite, adenine, is capable of undergoing oxidation leading to the 

### 18 formation of UA which, if present in high concentrations, could activate inflammasomes 

### 19 and result in caspase 1 activation and the maturation and secretion of IL

- 1β and IL

- 18 

### 20 ( Doster t et al., 2008 ) . A recent study in exercising human subjects exposed for 2 hours to 

### 21 400 ppb O 3 demonstrated a correlation between ATP metabolites and inflammatory 

### 22 markers ( Esther et al., 2011 ) , which provides some support for this mechanism. 

### 23

Several recent studies have focused on the role of toll

- like receptor (TLR) and its related 

### 24 adapto r protein MyD88 in mediating O 3 - induced neutrophilia. While Hollingsworth et al. 

### 25 ( 2004 ) demonstrated airways neutrophilia which was TLR4

- independent following acute 

### 26 (2 ppm, 3 hours) and subchronic (0.3 ppm, 72 hours) O 3 exposure in a mouse model, 

### 27 William s et al. ( 2007b ) found that MyD88 was important in mediating O 3 - induced 

### 28 neutrophilia in mice (3 ppm, 3 hours), with TLR4 and TLR2 contributing to the speed of 

### 29 the response. Moreove r, MyD88, TLR2 and TLR4 contributed to inflammatory gene 

### 30 expression in this model and O 3 upregulated MyD88, TLR4 and TLR4 gene expression 

### 31 ( Williams et al., 2007a ) 

### 32
Hyaluronan was found to mediate a later phase (24 hours) of O 3 - induced inflammation in 

### 33 mice ( Garantziotis et al., 2010 ; Garantziotis et al., 2009 ) . Hyaluronan is an extracellular 

### 34 matrix compon ent which is normally found in the ELF as a large polymer. Exposure to 

### 35
2 ppm O 3 for 3 hours resulted in elevated levels of soluble low molecular weight 

### 36 hyaluronan in the BALF 24

- hours postexposure ( Garantziotis et al., 2010 ; Garantziotis et 

### 37Draft

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- 34 September 2011 al., 2009 ) . Ozone may have caused the depolymerization of hyaluronan to soluble 

### 1 fragments which are known to be endogenous ligands of the CD44 receptor and TLR4 in 

### 2 the macrophage ( Jiang et al., 2005 ) . Binding of hyaluronan fragments to the CD44 

### 3 receptor activates hyaluronan clearance, while binding to TLR4 results in signaling 

### 4 through MyD88 to produce chemokines that stimulate the influx of inflammatory cells 

### 5 ( Jiang et al., 2005 ) . Activation of NF

- κB occurred in both airway epithelia and alveolar 

### 6 macrophages 24

- hours postexposure to O 3 . Increases in BALF pro

- inflamm atory factors 

### 7 KC, IL

- 1β, MCP

- 1, TNF

- α and IL

- 6 observed 24 hours following O 3 exposure were 

### 8 found to be partially dependent on TLR4 ( Garantziotis et al., 2010 ) while increases in 

### 9 BAL inflammatory cells, which consisted mainly of macr ophages, were dependent on 

### 10 CD44 ( Garantziotis et al., 2009 ) . BAL inflammatory cells number and injury markers 

### 11 following O 3 exposure were similar in wild

- type and TLR4

- deficient animals 

### 12 ( Garantziotis et al., 2010 )

. 

### 13
Since exposure to O 3 leads to airways inflammation characterized by neutrophilia, and 

### 14 since neutrophil

- derived oxidants often scavenge ELF antioxidants, concentrations of 

### 15 ELF antioxidants have been examined during airways neutrophilia ( Long et al., 2001 ; 

### 16 Gu nnison and Hatch, 1999 ; Mudway et al., 1999b ) . In exercising humans exposed to 200 

### 17 ppb O 3 for 2 hours, UA, GSH and α

- TOH levels remained unchanged in BALF 6

- hours 

### 18 postexposure while AH2 was decreased significantly in both BALF and plasma ( Mudway 

### 19 et al., 1999b ) . A second study involving the same protocol reported a loss of AH2 from 

### 20 bronchial wash fluid and BALF, representing proximal and distal airway ELF 

### 21 respectively, as well as a n increase in oxidized GSH in both compartments ( Mudway et 

### 22 al., 2001 ) . No change was observed in ELF UA levels in response to O 3 ( Mudway et al., 

### 23 2001 ) . Further, O 3 exposure (0.8 ppm, 4 hours) in female rats resulted in a 50% decrease 

### 24 in BALF AH2 immediately postexposure ( Gunnison and Hatch, 1999 ) . These studies 

### 25 suggested a role for AH2 and GSH in protecting against the oxidative stress associated 

### 26 with inflammation. 

### 27

5.3.4 Alteration of epithelial barrier function

Following O 3 exposure, injury and inflammation can lead to altered airway barrier 

### 28 function. Histologic analysis has demonstrated damage to tight junctions between 

### 29 epithelial cells, suggesting an increase in epithelial permeability. In addition, the presence 

### 30 of shed epithelial cells in the BALF and increased epithelial permeability, which is 

### 31 measured as the flux of small solutes, have been observed and are indicative of epithelial 

### 32 injury. Increases in vascular permeability, as measured by BALF protein and albumin, 

### 33 have also been demonstrated ( Costa et al., 1985 ; Hu et al., 1982 )

. 

### 34Draft

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- 35 September 2011 An early study in sheep measured changes in airway permeability as the flux of inhaled 

### 1 radiolabeled histamine into the plasma ( Abraham et al., 1984 ) . Exposure of sheep to 0.5 

### 2 ppm O 3 for 2 hours via an endotracheal tube resulted i n an increased rate of histamine 

### 3 appearance in the plasma at 1 day postexposure. Subsequently, numerous studies have 

### 4 measured epithelial permeability as the flux of the small solute 99m TcDTPA which was 

### 5 introduced into the air spaces in different regions of the respiratory tract. Increased 

### 6 pulmonary epithelial permeability, measured as the clearance of 99m Tc

- DTPA, was 

### 7 demonstrated in humans 1

- 2 hours following a 2

- hour exposure to 400 ppb O 3 while 

### 8 exercising moderately ( Kehrl et al., 1987 ) . Another study in human subjects found 

### 9 increased epithelial permeability 19

- hours postexposure to 240 ppb O 3 for 130 minutes 

### 10 while exercising ( Foster and Stetkiewicz, 1996 ) . Increased bronchial permeability wa s 

### 11 also observed in dogs 1

- day postexposure (0.4 ppm O 3 by endotracheal tube for 6 hours) 

### 12 and did not resolve for several days ( Foster and Freed, 1999 )

. 

### 13
A role for tachykinins in mediating airway epithelial injury and decreased barrier function 

### 14 has been suggested. Nishiyama et al. ( 1998 ) demonstrated that capsaicin, which depletes 

### 15 nerve fibers of substance P, blocked the O 3 - induced increase in permeability of guinea 

### 16 pig tracheal mucosa (0.5

- 3 ppm O 3 , 0 .5 hours). Pretreatment with propranolol or atropine 

### 17 failed to inhibit this response, suggesting that adrenergic and cholinergic pathways were 

### 18 not involved. In another study, tachykinins working through NK

- 1 and CGRP receptors 

### 19 were found to contribute to a irway epithelial injury in O 3 - exposed rats (1 ppm, 8 hours) 

### 20 ( Oslund et al., 2009
, 2008 )

. 

### 21
Kleeberger et al. ( 2000 ) evaluated genetic susceptibility to O 3 - induced altered barrier 

### 22 function in recombinant inbred strains of mice. Lung hyperpermeability, measured as 

### 23 BALF protein, was evaluated 72 hours after exposure to 0.3 ppm O 3 and found to be 

### 24 associated with a functioning TLR4 gene. This study concluded th at Tlr4 was a strong 

### 25 candidate gene for susceptibility to hyperpermeability in response to O 3 ( Kleebe rger et 

### 26 al., 2000 ) . A subsequent study by these same investigators found that Tlr4 modulated 

### 27 Nos2 mRNA levels and suggested that the gene product of Nos2, iNOS, plays an 

### 28 important role in O 2 - induced lung hyperpermeability (0.3 ppm, 72 hours) ( Kleeberger et 

### 29 al., 2001 ) . More recently, HSP70 was identified as part of the TLR4 signaling pathway 

### 30 (0.3 ppm, 6

- 72 hours) ( Bauer et al., 2011 )

. 

### 31

Antioxidants have been shown to confer resistance to O 3 - induced injury. In a recent 

### 32 study, lung hyperpermeability in response to O 3 (0.3 ppm, 48 hours) was unexpectedly 

### 33 reduced in mice deficient in the glutamate

- cysteine ligase modifier subunit gene 

### 34

co mpared with sufficient mice ( Johansson et al., 2010 ) . Since the lungs of these mice 

### 35 exhibited 70% glutathione depletion, protection against O 3 - induced injury was 

### 36 unexpected ( Johansson et al., 2010 ) . However it was found that several other antioxidant 

### 37Draft

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- 36 September 2011 defenses, including metallothio nein, were upregulated in response to O 3 to a greater 

### 1 degree in the glutathione

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

### 2 2010 ) . The authors suggested that resistance to O 3 - induced lung injury was due to 

### 3 compensatory augmentation of antioxidant defenses ( Johansso n et al., 2010 ) . Antioxidant 

### 4 effects have also been attributed to Clara cell secretory protein (CCSP) and surfactant 

### 5 protein A (SP

- A). CCSP was found to modulate the susceptibility of airway epithelium to 

### 6 injury in mice exposed to O 3 (0.2 or 1 ppm for 8 hours) by an unknown mechanism 

### 7 ( Plopper et al., 2006 ) . SP

- A protected against O 3 - induced airways inflammation and 

### 8 injury in mice (2 ppm, 3 hours), possibly by acting as a sacrificial substrate ( Haque et al., 

### 9 2007 )

. 

### 10
Increased epithelial permeability has been proposed to play a role in allergic sensitization 

### 11 ( Matsumura, 1970 ) , in activation of neural reflexes and in stimulation of smooth muscle 

### 12 receptors ( Dimeo et al., 1981 ) . Abraham et al. ( 1984 ) reported a correlation between 

### 13 airway permeability and airways hyperresponsiveness (AHR) in O 3 - exposed sheep. 

### 14
However a recent study in human subjects exposed to 220 ppb O 3 for 135 minutes while 

### 15 exercising did not find a relationship between O 3 - induced changes in airway permeability 

### 16 and AHR ( Que et al. )

. 

### 17

5.3.5 Sensitization of bronchial smooth muscle

Bronchial reactivity is general ly determined in terms of a response to a challenge agent. 

### 18
Non

- specific bronchial reactivity in humans is assessed by measuring the effect of 

### 19 inhaling increasing concentrations of a bronchoconstrictive drug on lung mechanics 

### 20 (sRaw or FEV 1 ). Methacholine is most commonly employed but histamine and other 

### 21 agents are also used. Specific bronchial reactivity is assessed by measuring effects in 

### 22 response to an inhaled allergen in individuals (or animals) already sensitized to that 

### 23 allergen. An increase in sRaw in response to non

- specific or specific challenge agents 

### 24 indicates AHR. 

### 25
In addition to causing mild airway obstruction as discussed above, acute O 3 exposure 

### 26 results in reversible increases in bronchial reactivity by mechanisms which are not well 

### 27 und e rstood. I n one study, bronchial reactivity of healthy subjects was significantly 

### 28 increased 19

- hours postexposure to O 3 (120

- 240 ppb O 3 for 2 hours with intermittent 

### 29 exercise) ( Foster et al., 2000 ) . These effects may be more significant in human subjects 

### 30 with already compromised airways (Section 5.4.2.2). 

### 31
Ozone may sensitize bronchial smooth muscle to stimulation through a direct effect on 

### 32 smooth muscle or through effects on the sensory nerves in the epithelium or on the motor 

### 33 nerves innervating the smooth muscle ( O'Byrne et al., 1984 ; O'Byrne et al., 1983 ; 

### 34Draft

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- 37 September 2011 Holtzman et al., 1979 ) . It is also recognized that increased bronchial reactivity can be 

### 1 both a rapidly occurring and a persistent response to O 3 ( Foster and Freed, 1999 )

. 

### 2 Tachykinins and secondary oxidation products of O 3 have been proposed as mediators of 

### 3 the early response and inflammation

- derived products have been proposed as mediators 

### 4 of the later response ( Foster and Freed, 1999 )

. 

### 5

Ozone

- induced increases in epithelial permeability, which could improve access of 

### 6 agonist to smooth muscle receptors, may be one me chanism of sensitization through a 

### 7 direct effect on bronchial smooth muscle ( Holtzman et al., 1979 ) . As noted above, a 

### 8 correlation between airway permeability and AHR has been reported in O 3 - exposed 

### 9 sheep ( Abraham et al., 1984 ) but not in O 3 - exposed human subjects ( Que et al. )

. 

### 10
Neurally

- mediated sensitization has been demonstrated. In human subjects exposed for 2 

### 11 hours to 600 ppb O 3 while exercising, pretreatment with atropine inhib ited O 3 - induced 

### 12 AHR, suggesting the involvement of cholinergic postganglionic pathways ( Holtzman et 

### 13 al., 1979 ) . Animal studies have demonstrated that O 3 - induced AHR involved v agally



### 14 mediated responses (rabbits, 0.2 ppm O 3 , 72 hours) ( Freed et al., 1996 ) and local axon 

### 15 reflex responses through bronchopulmonary C

- fiber

- mediated release of SP (guinea pigs, 

### 16 0.8 ppm O 3 , 2 hours) ( Joad et al., 1996 ) . Fur ther, pretreatment with capsaicin to deplete 

### 17 nerve fibers of SP blocked O 3 - mediated AHR (guinea pigs, 1

- 2 ppm O 3 , 2

- 2.25 hours) 

### 18 ( Tepper et al., 1993 ) . Other investigators demonstrated that SP released from airway 

### 19 nocice ptive neurons in ferrets contributed to O 3 - induced AHR (2 ppm O 3 , 3 hours) ( Wu 

### 20 et al., 2008b ; Wu et al., 2003 )

. 

### 21
Some evidence suggests the involvement of arachidonic acid metabolites and neutrophils 

### 22 in mediating O 3 - induced AHR ( Seltzer et al., 1986 ; Fabbri et al., 1985 ) . Increased BAL 

### 23 neutrophils and cyclooxygenase products were found in one study demonstrating AHR in 

### 24 exercising humans (600 ppb for 2 hours) immediately postexposure to ( Seltzer et al., 

### 25 1986 ) . Another study found that ibuprofen pretreatment had no effect on AHR in 

### 26 exercising humans following exposure to 400 ppb O 3 for 2 hours, although spirometric 

### 27 responses were blunted ( Hazucha et al., 1996 ) . This study indicated that the arachidonic 

### 28 acid metabolites whose g eneration was blocked by ibuprofen, (i.e. prostaglandins, 

### 29 thromboxanes and some leukotrienes) did not play a role in AHR. Experiments in dogs 

### 30 exposed for 2 hours to 2.1 ppm O 3 demonstrated a close correlation between O 3 - induced 

### 31 AHR and airways neutrophilic inflammation measured in tissue biopsies ( Holtzman et 

### 32 al., 1983 ) . Furthermore, the increased AHR observed in dogs following O 3 exposure (3 

### 33 ppm, 2 hours) was inhibited by neutrophil depletion ( O'Byrne et al., 1983 ) and by pre



### 34 treatment with inhibitors of arachidonic acid metabolism. In one of these studies, 

### 35 indomethacin pre

- treatment did not prevent airways neutrophilia in response to O 3 (3 

### 36 ppm, 2 hours) providing evidence that the subset of arachidonic acid metabolites whose 

### 37Draft

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- 38 September 2011 generation was i nhibitable by the cyclooxygenase inhibitor indomethacin (i.e., 

### 1 prostaglandins and thromboxanes) was not responsible for neutrophil influx ( O' Byrne et 

### 2 al., 1984 ) . Taken together, these findings suggest that arachidonic acid metabolites, but 

### 3 probably not prostaglandins or thromboxanes, may be involved in the AHR response 

### 4 following O 3 exposure in dogs. Studies probing the role of neutrophils in mediating the 

### 5 AHR response have provided inconsistent results ( Al

- Hegelan et al., 2011 )

. 

### 6
Evidence for cytokine and chemokine involvement in the AHR resp onse to O 3 has been 

### 7 described. Some studies have suggested a role for TNF

- α (mice, 0.5 and 2 ppm O 3 , 3 

### 8 hours) ( Cho et al., 2001 ; Shore et al., 2001 ) and IL

- 1 (mice and ferrets, 2 ppm O 3 , 3 

### 9 hours) ( Wu et al., 2008b ; Park et al., 2004 ) . The latter study found that SP expression in 

### 10 airway neurons was upregulated by IL

- 1 which was released in response to O 3 . Other 

### 11 studies in mice have demonstrated a key role for CXCR2, the chemokine receptor for the 

### 12 neutrophil chemokines KC and MIP

- 2, but not for IL

- 6 in O 3 - mediated AHR ( 1 ppm O 3 , 

### 13 3 hours) ( Johnston et al., 2005a ; Johnston et al., 2005b ) . In contrast, CXCR2 and IL

- 6 

### 14 were both required for neutrophil influx in this model ( Johnston et al., 2005a ; Johnston et 

### 15 al., 2005b ) , as discussed above. Williams et al. ( 2008a ) demonstrated that the Th2 

### 16 cytokine IL

- 13 contributed to AHR, as well as to airways neutrophilia, in mice (3 ppm 

### 17
O 3 , 3 hours). 

### 18

Other studies have focused on the role of TLR4. Hollingsworth et al. ( 2004 ) measured 

### 19 AHR, as well as airways neutrophilia, in mice 6 and 24 hours following acute (2 ppm O 3 

### 20 for 3 hours) and subchronic (0.3 ppm for 3 days) exposure to O 3 . TLR4 is a key 

### 21 component of the innate immune system and is responsible for the immediate 

### 22 inflammatory response seen following challenge with endotoxin and other pathogen



### 23 associated substances. In this study, a functioning TLR4 was required for the full AHR 

### 24 response following O 3 exposure but not for airways neu trophilia ( Hollingsworth et al., 

### 25 2004 ) . These findings are complemented by an older study demonstrating that O 3 effects 

### 26 on lung hyperpermeability required a functioning TLR4 (mice, 0.3 ppm O 3 , 72 hours) 

### 27 ( Kleeberger et al., 2000 ) . Williams et al. ( 2007b ) found that TLR2, TLR4 and the TLR 

### 28 adaptor protein MyD88 contributed to AHR in mice (3 ppm O 3 , 3 hours). Ozone was also 

### 29 found to upregulate MyD88, TLR4 and TLR4 gene expression in this model ( Willi ams et 

### 30 al., 2007b )

. 

### 31
A newly recognized mechanistic basis for O 3 - induced AHR is provided by studies 

### 32 focusing on the role of hyaluronan following O 3 exposure in mice ( Garantziotis et al., 

### 33 2010 ; Ga rantziotis et al., 2009 ) . Hyaluronan is an extracellular matrix component which 

### 34 is normally found in the ELF as a large polymer. Briefly, TLR4 and CD44 were found to 

### 35 mediate AHR in response to O 3 and hyaluronan. Exposure to 2 ppm O 3 for 3 hours 

### 36 resulted in enhanced AHR and elevated levels of soluble low molecular weight 

### 37Draft

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- 39 September 2011 hyaluronan in the BALF 24

- hours postexposure ( Garantziotis et al., 2010 ; Garantziotis et 

### 1 al., 2009 ) . Ozone may have caused the depolymerization of hyaluronan to soluble 

### 2 fragments which are known to be endogenous ligands of the CD44 receptor and TLR4 in 

### 3 the macrophage ( Jiang et al., 2005 ) . In the two recent studies, O 3 - induced AHR was 

### 4 attenuated in CD44 and TLR4

- deficient mice ( Garantziotis et al., 2010 ; Garantziotis et 

### 5 al., 2009 ) . Hyaluronan fragment

- mediated stimulation of AHR was found to require 

### 6 functioning CD44 receptor and TLR4 ( Garantziotis et al., 2010 ; Garantziotis et al., 2009 )

. 

### 7 In contrast, high

- molecular

- weight hyaluronan blocked AHR in response to O 3 

### 8 ( Garantziotis et al., 2009 ) . In another study high

- molecular

- weight hyaluronan enhanced 

### 9 repair of epithelial injury ( Jiang et al., 2005 ) . These studies provide a link between innate 

### 10 immunity and the development of AHR following O 3 exposure, and indicate a role for 

### 11 TLR4 in increasing airways responsiveness. While TLR4

- dependent responses usually 

### 12 involve activation of NF

- κB and the upregulation of proinflammatory factors, the precise 

### 13 mechanisms leading to AHR are unknown ( Al

- Hegelan et al., 2011 )

. 

### 14
In guinea pigs, AHR was found to be mediated by different pathways at 1

- and 3

- days 

### 15 postexposure to a single dose of O 3 (2 ppm for 4 hours ) ( Verhein et al., 2011 ; Yost et al., 

### 16 2005 ) . At 1 day, AHR was due to activation of airway parasympathetic nerves rather than 

### 17 to a direct effect on smooth muscle ( Yost et al., 2005 ) . This effect occurred as a result of 

### 18 O 3 - stimulated release of major basic protein from eosinophils ( Yost et al., 2005 ) . Major 

### 19 basic protein is known to block inhibitory M2 muscarinic receptors which normally 

### 20 dampen acetylcholine release from parasympathetic nerves ( Yost et al., 2005 ) . The 

### 21 resulting increase in acetylcholine release caused an increase in smooth muscle 

### 22 contraction following O 3 exposure ( Yost e t al., 2005 ) . Eosinophils played a different role 

### 23 3

- days postexposure to O 3 in guinea pigs ( Yost et al., 2005 ) . Ozone

- medi ated influx of 

### 24 eosinophils into lung airways resulted in a different population of cells present 3

- days 

### 25 postexposure compared to those present at 1 day ( Yost et al., 2005 ) . At this time point, 

### 26 eosinophil

- derived major basic protein increased smooth muscle responsiveness to 

### 27 acetylcholine which also contributed to AHR ( Yost et al., 2005 ) . However, the major 

### 28 effect of eosinophils was to protect against vagal hyperreactivity ( Yost et al., 2005 ) . The 

### 29 authors suggested that these beneficial effects were due to the production of nerve growth 

### 30 factor ( Yost et al., 2005 ) . Further work by these investigators demonstrated a key role for 

### 31
IL

- 1β in mediating AHR 3

- days postexposure to O 3 ( Verhein et al., 2011 ) . In this study, 

### 32
IL

- 1β increased nerve growth factor and SP which acted through the NK1 receptor to 

### 33 cause vagally

- mediated bronchoconstriction ( Verhein et al., 2011 ) . The mechanism by 

### 34 which SP caused acetylcholine release from parasympathetic nerves f ollowing O 3 

### 35 exposure was not determined ( Verhein et al., 2011 ) . Taken together, the above study 

### 36 results indicate that mechanisms involved in O 3 - mediated AHR can vary over time 

### 37 postexposure and that eosinophils and SP can play a role. Results of this a nimal model 

### 38Draft

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- 40 September 2011 may provide some insight into allergic airways disease in humans which is characterized 

### 1 by eosinophilia (Section 5.4.2.2). 

### 2

5.3.6 Modification of innate/adaptive immune system responses

Host defense depends on effective barrier function and on innate immunity and adaptive 

### 3 immunity ( Al

- Hegelan et al., 2011 ) . Ozone ’ s effect on barrier function in the airways was 

### 4 discussed above (Section 5.3.4). This sect ion focuses on the mechanisms by which O 3 

### 5 impacts innate and adaptive immunity. Both tissue damage and foreign pathogens are 

### 6 triggers for the activation of the innate immune system. This results in the influx of 

### 7 inflammatory cells such as neutrophils, mast cells, basophils, eosinophils, monocytes and 

### 8 dendritic cells and the generation of cytokines such as TNF

- α, IL

- 1, IL

- 6, KC and IL

- 17 . 

### 9 Further, innate immunity encompasses the actions of complement and collectins and the 

### 10 phagocytic functions of macrophage s, neutrophils and dendritic cells. Airway epithelium 

### 11 also contributes to innate immune responses. Innate immunity is highly dependent on cell 

### 12 signaling networks involving TLR4. Adaptive immunity provides immunologic memory 

### 13 through the actions of B and T c ells. Important links between the two systems are 

### 14 provided by dendritic cells and antigen presentation. Recent studies demonstrate that 

### 15 exposure to O 3 modifies cells and processes which are required for innate immunity, 

### 16 contributes to innate

- adaptive immun e system interaction and primes pulmonary immune 

### 17 responses to endotoxin. 

### 18
Ozone exposure of human subjects resulted in recruitment of activated innate immune 

### 19 cells to the airways. Healthy individuals were exposed to 80 ppb O 3 for 6.6 hours with 

### 20 intermitten t exercise and airways inflammation was characterized in induced sputum 18

- 

### 21 hours postexposure ( Alexis et al., 2010 ) . Previous studies demonstrated that induced 

### 22 sputum contains liquid and cellular constituents of the ELF from central conducting 

### 23 airways ( Alexis et al., 2001b ) and also identified these airways as a site of preferential O 3 

### 24 absorption during exercise ( Hu et al., 1994 ) . Ozone exposure resulted in increased 

### 25 numbers of neutrophils, airway monocytes and dendritic

- like cells in sputum ( Alexis et 

### 26 al., 2010 ) . In addition, i ncreased expression of cell surface markers characteristic of 

### 27 innate immunity and antigen presentation (i.e. CD

- 14 and HLA

- DR) was demonstrated 

### 28 on airway monocytes ( Alexis et al., 2010 ) . Enhanced antigen presentation contributes to 

### 29 exaggerated T cell responses and promotes Th2 inflammation and an allergic phenotype 

### 30 ( Lay et al., 2007 ) . Upregulation of pro

- inflammatory cytokines was also demonstrated in 

### 31 sputum of O 3 - exposed subjects ( Alexis et al., 2010 ) . One of these cytokines, IL

- 12p70, 

### 32 correlated with numbers of dendritic

- like cells in the sputum, and is an indicator of 

### 33 dendritic cell activation ( Alexis et al., 2010 ) . These authors have previously reported that 

### 34 exposure of exercising human subjects to 400 ppb O 3 for 2 hours resulted in activation of 

### 35Draft

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- 41 September 2011 monocytes and macrophages ( Lay et al., 2007 ) , which could play a role in exacerbating 

### 1 existing asthma by activating allergen

- specific memory T cells. The current study 

### 2 confirms these findings and extends them by suggesting a potential mechanism whereby 

### 3 O 3 - activated dendritic cells could stimulate naïve T

- cells to promote the development of 

### 4 asthma ( Alexis et al., 2010 ) . A companion study by these same investigators (described in 

### 5 detail in Section 5.4.2.1) provides evidence of dendritic cell activation, measured as 

### 6 increased expression of HLA

- DR, in a subset of the human subjects (GSTM1 null) 

### 7 exposed to 400 ppb O 3 for 2 hours with intermittent exercise ( Alexis et al., 2009 ) . Since 

### 8 dendritic cells are a link between innate and adaptive immunity, these studies provide 

### 9 evidence for an O 3 - mediated intera ction between the innate and adaptive immune 

### 10 systems. 

### 11
Another recent study linked O 3 - mediated activation of the innate immune system to the 

### 12 development of non

- specific AHR in a mouse model ( Pichavant et al., 2008 ) . Repeated 

### 13 exposure to 1 ppm O 3 for 3 hours (3 days over a 5 day period) induced non

- specific AHR 

### 14 measured 24 hours following the last exposure ( Pichavant et al., 2008 ) . This response 

### 15 was fo und to require NKT cells, which are effector lymphocytes of innate immunity, as 

### 16 well as IL

- 17 and airways neutrophilia ( Pichavant et al., 2008 ) . Since glycolipids such as 

### 17 galactosyl ceramide are ligands for the invariant CD1 receptor on NKT cells and serve as 

### 18 endogenous activators of NKT cells, a role for O 3 - oxidized lipids in a ctivating NKT cells 

### 19 was proposed ( Pichavant et al., 2008 ) . The authors con trasted this innate immunity 

### 20 pathway with that of allergen

- provoked specific AHR which involves adaptive immunity, 

### 21 the cytokines IL

- 4, IL

- 13, IL

- 17, and airways eosinophilia ( Pichavant et al., 2008 )

. 

### 22 Interestingly, NKT cells were required for both the specific AHR provoked by allergen 

### 23 and the non

- specific AHR provoked by O 3 ( Pichavant et al., 2008 ) . Different cytokine 

### 24 profiles of the NKT cells from allergen and O 3 - exposed mice was proposed to account 

### 25 for the different pathways ( Pichavant et al., 2008 ) . More recently, NKT cells have been 

### 26 found to function in both innate and adaptive immunity ( Vivier et al., 2011 )

. 

### 27
An interaction between allergen and O 3 in the induction of nonspecific AHR was shown 

### 28 in another animal study ( Larsen et al., 2010 ) . Mice were sensitized with the aerosolized 

### 29 allergen OVA on 10 consecutive days followed by exposure to O 3 (0.1

- 0.5 ppm for 3 

### 30 hours) ( Larsen et al., 2010 ) . While allergen sensitization alone did not alter airways 

### 31 responsiveness to a nonspecific challenge, O 3 exposure of sensitized mice resulted in 

### 32 nonspecific AHR at 6

- and 24

- hours postexposure ( Larsen et al., 2010 ) . The effects of O 3 

### 33 on AHR were independent of airways eosinophilia and neutrophilia ( Larsen et al., 2010 )

. 

### 34 However, OVA pretreatment led to goblet cell metaplasia which was enhanced by O 3 

### 35
exposure ( Larsen et al., 2010 ) . It should be noted that OVA sensitization using only 

### 36 aerosolized antigen in this study is less common than the usual procedure for OVA 

### 37 sensitization achieved by one or more initial systemic injections of OVA and adjuvant 

### 38Draft

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- 42 September 2011 followed by repeated inhalation exposure to OVA. This study also points to an interaction 

### 1 between innate and adaptive immune systems in the development of the AHR response. 

### 2

Furthermore, O 3 was f ound to act as an adjuvant for allergic sensitization ( Hollingsworth 

### 3 et al., 2010 ) . Oropharyngeal aspiration of OVA on day 0 and day 6 failed to lead to 

### 4 allergic sensitization unless mice were first exposed to 1 ppm O 3 for 2 hours 

### 5 ( Hollingsworth et al., 2010 ) . The O 3 - mediated response involved Th2 (IL

- 4, IL

- 5 and IL



### 6 9) and Th17 cytokines (IL

- 17) and wa s dependent on a functioning TLR4 ( Hollingsworth 

### 7

et al., 2010 ) . Ozone exposure also activated OVA

- bearing dendritic cells in the thoracic 

### 8 lymph nodes, as measured by the presence of the CD86 surface marker, which suggests 

### 9 naïve T cell stimulation and the involvement of Th2 pathways ( Hollingsworth et al., 

### 10 2010 ) . Thus the adjuvant effects of O 3 may be du e to activation of both innate and 

### 11 adaptive immunity. 

### 12
Priming of the innate immune system by O 3 was reported by Hollingsworth et al. ( 2007 )

. 

### 13 In this study, exposu re of mice to 2 ppm O 3 for 3 hours led to nonspecific AHR at 24

- 

### 14 and 48

- hours postexposure, an effect which subsided by 72 hours ( Hollingsworth et al., 

### 15 2007 ) . However, in mice treated with aerosolized endotoxin immediately following O 3 

### 16 exposure, AHR was greatly enhanced at 48

- and 72

- hours postexposure ( Hollings worth et 

### 17 al., 2007 ) . In addition, O 3 pre

- exposure was found to reduce the number of inflammatory 

### 18 cells in the BALF, to increase cytokine production and total protein in the BALF and to 

### 19 increase systemic IL

- 6 following exposure to endotoxin ( Hollingsworth et al., 2007 )

. 

### 20 Furthermore, O 3 stimulated the apoptosis of alveolar macrophages 24

- hours 

### 21 postexposure, an effect which was greatly enhanced by endotoxin tr eatment. Apoptosis of 

### 22 circulating blood monocytes was also observed in response to the combined exposures 

### 23 ( Hollingsworth et al., 2007 ) . Ozone pre

- exposure en hanced the response of lung 

### 24 macrophages to endotoxin ( Hollingsworth et al., 2007 ) . Taken together, these findings 

### 25 demonstrated that O 3 exposure increased inn ate immune responsiveness to endotoxin. 

### 26 The authors attributed these effects to the increased surface expression of TLR4 and 

### 27 increased signaling in macrophages observed in the study ( Hollingsworth et al., 2007 ) . It 

### 28 was proposed that the resulting decrease in airway inflammatory cells could account for 

### 29 O 3 - mediated decreased clearance of bacterial pathogens observed in numerous animal 

### 30 models ( Hollingsworth et al., 2007 )

. 

### 31
More recently, these authors demonstrated that hyaluronan contributed to the O 3 - primed 

### 32 response to endotoxin ( Li et al., 2010 ) . In this study, exposure of mice to 1 ppm O 3 for 3 

### 33 hours resulted in enhanced responses to endotoxin, which was mimicked by intratracheal 

### 34 instillation of hyaluronan fragments ( Li et al., 2010 ) . Hyaluronan, like O 3 , was also found 

### 35 to induce TLR4 receptor peripheralization in the macrophage membrane ( Li et al., 2010 ; 

### 36 Hollingsworth et al., 2007 ) , an effect which is asso ciated with enhanced responses to 

### 37Draft

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- 43 September 2011 endotoxin. This study and previous ones by the same investigators showed elevation of 

### 1 BALF hyaluronan in response to O 3 exposure ( Garantziotis et al., 2010 ; Li et al., 2010 ; 

### 2 Garantziotis et al., 2009 ) , providing evi dence that the effects of O 3 on innate immunity 

### 3 are at least in part mediated by hyaluronan fragments. The authors note that excessive 

### 4 TLR4 signaling can lead to lung injury and suggest that O 3 may be responsible for an 

### 5 exaggerated innate immune response which may underlie lung injury and decreased host 

### 6 defense ( Li et al., 2010 )

. 

### 7
Activation or upregulation of the immune system has not been reported in all studies. 

### 8 Impaired antigen

- specific immunity was demonstrated following subacute O 3 exposure 

### 9 (0.6 ppm, 10 h/day for 15 days) in mice ( Feng et al., 2006 ) . Specifically, O 3 exposure 

### 10 altered the lymphocyte subset and cytokine profile and impacted thymoc yte early 

### 11 development leading to immune dysfunction. Further, recent studies demonstrated SP

- A 

### 12 oxidation in mice exposed for 3

- 6 hours to 2 ppm O 3 . SP

- A is an important innate 

### 13 immune protein which plays a number of roles in host defense including acting as 

### 14 opsonin for the recognition of some pathogens ( Haque et al., 2009 ) . These investigations 

### 15 found that O 3 - mediated ca rbonylation of SP

- A was associated with impaired macrophage 

### 16 phagocytosis in vitro ( Mikerov et al., 2008b ) . Furthermore, O 3 exposure (2 ppm for 3 

### 17 hours) in mice was found to increase susceptibility to pneumonia infection in mice 

### 18 through an impai rment of SP

- A dependent phagocytosis ( Mikerov et al., 2008a ; Mikerov 

### 19 et al., 2008c )

. 

### 20
Taken together, results of recent studies provide evidence that O 3 alters host 

### 21 immunologic response and leads to immune system dysfunction through its effects on 

### 22 innate and adaptive immunity. 

### 23

5.3.7 Airways remodeling

As noted above, the degree of airways inflammation due to O 3 may have important long



### 24 term consequences since airways inflammation is often accompanied by tissue injury 

### 25 ( Balmes et al., 1996 ) . The nasal airways, conducting airways and distal airways ( i.e. 

### 26 respiratory bronchioles or centriacinar region depending on the species) have all been 

### 27 identified as sites of O 3 - mediated injury and inflammation ( Mudway and Kelly, 2000 ) . At 

### 28 all levels of the respiratory tract, loss of sensitive epithelial cells, degranulation of 

### 29 secretory cells, proliferation of resistant epithelial cells and neutrophilic influx have been 

### 30

o bserved as a result of O 3 exposure ( Mu dway and Kelly, 2000 ; Cho et al., 1999 ) . An 

### 31 important study ( Plopper et al., 1998 ) conducted in adult rhesus monkeys (0.4 and 

### 32 1.0 ppm O 3 for 2 hours) found that 1 ppm O 3 resulted in the greatest epithelial injury in 

### 33 the respiratory bronchioles immediately postexposure although injury was observed at all 

### 34Draft

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- 44 September 2011 of the RT sites studied except for the lung parenchyma. Exposure to 0.4 ppm O 3 resulted 

### 1 in epithelial injury only in the respiratory bronchioles. 

### 2
Persistent inflammation and injury, observed in animal models of chronic and intermittent 

### 3 exposure to O 3 , are associated with airways remodeling, including mucous cell 

### 4 metaplasia of nasal transitional epithelium ( Harkema et al., 1999 ; Hotchkiss et al., 1991 ) 

### 5 and bronchiolar metaplasia of alveolar ducts ( Mudway and Kelly, 2000 ) . Fibrotic changes 

### 6 such as deposition of collagen in the airways and sustained lung function decrements 

### 7 especially in small airways have also been demonstrated as a response to chronic O 3 

### 8
exposure ( Mudway and Kelly, 2000 ; Chang et al., 1992 )

. These effects, described in 

### 9 detail in Section 7.2.3.1, have been demonstrated in rats exposed to levels of O 3 as low as 

### 10 0.25 ppm. Mechanisms responsible for the resolution of inflammation and the repair of 

### 11 injury remain to be clarified and there is only a limited understanding of the biological 

### 12 processes underlying long

- term morphological changes. However, a recent study in mice 

### 13 demonstrated a key role for the TGF

- β signaling pathway in the deposition of collagen in 

### 14 the airw ays wall following chronic intermittent exposure to 0.5 ppm O 3 ( Katre et al., 

### 15 2011 )

. 

### 16
It should be noted that repeated exposure to O 3 results in attenuation of some O 3 - 

### 17 induced responses, including those associated with the activation of neural reflexes (e.g. 

### 18 decrements in p ulmonary function), as discussed in Section 5.3.2. However, numerous 

### 19 studies demonstrate that some markers of injury and inflammation remain increased 

### 20 during multi

- day exposures to O 3 . Mechanisms responsible for attenuation, or the lack 

### 21 thereof, are incomp letely understood. 

### 22

5.3.8 Systemic inflammation and oxidative/nitrosative stress

Extrapulmonary effects of O 3 have been noted for decades ( U.S. EPA, 2006b ) . It has 

### 23 been proposed that lipid oxidation products resulting from reaction of O 3 with lipids 

### 24 and/or cellular membranes in the ELF are responsible for systemic effects, however it is 

### 25 not known whether they gain access to the vascular space ( Chuang et al., 2009 )

. 

### 26 Alternatively, ext rapulmonary release of diffusible mediators may initiate or propagate 

### 27 inflammatory responses in the vascular or in systemic compartments ( Cole and Freeman, 

### 28 2009 ) . A role for O 3 in modulating endothelin, a potent vasoconstrictor, has also been 

### 29 proposed. Studies in rats found that exposure to 0.4 and 0.8 ppm O 3 induced endothelin 

### 30 system genes in the lung and increased circulating levels of endothelin ( Thomson et al., 

### 31 2006 ; Thomson et al., 2005 ) . Systemic oxidative stress is suggested by studies in humans 

### 32 which reporte d associations between O 3 exposure and levels of plasma 8

- isoprostanes 

### 33Draft

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- 45 September 2011 and the presence of peripheral blood lymphocyte micronuclei ( Chen et al., 2007 ; Chen et 

### 1 al., 2006a )

. 

### 2

Ozone

- induced perturbations of the cardiovascular system were recently investigated in 

### 3 young mice and monkeys ( C huang et al., 2009 ) and in rats ( Kodavanti et al., 2011 ; 

### 4 Perepu et al., 2010 ) (see Sections 6.3.3.2 and 7.3.1.2). These are the first studies to 

### 5 suggest that systemic oxidative stress and inflammation play a mechanistic role in O 3 - 

### 6 induced effects on the systemic vascular and heart. Exposure to 0.5 ppm O 3 for 5 days 

### 7 resulted in oxid ative/nitrosative stress, vascular dysfunction and mitochondrial DNA 

### 8 damage in the aorta ( Chuang et al., 2009 ) . Chronic exposure to 0.8 ppm O 3 resulted in an 

### 9 enhancement of inflammation and lipi d peroxidation in the heart following an ischemia



### 10 reperfusion challenge ( Perepu et al., 2010 ) . In addition, chronic intermittent exposure to 

### 11 0.4 ppm O 3 increased aortic levels of mRNA for biomarkers of oxidative stress, 

### 12 thrombosis, vasoconstriction and proteolysis and aortic lectin

- like oxidized

- low density 

### 13 lipoprotein receptor

- 1( LOX

- 1) mRNA and protein levels ( Kodavanti et al., 2011 ) . The 

### 14 latter study suggests a role for circulating oxidized lipids in mediating the effects of O 3 . 

### 15
Systemic inflammation and oxidative/nitrosative stress may similarly affect other organ 

### 16 systems as well as the plasma compartment. Circulating cytokines have the potential to 

### 17 enter the brain through diffusion and active trans port and to contribute to 

### 18 neuroinflammation, neurotoxicity, cerbrovascular damage and a break

- down of the blood 

### 19 brain barrier ( Block and Calderón

- Garcidueñas, 2009 ) (see Sectio ns 6.4 and 7.5). They 

### 20 can also activate neuronal afferents ( Block and Calderón

- Garcidueñas, 2009 ) . Vagal 

### 21 afferent pathways originating in the respiratory tract may also be resp onsible for O 3 - 

### 22 mediated activation of nucleus tractus solitarius neurons which resulted in neuronal 

### 23 activation in stress

- responsive regions of the CNS in rats (0.5 or 2 ppm O 3 for 1.5

- 120 

### 24 hours) ( Gackière et al., 2011 ) . Recent studies have demonstrated O 3 - induced brain lipid 

### 25 peroxidation, cytokine production in the brain and upregulated expression of VEGF in 

### 26 rats (0.5 ppm O 3 , 3 hours or 0.25

- 0.5 ppm O 3 , 4 h/day, 15

- 60 days) ( Guevara

- Guzmán et 

### 27 al., 2009 ; Araneda et al., 2008 ; Pereyra

- Muñoz et al., 2006 ) . Further, O 3 - induced 

### 28 oxidative stress resulted in increased plasma lipid peroxides (0.25 ppm, 4h/day, 15

- 60 

### 29 days) ( Santiago

- López et al., 2010 ) , which was correlated with damage to specific brain 

### 30 regions ( Pereyra

- Muñoz et al., 2006 )

. 

### 31
Oxidative stress is one mechanism by which testicular and sperm function is disrupted 

### 32 (see Section 7.4.1). Oxidative stress may inhibit testicular steroidogenesis leading to 

### 33 decrea sed testosterone levels ( Diemer et al., 2003 ) . It may decrease sperm quality by lipid 

### 34 peroxidation of sperm plasma membrane which leads to impaired sperm mobility 

### 35 ( Agarwal et al., 2003 ) . Further, it may damage DNA in the sperm nucleus leading to 

### 36 apoptosis and a decline in sperm counts ( Agarwal et al., 2003 ) . Since oxidative stress is a 

### 37Draft

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- 46 September 2011 key event underlying many of the health effects of O 3 , it is possible that sperm quality 

### 1 and quantity may be impact ed by this mechanism ( Sokol et al., 2006 )

. 

### 2
A role for plasma antioxidants in modulating O 3 - induced respiratory effects was 

### 3 suggested by a recent study ( Aibo et al., 2010 ) . In this study, pretreatment of rats with a 

### 4 high dose of acetaminophen resulted in increased levels of plasma cytokines and the 

### 5 influx of inflammatory cells into the lung following O 3 exposure (0.25

- 0.5 ppm, 6 hours) 

### 6 ( Aibo et al., 2010 ) . These effects were not observed in response to O 3 alone. 

### 7
Fu rthermore, acetaminophen

- induced liver injury was exacerbated by O 3 exposure. A 

### 8 greater increase in hepatic neutrophil accumulation and greater alteration in gene 

### 9 expression profiles was observed in mice exposed to O 3 and acetaminophen compared 

### 10 with either exposure alone ( Aibo et al., 2010 ) . Although not measured in this study, 

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

### 12 glutathione is the source of plasma glutathione, acetaminophen treatment may have 

### 13 lowered plasma glutat hione levels and altered the redox balance in the vascular 

### 14 compartment. These findings indicate interdependence between respiratory tract, plasma 

### 15 and liver responses to O 3 , possibly related to glutathione status. 

### 16

5.3.9 Impaired alveolar

- arterial O 2 

### transfer

O 3 may impair alveolar

- arterial oxygen transfer and reduce the supply of arterial oxygen 

### 17 to the myocardium. This may have a greater impact in individuals with compromised 

### 18 cardiopulmonary systems. Gong et al. ( 1998 ) provided evidence of a small decrease in 

### 19 arterial oxygen saturation in human subjects exposed for 3 hours to 300 ppb O 3 while 

### 20 exercising. In addition, Delaunois et al. ( 1998 ) demonstrated pulmonary vasoconstriction 

### 21 in O 3 - expos ed rabbits (0.4 ppm, 4 hours). Although of interest, the contribution of this 

### 22 pathway to O 3 - induced cardiovascular effects remains uncertain. 

### 23

5.3.10 Summary

This section summarizes the modes of action and toxicity pathways resulting from O 3 

### 24 inhalation (Figure 5

- 9). These pathways provide a mechanistic basis for the health effects 

### 25 which are described in detail in Chapters 6 and 7. Three distinct short

- term responses 

### 26 have been well

- characterized in humans challenged with O 3 : decreased pulmonary 

### 27 function, airways in flammation, and increased bronchial reactivity. In addition, O 3 

### 28 exposure exacerbates, and possibly also causes, asthma and allergic airways disease in 

### 29 humans. Animal studies have demonstrated airways remodeling and fibrosis in response 

### 30 to chronic and inter mittent O 3 exposures and a wide range of other responses. While the 

### 31Draft

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- 47 September 2011 respiratory tract is the primary target tissue, cardiovascular and other organ effects occur 

### 1 following short

- and long

- term exposures of animals to O 3 . 

### 2

Figure 5

- 9 The modes of action/po ssible pathways underlying the health effects resulting from inhalation exposure to O 3 

### .

The initial key event in the toxicity pathway of O 3 is the formation of secondary 

### 3 oxidation products in the respiratory tract. This involves direct reactions with components 

### 4 of the ELF and/or plasma membranes of cells residing in the respiratory tract. The 

### 5 resulting secondary oxidation products transmit signals to the epithelium, nociceptive 

### 6 sensory nerve fibers and, if present, dendritic cells, mast cells and eosin ophils. Thus, O 3 

### 7 effects are mediated by components of ELF and by the multiple cell types found in the 

### 8 respiratory tract. Further, oxidative stress is an implicit part of this initial key event. 

### 9
Another key event in the toxicity pathway of O 3 is the activ ation of neural reflexes which 

### 10 lead to decrements in pulmonary function (see Section 6.2.1). Evidence is accumulating 

### 11 that secondary oxidation products are responsible for this effect. Eicosanoids have been 

### 12 implicated in humans, while both eicosanoids and aldehydes are effective in animal 

### 13 models. Different receptors on bronchial C

- fibers have been shown to mediate separate 

### 14 Formation of secondary oxidation products 

### Ozone + Respiratory Tract

Systemic inflammation and

o xidative/ nitrosative stress Activation of neural reflexes Airways remodeling Sensitization of bronchial

s mooth muscle Alteration of epithelial barrier function Initiation of inflammation Modification of innate and adaptive immunity

Extrapulmonary Effects Decrements in pulmonary function Pulmonary inflammation/oxidative stress Increases in airways permeability Airways hyperresponsiveness Exacerbation/induction of asthma Decreased host defenses Epithelial metaplasia and fibrotic airways Altered lung developmentDraft

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- 48 September 2011 effects of O 3 on pulmonary function. Nociceptive sensory nerves are involved in the 

### 1 involuntary truncation of respiration which results in decreases in FVC, FEV 1 , tidal 

### 2 volume and pain upon deep inspiration. Opioids block these responses while atropine has 

### 3 only a minimal effect. New evidence in an animal model suggests that TRPA1 receptors 

### 4 on bronchial C

- fibers mediate this pathway. Ozone exposure also results in activation of 

### 5 vagal sensory nerves and a mild increase in airway obstruction measured as increased 

### 6 sRaw. Atropine and β

- adrenergic agonists greatly inhibit this response in humans 

### 7 indicating that the airway obstruction is due to br onchoconstriction. Other studies in 

### 8 humans implicated SP release from bronchial C

- fibers resulting in airway narrowing due 

### 9 to either neurogenic edema or bronchoconstriction. New evidence in an animal model 

### 10 suggests that the SP

- NK receptor pathway caused br onchoconstriction following O 3 

### 11
exposure. 

### 12
Initiation of inflammation is also a key event in the toxicity pathway of O 3 . Secondary 

### 13 oxidation products, as well as chemokines and cytokines elaborated by airway epithelial 

### 14 cells and macrophages, have been impli cated in the initiation of inflammation. Vascular 

### 15 endothelial adhesion molecules may also play a role. Work from several laboratories in 

### 16 using human subjects and animal models suggest that O 3 triggers the release of 

### 17 tachykinins such as SP from airway senso ry nerves which could contribute to 

### 18 downstream effects including inflammation (see Sections 6.2.3 and 7.2.4). Airways 

### 19 neutrophilia has been demonstrated in BALF, mucosal biopsy and induced sputum 

### 20 samples. Influx of mast cells, monocytes and macrophages als o occur. Inflammation 

### 21 further contributes to O 3 - mediated oxidative stress. Recent investigations show that O 3 

### 22 exposure leads to the generation of hyaluronan fragments from high molecular weight 

### 23 polymers of hyaluronan normally found in the ELF in mice. Hyal uronan activates TLR4 

### 24 and CD44

- dependent signaling pathways in macrophages, and results in an increased 

### 25 number of macrophages in the BALF. Activation of these pathways occurs later than the 

### 26 acute neutrophilic response suggesting that they may contribute to longer

- term effects of 

### 27 O 3 . The mechanisms involved in clearing O 3 - provoked inflammation remain to be 

### 28 clarified. It should be noted that inflammation, as measured by airways neutrophilia, is 

### 29 not correlated with decrements in pulmonary function as measured by spirometry. 

### 30
A fourth key event in the toxicity pathway of O 3 is alteration of epithelial barrier 

### 31 function. Increased permeability occurs as a result of damage to tight junctions between 

### 32 epithelial cells subsequent to O 3 - induced injury and inflammation. It may play a role in 

### 33 allergic sensitization and in AHR (see Sections 6.2.2, 6.2.6, and 7.2.5). Tachykinins 

### 34 mediate this response while antioxidants confer protection. Genetic susceptibility has 

### 35 been associated with a functioning TLR4 gene and with iNOS. 

### 36Draft

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- 49 September 2011 A fifth key event in the toxicity pathway of O 3 is the sensitization of bronchial smooth 

### 1 muscle. 

### 2
Increased bronchial reactivity can be both a rapidly occurring and a persistent response. 

### 3 The mechanisms responsible for early and later AHR are not well

- unde rstood (see 

### 4 Section 6.2.2). One proposed mechanism of sensitization, O 3 - induced increases in 

### 5 epithelial permeability, would improve access of agonist to smooth muscle receptors. The 

### 6 evidence for this mechanism is not consistent. Another proposed mechanism, for which 

### 7 there is greater evidence, is neurally

- mediated sensitization. In humans exposed to O 3 , 

### 8 atropine blocked the early AHR response indicating the involvement of cholinergic 

### 9 postganglionic pathways. Animal studies demonstrated that O 3 - induced AHR in volved 

### 10 vagally

- mediated responses and local axon reflex responses through bronchopulmonary 

### 11 C

- fiber

- mediated release of SP. Later phases of increased bronchial reactivity may 

### 12 involve the induction of IL

- 1β which in turn upregulates SP production. In guinea pigs, 

### 13 eosinophil

- derived major basic protein contributed to the stimulation of cholinergic 

### 14 postganglionic pathways. A novel role for hyaluronan in mediating the later phase effects 

### 15 O 3 - induced AHR has recently been demonstrated. Hyaluronan fragments stimula ted AHR 

### 16 in a TLR4

- and CD44 receptor

- dependent manner. Tachykinins and secondary oxidation 

### 17 products of O 3 have been proposed as mediators of the early response and inflammation



### 18 derived products have been proposed as mediators of the later response. Inhibit ion of 

### 19 arachidonic acid metabolism was ineffective in blocking O 3 - induced AHR in humans 

### 20 while in animal models mixed results were found. Other cytokines and chemokines have 

### 21 been implicated in the AHR response to O 3 in animal models. 

### 22
A sixth key event in t he toxicity pathway of O 3 is the modification of innate/adaptive 

### 23 immunity. While the majority of evidence for this key event comes from animal studies, 

### 24 there are several studies suggesting that this pathway may also be relevant in humans. O 3 

### 25 exposure of hu man subjects resulted in recruitment of activated innate immune cells to 

### 26 the airways. This included macrophages and monocytes with increased expression of cell 

### 27 surface markers characteristic of innate immunity and antigen presentation, the latter of 

### 28 which could contribute to exaggerated T cell responses and the promotion of an allergic 

### 29 phenotype. Evidence of dendritic cell activation was observed in GSTM1 null human 

### 30 subjects exposed to O 3 , suggesting O 3 - mediated interaction between the innate and 

### 31 immune sys tems. Animal studies further linked O 3 - mediated activation of the innate 

### 32 immune system to the development of nonspecific AHR, demonstrated an interaction 

### 33 between allergen and O 3 in the induction of nonspecific AHR, and found that O 3 acted as 

### 34 an adjuvant fo r allergic sensitization through the activation of both innate and adaptive 

### 35 immunity. Priming of the innate immune system by O 3 was reported in mice. This 

### 36 resulted in an exaggerated response to endotoxin which included enhanced TLR4 

### 37 signaling in macrophage s. Ozone

- mediated impairment of the function of SP

- A, an innate 

### 38Draft

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- 50 September 2011 immune protein, has also been demonstrated. Taken together these studies provide 

### 1 evidence that O 3 can alter host immunologic response and lead to immune system 

### 2 dysfunction. These mechanisms ma y underlie the exacerbation and induction of asthma 

### 3 (see Sections 6.2.6 and 7.2.1), as well as decreases in host defense (see Sections 6.2.5 and 

### 4 7.2.6). 

### 5
Another key event in the toxicity pathway of O 3 is airways remodeling. Persistent 

### 6 inflammation and inj ury, which are observed in animal models of chronic and 

### 7 intermittent exposure to O 3 , are associated with morphologic changes such as mucous 

### 8 cell metaplasia of nasal epithelium, bronchiolar metaplasia of alveolar ducts and fibrotic 

### 9 changes in small airways (see Section 7.2.3). Mechanisms responsible for these responses 

### 10 are not well

- understood. However a recent study in mice demonstrated a key role for the 

### 11 TGF

- β signaling pathway in the deposition of collagen in the airway wall following 

### 12 chronic intermittent exposure to O 3 . 

### 13
Systemic inflammation and vascular oxidative/nitrosative stress are also key events in the 

### 14 toxicity pathway of O 3 . Extrapulmonary effects of O 3 occur in numerous organ systems, 

### 15 including the cardiovascular, central nervous, reproductive an d hepatic systems (see 

### 16 Sections 6.3 to 6.5 and 7.3 to 7.5). It has been proposed that lipid oxidation products 

### 17 resulting from reaction of O 3 with lipids and/or cellular membranes in the ELF are 

### 18 responsible for systemic responses, however it is not known wh ether they gain access to 

### 19 the vascular space. Alternatively, release of diffusible mediators from the lung into the 

### 20 circulation may initiate or propagate inflammatory responses in the vascular or in 

### 21 systemic compartments. Systemic oxidative stress is sugge sted by studies in humans 

### 22 which reported associations between O 3 exposure and levels of plasma 8

- isoprostanes 

### 23 and the presence of peripheral blood lymphocyte micronuclei. 

### 24



## 5.4 Interindividual Variability in Response

Responses to O 3 exposure are variable within the population and the basis for this 

### 25 variability is not clear ( Mudway and Kelly, 2000 ) . Both dosimetric and mechanistic 

### 26 factors are likely to contribute to this variability and are discussed below. 

### 27

5.4.1 Dosimetric Considerations

Two studies have investigated the correlation of O 3 uptake with the pulmonary function 

### 28 responses to O 3 expo sure ( Reeser et al., 2005 ; Gerrity et al., 1994 ) . These studies found 

### 29 that the large subject

- to

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

### 30Draft

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- 51 September 2011 have a dosimetric explanation. Reeser et al. ( 2005 ) found no significant relationship 

### 1 between %ΔFEV 1 and fractional absorption o f O 3 using the bolus method. Contrary to 

### 2 previous findings, the percent change in dead space volume of the respiratory tract 

### 3 (%ΔV D ) did not correlate with O 3 uptake, possibly due to the contraction of dead space 

### 4 caused by airway closure. Gerrity et al. ( 1994 ) found that intersubject variability in FEV 1 

### 5 and airway resistance was not related to differences in the O 3 dose delivered to the lower 

### 6 airways, whereas minute ventilation was predictive of FEV 1 decrement. No study has yet 

### 7 demonstrated that subjects show a consistent pattern of O 3 retention when re

- exposed 

### 8 over weeks of time, as has been shown to be the case for the FEV 1 response, or that 

### 9 within

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

### 10
A delay in onset of O 3 - induced pulmonary function responses has been noted in 

### 11 numerous studies. Recently the delay was characterized in terms of changes in f B 

### 12 ( Schelegle et al., 2007 ) . In humans exposed for 1

- 2 hours to 120

- 350 ppb O 3 while 

### 13 exercising, no change in f B was observed until a certain cumulative inhaled dos e of O 3 

### 14 had been reached. Subsequently, the magnitude of the change in f B was correlated with 

### 15 the inhaled dose rate ( Schelegle et al., 2007 ) . These investigators proposed that initial 

### 16 reactions of O 3 with ELF resulted in a time

- dependent depletion of ELF antioxidants, and 

### 17 that activation of neural reflexes occurred only after the antioxidant d efenses were 

### 18 overwhelmed ( Schelegle et al., 2007 )

. 

### 19
Other studies investigated the relati onship between O 3 dose and cellular injury. In two 

### 20 studies, the initial cellular injury was found to correlate with the site

- specific O 3 dose. 

### 21 Contained within the CAR, the respiratory bronchioles were confirmed as the site 

### 22 receiving the greatest O 3 dose ( 18 O mass/lung weight) and sustained the greatest initial 

### 23 cellular injury in O 3 (0.4 and 1.0 ppm for 2 hours) exposed resting rhesus monkeys 

### 24 ( Plopper et al., 1 998 ) . The respiratory bronchioles, having the highest concentration of 

### 25 local O 3 dose, were also the site of significant GSH reduction. In addition, a study in 

### 26 isolated perfused rat lungs found greater injury in conducting airways downstream of 

### 27 bifurcations where local doses of O 3 were higher ( Postlethwait et al., 2000 )

. 

### 28
Further, the degree of inflammation in rats has been correlated with 18 O

- labeled O 3 dose 

### 29 markers in the lower lung. In female rats exposed to 0.8 ppm O 3 for 4 hours, BAL 

### 30 neutrophil number and 18 O reaction product were directly proportional ( Gunnison and 

### 31 Hatch, 1999 ) . Kari et al. ( 1997 ) observed that a 3

- week caloric restriction (75%) in rats 

### 32 abrogated the toxicity of O 3 (2 ppm, 2 hours), measured as BALF increases in protei n, 

### 33 fibronectin and neutrophils, which was seen in normally fed rats. Accompanying this 

### 34 resistance to O 3 toxicity was a reduction (30%) in the accumulation of 18 O reaction 

### 35 product in the lungs. These investigations also demonstrated an inverse relationship 

### 36 between AH2 levels and O 3 dose and provided evidence for AH2 playing a protective 

### 37Draft

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- 52 September 2011 role following O 3 exposure in these studies. Pregnant and lactating rats had lower AH2 

### 1 content in BALF and exhibited a greater increase in accumulation of 18 O reaction 

### 2 produc ts compared with pre

- pregnant rats in response to O 3 exposure ( Gunnison and 

### 3 Hatch, 1999 ) . In the calorie restricted model, a 30% higher basal BALF AH2 

### 4 concentration and a rapid accumulation of AH2 into the lungs to levels 60% above 

### 5 normal occurred as result of O 3 exposure ( Kari et al., 1997 ) . However, this relationship 

### 6 between AH2 levels and O 3 dose did not hold up in every study. Aging rats (9 and 24 

### 7 months old) had 49% and 64% lower AH2 in lung tissue compared with month

- old rats 

### 8 but the aging

- induced AH2 loss did not increase the accumulation of 18 O reaction 

### 9 products following O 3 exposure (0. 4

- 0.8 ppm, 2

- 6 hours) ( Vincent et al., 1996a )

. 

### 10
Interindividual variability in the neutrophilic response has been noted in human subjects 

### 11 ( Holz et al., 1999 ; Devlin et al., 1991 ; Schelegle et al., 1991 ) . One study demonstrated a 

### 12 threefold difference in airways neutrophilia, measured as percent of total cells in 

### 13 proximal BALF, among human subjects exp osed to 300 ppb O 3 for 1 hour while 

### 14 exercising ( Schelegle et al., 1991 ) . Another study reported a 20

- fold difference in BAL 

### 15 neutrophils following exposure t o 80

- 100 ppb O 3 for 6.6 hours in exercising human 

### 16 subjects ( Devlin et al., 1991 ) . Reproducibility of intra

- individual responses to 1

- hour 

### 17 exposure to 250 ppb O 3 , measured as sputum neutrophilia, was demonstrated by Holz 

### 18 ( 1999 ) . Few studies have examined the dose

- or concentration

- re sponsiveness of airways 

### 19 neutrophilia in O 3 - exposed humans ( Holz et al., 1999 ; Devlin et al., 1991 ) . No 

### 20 concentration

- responsiveness was observed in healthy human subjects exposed for 1 hour 

### 21 to 125

- 250 ppb O 3 and a statistically significant increase in sputum neutrophil ia was 

### 22 observed only at the higher dose ( Hol z et al., 1999 ) . However, concentration

- dependent 

### 23 and statistically significant increases in BAL neutrophils and the inflammatory mediator 

### 24
IL

- 6 were reported following exposure to 80 and 100 ppb O 3 for 6.6 hours in exercising 

### 25 humans ( Devlin et al., 1991 )

. Additional evidence is provided by a meta

- analysis of the 

### 26 O 3 dose

- inflammatory response in controlled human exposure studies involving exposure 

### 27 to 80

- 600 ppb O 3 for 60

- 396 minutes ( Mudway and Kelly, 2004b ) . Results demonstrated 

### 28 a linear relationship between inhaled O 3 dose (determined as the product of 

### 29 concentration, ventilation and time) and BAL neutrophils at 0

- 6 hours and 18

- 24 hours 

### 30 following O 3 exposure ( Mudway and Kelly, 2004b )

. 

### 31
Collectively these studies demonstrate a correlation between dose and response for some 

### 32 O 3 - induced effects and suggest a role for ELF antioxidants in modulating the dose to 

### 33 tissue. The lack of correlation between O 3 - induced effects and calculated O 3 dose may be 

### 34 a result of interindividual differences in TB volume. 

### 35Draft

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- 53 September 2011 

### 5.4.2 Mechanistic Considerations

There was a large range of pulmonary function responses to O 3 among healthy young 

### 1 adults exposed for 4 hours to 200 ppb O 3 or for 1.5 hours to 420 ppb O 3 while exercis ing 

### 2 ( Hazucha et al., 2003 ; Ba lmes et al., 1996 ) . Since individual responses were relatively 

### 3 consistent across time, it was thought that responsiveness reflected an intrinsic 

### 4 characteristic of the subject ( Mudway and Kelly, 2000 ) . Older adults were generally not 

### 5 responsive to O 3 ( Hazucha et al., 2003 ) , while obese young women may have been more 

### 6 responsive than lean young women (420 ppb, 1.5 hours, while exercising) ( Bennett et al., 

### 7 2007 ) . The lack of spirometric responsiveness was not attributable to the presence of 

### 8 endogenous endorphins, which could potentially block C

- fiber stimulation by O 3 , as 

### 9 demonstrated in a study involving intravenous administration of naloxone immedi ately 

### 10 following the O 3 exposure (420 ppb, 2 hours, while exercising) to weak responders 

### 11 ( Passannante et al., 1998 ) . Inflammation and other responses to O 3 were also 

### 12 characterized by a large degree of interindividual variability. Currently, the mechanisms 

### 13 underlying this variability are not known. It has been proposed that some of the variation 

### 14 in responses may be genetically determined ( Yang et al., 2005a ) . The role of gene



### 15 environment interactions, pre

- existing diseases and conditions, nutritional status, 

### 16 lifestage, attenuation, and co

- exposures in modulating responses to O 3 are discussed 

### 17 below. 

### 18

5.4.2.1 Gene

- Environment Interactions

The significant interindividu al variation in responses to O 3 infers that genetic background 

### 19 is an important determinant of susceptibility to O 3 ( C ho and Kleeberger, 2007 ; 

### 20 Kleeberger et al., 1997 ) (see also Section 8.4). Strains of mice which are prone or 

### 21 resistant to O 3 - induced effects have been used to systematically identify candidate 

### 22 susceptibility genes. Genome wide linkage analyses (also known as positional cloning) 

### 23 demonst rated quantitative trait loci for O 3 - induced lung inflammation and 

### 24 hyperpermeability on chromosome 17 ( Kleeberger et al., 1997 ) and chromosome 4 

### 25 ( Kleeberger et al., 2000 ) , respectively, using these recombinant inbred strains of mice and 

### 26 exposures to 0.3 ppm O 3 for up to 72 hours. More specifically, these studies found that 

### 27 Tnf, whose protein product is the inflammatory cytokine TNF

- α, and Tlr4, whose protein 

### 28 product is TLR4, were candidate susceptibility genes ( Kleeberger et al., 2000 ; Kleeberger 

### 29 et al., 1997 ) . Other studies, which used targeted deletion, identified genes encoding iNOS 

### 30 and heat shock proteins as TLR4 effector genes ( Bauer et al., 2011 ; Kleeberger et al., 

### 31 2001 ) and found that IL

- 10 protects against O 3 - induced pulmonary inflammation 

### 32 ( Backus et al., 2010 ) . Invest igations in inbred mouse strains found that differences in 

### 33 expression of certain proteins, such as CCSP (1.8 ppm O 3 for 3 hours) ( Broeckaert et al., 

### 34Draft

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- 54 September 2011 2003 ) and MARCO (0.3 ppm O 3 for up to 48 hours) ( Dahl et al., 2007 ) , were responsible 

### 1 for phenotypic characteristics, such as epithelial permeability and scavenging of oxidized 

### 2 lipids, respectively, which confer sensitivity to O 3 . 

### 3
Genetic polymorphisms have received increasing attention as modulators of O 3 - m ediated 

### 4 effects. Functionally relevant polymorphisms in candidate susceptibility genes have been 

### 5 studied at the individual and population level in humans, and also in animal models. 

### 6 Genes whose protein products are involved in antioxidant defense/oxidative stress and 

### 7 xenobiotic metabolism, such as glutathione

- S

- transferase M1 (GSTM1) and 

### 8 NADPH:quinone oxidoreductase 1 (NQO1), have also been a major focuses of these 

### 9 efforts. This is because oxidative stress resulting from O 3 exposure is thought to 

### 10 contribute to the pathogenesis of asthma, and because xenobiotic metabolism detoxifies 

### 11 secondary oxidation products formed by O 3 which contribute to oxidative stress ( Islam et 

### 12 al., 2008 ) . TNF

- α is of interest since it is linked to a candidate O 3 susceptibility gene and 

### 13 since it plays a key role in initiating airways inflammation ( Li et al., 200 6d )

. 

### 14 Polymorphisms of genes coding for GSTM1, NQO1 and TNF

- α have been associated 

### 15 with altered susceptibility to O 3 - mediated effects ( Li et al., 2006d ; Yang et al., 2005a ; 

### 16 Romieu et al., 2004a ; Corradi et al., 2002 ; Bergamaschi et al., 2001 ) . Additional studies 

### 17 have focused on functional variants in other genes involved in anti oxidant defense such 

### 18 as catalase (CAT), myeloperoxidase, heme oxygenase (HMOX

- 1) and ma nganese 

### 19 superoxide dismutase (Mn SOD) ( Wenten et al., 2009 ; Islam et al., 2008 ) . These studies 

### 20 are discussed below. 

### 21
GSTM1 is a phase II antioxidant enzyme which is transcriptionally regulated by NF

- E2

- 

### 22 related factor 2

- antioxidant response ele ment (Nrf2

- ARE) pathway. A large proportion 

### 23 (40

- 50%) of the general public (across ethnic populations) has the GSTM1

- null genotype, 

### 24 which has been linked to an increased risk of health effects due to exposure to air 

### 25 pollutants ( London, 2007 ) . A role for GSTs in metabolizing electrophiles such as 4

- 

### 26 hydroxynonenal, which is a secondary oxidation product formed following O 3 exposure, 

### 27 has been demonstrated ( Awasthi et al., 2004 ) . A recent study found that the GSTM1 

### 28 genotype modulated the time course of the neutrophilic inflammatory response following 

### 29 acute O 3 exposure (400 ppb for 2 hours with intermittent exercise) in healthy adults 

### 30 ( Alexis et al., 2009 ) . In GSTM1

- null and

- sufficient subjects, O 3 - induced sputum 

### 31 neutrophilia was similar at 4 hours. However, neutrophilia resolved by 24 hours in 

### 32 sufficient subjects but not in GSTM1

- null subjects. In contrast, no differences in 24 hour 

### 33 sputum neutrophilia were observed between GSTM1

- null and

- sufficient human subjec ts 

### 34 exposed to 60 ppb O 3 for 2 hours with intermittent exercise ( Kim et al., 2011 ) . It is not 

### 35 known whether the effect seen at the higher exposure level ( Alexis et al., 2009 ) was due 

### 36 to the persistence of pro

- inflammatory stimuli, impaired production of downregulators or 

### 37 impaired neutrophil apoptosis and clearance. However, a subsequent in vitro study by 

### 38Draft

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- 55 September 2011 these same investigators found that GSTM1 deficiency in airway epithelial cells 

### 1 enhanced IL

- 8 production in response to 0.4 ppm O 3 for 4 hours ( Wu et al., 2011 )

. 

### 2 Furthermore, NF

- κB activation was required for O 3 - induced IL

- 8 production ( Wu et al., 

### 3 2011 ) . Since IL

- 8 is a potent neutrophil activator and chemotaxin, this study provides 

### 4 additional evidence for the role of GSTM1 as a modulator of inflammatory responses due 

### 5 to O 3 exposure. 

### 6

In addition, O 3 exposure increased the expression of the surface ma rker CD14 in airway 

### 7 neutrophils of GSTM1

- null subjects compared with sufficient subjects ( Alexis et al., 

### 8 2009 ) . Furthermore, differences in airway macrophages were noted between the GSTM1

- 

### 9 sufficient and

- null subjects. Nnumbers of airway macrophages were decreased at 4 and 

### 10 24 hou rs following O 3 exposure in GSTM1

- sufficient subjects ( Alexis et al., 2009 )

. 

### 11 Airway macrophages in GSTM1

- null subjects were greater in number and found to have 

### 12 greater oxidative burst and phagocytic capability than those of sufficient subjects. Airway 

### 13 macrophages and dendritic ce lls from GSTM1

- null subjects exposed to O 3 expressed 

### 14 higher levels of the surface marker HLA

- DR, suggesting activation of the innate immune 

### 15 system ( Alexis et al., 2009 ) . These differences in inflammatory responses between the 

### 16 GSTM1

- null and

- sufficient subjects may provide biolog ical plausibility for the 

### 17 differences in O 3 - mediated effects reported in controlled human exposure studies 

### 18 ( Corradi et al., 2002 ; Bergamaschi et al., 2001 ) . It should also be noted that GSTM1 

### 19 genotype did not affect the acute pulmonary function (i.e. spirometric) response to O 3 

### 20 which provides additional evidenc e for separate mechanisms underlying O 3 ’ s effects on 

### 21 pulmonary function and inflammation in adults ( Alexis et al., 2009 ) . However, GSTM1

- 

### 22 null asthmatic children were previously found to be more at risk of O 3 - induced effects on 

### 23 pulmonary function than GSTM1

- sufficient asthmatic ch ildren ( Romieu et al., 2004a )

. 

### 24
Another enzyme involved in the metabolism of secondary oxidation products is NQO1. 

### 25 NQO1 catalyzes the 2

- electron reduction b y NADPH of quinones to hydroquinones. 

### 26 Depending on the substrate, it is capable of both protective detoxification reactions and 

### 27 redox cycling reactions resulting in the generation of reactive oxygen species. A recent 

### 28 study using NQO1

- null mice demonstrated that NQO1 contributes to O 3 - induced 

### 29 oxidative stress, AHR and inflammation following a 3

- hour exposure to 1 ppm O 3 

### 30 ( Voynow et al., 2009 ) . These experimental results may provide biological plausibility for 

### 31 the increased biomarkers of oxidative stress and increased pulmonary function 

### 32 decrements observed in O 3 - exposed i ndividuals bearing both the wild

- type NQO1 gene 

### 33 and the null GSTM1 gene ( Corradi et al., 2002 ; Bergamaschi et al., 2001 )

. 

### 34
Besides enzymes, other mechanisms participate in the removal of secondary oxidation 

### 35 products formed as a result of O 3 inhalation. One involves scavenging of oxidized lipids 

### 36 via the macrophage receptor with collagenous structure (MARCO) expressed on the cell 

### 37Draft

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- 56 September 2011 surface of alveolar macrophages. A recent study demonstrated increased gene expression 

### 1 of MARCO in the lungs of an O 3 - resistant C3H mouse strain (HeJ) b ut not in an O 3 - 

### 2 sensitive, genetically similar strain (OuJ) ( Dahl et al., 2007 ) . Upregulation of MARCO 

### 3 occurred in mice exposed to 0.3 ppm O 3 for 24

- 48 hours; inhalation exposure for 6 hours 

### 4 at this concentration was insufficie nt for this response. Animals lacking the MARCO 

### 5 receptor exhibited greater inflammation and injury, as measured by BAL neutrophils, 

### 6 protein and isoprostanes, following exposure to 0.3 ppm O 3 ( Dahl et al., 2007 ) . MARCO 

### 7 also prot ected against the inflammatory effects of oxidized surfactant lipids ( Dahl et al., 

### 8 2007 ) . Scavenging of oxidized lipids may limit O 3 - induced injury since ozonized 

### 9 cholesterol species formed in the ELF (mice, 0.5

- 3 ppm O 3 , 3 hou rs) ( Pulfer et al., 2005 ; 

### 10 Pulfer and Murphy, 2004 ) stimulated apoptosis and cytotoxicity in vitro ( Gao et al., 

### 11 2009b ; Sathishkumar et al., 2009 ; Sathishkumar et al., 2007a ; Sathishkumar et al., 

### 12 2007b )

. 

### 13
Two studies reported relationships between TNF promoter variants and O 3 - induced 

### 14 effects in humans. In one study, O 3 - induced change in lung function was significantly 

### 15 lower in adult subjects with TNF promoter variants

- 308A/A and

- 308G/A compared with 

### 16 adult subjects with the variant

- 308G/G ( Yang et al., 2005a ) . This response was 

### 17

mo dulated by a specific polymorphism of LTA ( Yang et al., 2005a ) , a previously 

### 18 identified candidate susceptibility gene whose protein product is lymphotoxin

- α 

### 19 ( Kleeberger et al., 1997 ) . In the second study, an association between the TNF promoter 

### 20 variant

- 308G/G and decreased risk of asthma and lifetime wheezing in children was 

### 21 found ( Li et al., 2006d ) . The protective effect on wheezing was modulated by ambient O 3 

### 22 levels and by GSTM1 and GSTP1 polymorphisms. The authors suggested that the 

### 23
TNF

- 308 G/G genotype may have a protective role in the development of childhood 

### 24 asthma ( Li et al., 2006d )

. 

### 25
Similarly, a promoter variant of the gene HMOX

- 1, consisting of a smaller number of 

### 26 (GT)n repeats, was associated with a reduced risk for new

- onset asthma in non

- Hispanic 

### 27 white children ( Islam et al., 2008 ) . The number of (GT) n repeats in this promoter has 

### 28 been shown to be inversely related to the inducibility of HMOX

- 1. A modulatory effect 

### 29 of O 3 was demonstrated since the beneficial effects of this polymorphism were seen only 

### 30 in children living in low O 3 communities ( Islam et al., 2008 ) . This study also identified 

### 31 an association between a polymorphism of the CAT gene and increased risk of new

- onset 

### 32 asthma in Hispanic children; however no modulation by O 3 was seen ( Islam et al., 2008 )

. 

### 33 No association was observed in this study between a MnSOD polymorphism and asthma 

### 34 ( Islam et al., 2008 )

. 

### 35Draft

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- 57 September 2011 Studies to date indicate that some variability in individual responsiveness to O 3 may be 

### 1 accounted for by functional genetic polymorphisms. Further, the effects of gene



### 2 environment interactions may be different in children and adults. 

### 3

5.4.2.2 Pre

- existing Diseases and Conditions

Pre

- existing diseases and conditions can alter the response to O 3 exposure. For example, 

### 4 responsiveness to O 3 , as measured by spirometry, is decreased in smokers and individuals 

### 5 with COPD ( U.S. EPA, 2006b ) . Asthma and allergic diseases are of major importance in 

### 6 this discussion. In individuals with asthma, there is increased responsiveness to 

### 7 bronchoconstrictor challenge. This results from a combination of structural and 

### 8 physiological fa ctors including increased airway inner

- wall thickness, smooth muscle 

### 9 responsiveness and mucus secretion. Although inflammation is likely to contribute, its 

### 10 relationship to AHR is not clear ( U.S. EPA, 2006b ) . However, some asthmatics have 

### 11 higher baseline levels of neutrophils, lymphocytes, eosinophils and mast cells in 

### 12 bronchial washes and bronchial biopsy tissue ( Stenfors et al., 2002 ) . It has been proposed 

### 13 that enhanc ed sensitivity to O 3 is conferred by the presence of greater numbers of 

### 14 resident airway inflammatory cells in disease states such as asthma ( Mudway and Kelly, 

### 15 2000 )

. 

### 16
In order to determine whether asthmatics exhibit greater responses to O 3 , several older 

### 17 studies compared pulmonary function in asthmatic and non

- asthmatic subjects following 

### 18 O 3 exposure. Some also pro bed mechanisms which could account for enhanced 

### 19 sensitivity. While the majority focused on measurements of FEV 1 and FVC and found no 

### 20 differences between the two groups following exposures of 2

- 4 hours to 125

- 250 ppb O 3 

### 21 or to a 30

- minute exposure to 120

- 180 ppb O 3 by mouthpiece while exercising ( Stenfors 

### 22 et al., 2002 ; Mudway et al., 2001 ; Holz et al., 1999 ; Scannell et al., 1996 ; Koenig et al., 

### 23 1987 ; Linn et al., 1978 ) , there were notable exceptions. In one study, greater airway 

### 24 obstruction in asthmatics compared with non

- asthmatic subjects was observed 

### 25 immediately following a 2

- hour exposure to 400 ppb O 3 with intermittent exercise ( Kreit 

### 26 et al., 1989 ) . These c hanges were measured as statistically significant greater decreases in 

### 27
FEV 1 and in FEF 25

- 75 (but not in FVC) in the absence of a bronchoconstrictor challenge 

### 28 ( Kreit et al., 1989 ) . These results suggest that this group of asthmatics responded to 

### 29 O 3 - exposure with a greater degree of vagally

- mediated bronchoconstriction compared 

### 30 with the non

- asthmatics. A second study demonstrated a statistically significant greater 

### 31 decrease in FEV 1 and in FEV 1 /FVC (but not in FVC) in asthmatics compared with non



### 32 asthmatics exposed to 160 ppb O 3 for 7.6 hours with light exercise ( Horstman et al., 

### 33 1995 ) . These respons es were accompanied by wheezing and inhaler use in the asthmatics 

### 34 ( Horstman et al., 1995 ) . Aerosol bolus dispersion measurements demonstrated a 

### 35Draft

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- 58 September 2011 statistically significant greater change in asthmatics compared with non

- asthmatics, 

### 1 which was suggestive of O 3 - induced small airway dysfunction ( Horstman et al., 1995 )

. 

### 2 Furthermore, a statistically significant correlation was observed between the degree of 

### 3 baseline airway status and the FEV 1 response to O 3 in the asthmatic subjects ( Horstman 

### 4 et al., 1995 ) . A third study found similar decreases in FVC and FEV 1 in both asthmatics 

### 5 and non

- asthmatics exposed to 400 ppb O 3 for 2 hours with mild exercise ( Alexis et al., 

### 6 2000 ) . However, a statistically significant decrease in FEF 75 , a measure of small airway 

### 7 function, was observed in asthmatics but not in non

- asthmatics ( Alexis et al., 2000 )

. 

### 8 Taken together, these latter studies indicate that while the magnitude of restrictive type 

### 9 spirometric decline was similar in asthmatics and non

- asthmatics, that obstructive type 

### 10 changes (i.e. bronchoconstriction) were greater in asthmatics. Further, asthmatics 

### 11 exhibited greater sensitivity to O 3 in terms of small airways function. 

### 12
Since asthma exacerbations occur in response to allergens and/or other triggers, some 

### 13 studies have focused on O 3 - induced changes in AHR following a bronchoconstrictor 

### 14 challenge. No difference in sensitivity to methacholine bronchoprovocation was observed 

### 15 between asthmatics and non

- asthmatics exposed to 400 ppb O 3 for 2 hours with moderate 

### 16 exercise ( Kreit et al., 1989 ) . However, increased bronchial reactivity to inhaled allergens 

### 17 was demonstrated in mild allergic asthmatics exposed to 160 ppb for 7.6 hours, 250 ppb 

### 18 for 3 hours and 120 ppb for 1 hour while exercising ( Kehrl et al., 1999 ; Jorres et al., 

### 19 1996 ; Molfino et al., 1991 ) and in allergen

- sensitized guinea pigs following O 3 exposure 

### 20 (1 ppm, 1 hour) ( Sun et al., 1997 ) . Similar, but modest, responses were reported for 

### 21 individuals with allergic rhinitis ( Jorres et al., 1996 ) . Further, the contractile response of 

### 22 isolated airways from human donor lung tissue, which were sensitized and challenged 

### 23 with allergen, was increased by pre

- exposure to 1 ppm O 3 for 20 ( Roux et al., 1999 )

. 

### 24 These studies provide support for O 3 - mediated enhancement of responses to allergens in 

### 25 allergic sub jects. 

### 26
In terms of airways neutrophilia, larger responses were observed in asthmatics compared 

### 27

to non

- asthmatics subjects exposed to O 3 in some ( Balmes et al., 1997 ; Scannell et al., 

### 28 1996 ; Basha et al., 1994 ) but not all ( Mudway et al., 2001 ) of the older studies. While 

### 29 each of these studies involved exposure of exercising human subjects to 200 ppb O 3 , the 

### 30 duration of exposure was longer (i.e. 4

- 6 hours) in the former studies than in the latter 

### 31 study (2 hours). Further, statistically significantly increases in myeloperoxidase levels (an 

### 32 indicator of neutrophil activation) in bronchial washes was observed in mild asthmatics 

### 33 compared with non

- asthmatics, despite no difference in O 3 - s timulated neutrophil influx 

### 34 between the 2 groups following exposure to 200 ppb O 3 for 2 hours with mild exercise 

### 35 ( Stenfors et al., 2002 ) . A more recent study found that atopic asthmatic subjects exhibited 

### 36 an enhanced inflammatory response to O 3 (400 ppb, 4 hours, with exercise) ( Hernandez 

### 37 et al., 2010 ) . This response was characterized by greater numbers of neutrophils, higher 

### 38Draft

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- 59 September 2011 levels of IL

- 6, IL

- 8 and IL

- 1β and greater macrophage cell

- surface expres sion of TLR4 

### 1 and IgE receptors in induced sputum compared with healthy subjects. This study also 

### 2 reported a greater increase in hyaluronan in atopic subjects and atopic asthmatics 

### 3 compared with healthy subjects following O 3 exposure. Animal studies have pr eviously 

### 4 reported that hyaluronic acid activates TLR4 signaling and results in AHR (see Section 

### 5 5.3.5). Furthermore, levels of IL

- 10, a potent anti

- inflammatory cytokine, were greatly 

### 6 reduced in atopic asthmatics compared to healthy subjects. These results provide 

### 7 evidence that innate immune and adaptive responses are different in asthmatics and 

### 8 healthy subjects exposed to O 3 . 

### 9
Eosinophils may be an important modulator of responses to O 3 in asthma and allergic 

### 10 airways disease. Eosinophils and associated pro teins are thought to affect muscarinic 

### 11 cholinergic receptors which are involved in vagally

- mediated bronchoconstriction 

### 12 ( Mudway and Kelly, 2000 ) . Studies described in Section 5.3.5 which demonstrated a key 

### 13 role of eosinophils in O 3 - mediated AHR may be relevant to human allergic airways 

### 14 disease which is characterized by airways eosinophilia ( Yost et al., 2005 ) . Furthermore, 

### 15 O 3 exposure sometimes results in airways eosinophilia in allergic subjects or animal 

### 16 models. For example, eosinophilia of the nasal and other airways was observed in 

### 17 individuals with pre

- existing allergic disease following O 3 inhalation (270 and 400 ppb 

### 18 O 3 , 2 hours, with exercise) ( Vagaggini et al., 2002 ; Peden et al., 1995 ) . Further, O 3 

### 19 exposure (0.5 ppm, 8 hours/day for 1

- 3 days) increased allergic responses, such as 

### 20 eosinophilia and augmented intraepithelial mucosubstances, in the nasal airways of 

### 21 ovalbumin (OVA)

- sensitized rats ( Wagner et al., 2002 ) . In contrast, Stenfors ( 2002 ) found 

### 22 no stimulation of eosinophil influx measured in bronchial washes and BALF of mild 

### 23 asthmatics following exposure to a lower concentration (200 ppb, 2 hours, with exercise) 

### 24 of O 3 . 

### 25

The role of mast cells in O 3 - mediated asthma exacerbations has been investigated. Mast 

### 26 cells are thought to play a key role in O 3 - induced airways inflammation, since airways 

### 27 neutrophilia was decreased in mast cell

- deficient mice exposed to O 3 ( Kleeberger et al., 

### 28 1993 ) . However, another study found that mast cells were not involved in the 

### 29 development of increased bronc hial reactivity in O 3 - exposed mice ( Noviski et al., 1999 )

. 

### 30 Nonetheless, mast cells release a wide variety of important inflammatory mediators 

### 31 which may lead to asthma exacerbations ( Stenfors et al., 2002 ) . A large increase in mast 

### 32 cell number in bronchial submucosa was observed in non

- asthmatics and a significant 

### 33 decrease in mast cell number in bronchial epithelium was obse rved in mild asthmatics 6 

### 34 hours following exposure to 200 ppb O 3 for 2 hours during mild exercise ( Stenfors et al., 

### 35 2002 ) . While these results point to an O 3 - mediated flux in bronchial mast cell 

### 36 populations which differed between the non

- asthmatics and mild asthmatics, 

### 37Draft

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- 60 September 2011 interpretation of these findings is difficult. Furthermore, mast cell number did not change 

### 1 in airway lavages in either group in response to O 3 ( Stenfors et al., 2002 ) 

### 2
Cytokine profiles in the airways have been investigated as an indicator of O 3 sensitivity. 

### 3 Differences in epithelial cytokine expression were observed in bronchial biopsy samples 

### 4 in non

- as thmatic and asthmatic subjects both at baseline and 6

- hours postexposure to 200 

### 5 ppb O 3 for 2 hours ( Bosson et al., 2003 ) . The asthmatic subjects had a higher baseline 

### 6 expression of IL

- 4 and IL

- 5 compared to non

- asthmatics. In addition, expression of IL

- 5, 

### 7
IL

- 8, GM

- CSF, and ENA

- 78 in asthmatics was increased significantly following O 3 

### 8 exposure compared to non

- asthmatics ( Bosson et al., 2003 ) . Some of these (IL

- 4, IL

- 5 

### 9 and GM

- CSF) are Th2

- related cytokines or neutrophil chemoattractants, and play a role 

### 10 in IgE production, airways eosinophilia and suppression of Th 1

- cytokine production 

### 11 ( Bosson e t al., 2003 ) . These findings suggest a link between adaptive immunity and 

### 12 enhanced responses of asthmatics to O 3 . 

### 13
A further consideration is the compromised status of ELF antioxidants in disease states 

### 14 such as asthma ( Mudway and Kelly, 2000 ) . This could possibly be due to ongoing 

### 15 inflammation which causes antioxidant depletion or to abnormal antioxidant transp ort or 

### 16 synthesis ( Mudway and Kelly, 2000 ) . For example, basal levels of AH2 were 

### 17 significantly lower and basal levels of oxidized GSH and UA were significantly higher in 

### 18 bronchial wash fluid and BALF of mild asthmatics compared with healthy control 

### 19 subjects ( Mudway et al., 2001 ) . Differences in ELF antioxidant content have also been 

### 20 noted between species. These observations have led to the su ggestion that the amount and 

### 21 composition of ELF antioxidants, the capacity to replenish antioxidants in the ELF or the 

### 22 balance between beneficial and injurious interactions between antioxidants and O 3 may 

### 23 contribute to O 3 sensitivity, which varies between individuals and species ( Mudway et 

### 24 al., 2006 ; Mudway and Kelly, 2000 ; Mudway et al., 1999a ) . The complexity of these 

### 25 interactions was demonstrated by a study in which a 2

- hour exposure to 200 ppb O 3 , 

### 26 while exercising, resulted in similar increases in airway neutrophils and decreases in 

### 27 pulmonary function in both mild asthmatics and hea lthy controls, despite differences in 

### 28 ELF antioxidant concentrations prior to O 3 exposure ( Mudway et al., 2001 ) . Further, the 

### 29 O 3 - induced increase in oxidized GSH and decrease in AH2 observed in ELF of healthy 

### 30 controls was not observed in mild asthmatics ( Mudway et al., 2001 ) . While the authors 

### 31 concluded that basa l AH2 and oxidized GSH concentrations were not predictive of 

### 32 responsiveness to O 3 , they also suggested that the increased basal UA concentrations in 

### 33 the mild asthmatics may have played a protective role ( Mudway et al., 2001 ) . Thus 

### 34 compensatory mechanisms resulting in enhanced total an tioxidant capacity may play a 

### 35 role in modulating responses to O 3 . 

### 36Draft

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- 61 September 2011 Collectively these older and more recent studies provide insight into mechanisms which 

### 1 may contribute to enhanced responses of asthmatic and atopic individuals following O 3 

### 2 exposure. Greate r airways inflammation and/or greater bronchial reactivity have been 

### 3 demonstrated in asthmatics compared to non

- asthmatics. This pre

- existing inflammation 

### 4 and altered baseline bronchial reactivity may contribute to the enhanced 

### 5 bronchoconstriction seen in asthmatics exposed to O 3 . Furthermore, O 3 - induced 

### 6 inflammation may contribute to O 3 - mediated AHR. An enhanced neutrophilic response 

### 7 has been demonstrated in some asthmatics. A recent study in humans provided evidence 

### 8 for differences in innate immune respon ses related to TLR4 signaling between asthmatics 

### 9 and healthy subjects. Animal studies have demonstrated a role for eosinophil

- derived 

### 10 proteins in mediating the effects of O 3 . Since airways eosinophilia occurs in both allergic 

### 11 humans and allergic animal mod els, this pathway may underlie the exacerbation of 

### 12 allergic asthma by O 3 . In addition, differences have been noted in epithelial cytokine 

### 13 expression in bronchial biopsy samples of healthy and asthmatic subjects. A Th2 

### 14 phenotype, indicative of adaptive immu ne system activation and enhanced allergic 

### 15 responses, was observed before O 3 exposure and was increased by O 3 exposure in 

### 16 asthmatics. These findings support links between innate and adaptive immunity and 

### 17 sensitivity to O 3 - mediated effects in asthmatics and allergic airways disease. 

### 18
In addition to asthma and allergic diseases, obesity may alter responses to O 3 . While O 3 

### 19 is a trigger for asthma, obesity is a known risk factor for asthma ( Shore, 2007 ) . The 

### 20 relationship between obesity and asthma is not well understood but recent investigations 

### 21 have focused on alterations in endocrine function of adipose tissue in obesity. It is 

### 22 thought that the increases in serum levels of factors produced by adipocyte s (i.e. 

### 23 adipokines) such as cytokines, chemokines, soluble cytokine receptors and energy 

### 24 regulating hormones, may contribute to the relationship between obesity and asthma. 

### 25 Some of these same mechanisms may be relevant to insulin resistant states such as 

### 26

m etabolic syndrome. 

### 27
In a reanalysis of the data of Hazucha ( 2003 ) , increasing body mass index in young 

### 28 women was associated with increased O 3 responsiveness, as measured by spirometry 

### 29 following a 2

- hour exposure to 500 ppb O 3 while exercising ( Bennett et al., 2007 ) . In 

### 30 several mouse models of obesity, airways were fou nd to be innately more 

### 31 hyperresponsive and responded more vigorously to acute O 3 exposure than lean controls 

### 32 ( Shore, 2007 ) . Pulmonary inflammatory and injury in response to O 3 were also enhanced 

### 33 ( Shore, 2007 ) . It was postulated that oxidative stress resulting from obesity

- related 

### 34 hyperglycemia could account for these effects ( Shore, 2007 ) . However, responses to O 3 

### 35 in the different mouse models are somewhat variable and depend on whether exposures 

### 36 are acute or subacute. For example, diet

- induced obesity augmented inflammation and 

### 37 injury, as measured by BALF markers, and enhanced AHR in mice exposed acutely to O 3 

### 38Draft

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- 62 September 2011 (2 ppm, 3 hours) ( Johnston et al., 2008 ) . In contrast, the inflammatory response following 

### 1 sub

- acute exposure to O 3 was dampened by obesity in a different mouse model (0.3 ppm, 

### 2 72 hours) ( Shore et al., 2009 )

. 

### 3

5.4.2.3 Nutritional Status

Many investigations have focused on antioxidant deficiency and supplementation as 

### 4 modulators of O 3 - mediated effects. One study in m ice found that vitamin A deficiency 

### 5 enhanced lung injury induced by exposure to 0.3 ppm O 3 for 72 hours ( Paquette et al., 

### 6 1996 ) . Ascorbate deficiency was shown to increase the effects of acute (0.5

- 1 ppm for 4 

### 7 hours), but not subacute (0.2

- 0.8 ppm for 7 days), O 3 exposure in guinea pigs ( Kodavanti 

### 8 et al., 1995 ; Sl ade et al., 1989 ) . Supplementation with AH2 and α

- TOH was protective in 

### 9 healthy adults who were on an AH2

- deficient diet and exposed to 400 ppb O 3 for 2 hours 

### 10 while exercising ( Samet et al., 2001 ) . In this st udy, the protective effect consisted of a 

### 11 smaller reduction in FEV 1 following O 3 exposure ( Samet et al., 2001 ) . However the 

### 12 inflammatory response (influx of neutrophils and levels of IL

- 6) measured in BALF 1 

### 13 hour after O 3 exposure was not different between supplemented and non

- supplemented 

### 14 subjects ( Samet et al., 2001 ) . Other investigators found that AH2 and α

- TOH 

### 15 supplementation failed to ameliorate the pulmonary function decrements or airway s 

### 16 neutrophilia observed in humans exposed to 200 ppb O 3 for 2 hours ( Mudway et al., 

### 17 2006 ) . It was suggested that supplementation may be ineffe ctive in the absence of 

### 18 antioxidant deficiency ( Mudway et al., 2006 )

. 

### 19
In asthmatic adults, these same dietary antioxidants reduced O 3 - induced bronchial 

### 20 hyperresponsiveness (120 ppb, 45 min, with exercise) ( Trenga et al., 2001 ) . Furthermore, 

### 21 supplementation with AH2 and α

- tocopherol protected against pulmonary function 

### 22 decrements and nasal inflammatory responses which were associated with high levels of 

### 23 ambient O 3 in asthmatic children living in Mexico City ( Sienra

- Monge et al., 2004 ; 

### 24 Romieu et al., 2002 ) . Similarly, supplementation with ascorbate, α

- tocopherol and 

### 25 β

- carotene improved pulmonary function in Mexico City street workers ( Romieu et al., 

### 26 1998a )

. 

### 27
Protective effects of supplementation with α

- tocopherol alone have not been observed in 

### 28 humans experimentally exposed to O 3 ( Mudway and Kelly, 2000 ) . Alpha

- TOH 

### 29 supplementation also failed to protect against O 3 - induced effects in animal models of 

### 30 allergic rhinosinusitis and l ower airways allergic inflammation (rats, 1 ppm O 3 for 2 

### 31 days) ( Wagner et al., 2007 ) . However, protection in these same animal models was 

### 32 reported using γ

- TOH supplementation ( Wagner et al., 2009 ; Wagner et al., 2007 ) . Other 

### 33 investigators found that α

- TOH deficiency led to an increase in liver lipid peroxidation 

### 34Draft

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- 63 September 2011 (rats, 0.3 ppm 3 hours/day for 7 months) ( Sato et al., 1980 ) and a drop in liver α

- TOH 

### 1 levels following O 3 exposure (mice, 0.5 ppm, 6 hours/day for 3 days) ( Vasu et al., 2010 )

. 

### 2 A recent study used α

- TOH transfer protein null mice as a model of α

- TOH deficiency 

### 3 and demonstrated an altered adaptive response of the lung genome to O 3 exposure ( Vasu 

### 4 et al., 2010 ) . Taken together, these studies provide evidence that the tocopherol system 

### 5 modulates O 3 - induced responses. 

### 6

5.4.2.4 Lifestage

Responses to O 3 are m odulated by factors associated with lifestage. On one end of the 

### 7 lifestage spectrum is aging. The spirometric response to O 3 appears to be lost in humans 

### 8 as they age, as was demonstrated in two studies involving exposures of exercising human 

### 9 subjects to 42 0

- 450 ppb O 3 for 1.5

- 2 hours ( Hazucha et al., 2003 ; Drechsler

- Parks, 

### 10 1995 ) . In mice, physiological responses to O 3 (600 ppb, 2 hours) were diminished with 

### 11
age ( Hamade et al., 2010 ) . Mechanisms accounting for this effect have not been well



### 12 studied but could include altered number and sensitivity of receptors or altered signaling 

### 13 pathways involved in neural reflexes. 

### 14
On the other side of the lifestage spectrum is pre/postnatal develo pment. Critical 

### 15 windows of development during the pre/postnatal period are associated with an enhanced 

### 16 sensitivity to environmental toxicants. Adverse birth outcomes and developmental 

### 17 disorders may occur as a result. 

### 18
Adverse birth outcomes may result fro m stressors which impact transplacental oxygen 

### 19 and nutrient transport by a variety of mechanisms including oxidative stress, placental 

### 20 inflammation and placental vascular dysfunction ( Kannan et al., 2006 ) . These 

### 21 mechanisms may be linked since oxidative/ nitrosative stress is reported to cause vascular 

### 22 dysfunction in the placenta ( Myatt et al., 2000 ) . As described in Section 7.4, systemic 

### 23 inflammation and oxidative/nitrosative stress and modification of innate and adaptive 

### 24 immunity are key events underlying the health effects of O 3 and as such they may 

### 25 contribute to adverse birth outcomes. An animal t oxicology study showing that exposure 

### 26 to 2 ppm O 3 led to anorexia ( Kavlock et al ., 1979 ) (see Section 7.4.2) in exposed rat 

### 27 dams provide an additional mechanism by which O 3 exposure could lead to diminished 

### 28 transplacental nutrient transport. Disturbances of the pituitary

- adrenocortico

- placental 

### 29 system ( Ritz et al., 2000 ) may also impact normal intrauterine growth and development. 

### 30 Further, restricted fetal growth ma y result from pro

- inflammatory cytokines which limit 

### 31 trophoblast invasion during the early stages of pregnancy ( Hansen et al., 2008 ) . Direct 

### 32 effects on maternal health, such as susceptibility to infection, and on fe tal health, such as 

### 33 DNA damage, have also been proposed as mechanisms underlying adverse birth 

### 34Draft

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- 64 September 2011 outcomes ( Ritz et al., 2000 ) . In addition to restricted fetal growth, preterm birth may 

### 1 contribute to adverse birth outcomes. Preterm birth may result from the development of 

### 2 premature contractions and/or premature rupture of membranes as well as from disrupted 

### 3 implantation and placentation which results in suboptimal placental function ( Darrow et 

### 4 al., 2009 ; Ritz et al., 2000 ) . Genetic mutations are thought to be an important cause of 

### 5 placental abnormalities in the first t rimester, while vascular alterations may be the main 

### 6 cause of placental abnormalities in later trimesters ( Jalaludin et al., 2007 ) . Ozone



### 7 mediated systemic inflammation and oxidative stress/nitrosative stress may possibly be 

### 8 related to these effects although there is no firm evidence. 

### 9
Enhanced sensitivity to environmental toxicants during critical windows of development 

### 10 may also result in developmental disorders. For example, normal migration and 

### 11 differentiation of neural crest cells are imp ortant for heart development and are 

### 12 particularly sensitive to toxic insults ( Ritz et al., 2002 ) . Further, immune dysregulation 

### 13 and related pathologies are known to be associated with pre/postnatal environmental 

### 14
exposures ( Dietert et al., 2010 ) . Ozone exp osure is associated with developmental effects 

### 15 in several organ systems. These include neurobehavioral changes which could reflect 

### 16 O 3 ’ s effects on CNS plasticity or the hypothalamic

- pituitary axis ( Aute n and Foster, In 

### 17
Press ) (see Section 7.4.9). 

### 18
The majority of developmental effects due to O 3 have been described for the respiratory 

### 19 system (see Section 7.2.3 and 7.4.8). Since its growth and development take place during 

### 20 both the prenatal and early po stnatal periods, both prenatal and postnatal exposures to O 3 

### 21 have been studied. Maternal exposure to 0.4

- 1.2 ppm O 3 during gestation resulted in 

### 22 developmental health effects in the RT of mice ( Sharkhuu et al., 2011 ) . Recent studies 

### 23 involving postnatal exposure to O 3 have focused on differences between developing and 

### 24 adult animals in antioxidant defenses, respiratory physiology and sensitivity to cellular 

### 25 injury ( Auten and Foster, In Press ) . In particular, one set of studies in infant rhesus 

### 26 monkeys exposed to 0.5 ppm O 3 intermittently over 5 months has identified numerous 

### 27 O 3 - mediated perturbations in the developing lung and immune system ( Plopper et al., 

### 28 2007 ) . These investigations were prompted by the dramatic rise in the incidence of 

### 29 childhood asthma and focused on the possible role of O 3 and allergens in promoting 

### 30 remodeling of the epithelial

- mesenchymal trophic unit during postnatal development of 

### 31 the tracheobronchial airway wall. These and other studies have focused on mechanisms, 

### 32 such as lung structural changes, antigen sensitization, interaction with nitric oxide 

### 33 signaling, altered airway afferent innervation and loss of alveolar repair capac ity, by 

### 34 which early O 3 exposure could lead to asthma pathogenesis or exacerbations in later life 

### 35 ( Auten and Foster, In Press ) . Further, a recent study demonstrated that maternal exposure 

### 36 to particula te matter (PM) resulted in augmented lung inflammation, airway epithelial 

### 37 mucous metaplasia and AHR in young mice exposed chronically and intermittently to 1 

### 38Draft

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- 65 September 2011 ppm O 3 ( Auten et al., 2009 ) . Early life exposure to O 3 has also been found to modulate 

### 1 pulmonary and systemic innate immunity later in life in the infant rhesus monkey model 

### 2 ( Maniar

- Hew et al., 2011 )

. 

### 3

5.4.2.5 Attenuation of Responses

In responsive individuals, a striking degree of response attenuation occurred following 

### 4 repeated daily ex posures to O 3 . Generally, the young O 3 responder was no longer 

### 5 responsive on the fourth or fifth day of consecutive daily O 3 exposure (200

- 500 ppb O 3 

### 6 for 2

- 4 hours) and required days to weeks of non

- exposure in order for the subject to 

### 7 regain O 3 responsiveness ( Christian et al., 1998 ; Devlin et al., 1997 ; Linn et al., 1982a ; 

### 8 Horvath et al., 1981 ; Hackney et al., 1977 ) . This phenomena has been reported for both 

### 9 lung function and symptoms such as upper airway irritation, nonproductive cough, 

### 10 substernal discomfort and pain upon deep inspiration ( Linn et al., 1982a ; Horvath et al., 

### 11 1981 ; Hackney et al., 1977 ) . Repeated daily exposures also led to an attenuation of the 

### 12 sRaw response in exercising human subjects exposed for 4 hours to 200 ppb O 3 

### 13 ( Christian et al., 1998 ) and to a dampened AHR response compared with a single day 

### 14 exposure in exercising human subjects exposed for 2 hours to 400 ppb O 3 ( Dimeo et al., 

### 15 1981 ) . However, one group reported pe rsistent small airway dysfunction despite 

### 16 attenuation of the FEV 1 response on the third day of consecutive O 3 exposure (250 ppb, 

### 17 2 hours, with exercise) ( Frank et al., 2001 )

. 

### 18
Studies in rodents also indicated an attenuation of the physiologic response measured by 

### 19 breathing patterns and tidal volume following five consecutive days of exposure to 0.35

- 1 

### 20 ppm O 3 for 2.25 hours ( T epper et al., 1989 ) . Attenuation of O 3 - induced bradycardic 

### 21 responses, which also result from activation of neural reflexes, has been reported in 

### 22 rodents (0.5

- 0.6 ppm O 3 , 2

- 6 h/dy, 3

- 5 days ( Hamade and Tankersley, 2009 ; Watkinson et 

### 23 al., 2001 )

. 

### 24

Multi

- day exposure to O 3 has been found to decrease some markers of inflammation 

### 25 compared with a single day exposure ( Christian et al., 1998 ; Devlin et al., 1997 ) . For 

### 26 example, i n human subjects exposed for 4 hours to 200 ppb O 3 during moderate exercise, 

### 27 decreased numbers of BAL neutrophils and decreased levels of BALF fibronectin and IL



### 28 6 were observed after 4 days of consecutive exposure compared with responses after 1 

### 29 day ( Christian et al., 1998 ) . Results indicated an attenuation of the infl ammatory response 

### 30 in both proximal airways and distal lung. However markers of injury, such as lactate 

### 31 dehydrogenase (LDH) and protein in the BALF, were not attenuated in this study 

### 32 ( Christian et al., 1998 ) . Other investigators found that repeated O 3 exposure (200 ppb O 3 

### 33 for 4 hours on 4 consecutive days with intermitte nt exercise) resulted in increased 

### 34Draft

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- 66 September 2011 numbers of neutrophils in bronchial mucosal biopsies despite decreased BAL 

### 1 neutrophilia ( Jorres et al., 2000 ) . Other markers of inflammation, including BALF protein 

### 2 and IL

- 6 remained elevated following the multi

- day exposure ( Jorres et al., 2000 )

. 

### 3
In rats, the increases in BALF levels of prote ins, fibronectin, IL

- 6 and inflammatory cells 

### 4 observed after one day of exposure to 0.4 ppm O 3 for 12 hours were no longer observed 

### 5 after 5 consecutive days of exposure ( Van Bree et al., 2002 ) . A separate study in rats 

### 6 exposed to 0.35

- 1 ppm O 3 for 2.25 hours for 5 consecutive days demonstrated a lack of 

### 7 attenuation of the increase in BALF protein, persistence of macrophages in the 

### 8 centriacinar region and histological evidence of progressive tissue injury ( Tepper et al., 

### 9 1989 ) . Findings that injury, measured by BALF markers or by histopathology, pers ist in 

### 10 the absence of BAL neutrophila or pulmonary function decrements suggested that 

### 11 repeated exposure to O 3 may have serious long

- term consequences such as airway 

### 12 remodeling. In particular, the small airways were identified as a site where cumulative 

### 13 inj ury may occur ( Frank et al., 2001 )

. 

### 14

Some studies exami ned the recovery of responses which were attenuated by repeated O 3 

### 15 exposure. In a study of humans undergoing heavy intermittent exercise who were 

### 16 exposed for 2 hours to 400 ppb O 3 for five consecutive days ( Devlin et al., 1997 )
, 

### 17 recovery of the inflammatory responses which were diminished by repeated exposure 

### 18 required 10

- 20 days following the exposure ( Devlin et al., 1997 ) . In an animal st udy 

### 19 conducted in parallel ( Van Bree et al., 2002 ) , full susceptibility to O 3 challenge following 

### 20 exposure to O 3 for five consecutive days required 15

- 20 days recovery. 

### 21
Several mechanisms have been postulated to explain the attenuation of responses 

### 22 observed in human subjects and animal models following repeated exposure to O 3 . First, 

### 23 the upregulation of antioxidant defenses (or conversely, a decrease in critical O 3 - reactive 

### 24 substrates) may protect against O 3 - mediated adverse effects. Increases in antioxidant 

### 25 content of the BALF have been demonstrated in rats exposed to 0.25 and 0.5 ppm O 3 for 

### 26 several hours on consecutive days ( Devlin et al., 1997 ; Wiester et al., 1996a ; Tepper et 

### 27 al., 1989 ) . Second, IL

- 6 was demonstrated to be an important mediato r of attenuation in 

### 28 rats exposed to 0.5 ppm for 4 hours on two consecutive days ( McKinney et al., 1998 )

. 

### 29
T hird, a protective role for increases in mucus producing cells and mucus concentrations 

### 30 in the airways has been proposed ( Devlin et al., 1997 ) . Fourth, epithelial hyperplasia or 

### 31 metaplasia may dec rease susceptibility to subsequent O 3 challenge ( Carey et al., 2007 ; 

### 32 Harkema et al., 1987a ; Harkema et al., 1987b ) . These morphologic changes have been 

### 33 observed in nasal and lower airways in monkeys exposed chronically to 0.15

- 0.5 ppm O 3 . 

### 34 Although there is some evidence to support these possibilities, there is no consensus on 

### 35 mechanisms underlying response attenuation. Recent studies demonstrating that O 3 

### 36Draft

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- 67 September 2011 activates TRP receptors suggest that modulation of TRP receptor number or sensitivity by 

### 1 repeated O 3 exposures may also contribute to the attenuation of responses. 

### 2

5.4.2.6 Co

- Exposures with Particulate Matter

Numerous studies have investigated the effects of co

- exposure to O 3 and PM because of 

### 3 the prevalence of these pollutants in am bient air. Results are highly variable and depend 

### 4 on whether exposures are simultaneous or sequential, the type of PM employed and the 

### 5 endpoint examined. Additive and interactive effects have been demonstrated. For 

### 6 example, simultaneous exposure to O 3 (120 ppb for 2 hours at rest) and concentrated 

### 7 ambient particles (CAPs) in human subjects resulted in a diminished systemic IL

- 6 

### 8 response compared with exposure to CAPs alone ( Urch et al., 2010 ) . However, exposure 

### 9 to O 3 alone did not alter blood IL

- 6 levels ( Urch et al., 2010 ) . The authors provided 

### 10 evidence t hat O 3 mediated a switch to shallow breathing which may have accounted for 

### 11 the observed antagonism ( Urch et al., 2010 ) . Further, simultaneous exposure to O 3 (114 

### 12 ppb for 2 hours at rest) and CAPs but not exposure to either alone, resulted in increased 

### 13 diastolic blood pressure in human subjects ( Fakhri et al., 2009 ) . Mechanisms underlying 

### 14 th is potentiation of response were not explored. In some strains of mice, pre

- exposure to 

### 15 O 3 (0.5 ppm for 2 hours) modulated the effects of carbon black PM on heart rate, HRV 

### 16 and breathing patterns ( Hamade and Tankersley, 2009 ) . Another recent study in mice 

### 17 demonstrated that treatment with carbon nanotubes followed 12 hours lat er by O 3 

### 18 exposure (0.5 ppm for 3 hours) resulted in a dampening of some of the pulmonary effects 

### 19 of carbon nanotubes measured as markers of inflammation and injury in the BALF ( Han 

### 20 et al., 2008 ) . Lastly, Harkema et al. ( 2005 ) found that epithelial and inflammatory 

### 21 responses in the airways of rats were enhanced by co

- exposure to O 3 (0.5 ppm for 3 days) 

### 22 and LPS (used as a model of biogenic PM) or to O 3 (1 ppm for 2 days) and OVA (used as 

### 23 a model of an aeroallergen). Many of the demonstrated responses were more

- than



### 24 additive. Overall, these findings are hard to interpret but demonstrate the complexity of 

### 25 responses following combined exposure to PM and O 3 . 

### 26

5.4.2.7 Summary

Collecti vely, these older and more recent studies provide evidence for mechanisms which 

### 27 may underlie the variability in responsiveness seen among individuals (Figure 5

- 10). 

### 28 Certain functional genetic polymorphisms, pre

- existing conditions and diseases, 

### 29 nutritional status, lifestage and co

- exposures contribute to altered risk of O 3 - induced 

### 30 effects. Attenuation of responses may also be important, but it is incompletely 

### 31 understood, both in terms of the pathways involved and the resulting consequences. 

### 32Draft

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- 68 September 2011 

### Figure 5

- 10 Factors which contribute to the interindividual variability in responses resulting from inhalation exposure to ozone.



## 5.5 Species Homology and Interspecies Sensitivity

The previous O 3 AQCDs discussed the suitability of animal models for comparison with 

### 1 human O 3 exposure and concluded that the acute and chronic functional responses of 

### 2 laboratory animals to O 3 appear qualitatively homologous to human responses. Thus, 

### 3 animal studies can provide important data in determining cause

- effect relationships 

### 4 between expos ure and health outcome that would be impossible to collect in human 

### 5 studies. Still, care must be taken when comparing quantitative dose

- response 

### 6 relationships in animal models to humans due to obvious interspecies differences. This 

### 7 section will describe ba sic concepts in species homology concerning both dose and 

### 8 response to O 3 exposure. This will not be a quantitative extrapolation of doses where O 3 

### 9 effects have been observed. Overall, there have been few new publications examining 

### 10 interspecies differences in dosimetry and response to O 3 since the last AQCD. These 

### 11 studies do not overtly change the conclusions discussed in the previous document. 

### 12 Ozone + Respiratory Tract

Formation of secondary oxidation products

Systemic inflammation and

o xidative/ nitrosative stress Activation of neural reflexes Airways remodeling Sensitization of bronchial

s mooth muscle Alteration of epithelial barrier function Initiation of inflammation Modification of innate and adaptive immunity

Extrapulmonary Effects Respiratory System Effects Dosimetric factors

Nutritional status Lifestage Attenuation factors Co

- exposures Gene

- environment interactions Pre

- existing diseases/conditions COPD/smoking status Asthma/allergic airways disease

Obesity/metabolic syndrome

Obesity/ Metabolic Stress Lifestage Attenuation factorsDraft

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- 69 September 2011 

### 5.5.1 Dosimetry

As discussed in Section 5.2.1, O 3 uptake depends on complex interactions between RT 

### 1 morphology, breathin g route, rate, and depth, physicochemical properties of the gas, 

### 2 physical processes of gas transport, as well as the physical and chemical properties of the 

### 3 ELF and tissue layers. Understanding differences in these variables between humans and 

### 4 experimental animals is important to interpreting delivered doses in animal and human 

### 5 toxicology studies. 

### 6
Physiological and anatomical differences exist between experimental species. The 

### 7 structure of the URT is vastly different between rodents and humans and scales a ccording 

### 8 to body mass. The difference in the cross

- sectional shape and size of the nasal passages 

### 9 affects bulk airflow patterns such that major airflow streams are created. The nasal 

### 10 epithelium is lined by squamous, respiratory, or olfactory cells, dependi ng on location. 

### 11 The differences in airflow patterns in the URT mean that not all nasal surfaces and cell 

### 12 types receive the same exposure to inhaled O 3 leading to differences in local absorption 

### 13 and potential for site

- specific tissue damage. The morphology of the LRT also varies 

### 14 within and among species. Rats and mice do not possess respiratory bronchioles; 

### 15 however, these structures are present in humans, dogs, ferrets, cats, and monkeys. 

### 16 Respiratory bronchioles are abbreviated in hamsters, guinea pigs, shee p, and pigs. The 

### 17 branching structure of the ciliated bronchi and bronchioles also differs between species 

### 18 from being a rather symmetric and dichotomous branching network of airways in humans 

### 19 and primates to a more monopodial branching network in other mamm als. In addition, 

### 20 rodents have fewer terminal bronchioles due to a smaller lung size compared to humans 

### 21 or canines ( McBride, 1992 ) . Th e cellular composition in the pulmonary region is similar 

### 22 across mammalian species; at least 95% of the alveolar epithelial tissue is composed of 

### 23 Type I cells. However, significant differences exist between species in the number and 

### 24 type of cells in the TB airways. Differences also exist in breathing route and rate. 

### 25 Primates are oronasal breathers, while rodents are obligate nasal breathers. Past studies of 

### 26 the effect of body size on resting oxygen consumption also suggest that rodents inhale 

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

### 28 in nasal structure between primates and rodents could affect the amount of O 3 uptake. 

### 29

As O 3 absorption and activity relies on ELF antioxidant substances as described in 

### 30 Section 5. 2.3, variability in antioxidant concentrations and metabolism between species 

### 31 may affect dose and O 3 - induced health outcomes. The thickness of the ELF in the TB 

### 32 airways varies among species. Mercer et al. ( 1992 ) found that the human ELF thickness 

### 33 in bronchi and bronchioles was 6.9 and 1.8 μm, respectively, compared to 2.6 and 1.9 μm 

### 34 for the same locations in the rat. Guinea pigs and mice have a lower basal activity of 

### 35 GSH transferase and GSH peroxidase, and lower α

- TOH levels in the lung compared to 

### 36Draft

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- 70 September 2011 rats ( Ichinose et al., 1988 ; Sagai et al., 1987 ) . Nasal lavage fluid analysis shows that 

### 1 humans have a higher proportion of their nasal antioxidants as UA and low levels of AH 2 

### 2 whereas mice, rats, or guinea pigs have high levels of AH 2 and undetectable levels of UA 

### 3 (Figure 5

- 11a ). GSH is not detected in the nasal lavage of most of these species, but is 

### 4 present in monkey nasal lavage. Guinea pigs and rats have a higher antioxidant to protein 

### 5 ratio in nasal lavage and BALF than humans ( Hatch, 1992 ) . The BALF profile differs 

### 6 from the nasal lavage (Figure 5

- 11b). Humans have a higher proportion of GSH and less 

### 7
AH 2 making up their BALF cont ent compared to the guinea pigs and rats ( Slade et al., 

### 8 1993 ; Hatch, 1992 ) . Similar to the nose, rats have the highest antioxidant to protein mass 

### 9 ratio found in BALF ( Slade et al., 1993 ) . Antioxidant defenses also vary with age 

### 10 ( Servais et al., 2005 ) and exposure history ( Duan et al., 1996 ) . Duan et al. ( 1996 ; 1993 ) 

### 11 reported that differences in antioxidant levels between species and lung regions did not 

### 12 appear to be the primary factor in O 3 induced tissue injury. However, a close association 

### 13 between site

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

### 14 depletion was later revealed in monkeys ( Plopper et al., 1998 )

. 

### 15
Humans and animals are similar in the pattern of regional O 3 dose distribution. As 

### 16 discussed for humans in Section 5.2.2, O 3 flux to the air

- li quid interface of the ELF 

### 17 slowly decreases distally in the TB region and then rapidly decreases distally in the 

### 18 alveolar region ( Miller et al., 1985 ) . Modeled tissue dose in the human RT, representing 

### 19 O 3 flux to the liquid

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

### 20 in the CAR, and then rapidly decreases distally in the alveolar region (Figure 5

- 12). 

### 21 Similar patterns of O 3 tissue dose profiles normalized to inhaled O 3 concentration were 

### 22 predicted for rat, guinea pig, and rabbit ( Miller et al., 1988 ; Overton et al., 1987 ) (Figure 

### 23 5

- 12a). Overton et al. ( 1987 ) modeled rat and guinea pig O 3 dose distribution and found 

### 24 that after comparing two different morphomet rically based anatomical models for each 

### 25 species, considerable difference in predicted percent RT and alveolar region uptakes were 

### 26 observed. This was due to the variability between the two anatomical models in airway 

### 27 path distance to the first alveolated d uct. As a result, the overall dose profile was similar 

### 28 between species however the O 3 uptake efficiency varied due to RT size and path length 

### 29 (Section 5.2.2). A similar pattern of O 3 dose distribution was measured in monkeys 

### 30 exposed to 0.4 and 1.0 ppm 18 O 3 ( Plopper et al., 1998 ) (Figure 5

- 12b). Less 18 O was 

### 31 measured in the trachea, proximal bronchus, and distal bronchus than was observed in the 

### 32 respiratory b ronchioles. Again indicating the highest concentration of O 3 tissue dose to 

### 33 be localized to the CAR, which are the respiratory bronchioles in nonhuman primates. In 

### 34 addition, the lowest 18 O detected in the RT was in the parenchyma (i.e. alveolar region), 

### 35 mimicking the rapid decrease in tissue O 3 dose predicted by models for the alveolar 

### 36 regions of humans and other animals. 

### 37Draft

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- 71 September 2011 a



### .

b.

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


### Figure 5

- 11 Species comparison of antioxidant / protein ratios of: (a) nasal lavage fluid and, (b) bronchoalveolar lavage fluid. Rat

Guinea pig

Human

0 200 400 600 800 1000 Antioxidant / Protein, nanomoles / gram Ascorbic acid Uric acid Glutathione

Guinea pig Rat

Human

0 50 100 150 200 250 Antioxidant / Protein, nanomoles / gram Ascorbic acid Uric acid GlutathioneDraft

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- 72 September 2011 Source: Panel (a) U.S. EPA ( 1996a ) (b) Plopper et al. ( 1998 )


### Figure 5

- 12 Humans and animals are similar in the regional pattern of O 3 

### tissue dose di stribution. Panel (a) presents the predicted tissue dose of O 3 

### (as μg of O 3 

### per cm 2 

### of segment surface area per min, standardized to a tracheal O 3 

### value of 1 μg/m 3 

### ) for various regions of the rabbit, guinea pig, rat, and human RT. TB = tracheobronchial
re gion, A = alveolar region. Panel (b) presents a comparison of
excess 18 

### O in the five regions of the TB airways of rhesus monkeys exposed to O 3 

### for 2h. *p<0.05 comparing the same O 3 

### concentration across regions. **p<0.05 comparing different O 3 

### concentration s in the same region. 0.4 ppm O 3 1.0 ppm O 3Oxygen-18-Content(μg/gdry weight)

0 10 20 30 40 50

TRACHEA PROXIMAL BRONCHUS DISTAL BRONCHUS RESPIRATORY BRONCHIOLE PARENCHYMA 

### a.

b. ADraft

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- 73 September 2011 Humans and animal models are similar in the pattern of regional O 3 dose, but absolute 

### 1 values differ. Hatch et al. ( 1994 ) reported that exercising humans exposed to oxygen

- 18 

### 2 labeled O 3 (400 ppb) accumulated 4

- 5 ti mes higher concentrations of O 3 reaction product 

### 3 in BAL cells, surfactant and protein fractions compared to resting rats similarly exposed 

### 4 (0.4 ppm) (Figure 5

- 13). It was necessary to expose resting rats to 2 ppm O 3 to achieve 

### 5 the same BALF accumulation of 18 O reaction product that was observed in humans 

### 6 exposed to 400 ppb with intermittent heavy exercise (MV ~60 L/min). The concentration 

### 7 of 18 O reaction product in BALF paralleled the accumulation of BALF protein and 

### 8 cellular effects of the O 3 exposure obse rved such that these responses to 2.0 ppm O 3 were 

### 9 similar to those of the 400 ppb O 3 in exercising humans. This suggests that animal data 

### 10 obtained in resting conditions would underestimate the dose to the RT and presumably 

### 11 the resultant risk of effect for humans. 

### 12

Source: Hatch et al. ( 1994 )


### Figure 5

- 13 Oxygen

- 18 incorporation into different fractions of BALF from humans and rats exposed to 0.4 and 2.0 ppm 18 

### O 3 

### . The excess 18 

### O in each fraction is expressed relative to the dry weight of that fraction. Fractions assayed include cells, high speed pellet (HSP), high speed supernatant (HSS), and lavaged lung homogenates.Draft

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- 74 September 2011 Recently, a quantitative comparison of O 3 transport in the airways of rats, dogs, and 

### 1 humans was conducted using a three

- c ompartment airways model, based on upper and 

### 2 lower airway casts and mathematical calculation for alveolar parameters ( Tsujino et al., 

### 3 2005 ) . This model examin ed how interspecies anatomical and physiological differences 

### 4 affect intra

- airway O 3 concentrations and the amount of gas absorbed. The model was 

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

- dimensional gas movement 

### 6 and no airway branching pat terns. Peak, real

- time, and mean O 3 concentrations were 

### 7 higher in the upper and lower airways of humans compared to rats and dogs, but lowest 

### 8 in the alveoli of humans. The amount of O 3 ab s orbed was lowest in humans when 

### 9 normalized by body weight. The intra

- airway concentration decreased distally in all 

### 10 species. Sensitivity analysis demonstrated that V T , f B , and upper and lower airways 

### 11 surface area had a significant impact on model results. The model is limited in that it did 

### 12 not account for chemical reactio ns in the ELF or consider gas diffusion as a driving force 

### 13 for O 3 transport. Also, the model was run at a respiratory rate of 16/min simulating a 

### 14 resting individual, however exercise may cause a further deviation from animal models as 

### 15 was seen in Hatch et al. ( 1994 )

. 

### 16
Overall, animal models exhibit qualitatively similar patterns of O 3 net and tissue dose 

### 17 distribution with the largest tissue dose delivered to the CAR. However, due t o 

### 18 anatomical and biochemical RT differences the absolute values of O 3 dose delivered 

### 19 differs. Past results suggest that animal data obtained in resting conditions would 

### 20 underestimate the dose to the RT and presumably the resultant risk of effect for humans
, 

### 21 especially for humans during exercise. 

### 22

5.5.2 Homology of Response

Risk of heath effects from O 3 varies between and within species, as well as between 

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

### 24 less sensitive to change s in pulmonary function test than humans ( U.S. EPA, 1996a )

. 

### 25 However, rats experience attenuation of pulmonary function and tachypneic ventilatory 

### 26 responses, similar to humans ( Wiester et al., 1996a ) . Hatch et al. ( 1986 ) reported that 

### 27 guinea pigs were the most responsive to O 3 - induced inflammatory cell and protein influx. 

### 28 Rabbits were the least responsive and rats, hamsters, and mice were intermediate 

### 29 responders. Furth er analysis of this study by Miller et al. ( 1988 ) found that the protein 

### 30 level s in guinea pigs increased more rapidly with predicted pulmonary tissue dose than in 

### 31 rats and rabbits. Alveolar macrophages isolated from guinea pigs and humans mounted 

### 32 similar qualitative and quantitative cytokine responses to in vitro O 3 (0.1

- 1.0 ppm for 60 

### 33 minutes) exposure ( Arsalane et al., 1995 )

. 

### 34Draft

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- 75 September 2011 Also, because of their higher body surface to volume ratio, rodents can rapidly lower 

### 1 body temperature during exposure leading to lowered O 3 dose and toxicity ( Watkinson et 

### 2 al., 2003 ; Iwasaki et al., 1998 ; Slade et al., 1997 ) . In addition to lowering the O 3 dose to 

### 3 the lungs, this hypothermic response may cause: (1) lower metabolic rate, (2) al tered 

### 4 enzyme kinetics, and (3) altered membrane function. The thermoregulatory mechanisms 

### 5 also may affect disruption of heart rate which may lead to: (1) decreased cardiac output, 

### 6 (2) lowered blood pressure, and (3) decreased tissue perfusion ( Watkinson et al., 2003 )

. 

### 7 These responses have not been observed in humans except at very high exposures, thus 

### 8 further complicating extrapolation of effects from animals to humans. 

### 9
Recently, the three

- dimensio nal detail of the nasal passages of immature Rhesus macaque 

### 10 monkeys was analyzed for developing predictive dosimetry models and exposure

- dose



### 11 response relationships ( Carey et al., 2007 ) . In doing so the authors reported that the 

### 12 relative amounts of the five epithelial cell types in the nasal airways of monkeys remain s 

### 13 consistent between infancy and adulthood (comparing to ( Gross et al., 1987 ; Gross et al., 

### 14 1982 ) . Ozone exposures (0.5 ppm, 8 h/day under acute [5 days] and episodic conditions 

### 15 [5 replicates of the acute paradigm spaced a week apart]) confirmed that the ciliated 

### 16 respiratory and transitional epithelium were the most sensitive cell types in the nasal 

### 17 cavity to O 3 exposu re, showing 50

- 80% decreases in epithelial thickness and epithelial 

### 18 cell volume. The character and location of nasal lesions resulting from O 3 exposure were 

### 19 similar between adult and infant monkeys similarly exposed. However, infant monkeys 

### 20 did not undergo nasal airway epithelial remodeling or adaptation that occurs in adult 

### 21 animals and they may develop persistent necrotizing rhinitis following episodic longer



### 22 term exposures. 

### 23
To further understand the genetic basis for age

- dependent differential response to O 3 , 

### 24 adult (15 week old) and neonatal (15

- 16 day old) mice from 8 genetically diverse strains 

### 25 were examined for O 3 - induced (0.8 ppm for 5 hours) pulmonary injury and lung 

### 26 inflammation ( Vancza et al., 2009 ) . Ozone exposure increased polymorphonuclear 

### 27 leukocytes (PMN) influx in all strains of neonatal mice tested, but significantly greater 

### 28
PMNs occu rred in neonatal compared to adult mice for only some sensitive strains, 

### 29 suggesting a genetic background effect. This strain difference was not due to differences 

### 30 in delivered dose of O 3 to the lung, evidenced by 18 O lung enrichment. The sensitivity of 

### 31

str ains for O 3 - induced increases in BALF protein and PMNs was different for different 

### 32 strains of mice suggesting that genetic factors contributed to heightened responses. 

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

### 34 of O 3 than corresponding strain neonates. Thus, it appeared that the infant mice showed a 

### 35

two

- to threefold higher response than the adults when expressed relative to the 

### 36 accumulated O 3 reaction product in their lungs. The apparent decrease in delivered O 3 

### 37Draft

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- 76 September 2011 dose in neonates could be a result of a more rapid loss of body temperature in infant 

### 1 rodents incident to maternal separation and chamber air flow. 

### 2

Further, O 3 - induced injury and inflammation responses are variable between species. For 

### 3 example, Dormans et al. ( 1999 ) found that rats, mice, and guinea pigs all exhibited 

### 4 O 3 - induced (0.2

- 0.4 ppm for 3

- 56 days) inflammation; however, guinea pigs were the 

### 5 most sensitive with respect to alveolar macrophage elicitation and pulmonary cell density 

### 6 in t he centriacinar region. Mice were the most sensitive to bronchiolar epithelial 

### 7 hypertrophy and biochemical changes (e.g. LDH, glutathione reductase, glucose

- 6

- 

### 8 phosphate dehydrogenase activity), and had the slowest recovery from O 3 exposure. All 

### 9 species dis played increased collagen in the ductal septa and large lamellar bodies in Type 

### 10 II pneumocytes at the longest exposure and highest concentration; whereas this response 

### 11 occurred in the rat and guinea pig at lower O 3 levels (0.2 ppm) as well. Overall, the 

### 12

au thors rated mice as most sensitive, followed by guinea pigs, then rats ( Dormans et al., 

### 13 1999 ) . Rats were also less sensitive to epithelial necrosis and inflammatory responses 

### 14 from O 3 (1.0 ppm for 8 hours) than monkeys and ferrets, which manifeste d a similar 

### 15
response ( Sterner

- Kock et al., 2000 ) . These data suggest that ferrets may be a good 

### 16 animal model for O 3 - induced airway effects due to the similarities in pulmonary structure 

### 17 between primates and ferrets. However, this study provided no dose metric and, it is 

### 18 possible that some of these differences may be attributable to disparate total inhaled dose 

### 19 or local organ dose. 

### 20

5.5.3 Summary

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

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

- 5 times higher exposure to O 3 to 

### 22 achieve comparable increases in BALF protein and PMNs to exercising humans. New 

### 23 studies have shown that varied O 3 response in different mouse strains was not due to 

### 24 differences in delivered dose of O 3 to the lung but more likely genetic sensitivity, and that 

### 25 infant mice show greater toxicity relative to their s maller lung dose than adults. Even 

### 26 though interspecies differences limit quantitative comparison between species, the acute 

### 27 and chronic functional responses of laboratory animals to O 3 appear qualitatively 

### 28 homologous to those of the human making them a use ful tool in determining mechanistic 

### 29 and cause

- effect relationships with O 3 exposure. 

### 30Draft

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- 77 September 2011 

## 5.6 Chapter Summary

Ozone is a highly reactive gas and a powerful oxidant with a short half

- life. Both O 3 

### 1 uptake and responses are dependent upon the formation of secondary re action products in 

### 2 the ELF; however more complex interactions occur. Uptake in humans at rest is 80

- 95% 

### 3 efficient and it is influenced by a number of factors including RT morphology, breathing 

### 4 route, frequency, and volume, physicochemical properties of the gas, physical processes 

### 5 of gas transport, as well as the physical and chemical properties of the ELF and tissue 

### 6 layers. The primary uptake site of O 3 delivery to the lung epithelium is believed to be the 

### 7 CAR, however changes in a number of factors (e.g. p hysical activity) can alter the 

### 8 distribution of O 3 uptake in the RT. Ozone uptake is chemical reaction

- dependent and the 

### 9 substances present in the ELF appear in most cases to limit interaction of O 3 with 

### 10 underlying tissues and to prevent penetration of O 3 distally into the RT. Still, reactions of 

### 11 O 3 with soluble ELF components or plasma membranes result in distinct products, some 

### 12 of which are highly reactive and can injure and/or transmit signals to RT cells. 

### 13
Thus, in addition to contributing to the driving force for O 3 uptake, formation of 

### 14 secondary oxidation products initiates pathways that provide the mechanistic basis for 

### 15 health effects which are described in detail in Chapters 6 and 7 and which involve the RT 

### 16 as well as extrapulmonary systems. These pat hways include activation of neural reflexes, 

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

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

### 19 remodeling, and systemic inflammation and ox idative/nitrosative stress. With the 

### 20 exception of airways remodeling, these pathways have been demonstrated in both 

### 21 animals and human subjects in response to the inhalation of O 3 . 

### 22
Both dosimetric and mechanistic factors contribute to the understanding of 

### 23 interindividual variability in responses to O 3 . Interindividual variability is influenced by 

### 24 variability in RT volume and thus surface area, certain genetic polymorphisms, pre



### 25 existing conditions and disease, nutritional status, lifestages, attenuation, an d co



### 26 exposures. Some of these factors are also influential in understanding species homology 

### 27 and sensitivity. Qualitatively, animal models exhibit similar patterns of O 3 net and tissue 

### 28 dose distribution with the largest tissue dose delivered to the CAR. Ho wever, due to 

### 29 anatomical and biochemical RT differences, the absolute value of delivered O 3 dose 

### 30 differs, with animal data obtained in resting conditions underestimating the dose to the 

### 31 RT and presumably the resultant risk of effect for humans, especially humans during 

### 32 exercise. Even though interspecies differences limit quantitative comparison between 

### 33 species, the acute and chronic functional responses of laboratory animals to O 3 appear 

### 34 qualitatively homologous to those of the human making them a useful to ol in determining 

### 35 mechanistic and cause

- effect relationships with O 3 exposure. 

### 36Draft

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- 78 September 2011 

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