### Draft

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- 1 June 2012 

## 5 DOSIMETRY AND MODE O F ACTION

5.1 Introduction

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

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

### 2
modes of action leading to the health effects that will be presented in Chapters 6 and 7. 

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

### 4

basic con cepts derived 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 studies 

### 8
published since the last AQCD, the reader is referred to previous documents ( U.S. EPA, 

### 9

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 exerts its actions. Since 

### 14

common mechanisms lea d 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 related 

### 16
sections of Chapt ers 6 and 7 are indicated. Earlier studies that represent the current state 

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

- relevant 

### 18

concentrations of O 3 are of great er interest, since mechanisms responsible for effects at 

### 19

low O 3 concentrations may not be identical to those occurring at high O 3 concentrations. 

### 20
Some studies at higher concentrations are included if they were early demonstrations of 

### 21
key mechanisms or if t hey are recent demonstrations of potentially important new 

### 22
mechanisms. The topics of dosimetry and mode of action are bridged by reactions of O 3 

### 23
with components of the extracellular lining fluid (ELF), which play a role in both O 3 

### 24
uptake and biological res ponses ( Figure 5

- 1 ). 

### 25

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

### 26
related to species comparison of doses and responses (Section 5.4 and Section 5.5 ). These 

### 27
topics are included in this chapter because they are influenced by both dosimetric and 

### 28

mechanistic considerations. 

### 29

### Draft

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- 2 June 2012 Figure 5

- 1 Schematic of the ozone exposure and response pathway. Ozone transport follows a path from exposure concentration, to inhaled dose, to net dose, to the local tissue dose. Chapter 5 discusses the concepts of dose and modes of action that result in the health effects discussed in Chapters 6 and 7.



## 5.2 Human and Ani mal Ozone Dosimetry

5.2.1 Introduction

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

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

- 1 

### 2
illustrates the transport of O 3 or its reaction products from exposure to dose to the 

### 3
development of health effects. Ozone exposure has been defined in Section 4.2 and 

### 4

consists of contact between the human or animal and O 3 at a specific concentration for a 

### 5
specified period of time (i.e., exposure = concentration × time). The amount of O 3 p resent 

### 6
in a given volume of air for which animals and individuals are exposed is termed 

### 7
exposure concentration. Ozone exposure will result in some amount (dose) of O 3 crossing 

### 8
an exposure surface to enter a target area. The initial measure of dose after O 3 enters the 

### 9

RT is inhaled dose and is the amount or rate of O 3 that crosses the outer RT surface 

### 10

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

### 11
phase across the ELF interface where net dose may be measured. Net dose is the amount 

### 12
or rate of entry of O 3 across the gas/ELF interface. In modeling studies, the dose rate is 

### 13
often expressed as a flux per unit of surface area of a region of respiratory epithelium. 

### 14
Finally, O 3 or its reaction products may reach the tissues and tissue dose of O 3 can be 

### 15
reported. Tissue dose is the amount of O 3 or its reaction products absorbed and available 

### 16
for reacting with tissues and is difficult and rarely measured. In the literature, the 

### 17
exposure concentration and various measures of dose (i .e., net dose and inhaled dose) are 

### 18
often used as surrogates for tissue dose. However, ambient or exposure concentrations are 

### 19 O 3 exposure Inhaled

O 3 dose Net O 3 dose

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

### Draft

– Do Not Cite or Quote 5

- 3 June 2012 not a true measure of dose so understanding the relationship between ambient 

### 1
concentrations and tissue dose allows for a greater a ppreciation of the dose

- response from 

### 2

O 3 exposure. 

### 3

Note: Structures are anterior nasal passages, ET 1 ; oral airway and posterior nasal passages, ET 2 ; bronchial airways, BB; bronchioles, bb; and alveolar interstitial, AI.

Source: Based on ICRP (1994 )

.



### Figure 5

- 2 Representation of respiratory tract regions in humans.

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

### 4

The latter feature is believed to be the reason w hy it is able to penetrate into targets in the 

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

- 2 presents the basic structure of the human 

### 6
respiratory tract ( RT). The lung can be divided into three major regions: the extrathoracic 

### 7
(ET) region or upper respiratory tract (URT, from the nose/mouth to larynx); the 

### 8

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

### 9

### Draft

– Do Not Cite or Quote 5

- 4 June 2012 pulmonary r egion (from the respiratory bronchioles to the terminal alveolar sacs). The 

### 1
latter two regions comprise the LRT. Although the structure varies, the illustrated 

### 2
anatomic regions are common to all mammalian species with the exception of the 

### 3
respiratory bronc hioles. Respiratory bronchioles, the transition region between ciliated 

### 4
and fully alveolated airways, are found in humans, dogs, ferrets, cats, and monkeys. 

### 5
Respiratory bronchioles are absent in rats and mice and abbreviated in hamsters, guinea 

### 6
pigs, sheep , and pigs. The branching structure of the ciliated bronchi and bronchioles also 

### 7
differs between species from being a rather symmetric and dichotomous branching 

### 8
network of airways in humans to a more monopodial branching network in other 

### 9

mammals. 

### 10

Figure 5

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

### 11
the TB region to the alveolus in the alveolar region. The fact that O 3 is so chemically 

### 12
reactive has suggested to some that its tissue dose at the target sites exists in the form of 

### 13
oxidation products such as aldehydes and peroxides (see Section 5.2.3 ). Reaction 

### 14
products are formed when O 3 interacts with components of the ELF such as lipids and 

### 15
antioxidants. The ELF varies throughout the length of the RT with the bronchial tree 

### 16

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

### 17

surfactant solution ( Figure 5

- 3 b). Ozone dose is directly related to the co upled diffusion 

### 18
and chemical reactions occurring in ELF, a process termed “reactive absorption.” Thus, 

### 19

the O 3 dose depends on both the concentration of O 3 as well as the availability of 

### 20

substrates within the ELF. 

### 21

Ozone dose is affected by complex interact ions between a number of other major factors 

### 22
including RT morphology, breathing route, frequency, and volume, physicochemical 

### 23
properties of the gas, physical processes of gas transport, as well as the physical and 

### 24
chemical properties of the ELF and tissue layers ( Figure 5

- 3 c). The role of these 

### 25
processes varies throughout the length of the RT and as O 3 moves from the gas to liquid 

### 26
compartments of the RT

. 

### 27

### Draft

– Do Not Cite or Quote 5

- 5 June 2012 Note: (a) Illustrates basic airway anatomy. Structures are epithelial cells, EP; basement membrane, BM; smooth muscle cells, SM; and fibrocartilaginous coat, FC. (b) Illustrates the relative amounts of liquid, tissue, and blood with distal progression . In the bronchi there is a thick surface lining over a relatively thick layer of tissues. With distal progress, the lining diminishes allowin g increased access of compounds crossing the air

- liquid interface to the tissues and the blood. (c) Presents the f actors acting in the gas and liquid phases of O 3 transport. Source: Panel (a) reprinted with permission of McGraw

- Hill ( Weibel, 1980 )

.



### Figure 5

- 3 Structure of lower airways with progression from the large airways
to the alveolus.

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

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

### 2

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

### 3
present in tissues (termed “reactants”). The results of the above measurements have been 

### 4
incorporated into mathematical models for the p urpose of explaining, predicting, and 

### 5 c. Transport Factors
Gas Phase

Convection
Diffusion Dispersion

Liquid Phase

Solubility
Diffusion
Chemical Reaction

Convection O 3 transport

### Draft

– Do Not Cite or Quote 5

- 6 June 2012 extrapolating O 3 dose in different exposure scenarios. Few new studies have investigated 

### 1
the uptake of O 3 in the RT since the last O 3 assessment ( U.S. EPA, 2006b ) . The studies 

### 2
that have been conducted generally agree with the results presented in the past and do not 

### 3
change the do simetry conclusions of the last document. 

### 4

5.2.2 Ozone Uptake

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

### 5

the state of the science in O 3 dosimetry. Measurements of O 3 dose have been inferred 

### 6

from simultaneous measureme nts of airflow and O 3 concentration at the airway opening 

### 7

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

### 8
internal sampling catheters ( Gerrity et al., 1995 ; Gerrity et al., 1988 ) . One method of 

### 9
quantifying O 3 dose is to measure the amount of O 3 removed from the air stream during 

### 10
breathing (termed “uptake”). The difference in the amount of O 3 inhaled and exhaled 

### 11

relative to the amount of inhaled O 3 is termed fractional absorption. Uptake efficiency is 

### 12
also reported and refers to the fraction of O 3 absorbed in a region as a function of the total 

### 13

amount of O 3 entering the given region. Uptake studies have utilized bolus and 

### 14

continuous O 3 breathing techniques as well as modeling to investigate these measures of 

### 15
uptake and the distribution of O 3 uptake between the upper and lower respiratory tract. A 

### 16

number of the studies that have measured the f ractional absorption and uptake efficiency 

### 17

of O 3 in the human RT, URT, and LRT are presented in Table 5

- 1 . For bolus exposure 

### 18
studies that reported fra ctional absorption, the total RT uptake efficiency was estimated 

### 19
as the sum of the products of the experimental bolus absorption and incremental volume 

### 20
of a bolus into a breath divided by the tidal volume of the breath, or where available, was 

### 21

taken from T able 1A of Schlesinger et al. (1997 )

. 

### 22

### Draft

– Do Not Cite or Quote 5

- 7 June 2012 Table 5

- 1 Human respiratory tract uptake efficiency data

Reference Mouth/ Nose a Inspiratory Flow (mL/sec) V T (mL) f B (bpm) b Uptake Efficiency

URT, complete breath URT, inspiration LRT, complete breath Total RT, tidal breath

Continuous Exposure

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

N 456 754 18 0.36 0.91

OR/N 500 800 18 0.43 0.91

OR/N 350 832 12 0.41 0.93

OR/N 634 778 24 0.38 0.89
Gerrity et al. (1994 ) OR 1,360 1,650 25 0.37 0.43 0.81

OR 1,360 1,239 35 0.41 0.36 0.78
Gerrity et al. (1995 ) OR 330 825 12 0.27 0.95 0.91

Wies ter et al. (1996a ) OR 539 631 16 0.76

N 514 642 16 0.73
Rigas et al. (2000 ) Face mask 480 1,100 27.6 0.86

Santiago et al. (2001 ) N 50 0.80 c

N 250 0.33

Bolus Exposure

Hu et al. (1992 ) Mouth

piece 250 500 0.46 0.88

Kabel et al. (1994 ) Mouth

piece 250 500 0.50 0.88

Mouth

piece 250 500 0.53 0.88

N 250 500 0.78 0.94
Hu et al. (1994 ) Mouth

piece 150 500 0.65 0.91

Mouth

piece 250 500 0.51 0.87

Mouth

piece 500 500 0.26 0.82

Mouth

piece 750 500 0.16 0.78

Mouth

piece 1,000 500 0.11 0.76

Ultman et al. (1994 ) Mouth

piece 250 500 d 15 0.30

Mouth

piece 250 500 15 0.47

### Draft

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- 8 June 2012 5.2.2.1 Gas Transport Principles

The three

- dimensional transport of O 3 in the lumen of an airway is governed by diffusion 

### 1

and bulk flow or convection. When modeled as a one

- dimensional process in which the 

### 2
radial profiles of axial velocity and O 3 concentration profiles are flat, O 3 transport along 

### 3
an airway lumen occurs by c onvection, axial diffusion and a coupled diffusion

- reaction 

### 4
process called dispersion. Simultaneously, O 3 diffuses into the ELF where it undergoes 

### 5

radial diffusion and chemical reaction ( Figure 5

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

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

### 7

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

### 8
mechanism for axial transport in the airway lumen. However, in the alveolar region of the 

### 9
lung, diffusion is the major gas transport mechanism. 

### 10

Gas transport in the TB region occurs by a combi nation of bulk flow and mixing 

### 11

( Ultman, 1985 ) . Mixing can occur by diffusion processes associated w ith the molecular 

### 12
nature of the gas and by convection, which depends on local velocity patterns. The 

### 13
complexity of the airway structure and surface affects the bulk airflow patterns so that not 

### 14
all nasal and lung surfaces receive the same O 3 exposure or do se ( Miller and Kimbell, 

### 15

1995 ) . For example, it has been reported that the larger surface

- to

- volume ratio 

### 16
associated with the smaller airways in women enhances local O 3 uptake and reduces the 

### 17
distal penetration volume of O 3 into the RT of women relative to men ( Ultman et al., 

### 18

2004 ) . Also, it was reported that changes in cro ss

- sectional area available for gas 

### 19

diffusion are related to overall O 3 retention ( Reeser et al., 2005 ; Ultman et al., 2004 )

. 

### 20 Reference Mouth/
Nose a Inspiratory Flow (mL/sec) V T (mL) f B (bpm) b Uptake Efficiency

URT, complete

breath URT, inspiration LRT, complete

breath Total RT, tidal breath

Bush et al. (1996 ) Mouth

piece 250 500 0.51 0.89

Nodelman and Ultman (1999 ) Nasal Cannula 150 500 18 0.90 0.92

Nasal Cannula 1,000 500 120 0.50 0.84

Mouth

piece 150 500 18 0.77 0.91

Mouth

piece 1,000 500 120 0.25 0.75

Ultman et al. (2004 ) OR 490 450 d 32.7 0.87

OR 517 574 27 0.91

a OR = oral exposure during spontaneous breathing; N = nasal exposure during spontaneous breathing; OR/N = pooled data from oral and nasal exposure; mouthpiece = exposure by mouthpiece.

b f B is either measured or is computed from flows and V T

.

c F URT from Santiago et al. (2001 ) represents nasal absorption (F nose ).

d V T is computed from flow and f B

.

### Draft

– Do Not Cite or Quote 5

- 9 June 2012 The principal influence on mixing in the TB region comes from the axial velocity profile 

### 1
and diffusion. When air flows through an airway, O 3 located near the tube center moves 

### 2

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

### 3

non

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

### 4
dispersion of the O 3 that operates in parallel with bul k flow and axial diffusion (a process 

### 5
caused by the ever

- present Brownian motion of individual O 3 molecules). The shape of 

### 6
the velocity profile is affected by the flow direction through bifurcating airway branches 

### 7

( Schroter and Sudlow, 1969 ) . The velocity profile is nearly p arabolic during inhalation 

### 8
but quite flat during exhalation. Thus, there tends to be greater axial dispersion during 

### 9
inhalation than during exhalation. Dispersion also depends on the nature of the flow, that 

### 10

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

### 11
fluctuations). Because turbulent flow flattens velocity profiles, it may actually diminish 

### 12
dispersion. In humans, turbulent flow persists only a few generations into the RT. The 

### 13
persistence of turbulence into th e RT also varies by species and flow rates. For example, 

### 14
airflow is nonturbulent in the rat nose at any physiologic flow rate but may be highly 

### 15
turbulent in the human nose during exercise ( M iller, 1995 ) . Diffusive forces and 

### 16
resistance vary along the RT. Diffusive resistance increases with distal penetration into 

### 17
the RT with a study reporting that the gas boundary layer contributes 53% of the overall 

### 18

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

### 19

( Hu e t al., 1994 )

. 

### 20

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

### 21

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

### 22

cross

- sectional area of th e airways rapidly increases and linear velocities decrease, 

### 23
leading to a greater role for molecular diffusion of gases. Gas molecules close to the 

### 24

alveolar

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

### 25

membrane, and this creat es a substantial boundary layer resistance to O 3 transfer across 

### 26
the gas

- eLF interface. 

### 27

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 

### 28
centriacinar region (CAR). The CAR refers to the zone at the junction of the TB airways 

### 29
and the gas exchange region. This area is also termed the proximal alveolar region (PAR) 

### 30
and is defined as the first generation distal to the terminal bronchioles. Contained within 

### 31
the CAR, the respiratory bronchioles were co nfirmed as the site receiving the greatest O 3 

### 32

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

### 33

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

### 34
vicinity of the respiratory bronchioles and was dependent on the delivered O 3 dose to 

### 35

### Draft

– Do Not Cite or Quote 5

- 10 June 2012 these tissues (see also Section 5.4.1 ). However, 18 O label was detected to a lesser extent 

### 1
in other regions of the TB airway tree, showing that O 3 is delivered to these 

### 2
compartments as well, although in a smaller dose. These studies agree with earlier model 

### 3
predictions showing that the tissue O 3 dose (O 3 flux to liquid

- tissue interface) was low in 

### 4

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

### 5
rapidly decreased in the alveolar region ( Miller et al., 1985 ) . It was also predicted that the 

### 6

net O 3 dose (total absorption, O 3 flux to air

- liquid interface) g radually decreased with 

### 7
distal progression from the trachea to the end of the TB region and then rapidly decreased 

### 8
in the alveolar region. Despite the exclusion of the URT and appreciable O 3 reactions 

### 9
with ELF constituents after the 16th generation, the re sults from the model agree with 

### 10
experimental results showing that the greatest O 3 tissue dose was received in the CAR 

### 11

( Miller et al., 1985 )

. 

### 12

Inhomogeneity in the RT structure may affect the dos e delivered to this target site. 

### 13
Models have predicted that the farther the PAR is from the trachea, the less the O 3 tissue 

### 14
dose to the region. Ultman and Anjilvel (1990 ) and Overton and Graham (1989 ) 

### 15
predicted approximately a 50 to 300% greater PAR dose for the shortest path relative to 

### 16
the longest path in humans and rats, respectively. In addition, Mercer et al. (1991 ) found 

### 17
that both path distance and ventilatory unit size affected dose. The variation of O 3 dose 

### 18
among anatomically equivalent ventilatory units was predicted to vary as much as 6

- fol d, 

### 19
as a function of path length from the trachea. This could have implications in regional 

### 20
damage to the LRT, such that even though the average LRT dose may be at a level where 

### 21
health effects would not be predicted , local regions of the RT may receive considerab ly 

### 22
higher than average doses and therefore be at greater risk of effects. 

### 23

5.2.2.3 Upper Respiratory Tract Ozone Removal and Dose

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

- 54% efficient 

### 24

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

### 25
ranges from 80

- 95% efficient ( Hu et al., 1992 )

. The URT provides a defense against O 3 

### 26
entering the lungs by removing half of the O 3 that will be absorbed from the airstream. In 

### 27

both animals and h umans, about 50% of the O 3 that was absorbed in the RT was removed 

### 28
in the head (nose, mouth, and pharynx), about 7% in the larynx/trachea, and about 43% in 

### 29
the lungs ( Hu et al., 1992 ; Hatch et al., 1989 ; Miller et al., 1979 ) . However, experimental 

### 30
studies in dogs have reported 75

- 100% uptake in the URT ( Yoko yama and Frank, 1972 ; 

### 31
Vaughan et al., 1969 ) . The fraction of O 3 taken up was inversely related to flow rate and 

### 32

to inlet O 3 concentration ( Yokoyama and Frank, 1972 ; Vaughan et al., 1969 ) . The 

### 33
limiting factors in nasal O 3 uptake were simultaneous diffusion and chemical reaction of 

### 34

O 3 in the nasal ELF layer ( Santiago et al., 2001 ) . The ELF layer in the nose is thicker 

### 35

### Draft

– Do Not Cite or Quote 5

- 11 June 2012 than in the rest of the RT, and mathematical estimates predicted that O 3 penetrates less 

### 1
than the thickness of the ELF layer; reac tion products are likely the agents damaging the 

### 2

nasal tissue and not O 3 itself. It was hypothesized that the nasal non

- linear kinetics of O 3 

### 3
uptake fraction result from the depleting substrates in the nasal ELF becoming the 

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

. 

### 5

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

- 1 ). 

### 6
Gerrity e t al. (1995 ) reported unidirectional uptake efficiencies of O 3 inhaled from a 

### 7
mouthpiece; of 17.6% from the mouth to vocal cords, 9.5% from the vocal cords to the 

### 8
upper trachea (totaling 27.1%), 8.4% from the upper trachea to the main bifurcation 

### 9

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

### 10
intermedius (totaling 32.5%). These values are lower than those calculated by Hu et al. 

### 11

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

### 12

in which an O 3 bolus pen etrated between the mouth and the vocal cords, the upper 

### 13
trachea, the main bifurcation carina, and the bronchus intermedius, respectively. The 

### 14

lower efficiencies seen in Gerrity et al. (1995 ) may hav e resulted because these 

### 15
investigators measurements were based on inhalation alone or was caused by O 3 

### 16
scrubbing by the mouthpiece. 

### 17

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

### 18
the LRT from damage from inspired O 3 ( Santiago et al., 2001 ; Gerrity et al., 1988 )

. 

### 19
Sawyer et al. (2007 ) further investigated nasa l uptake of O 3 in healthy adults during 

### 20

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

### 21
were taken on ten adults (8 women, 2 men) exposed to 200 ppb O 3 before and after 

### 22

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

### 23
was ~50% greater at 10 L/min compared to 20 L/min both pre

- and post

- exercise. 

### 24

However, the inhaled O 3 dose delivered to the LRT (i.e., flow rate × exposure 

### 25

concentration × (1

- nasal absorbed fraction)) was 1.6

- fold grea ter at the higher flow than 

### 26
at the lower flow (2.5 compared to 0.9 ppm∙L/min). These results are similar to those 

### 27
published earlier that found air pollutant retention increased with increasing airflow by 

### 28
more than what would be predicted by just the increa sed partial pressure difference of the 

### 29

gas ( Aharonson et al., 1974 ) . Prior exercise did not affect O 3 uptake at either flow rate, 

### 30

but did significa ntly increase nasal volume (Vn) and AR measurements of nasal 

### 31

cross

- sectional area (minimum c ross

- sectional area (MCA) that corresponds to the nasal 

### 32
valve, CSA2 that corresponds to the anterior edge of the nasal turbinates, and CSA3 that 

### 33
corresponds to the posterior edge of the nasal turbinates) (p ≤ 0.05) ( Sawyer et al., 2007 )

. 

### 34
Conversely, exercise decreased nasal resistance (Rn) (p <0.01) and NO production 

### 35

(non significa nt, p > 0.05). The change in Vn and CSA2:MCA ratio was correlated with 

### 36
the percent change in nasal uptake, however the overall effect was small and sensitive to 

### 37
elimination of outliers and gender segregation. 

### 38

### Draft

– Do Not Cite or Quote 5

- 12 June 2012 Overall, the majority of studies suggest that the URT removes about half of the O 3 that 

### 1
will be absorbed by reactions in the nasal ELF. The exact uptake efficiency will change 

### 2

due to variations in flow rate and inhaled concentration. 

### 3

5.2.2.4 Lower Respiratory Tract Ozone Uptake and Dose

Approximately 43% of the O 3 a bsorption occurs in the LRT of both humans and animals. 

### 4
Models predicted that the net O 3 dose decreases distally from the trachea toward the end 

### 5
of the TB region and then rapidly decreases in the alveolar region ( Miller et al., 1985 )

. 

### 6
Further, these models predicted low tissue O 3 dose in the trachea and large bronchi. 

### 7

Uptake efficiency depends on a number of variables, including O 3 exposure 

### 8
concentration, exposure time, and breathing pattern. Fo r breaths of similar waveforms, 

### 9
respiratory patterns are uniquely described by breathing frequency (f B ) and tidal volume 

### 10

(V T ); by minute ventilation ( V ̇ E = f B x V T ) and f B ; or by V ̇ E and V T . Simulations from the 

### 11

Overton et a l. (1996 ) single

- path anatomical respiratory tract model, where the upper and 

### 12
lower respiratory tracts were modeled but uptake by the URT was not considered, 

### 13
predicted that fractional uptake and PAR O 3 dose increased with V T when f B was held 

### 14

constant. L ikewise, experimental studies found that O 3 uptake was positively correlated 

### 15
with changes in V T ( Ultman et al., 2004 ; Gerrity et al., 1988 ) . Also, O 3 exposure led to a 

### 16

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

### 17

decreasing the dose delivered to the lung at a particular V ̇ E ( Gerrity et al., 1994 ) . Na sal O 3 

### 18
uptake efficiency was inversely proportional to flow rate ( Santiago et al., 2001 ) , so that 

### 19

an increase in V ̇ E will increase O 3 delivery to the lower airways. At a fixed V ̇ E , increasing 

### 20

V T (corresponding to decreasing f B ) drove O 3 deeper into the lungs and increased total 

### 21

re spiratory uptake efficiency ( Figure 5

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

### 22
Gerrity et al., 1988 ) . Modeling predicted a decrease in fractional uptake with increased f B 

### 23

when V T was held c onstant, but an increase in PAR dose with increased f B ( Overton et 

### 24

al., 1996 ) . Similarly, increased f B (80

- 160 bpm) and shallow breathing in rats decreased 

### 25

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

### 26

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

- induced alterations in 

### 27

contact time that affects the first

- order absorption rate for O 3 ( Postlethwait et al., 1994 )

. 

### 28

Also, an association of O 3 uptake efficiency was found with V ̇ E and exposure time. 

### 29

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

### 30

fraction of O 3 is absorbed in the URT and uptake shifts to the TB airways and respirator y 

### 31
airspaces ( Nodelman and Ultman, 1999 ; Hu et al., 1994 ; Ultman et al., 1994 )

. Hu et al. 

### 32

(1994 ) and Ultman et al. (1994 ) found that O 3 absorption inc reased with volumetric 

### 33
penetration (Vp) of a bolus of O 3 into the RT. Ozone uptake efficiency and Vp were not 

### 34

### Draft

– Do Not Cite or Quote 5

- 13 June 2012 affected by bolus O 3 concentration ( Kabel et al., 1994 ; Hu et al., 1992 ) , indicating that 

### 1
under these experimental conditions O 3 uptake was a linear absorption process, where the 

### 2

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

### 3
The absorption relationship would not be linear once interfacial mass transfer is 

### 4

saturated. As mentioned above, a wea k negative relationship between O 3 concentration 

### 5
and uptake efficiency was reported for the nasal cavities by Santiago et al. (2001 )

. Rigas 

### 6
et al. (2000 ) also found a weak but significa nt negative dependence of O 3 concentration 

### 7
on RT uptake effi ciency in exercising individuals. This study also found that exposure 

### 8

time had a small but significa nt influence on uptake efficiency; however, this negative 

### 9
dependence may be an artifact of progressive depletion of reactive substrates from the 

### 10

ELF. 

### 11

Note: Subjects breathed 250 ppb 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).
Source: Reprinted with permission of Heal th 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.

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

### 12
reproducible pattern of severity between daughter branches of individual bifurcations that 

### 13
is dependent on the O 3 concentration

- time profile of the inhaled gas. A 3

- D 

### 14
computational fluid dynamics model was created to investigate the O 3 transport in a 

### 15

### Draft

– Do Not Cite or Quote 5

- 14 June 2012 singl e airway bifurcation ( Taylor et al., 2007 ) . The model consisted of one parent branch 

### 1
and two symmetrical daughter branches with a branching angle of 90° and a sharp carinal 

### 2
ridge. Various flow scenarios were simulated using Reynolds numbers (Re) ranging from 

### 3
100 to 500. The Re that corresponds to a certain airway generation is dependent upon 

### 4

both lung size a nd V ̇ E , such that the range in Re from 100

- 500 would encompass 

### 5
generations 1

- 5, 3

- 7, and 6

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

### 6
heavy exercise, respectively, whereas the same Re range corresponds to generations 0

- 4, 

### 7

1

- 6, and 4

- 8 for a 4

- year

- old child. This model predicted velocity distributions that were 

### 8

consistent with earlier work of Schroter and Sudlow (1969 ) , and also reported O 3 

### 9
concentration and wall uptake distributions. The model predicted that during inspiration, 

### 10
the velocity and O 3 concentration distribution were axisymmetric throughout the parent 

### 11
branch, but skewed towards the inner wall within the daughter branches. During 

### 12
expiration, the model predicted that the velocity and O 3 concentration distribution was 

### 13
slightly skewed towards the outer walls of the daughter branches. Hot spots of wall flux 

### 14
existed at the carina during inspiration and expiration with Re >100. Additional hot spots 

### 15
were found during expiration on the parent b ranch wall downstream of the branching 

### 16
region. 

### 17

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

### 18

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

### 19
the lung is sensitive to changes i n V T , f B , and to a minor extent, exposure time. 

### 20

5.2.2.5 Mode of Breathing

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

### 21
airways volume had a weaker influence on O 3 absorption during nasal breathing 

### 22
compared to oral breathin g. This could be a result of O 3 scrubbing in the nasal 

### 23
passageways that are bypassed by oral breathing. Studies by Ultman and colleagues using 

### 24

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

### 25
than during oral breathi ng at each Vp (e.g., 0.90 during nasal breathing and 0.80 during 

### 26
oral breathing at 150 mL/sec and 0.45 during nasal breathing and 0.25 during oral 

### 27
breathing at 1,000 mL/sec) ( Nodelman and Ultman, 1999 ; Kabel et al., 1994 ; Ultman et 

### 28

al., 1994 ) . Therefore, oral breathing results in deeper penetration of O 3 into the RT with a 

### 29
higher absorbed fraction in the TB and alveolar airways ( Nodelman and Ultman, 1999 )

. 

### 30

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

### 31

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

### 32

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

. 

### 33
Overall, the mode of breathing may have a seemingly small effect on the RT uptake 

### 34

### Draft

– Do Not Cite or Quote 5

- 15 June 2012 efficiency; however, it does play an important role in the distribution of O 3 deposited in 

### 1
the distal airways. 

### 2

5.2.2.6 Interindividual Variability in Dose

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

### 3

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

### 4

accounted for between 10

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

### 5

( S antiago et al., 2001 ; Rigas et al., 2000 ) . When concentration, time, and V ̇ E were held 

### 6
constant, fractional absorption ranged from 0.80 to 0.91 ( Rigas et al., 2000 ) . It has been 

### 7
hypothesized that interindividual variation in O 3 induced response s such as FEV 1 is the 

### 8

result of interi ndividual variation in net dose or regional O 3 uptake among exposed 

### 9

individuals. 

### 10

Recent studies have reit erated the importance of intersubject variation in O 3 uptake. The 

### 11
intersubject variability in nasal O 3 uptake determined by Sawyer et al. (2007 ) ranged 

### 12
from 26.8 to 65.4% (pre

- and post

- exercise). A second study investigating the use of the 

### 13

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

### 14

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

. 

### 15

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

### 16
physiology, anatomy, and biochemistry. Since the URT and TB airways remove the 

### 17
majority of inhaled O 3 before it reach es the gas exchange region, the volume and surface 

### 18
area of these airways will influence O 3 uptake. Models predicted that fractional O 3 uptake 

### 19

and PAR dose (flux of O 3 to the PAR surfaces divided by exposure concentration) 

### 20
increase with decreasing TB volume and decreasing TB region expansion. On the 

### 21
contrary, alveolar expansion had minimal effect on uptake efficiency as relatively little 

### 22

O 3 reaches the peripheral lung ( Bush et al., 2001 ; Overton et al., 1996 ) . Ozone uptake 

### 23
was virtually complete by the time O 3 reaches the alveolar spaces of the lung 

### 24

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

### 25
volumes may ac count for 75% of the variation in absorption between subjects ( Ultman et 

### 26

al., 2004 ) . In support of this concept, regression analysis showed that O 3 absorption was 

### 27
positively correlated with anatomical dead space (V D ) and TB volume (i.e., V D minus 

### 28

V URT ), but not total lung capacity (TLC), forced vital ca pacity (FVC), or functional 

### 29
residual capacity (FRC) ( Ultman et al., 2004 ; Bush et al., 1996 ; Hu et al., 1994 ; 

### 30

Postlethwait et al., 1994 ) . Variability in V D was correlated more with the variability in the 

### 31
TB volume than the URT volume. Similarly, uptake was correlated with changes in 

### 32

individual bronchial cross

- sectional area, indicating that changes in cross

- sectional area 

### 33
available for gas diffusion are related to overall O 3 retention ( Reeser et al., 2005 ; Ultman 

### 34

### Draft

– Do Not Cite or Quote 5

- 16 June 2012 et al., 2004 ) . When coupled, these results suggest that the larger surface

- to

- volume ratio 

### 1

associated with the smaller airways in women enhances local O 3 uptake, thereby reducing 

### 2
the distal penetration volume of O 3 into the female respiratory system. When absorption 

### 3
data were normalized to Vp/V D , variability attributed to gender differences were not 

### 4
distinguishable ( Bush et al., 1996 ) . These studies provide support to the RT anatomy, 

### 5
especially the TB volume and surface area, playing a key role in variability of O 3 uptake 

### 6

between individuals. 

### 7

In addition, variability between in dividuals is influenced by age. Overton and Graham 

### 8

(1989 ) p redicted that the total mass of O 3 absorbed per minute (in units of: μg/min per 

### 9
[μg/m 3 of ambient O 3 ]) increased with age from birth to adulthood. This model predicted 

### 10
that during quiet breathing the LRT distribution of absorbed O 3 and the CAR O 3 tissue 

### 11
dose were not sensitive to age. However, during heavy exercise or work O 3 uptake was 

### 12
dependent on age. A physiologically based pharmacokinetic model simulating O 3 uptake 

### 13
predicted that regional extraction of O 3 was relatively insensitive to age, but extraction 

### 14
per unit surface area was 2

- fold to 8

- fold higher in infants compare d to adults, due to the 

### 15
fact that children under age 5 have much a much smaller airway surface area in the 

### 16
extrathoracic (nasal) and alveolar regions ( Sarangapani et al., 2003 ) . Additionally, 

### 17
children tend to have a greater oral breathing contribution than adults at rest and du ring 

### 18

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

### 19
lung surface area, the dose rate to the lower airways of children compared to adults is 

### 20
increased further because children breathe at higher minute ve ntilations relative to their 

### 21
lung volumes. 

### 22

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

### 23

the f ractional uptake of a bolus of O 3 in apparently healthy smokers with limited smoking 

### 24
history ( Bates et al., 2009 ) . Despite similar internal O 3 dose distribution, the smokers 

### 25
exhibited greater pulmonary responses to O 3 bolus exposures, measured as FEV 1 

### 26
decrements and increases in the normalized slope of the alveolar plateau (S N ). This was 

### 27
contrary to previous studies conducted in smokers with a greater smoking history that 

### 28

found decreased O 3 induced decrements in FEV 1 in smokers during continuous O 3 

### 29

exp osure ( Frampton et al., 1 997a ; Emmons and Foster, 1991 )

. 

### 30

5.2.2.7 Physical Activity

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

### 31
part, to the increased intake of air. Thus, human studies have used exercise, at a variety of 

### 32
activity levels, to enhance the effects of O 3 ( Table 5

- 2 ). Further explanation of the effects 

### 33
of physical activity on ventilation can be found in Chapters 4 and 6. Table 4

- 5 presents 

### 34

### Draft

– Do Not Cite or Quote 5

- 17 June 2012 the mean ventilation rates at different activity levels for different age groups. Table 6

- 1 

### 1
provides activity levels as detailed in specific human exposure studies. 

### 2

Table 5

- 2 General adult human inhalation rates by activity levels.

As exercise increases from a light to moderate level, V T increases. This increase in V T is 

### 3
achieved by encroaching upon both the inspiratory and expir atory reserve volumes of the 

### 4
lung ( Dempsey et al., 1990 ) . After V T reaches about 50% of the vital capacity, generally 

### 5
during heavy exercise, furt her increases in ventilation are achieved by increasing f B
. 

### 6
Ventilatory demands of very heavy exercise require airway flow rates that often exceed 

### 7
10 times resting levels and V T that approach 5 times resting levels ( Dempsey et al., 

### 8

2008 )

. 

### 9

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

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

### 11

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

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

### 13

necessitates a lower

- resistance path through the mouth. Modeling heavy exercise by 

### 14
increasing ventilatory parame ters from normal respiration levels predicted a 10

- fold 

### 15
increase in total mass uptake of O 3 ( Miller et al., 1985 )

. This model also predicted that as 

### 16
exercise and ventilatory demand increased, th e maximum tissue dose, the O 3 reaching the 

### 17
tissues, moved distally into the RT ( Figure 5

- 5 ). By increasing flow to what is common 

### 18
in moderate or heavy exercise (respiratory flow = 45

- 60 L/min compared to 15 L/min), 

### 19

the URT absorbed a smaller fraction of the O 3 (~0.50 at low flow rate to 0.10 at high flow 

### 20
rate); however, the trachea and more distal TB airways received higher doses during 

### 21
higher flow rate s than at lower flow rates (0.65 absorbed in the lower TB airways, and 

### 22
0.25 absorbed in the alveolar zone with high flow compared to 0.5 in the TB with almost 

### 23

no O 3 reaching the alveolar zone at low flow) ( Hu et al., 1994 ) . The same shift in the O 3 

### 24
dose distribution more distally in the lung occurred i n other studies mimicking the effects 

### 25

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

### 26
age only under exercise conditions ( Overton and Graham, 1 989 ) . The total mass of O 3 

### 27 Activity Level Inhalation Rate

Light 2 to 3 × resting V ̇ E a

Moderate 4 to 6 × resting V ̇ E
Heavy 7 to 8 × resting V ̇ E
Very Heavy >9 × resting V ̇ E

a Resting V ̇ E approximates 8 L/min

Source: U.S. EPA (1986 )

.

### Draft

– Do Not Cite or Quote 5

- 18 June 2012 absorbed per minute (μg/min per [μg/m 3 of ambient O 3 ]) was predicted to increase with 

### 1
age during heavy work or exercise. A recent study by Sawyer et al. (2007 ) approximated 

### 2
that doubling minute ventilation led to only a 1.6

- fold higher delivered dose rate of O 3 to 

### 3
the lung (delivered dose was calcula ted as: flow rate × [O 3 ppm] × (100

- percent nasal O 3 

### 4
uptake)). Past models have predicted the increase in uptake during exercise is distributed 

### 5
unevenly in the RT compartments and regions. Tissue and mucus layer dose in the TB 

### 6
region increased ~1.4

- fold du ring heavy exercise compared to resting conditions, whereas 

### 7
the alveolar region surfactant and tissue uptake increased by factors of 5.2 and 13.6, 

### 8
respectively ( Miller et al., 1985 )

. 

### 9

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

.



### Figure 5

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

### Draft

– Do Not Cite or Quote 5

- 19 June 2012 5.2.2.8 Summary

In s ummary, O 3 uptake is affected by complex 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

chemi cal 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 the RT structure may affect the dose delivered to 

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

### 8
could have implications in regional damage to the LRT, such that even though the 

### 9
average LRT dose may be at a level where health effects would not be predicted , local 

### 10
regions of the RT may receive considerably higher than average doses and therefore be at 

### 11
greater risk of effects. Recent studies have provided evidence for hot spots of O 3 flux 

### 12
around bifurcations in airways. Experimental studies and models have suggested that the 

### 13

net O 3 dose gradually decreases distally from the trachea toward the end of the TB region 

### 14
and then rapidly decreases in the alveolar region. However, the tissu e O 3 dose is low in 

### 15

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

### 16
rapidly decreases distally into the alveolar region. 

### 17

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

### 18

decrease wi th increased flow and increase proportional to V T , so that at a fixed V ̇ E , 

### 19
increasing V T (or decreasing f B ) drives O 3 deeper into the lungs and increases total 

### 20
respiratory uptake efficiency. Individual total airway O 3 uptake efficiency is also 

### 21

sensitive to large changes in O 3 concentration, exposure time, and V ̇ E . Major sources of 

### 22

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

### 23
but the interindividual variation is the greatest so urce of variability uptake efficiency. The 

### 24
majority of this interindividual variability is due to differences in TB volume and surface 

### 25

area. 

### 26

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

### 27
changes and a switch to oronas al breathing during exercise results in deeper penetration 

### 28

of O 3 into the lung with a higher absorbed fraction in the ET, TB, and alveolar airways. 

### 29
For these reasons, increased physical activity acts to move the maximum tissue dose of 

### 30

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

### 31

### Draft

– Do Not Cite or Quote 5

- 20 June 2012 5.2.3 Ozone Reactions and Reaction Products

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

### 1

result from O 3 exposure. The process by which O 3 moves from the airway lumen and into 

### 2

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

### 3
called “reactive absorption”. Ozone is chemically reactive with a wide spectrum of 

### 4
biomolecules and numerous studies have evaluated the loss of specific molecules such as 

### 5

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

### 6
studies contribute to the understanding of O 3 reactions and reaction products. 

### 7

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

### 8
neutral lipid s like cholesterol, free fatty acids, proteins, and low molecular weight 

### 9

antioxidants as has been demonstrated in in vitro studies ( Per ez

- Gil, 2008 ; Uppu et al., 

### 10

1995 ) . It was estimated that 88% of the O 3 that does not come in contact with 

### 11
antioxidants will react with unsaturated fatty acids in the ELF including those contained 

### 12
within phospholipids or neutral lipids ( Uppu et al., 1995 ) . Ozone r eacts with the double 

### 13
bond of unsaturated fatty acids to form stable and less reactive ozonide, aldehyde, and 

### 14
hydroperoxide reaction products via chemical reactions such as the Criegee ozonolysis 

### 15

mechanism ( Figure 5

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

### 16
aldehydes hexanal, heptanal, and nonanal, have been recovered after O 3 exposure in 

### 17

human BAL fluid (BALF), rat BALF, iso lated rat lung, and in vitro systems ( Frampton et 

### 18

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

### 19

4

- hydroxynonenal were found in human alveolar macrophages after O 3 exposure in vivo 

### 20

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

### 21
availability of O 3 since lipids are so abundant in the ELF. Yields of O 3 - induced aldeh ydes 

### 22
were increased by the decrease in other substrates such as ascorbic acid (AH 2 ) 

### 23

( Postlethwait et al., 1998 ) . Free radicals are also generated during O 3 - mediated oxidation 

### 24

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

### 25
lipid

- soluble free radical scavenger α

- tocopherol (α

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

### 26

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

### 27
account for acute cell injury, however only modest amounts of products may be 

### 28
necessary to induce cytotoxicity ( Postlethwait and Ultman, 2001 ; Postlethwait et al., 

### 29

1998 )

. 

### 30

### Draft

– Do Not Cite or Quote 5

- 21 June 2012 Note: Not all secondary reaction products are shown.

Source: U.S. EPA (2006b )

.



### Figure 5

- 6 Schematic overview of ozone interaction with PUFA in ELF and lung cells.

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

### 1

with O 3 results in biologically active cholesterol products such as the oxysterols, 

### 2
β

- epoxide and 6

- oxo

- 3,5

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

### 3
Murp hy, 2004 ) . Product yields depend on ozonolysis conditions, however cholesterol 

### 4
ozonolysis products form in similar abundance to phospholipid

- derived ozonolysis 

### 5
products in rat ELF ( Pulfer and Murphy, 2004 )

. 

### 6

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

### 7
surface epithelial cells. Ozone reactions with proteins have been studied by their in vitro 

### 8

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

### 9
are cystein e, histidine, methionine, tyrosine, and tryptophan). Ozone preferentially reacts 

### 10
with biomolecules in the following order: thiosulfate >ascorbate >cysteine ≈ methionine 

### 11
>glutathione ( Kanofsky and Sima, 1995 ) . Rat e constants for the reaction of amino acids 

### 12

with O 3 vary between studies due to differing reaction conditions and assumptions; 

### 13
however aliphatic amino acids were consistently very slow to react with O 3 (e.g., alanine: 

### 14

25

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

### 15
Pryor et al., 1984 ; Hoigné and Bader, 1983 )

. Uppu et al. (1995 ) predicted that 12% of 

### 16

inhaled O 3 that does not react with antioxidants will react with proteins in the E LF. 

### 17

Reactions of O 3 with low molecular weight antioxidants have been extensively studied. 

### 18
The consumption of antioxidants such as uric acid (UA), ascorbate (AH 2 ), and reduced 

### 19
glutathione (GSH) by O 3 was linear with time and positively correlated with initi al 

### 20

sub strate concentration and O 3 concentration ( Mudway and Kelly, 1998 ; Mudway et al., 

### 21

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

### 22
of the human airways (obt ained as BALF) and display high (but not equal) intrinsic 

### 23

### Draft

– Do Not Cite or Quote 5

- 22 June 2012 reactivities toward O 3 . In individual and in limited composite mixtures, UA was the most 

### 1
reactive antioxidant tested, followed by AH 2 ( Mudway and Kelly, 1998 ) . GSH was 

### 2
consistently less reactive than UA or AH 2 ( Mudway and Kelly, 1998 ; Mudway et al., 

### 3

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

### 4
evaluated the interfacial exposure of aqueous solutions of UA, AH 2 , and GSH 

### 5

(50

- 200 μM) with O 3 (1

- 5 ppm). Similar to the resu lts of Mudway and Kelly (1998 ) , this 

### 6
study found the hierarchy in reactivity between O 3 and these antioxidants to be 

### 7

UA≥ AH 2 >>GSH. UA and AH 2 shared a 1:1 stoichiometry with O 3 , whereas 2.5 moles of 

### 8
GSH were consumed per mole of O 3 . Using these stoichiometries, reaction rate constants 

### 9

were derived (5.8×10 4 M - 1 sec - 1 , 5.5×10 4 M - 1 sec - 1 , and 57.5 M
- 0.75 sec - 1 [20.9 M - 1 sec - 1 ] for 

### 10

the reaction of O 3 with UA, AH 2 , and GSH, respectively). Other studies report reactive 

### 11
rate constants that are two to three orders of magnitude larger, however these studies used 

### 12
higher concentrations of O 3 and antioxidants under less physiologically relevant 

### 13
experimental conditions ( Kanofsky and Sima, 1995 ; Giamalva et al., 1985 ; Pryor et al., 

### 14

1984 ) . However, O 3 acts through competition kinetics so the effective concentration of 

### 15
the reactants present in the ELF will determine the reactions that occur in vivo. For 

### 16
example, the pKa of GSH is about 8.7 so that at physiological pH very little is in the 

### 17

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

### 18
it exists almost entirely as ascorbate (AH - ) in the ELF. Thus, the effective concentration 

### 19

of GSH that is available to react with O 3 will be much lower than that of ascorbate in 

### 20

ELF. 

### 21

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

### 22

from initial air

- liquid interface interactions of O 3 with ELF components 

### 23
(e.g., antioxidants and proteins) in ~1 millisecond ( Enami et al., 2009a

, b

, c , 2008a

, b )

. 

### 24
Solutions of aqueous UA, AH 2 , GSH, α

- TOH, and protein cysteines (CyS) were sprayed 

### 25
as microdroplets in O 3 /N 2 m ixtures at atmospheric pressure and analyzed by electrospray 

### 26
mass spectrometry. These recent studies demonstrated different reactivity toward AH 2 , 

### 27
UA, and GSH by O 3 when the large surface to volume ratio of microdroplets promote an 

### 28

interfacial reaction com pared to previous studies using bulk liquid phase bioreactors. This 

### 29
artificial system does not recapitulate the lung surface so caution must be taken in 

### 30
translating the results of these studies to in vivo conditions. 

### 31

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

### 32
hierarchy of reactivity of these ELF components with O 3 was determined to be AH 2 ≈ UA 

### 33

> CyS >GSH. There was some variance between the reaction rates and product fo rmation 

### 34

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

### 35
bulk liquid phase bioreactors as described previously. UA was more reactive than AH 2 

### 36

toward O 3 in previous studies, but in reactions with O 3 with microdroplets, these 

### 37
antioxidants had equivalent reactivity ( Enami et al., 2008b ) . As O 3 is a kinetically slow 

### 38

### Draft

– Do Not Cite or Quote 5

- 23 June 2012 one

- electron acceptor but very reactiv e O

- atom donor, products of the interaction of O 3 

### 1

with UA, AH 2 , GSH, CyS, and α

- TOH result from addition of n O

- atoms ( n = 1

- 4). These 

### 2
products included epoxides (e.g., U

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

- O 2 - ), and ozonides 

### 3
(e.g., U

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

- ), not 

### 4
glutathione disulfide (GSSG) by O 3 ( Enami et al., 2009b ) . However, it is possible th at 

### 5
other oxidative species are oxidizing GSH in vivo, since sulfonates are not detected in O 3 

### 6
exposed ELF whereas GSSG is. This is also supported by the fact that O 3 is much less 

### 7

reactive with GSH than other antioxidants, such that <3% of O 3 will be scaven ged by 

### 8
GSH when in equimolar amounts with AH 2 ( Enami et al., 2009b )

. 

### 9

This series of studies also demonstrated that ozonol ysis product yields and formation 

### 10
were affected by pH. Acidified conditions (pH ≈ 3

- 4), such as those that may result from 

### 11
acidic particulate exposure or pathological conditions like asthma (pH ≈ 6), decreased the 

### 12
scavenging ability of UA and GSH for O 3 ; s uch that at low pH, the scavenging of O 3 

### 13
must be taken over by other antioxidants, such as AH 2 ( Enami et al., 2009b

, 2008b )

. 

### 14

Also, under acidic conditions (pH ≈ 5), the ozonolysis products of AH 2 shifted from the 

### 15
innocuous dehydroascorbic acid to the more persistent products, AH 2 ozonide and 

### 16

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

### 17
acidic copollutant exposure will increase the toxicity of O 3 by preven ting some 

### 18
antioxidant reactions and shifting the reaction products to more persistent compounds. 

### 19

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

### 20
mixtures. Individual antioxidant consumption rates decreased as th e substrate mixture 

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

### 22
Kelly, 1998 ) . However, O 3 reactions with AH 2 predominated over the reaction with 

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

### 24
suggested that O 3 may react with other substrates once AH 2 concentrations within the 

### 25
reaction plane fall sufficiently. Additionally, once AH 2 was consumed, the absorption 

### 26
efficiency diminished, allowing inhaled O 3 to be distributed to more distal airways 

### 27

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

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

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

. 

### 30

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

### 31

6

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

### 32

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

### 33

O 3 - d erived aldehydes from exposing O 3 to RBC membranes ( Ballinger et al., 2005 ) . In 

### 34

addition, O 3 induced cell membrane oxidation that required interactions with AH 2 and 

### 35
GSH, but not UA or the vitamin E analog Trolox. Further, aqu eous phase reactions 

### 36

between O 3 and bovine serum albumin did not result in membrane oxidation ( Ballinger et 

### 37

### Draft

– Do Not Cite or Quote 5

- 2 4 June 2012 al., 2005 ) . The presence of UA or bovine serum albumin protected against lipid and 

### 1
protein oxidation resulting from the reaction of O 3 and AH 2 ( Ballinger et al., 2005 ) . This 

### 2
study provided evidence that antioxidants may paradoxically facilitate O 3 - mediated 

### 3
damage. This apparent contradiction should be viewed in terms of the 

### 4

concentration

- d ependent role of the ELF antioxidants. Reactions between O 3 and 

### 5
antioxidant species exhibited a biphasic concentration response, with oxidation of protein 

### 6
and lipid occurring at lower, but not higher, concentrations of antioxidant. In this way, 

### 7
endogenous reactants led to the formation of secondary oxidation products that were 

### 8
injurious and also led to quenching reactions that were protective. Moreover, the 

### 9
formation of secondary oxidation products mediated by some antioxidants was opposed 

### 10
by quenching reac tions involving other antioxidants. 

### 11

Alterations in ELF composition can result in alterations in O 3 uptake. Bolus O 3 uptake in 

### 12
human subjects can be decreased by previous continuous O 3 exposure (120

- 360 ppb), 

### 13
possibly due to depletion of compounds able to react with O 3 ( Rigas et al., 1997 ; Asplund 

### 14

e t al., 1996 ) . Conversely, O 3 (360 ppb) bolus uptake was increased with prior NO 2 

### 15

(360

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

### 16
this increased fractional absorption of O 3 could be due to increased production of reactive 

### 17

substrates in the ELF due to oxidant

- induced airway inflammation. 

### 18

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

### 19
appear to be an important cellular defense against O 3 including α

- TOH, albumin, 

### 20
ceruloplasmin, lactoferrin, mucins, and transferrin ( Mudway et al., 2006 ; Freed et al., 

### 21

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

### 22
of the RT, and can be altered by O 3 exposure. Mechanisms underlying the regional 

### 23
variability are not well

- u nderstood. It is thought that both plasma ultrafiltrate and locally 

### 24

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

### 25

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

### 26

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

### 27
sequential nasal lavage in human subjects ( Mudway et al., 1999a ) . When these subjects, 

### 28
exercising at a moderate level, were exposed to 200 ppb O 3 for 2 hours , nasal lavage 

### 29

fluid UA was significa ntly decreased while plasma UA levels were significa ntly 

### 30

increased ( Mudway et al., 1999a ) . The finding that UA, but not AH 2 or GSH, was 

### 31
depleted in nasal lavage fluid indicated that UA was the predominant antioxidant with 

### 32
respect to O 3 reactivity in the nasal ca vity ( Mudway et al., 1999a ) . However, in human 

### 33
BALF samples, the mean consumption of AH 2 was greater than UA ( Mudway et al., 

### 34

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

### 35
airways in human subjects, which suggested that increased mucosal gland secretions were 

### 36
an important source ( Peden et al., 199 3 ) . Using the O 3 - specific antioxidant capacity assay 

### 37
on human nasal lavage samples, Rutkowski et al. (2011 ) concluded that about 30% of the 

### 38

### Draft

– Do Not Cite or Quote 5

- 25 June 2012 antioxidant capacity of the nasal liquid lining layer was attributed to UA activity. 

### 1
Additionally, more than 50% of the subject

- to

- subject differences in antioxidan t capacity 

### 2
were driven by differences in UA concentration. However, day

- to

- day within

- subject 

### 3
variations in measured antioxidant capacity were not related to the corresponding 

### 4
variations in UA concentration in the nasal lavage fluid. Efforts to identify th e 

### 5
predominant antioxidant(s) in other RT regions besides the nasal cavity have failed to 

### 6
yield definitive results. 

### 7

Regulation of AH 2 , GSH and α

- TOH concentrations within the ELF is less clear than that 

### 8

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

- out of 

### 9

AH 2 and GSH occurred, indicating the absence of rapidly acting repletion mechanisms 

### 10

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

### 11
decreases in BALF and plasma AH 2 levels several hours following O 3 exposure (200 ppb 

### 12
for 2 h, while exercising at a moderate level) ( Mudway et al., 2001 ; Blomberg et al., 

### 13

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

### 14

oxidized AH 2 by several cell types leading to intracellu lar reduction and export of 

### 15

reduced AH 2 ( Welch et al., 1995 ) . Studies with rats exposed to 0.4

- 1.1 ppm O 3 for 

### 16

1

- 6 hours have shown consumption of AH 2 that correlates with O 3 exposure ( Gunnison 

### 17

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

. 

### 18

A body of evidence suggests that reaction of O 3 within the ELF limits its diffusive 

### 19
transport through the ELF; direct contact of O 3 with the apical membranes of the 

### 20
underlying epithelial cells therefore might be negligible ( Ballinger et al., 2005 ; Connor et 

### 21

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

### 22
computational analyses and in vitro studies. Direct confirm ation using in vivo studies is 

### 23
lacking. Nevertheless, when predicting exposure

- related outcomes across species and 

### 24

anatomic sites, whether O 3 directly contacts the apical membranes of the epithelial cells 

### 25
is an important consideration, given that the extra cellular surface milieu of the RT 

### 26
appreciab ly varies in terms of the types and concentrations of the substrates present and 

### 27

the thickness of the ELF. 

### 28

For O 3 or its reaction products to gain access to the underlying cellular compartments, O 3 

### 29

must diffuse a t the air

- liquid interface of the airway surface and travel through the ELF 

### 30
layer. In vitro experiments have shown that O 3 disappearance from the gas phase depends 

### 31

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

. 

### 32

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

### 33
and overlying mucus gel layer, and the alveolar surface lining that includes the subphase 

### 34
of liquid and vesicular surfactant and the continuous surfactant monolayer ( Bastacky et 

### 35

al., 1995 ) . There is a progressive decrease in ELF thickness and increase in int erfacial 

### 36
surface with progression from the TB region to the alveolus ( Mercer et al., 1992 ) . The 

### 37

### Draft

– Do Not Cite or Quote 5

- 26 June 2012 progressive thinning of the ELF while moving further down the RT decreases the radial 

### 1

distance O 3 or its rea ction products must travel to reach the cells lining the RT. 

### 2

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

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

### 4

transformed to other more lon g

- lived reactive species, thus initiating a reaction cascade 

### 5

( Pryor, 1992 ) . These calculations utilize the Einstein

- Smoluchowski equation ( Equation 

### 6

5

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

### 7
compare the half

- life of O 3 in the ELF layer to the time it takes, t , for O 3 to travel a 

### 8

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

### 9

t = d 2 

### /2D

Equation 5

- 1

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

### 10

The half

- life of O 3 can be approximated by dividing the pseudo

- first order rate constant, 

### 11

k 1 , into ln 2. Pryor (1992 ) assumed t he reaction rate constant 10 9 L/mol/sec for O 3 with 

### 12
GSH and the concentration of GSH equaled 1 mM in the ELF. Using these values and 

### 13
neglecting reactions of O 3 with other ELF species, the half

- life of O 3 would be 7×10
- 7 

### 14
seconds. Under these assumptions of GSH concentration and ELF thickness, the half

- life 

### 15

of O 3 is about one third of the time necessary for O 3 to diffuse through the ELF layer. 

### 16

Further, assuming that 0.5 ppm O 3 enters the trachea and the intrapulmonary gas

- phase 

### 17
concentration is reduced only 5 fold during transport to the terminal bronchioles, by using 

### 18
a Henry’s law constant and assuming equilibrium, the ELF O 3 concentration could be 

### 19

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

### 20

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

### 21
lipids and reactive proteins, the most facile reactants would equate to an approximately 

### 22

500,000

- fold excess over O 3 . If one then assumes a lumped reaction rate constant of 

### 23

10 7 M - 1 sec - 1 , any O 3 in solution would be consumed by reaction almost instantaneously, 

### 24
thereby constraining its diffusion as an unreacted species to within <0.1 μm, which is less 

### 25
than the thickness values estimated for distal airway ELF. If unsaturated lipids 

### 26

(~ 10 6 M - 1 sec - 1 ) and proteins (for which the rate constant will vary depending on low 

### 27
pKa thiolates and other amino acid

- reactive sites) are included, the penetration depth is 

### 28

further reduced. 

### 29

Similarly, model calculations of the nasal cavity based on diffusion equation s and 

### 30

reaction rates of O 3 with model substrates predict an O 3 penetration distance (0.5 μm) 

### 31
less than the thickness of the nasal lining layer (10 μm) ( Santiago et al., 2001 )

. 

### 32

A computational fluid dynamics model was able to predict experimentally measured 

### 33

### Draft

– Do Not Cite or Quote 5

- 27 June 2012 O 3 uptake when nasal m ucus layer thickness was considered ( Cohen

- Hubal et al., 1996 )

, 

### 1
reaffirming the importance of the resistance imparted by the ELF layer in dose and lesion 

### 2
patterns in the nasal passage

. 

### 3

Despite calculations and in vitro studies suggesting that reactions of O 3 wi th underlying 

### 4
epithelial cells may be negligible, there is some evidence that suggests direct interaction 

### 5

of O 3 with epithelial cells is possible. While moving distally in the lung, the ELF 

### 6
thickness decreases and becomes ultrathin in the alveolar region, possibly allowing for 

### 7

direct interaction of O 3 with the underlying epithelial cells. One definitive study 

### 8
conducted in excised rat lung measured alveolar lining layer thickness over relatively flat 

### 9
portions of the alveolar wall to be 0.14 μm, to be 0.89 μm at the alveolar wall junctions, 

### 10

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

- weighted 

### 11
average thickness of the alveolar lining fluid was found to be about 0.2 μm and the 

### 12
alveolar lining layer was continuous over the entire alveolar surface measured. The 

### 13
surface appeared smooth, and no epithelial surface feat ures or macrophage features 

### 14
protruded above the air

- liquid interface. It was noted that measurements of alveolar lining 

### 15
layer thickness were made in lungs prepared in a state of roughly 80% of total lung 

### 16
capacity, and as a result, the values reported would be approaching the lowest values 

### 17
possible during the respiratory cycle. However, 4% of the surface area in the alveolar 

### 18
compartment was covered by alveolar lining fluid layer of less than 20 nm ( Bastacky et 

### 19

al., 1995 ) , suggesting the possibility that unreacted O 3 could penetrate to the cell layer in 

### 20
this region. Further it remains a possibility that airways macrophages may protrude into 

### 21
the gas phase, allowing for direct contact between O 3 and airways epithelial cells. 

### 22

Still, direct reaction of O 3 with alveolar ep ithelial cells or macrophages may be limited by 

### 23
the presence of dipalmitoyl phosphatidylcholine (DPPC), the major component of 

### 24
surfactant, which has been shown in vitro to inhibit uptake of O 3 into an aqueous 

### 25
compartment containing ascorbate, glutathione, and uric acid ( Connor et al., 2004 )

. 

### 26

Further, the amount of O 3 available to the alveo lar compartment may be limited by 

### 27
uptake of O 3 in nasal and TB compartments ( Figure 5

- 5 ). In fact, the amount of 18 O 

### 28
reaction product was lower in the alveolar tissues than in TB tissues of rhesus monkeys 

### 29
immediately following a 2 hour exposure to 18 O

- labeled O 3 (0.4 and 1 ppm) ( Plopper et 

### 30

al., 1998 ) . These considerations illustrate the difficulty in determining whether O 3 reacts 

### 31
directly with cells in the alveolar compartment. 

### 32

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

### 33
perturbations, the precise reactiv e species that encounters the epithelia might or might not 

### 34
have specificity to O 3 per se or to its secondary oxidants. Many of the measureable 

### 35
products formed as a consequence of O 3 exposure have limited specificity to O 3 , such as 

### 36

4

- hydroxynonenal that is formed by autoxidation, an event that can be initiated by O 3 but 

### 37

### Draft

– Do Not Cite or Quote 5

- 28 June 2012 also by a multitude of other oxidants. Although some classes of lipid oxidation products 

### 1
(e.g., specific aldehydes, cholesterol products) are specific to O 3 , measurement in either 

### 2

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

### 3

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

### 4
dynamic compartment and, once formed, hydrophobic species can partition. Oxidation of 

### 5

membrane components might produce similar cellular outcomes regardless of the 

### 6
initiating oxidant. Lipid ozonides, which could be generated either within the ELF or 

### 7
from ozonation of cell membrane unsaturated lipids, could bind to receptors, activate 

### 8
signaling casc ades, and act in other ways, making differences between pure extracellular 

### 9
reaction and direct membrane reaction indistinguishable. Thus, in some cases 

### 10
documenting whether O 3 per se reacts directly with cellular constituents might be 

### 11
essential (despite the challenges of in vivo demonstrations), while in other cases precisely 

### 12

where O 3 reacts might be of less concern with regard to characterizing mechanisms of 

### 13

health outcomes. 

### 14

Thus, components of the ELF are major targets for O 3 and the resulting secondary 

### 15

ox idation products key mediators of toxicity in the airways (the role of reaction products 

### 16

in O 3 - induced toxicity is discussed in Section 5.3 ). The reaction cascade resulting from 

### 17

the interaction of O 3 with ELF substrates can then carry the oxidative burden deeper into 

### 18
cells lining the RT to elicit the health effects observed. 

### 19

5.2.3.1 Summ ary

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

### 20
barrier and target for inhaled O 3 ( Figure 5

- 7 ). The thi ckness of the lining fluid and mucus 

### 21
layer is an important determinant of the dose of O 3 to the tissues. The antioxidant 

### 22
substances present in the ELF appear in most cases to limit interaction of O 3 with 

### 23
underlying tissues and to prevent penetration of O 3 deeper into the lung. The formation of 

### 24
secondary oxidation products is likely related to the concentration of antioxidants present 

### 25
and the quenching ability of the lining fluid. Mechanisms are present to replenish the 

### 26
antioxidant substrate pools as well as to remove secondary reaction products from tissue 

### 27
interactions. Important differences exist in the reaction rates for O 3 and these ELF 

### 28
biomolecules and the reactivity of the resulting products. Overall, studies suggest that UA 

### 29

and AH 2 are more reactive wi th O 3 than GSH, proteins, or lipids. In addition to 

### 30
contributing to the driving force for O 3 uptake, formation of secondary oxidation 

### 31
products may lead to increased cellular injury and cell signaling (discussed in 

### 32

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

### 33
where the resulting secondary oxidation products might penetrate into the tissue layer and 

### 34
lead to p erturbations. 

### 35

### Draft

– Do Not Cite or Quote 5

- 29 June 2012 Note: Contents of this figure not discussed in Section 5.2 will be discussed in Section 5.3 . Clara cell secretory protein, CCSP; Surfactant Protein

- A, SP

- A; Platelet activating factor, PAF. Ozone will react with components of the ELF to produce reaction products that may lead to cel lular injury and cell signaling as discussed in Section 5.3

.



### Figure 5

- 7 Details of the ozone intera ction with the airway ELF to form secondary oxidation products.



## 5.3 Possible Pathways/Modes of Action

5.3.1 Introduction

Mode of action refers to a sequence of key events and processes that 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 cell injury and su bsequent events such as adverse 

### 5 Oxidized proteins Aldehydes Ozonized Cholesterol Species Lipid Peroxides
Eicosanoids and PAF Hyaluronan Fragments
Ozonized Radical Reactive Oxygen Species Liquid Layer Antioxidants

Uric Acid, Ascorbate, Glutathione, α

- Tocopherol Cellular Macromolecules

Plasma membrane proteins and phospholipids Free fatty acids and

carbohydrates

Mechanisms for Antioxidant Repletion

• Secretion by epithelial cells

• Transport from plasma

• Reduction of oxidized ascorbate Mechanisms for Reaction
Product Removal

• Quenching reactions by ELF

antioxidants and proteins

• Non

- enzymatic reactions with

cellular antioxidants

• Metabolism by cellular GST/NQO1

• Receptor

- mediated uptake by

macrophages 

# Ozone

ELF Macromolecules

Surfactant components e.g. proteins, phospholipids/ cholesterol, CCSP, Albumin, Hyaluronan , SP

- A



### Secondary Oxidation Products Mucus Layer

M ucins Phospholipids



### Cellular injury Cellular signaling

### Draft

– Do Not Cite or Quote 5

- 30 June 2012 health effects ( U.S. EPA, 2009f ) . The purpose of this section of Chapter 5 is to describe 

### 1
the key events and toxicity pathways that contribute to health effects resulting from short



### 2
term and long

- term exposures to O 3 . The extensive research carried out over several 

### 3
decades in humans and in laboratory animals has yielded numerous studi es on 

### 4
mechanisms by which O 3 exerts its effects. This section will discuss some of the 

### 5
representative studies with particular emphasis on studies published since the 2006 O 3 

### 6
AQCD and on studies in humans that inform biological mechanisms underlying 

### 7

respons es to O 3
. 

### 8

It is well

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

### 9

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

### 10
the respiratory system. These responses include the activation of neural reflexes, 

### 11
initiation of inflammation, alteration of epithelial barrier function, sensitization of 

### 12
bronchial smooth muscle, modification of innate/adaptive immunity and airways 

### 13
remodeling, as will be discussed below. These have the potential to resul t in effects on 

### 14
other organ systems such as the cardiovascular, central nervous, hepatic and reproductive 

### 15
systems or result in developmental effects. It has been proposed that lipid ozonides and 

### 16
other secondary oxidation products, which are bioactive and c ytotoxic in the respiratory 

### 17
system, are responsible for systemic effects. However it is not known whether they gain 

### 18
access to the vascular space ( Chuang et al., 2009 ) . Recent studies in anima l models show 

### 19

that inhalation of O 3 results in systemic oxidative stress. The following subsections 

### 20
describe the current understanding of potential pathways and modes of action responsible 

### 21
for the pulmonary and extrapulmonary effects of O 3 exposure. 

### 22

5.3.2 Activa tion of Neural Reflexes

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

### 23

activation of neural reflexes. The involvement of bronchial C

- fibers, a type of nociceptive 

### 24
sensory nerve, has been demonstrated in dogs exposed thr ough an endotracheal tube to 

### 25

2

- 3 ppm O 3 for 20

- 70 minutes ( Coleridge et al., 1993 ; Schelegle et al., 199 3 ) . This vagal 

### 26
afferent pathway was found to be responsible for O 3 - mediated rapid shallow breathing 

### 27
and other changes in respiratory mechanics in O 3 - exposed dogs ( Schelegle et al., 1993 )

. 

### 28
Ozone also triggers neural reflexes that stimulate the auto nomic nervous system and alter 

### 29
electrophysiologic responses of the heart. For example, bradycardia, altered HRV and 

### 30
arrhythmia have been demonstrated in rodents exposed for several hours to 0.1

- 0.6 ppm 

### 31

O 3 ( Hamade and Tankersley, 2009 ; Watkinson et al., 2001 ; Arito et al., 1990 ) . Another 

### 32
effect is hypothermia, which in rodents occurred subsequent to the activation of neural 

### 33
reflexes involving the parasympathetic nervo us system ( Watkinson et al., 2001 ) . Vagal 

### 34
afferent pathways originating in the RT may also be responsible for O 3 - mediated 

### 35

### Draft

– Do Not Cite or Quote 5

- 31 June 2012 activation of nucleus tractus solitarius neurons that resulted in neuronal activation in 

### 1

stress

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

- 2.0 ppm O 3 for 

### 2

1.5

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

. 

### 3

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

### 4
responses mediated by bronchopulmonary C

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

### 5

(30 μM solubilized) selectively activated a subset of C

- fiber receptors that are TRPA1 

### 6

ion channels ( Taylor

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

### 7
TRP family of ion channels, which are known to mediate the responses of sensory 

### 8
neurons to inflammatory mediators ( Caceres et al., 2009 ) . In addition to TRPA1 ion 

### 9
channels possibly playing a key role in O 3 - induced decrements in pulmonary function, 

### 10
they may mediate allergic asthma ( Caceres et al., 2009 ) . Activation of TRPA1 ion 

### 11
channels following O 3 exposure is likely initiated by secondary oxidation products such 

### 12

as a ldehydes and prostaglandins ( Taylor

- Clark and Undem, 2010 ) through covalent 

### 13
modification of cysteine and lysine residues ( Trevisani et al., 2007 ) . Ozonation of 

### 14
unsaturated fatty acids in the ELF was found to result in the generation of aldehydes 

### 15

( Frampton et al., 1999 ) such as 4

- hydroxynonenal and 4

- oxononenal ( Taylor

- Clark et al., 

### 16

2008 ; Trevi sani et al., 2007 ) . 4

- oxononenal is a stronger electrophile than 

### 17

4

- hydroxynonenal and exhibits greater potency towards the TRPA1 channels ( Taylor



### 18

Clark et al., 2008 ; Trevisani et al., 2007 ) . In addition, PGE 2 is known to s ensitize TRPA1 

### 19

channels ( Bang et al., 2007 )

. 

### 20

In human s exercising at a moderate level, the response to O 3 (500 ppb for 2 h) was 

### 21
characterized by substernal discomfort, especially on deep inspiration, accompanied by 

### 22
involuntary truncation of inspiration ( Hazucha et al., 1989 ) . This latter response led to 

### 23
decreased inspiratory capacity and to decreased forced vital capacity (FVC ) and forced 

### 24
expiratory volume in one second (FEV 1 ), as measured by spirometry. These changes, 

### 25
which occurred during O 3 exposure, were accompanied by decreased V T and increased 

### 26
respiratory frequency in human subjects. Spirometric changes in FEV 1 and FVC we re not 

### 27
due to changes in respiratory muscle strength ( Hazucha et al., 198 9 ) . In addition, 

### 28
parasympathetic involvement in the O 3 - mediated decreases in lung volume was minimal 

### 29

( Mudway and Kelly, 2000 ) , since changes in FVC or symptoms were not modified by 

### 30
treatment with bronchodilators such as atropine in human subjects exposed to 400 ppb O 3 

### 31

for 2 hours while exercising at a heavy level ( Beckett et al., 1985 ) . However, the loss of 

### 32
vital capacity was reversible with intravenous administration of the rapid

- acting opioid 

### 33
agonist, sufentanyl, in human subjects exercising at a moderate level and exposed to 

### 34

420 ppb O 3 for 2 hours, which indicated the involvement of opioid receptor

- containing 

### 35

nerve fiber s and/or more central neurons ( Passannante et al., 1998 ) . The effects of 

### 36
sufentanyl may be attributed to blocking C

- fiber stimulation by O 3 since activation of 

### 37
opioid receptors downregulated C

- fiber fun ction ( Belvisi et al., 1992 ) . Thus, nociceptive 

### 38

### Draft

– Do Not Cite or Quote 5

- 32 June 2012 sensory nerves, presumably bronchial C

- fibers, are responsible for O 3 - mediated 

### 1
responses in humans ( Passannante et al., 1998 ) . This vagal afferent pathway is 

### 2
responsible for pain

- related symptoms and inhibition of maximal inspiration in humans 

### 3

( Hazucha et al., 1989 )

. 

### 4

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

### 5
reflex since cyclooxygenase inhibition with indomethacin ( Alexis et al., 2000 ; Schelegle 

### 6

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

### 7

1996 ) , before exposure to O 3 significa ntly blunted the spirometric responses. These 

### 8
studies involved exposures of 1

- 2 hours to 350

- 400 ppb O 3 in human subjects exercising 

### 9
at light, moderate and heavy levels. In the latter study, ibuprofen treatment resulted in 

### 10

measurable decreases in BALF levels of PGE 2 and TXB 2 at 1

- hour postexposure 

### 11

( Hazucha et al., 1996 ) . Although an earlier study demonstrated that PGE 2 stimulated 

### 12

bronchial C

- fibers ( Coleridge et al., 1993 ; Coleridge et al., 1976 ) and suggested that 

### 13

PGE 2 mediated O 3 - induced decreases in pulmonary function, no correlation was observed 

### 14
between the degree of ibuprofen

- induced inhibition of BALF PGE 2 levels and blunting of 

### 15
the spirometric response to O 3 ( Hazucha et al., 1996 ) . These results point to the 

### 16
involvement of a lipoxygenase product. Further, as noted above, PGE 2 may play a role in 

### 17
the neural reflex by sensitizing TRPA1 channels. A recent study in human subjects 

### 18
exercising at a moderate to high level and exposed for 1 hour to 350 ppb O 3 also provided 

### 19

evidence that arachidonic acid metabolites, as well as oxidative stress, contribute to 

### 20
human responsiveness to O 3 ( Alfaro et al., 2007 )

. 

### 21

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

### 22
subjects exercising at a moderate level during O 3 exposure (500 ppb for 2 hours) 

### 23

( Hazucha et al., 1989 ) . This pulmonary function decrement is generally measured as 

### 24
specific a irway resistance (sRaw) which is the product of airway resistance and thoracic 

### 25
gas volume. In several studies involving human subjects exercising at a moderate to 

### 26
heavy level and exposed for 1

- 4 hours to 200

- 300 ppb O 3 , changes in sRaw correlated 

### 27

with chan ges in inflammatory and injury endpoints measured 18

- hours postexposure, but 

### 28
did not follow the same time course or change to the same degree as spirometric changes 

### 29

(i.e., FEV 1 , FVC) measured during exposure ( Balmes et al., 1996 ; Aris et al., 1993 ; 

### 30
Schelegle et al., 1991 ) . In addition, a small but persistent increase in airway resistance 

### 31
associated with narrowing of small peripheral airways (measured as changes in 

### 32

isovolumetric FEF 25

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

### 33

130 minutes, moderate exercise level) ( Weinmann et al., 1995c ; Weinmann et al., 1995b )

. 

### 34
A similar study (400 ppb O 3 for 2 hours in human subjects exercising at a heavy level) 

### 35

found decreases in FEF 25

- 75 concomitant with increases in residual volume, which is 

### 36

su ggestive of small airways dysfunction ( Kreit et al., 1989 ) . In separate studies, a 

### 37
statistically significa nt increase in residual volume (500 ppb for 2 hours) ( Hazucha et al., 

### 38

### Draft

– Do Not Cite or Quote 5

- 33 June 2012 1989 ) and a statistically significa nt decrease in FEF 25

- 75 (160 ppb for 7.6 hours) 

### 1

( Horstman et al., 1995 ) were observed following O 3 exposure in human subjects 

### 2
exercising at moderate and light levels, respectively, providing further suppor t for an 

### 3

O 3 - induced effect on small airways. 

### 4

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

### 5
are incompletely understood. Pretreatment with atropine decreased baseline sRaw and 

### 6
prevented O 3 - induced increases in sRaw in human subjects exercising at a heavy level 

### 7

(400 ppb for 0.5 hours) ( Beckett et al., 1985 ) , indicating the involvement of muscarinic 

### 8
cholinergic receptors of the parasympathetic nervous system. Interestingly, atropine 

### 9
pretreatment partially blocked the decrease in F EV 1 , but had no effect on the decrease in 

### 10
FVC, breathing rate, tidal volume or respiratory symptoms ( Beckett et al., 1985 ) . Using a 

### 11
β

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

### 12
mucus secretion, was responsible for O 3 - induc ed increases in airway resistance ( Beckett 

### 13

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

### 14
restrictive (such as decreased inspiratory capacity) and obstructive (such as 

### 15
bronchoconstriction) type changes in airway responses. This is consistent with findings of 

### 16

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

### 17

FEV 1 (r =

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

### 18

(r =

- 0.16, p = 0.10) in human subjects exercising at a heavy level and exposed for 

### 19

2.5 hours to 120

- 400 ppb O 3
. 

### 20

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

### 21

In addition to stimulating CNS reflexes, bronchopulmonary C

- fibers mediate local axon 

### 22
responses by releasing neuropeptides such as substance P (SP), neurokinin (NK) A and 

### 23
calcitonin gene

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

### 24

responses s uch as bronchoconstriction. Recent studies in animals demonstrated that NK

- 1 

### 25
receptor blockade had no effect on O 3 - stimulated physiologic responses such as V T and f B 

### 26

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

### 27
receptors contributed to vagally

- mediated bronchoconstriction in guinea pigs 3 days after 

### 28
a single 4

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

### 29
bronchial biopsies were performed and studied by immunohistochemistry, SP was 

### 30
substantially diminished in submucosal sensory nerves 6 hours following O 3 exposure 

### 31

(200 ppb for 2 hours, light exercise) ( Krishna et al., 1997 ) . A statistically significa nt 

### 32
correlation was observed between loss of SP immunoreactivity from neurons in the 

### 33
bronchial mucosa and changes in FEV 1 measured 1

- hour postexposure ( Krishna et al., 

### 34

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

### 35
immediately after O 3 challenge (250 ppb for 1 hour, heavy exercise) ( Hazbun et al., 

### 36

1993 ) . These results provide evidence that the increased airway resistance observed 

### 37

### Draft

– Do Not Cite or Quote 5

- 34 June 2012 following O 3 exposure is due to vagally

- mediated responses and possibly by local axon 

### 1
reflex responses through bronchopul monary C

- fiber

- mediated release of SP. 

### 2

A role for antioxidant defenses in modulating neural reflexes has been proposed given the 

### 3
delay in onset of O 3 - induced pulmonary function responses that has been noted in 

### 4
numerous studies. Recently, this delay was ch aracterized in terms of changes in f B 

### 5

( Schelegle et al., 2007 ) . In humans exposed for 1

- 2 hours to 120

- 350 ppb O 3 while 

### 6
exercising at a high level, no change in f B was observed until a certain cumulative inhaled 

### 7

dose of O 3 had been reached. Subsequently, the magnitude of the change in f B was 

### 8

correlated with the inhaled dose rate ( Schelegle et al., 2007 ) . These investigators 

### 9
proposed that initial reactions of O 3 with ELF resulted in a time

- dependent depletion of 

### 10
ELF antioxidants, and that activation of neural reflexes occurred only after the 

### 11

antioxidant defenses were overwhelmed ( Schelegle et al., 2007 )

. 

### 12

5.3.3 Initiation of inflammation

As described previously (Section 5.2.3 ), O 3 mainly reacts with components of the ELF 

### 13
and cellular membranes resulting in the generation of secondary oxidation products. 

### 14
Higher concentrations of these products may directly injure RT epithelium. Subsequent 

### 15
airways remod eling may also occur (Section 5.3.7 ) ( Mudway and Kelly, 2000 ) . Lower 

### 16
concentrations of secondary oxidation products may initiate cellular responses including 

### 17
cytokine generation, adhesion molecule expression, and modification of tight junctions 

### 18
leading to inflamm ation and increased permeability across airway epithelium 

### 19

(Section 5.3.4 ) ( Dahl et al., 2007 ; Mudway and Kelly, 2000 )

. 

### 20

A n important hallmark of acute O 3 exposure in humans and animals is neutrophilic 

### 21
airways inflammation. Although neutrophil influx into nasal airways has been 

### 22
demonstrated in human subjects (400 ppb O 3 2 hours, heavy exercise) ( Graham and 

### 23

Koren, 1990 ) , most studies of neutrophil influx have focused on the lower airways 

### 24

( Hazucha et al., 1996 ; Aris et al., 1993 ) . The time course of this response in the lower 

### 25
airways and its resolution appears to be slower than that of the decrements in pulmonary 

### 26

functio n in exercising human subjects ( Hazucha et al., 1996 ) . In several studies, air ways 

### 27
neutroph ilia was observed by 1

- 3 hours, peaked by 6 hours and was returning to baseline 

### 28

levels at 18

- 24 hours in human subjects exercising at a heavy level and exposed for 

### 29

1

- 2 hours to 300

- 400 ppb O 3 ( Schelegle et al., 1991 ; Koren et al., 1989 ; Seltzer et al., 

### 30

1986 )

. Neutrophils are thought to be injurious and a study in guinea pigs demonstrated 

### 31

that the influx and persistence of neutrophils in airways following O 3 exposure correlated 

### 32
with the temporal profile of epithelial injury (0.26

- 1 ppm O 3, 72 hours) ( Hu et al., 1982 )

. 

### 33
However, neutrophils have also been shown to contribute to repair of O 3 - injur ed 

### 34

### Draft

– Do Not Cite or Quote 5

- 35 June 2012 epithelium in rats exposed for 8 hours to 1 ppm O 3, possibly by removing necrotic 

### 1
epithelial cells ( Mudway and Kelly, 2000 ; Vesely et al., 1999 ) . Nonetheless, the degree 

### 2
of airways inflammation due to O 3 is thought to have more important long

- term 

### 3
consequences than the more quickly resolving changes in pulmonary function since 

### 4
airways inflammation is often accompanied by tissue injury ( Balmes et al., 1996 )

. 

### 5

Ozone exposure results in alterations in other airw ays inflammatory cells besides 

### 6
neutrophils, including lymphocytes, macrophages, monocytes and mast cells. Influx of 

### 7
some of these cells accounts for the later (i.e., 18

- 20 hours) phase of inflammation 

### 8
following O 3 exposure. Numbers of lymphocytes and total cells in BALF were decreased 

### 9
early after O 3 exposure in human subjects exercising at a light to moderate level and 

### 10
exposed for 2 hours to 200 ppb O 3 , which preceded the neutrophil influx ( Mudway and 

### 11
Kelly, 2000 ; Blomberg et al., 1999 ; Krishna et al., 1997 ) . The decrease in total cells was 

### 12
thought to reflect decreases in macrophages, although it was not clear whether the cells 

### 13
were necrotic or whether membrane adhesive properties were altered making them more 

### 14
difficult to obtain by l avage ( Mudway a nd Kelly, 2000 ; Blomberg et al., 1999 ; Mudway 

### 15

et al., 1999b ; Frampton et al., 1997b ; Pearson and Bhalla, 1997 ) . A recent study in human 

### 16
subjects exercising at a moderate level and exposed for 6.6 hours to 80 ppb O 3 

### 17
demonstrated an increase in numbers of sputum monocytes and dendritic

- li ke cells with 

### 18
increased expression of innate immune surface proteins and antigen presentation markers 

### 19

( Peden, 2011 ; Alexis et al., 2010 ) (see Section 6.2.3.1 ). An increase in submucosal mast 

### 20

cell s was observed 1.5 hours after a 2 hour

- exposure to 200 ppb O 3 ( Blomberg et al., 

### 21

1999 ) and an increase in BAL mast cell number was observed 18 hours after a 4

- hour 

### 22
exposure to 220 ppb O 3 exposure in human subjects exercising at a moderate level 

### 23

( Frampton et al., 1997b ) . Mast cells may play an import ant role in mediating neutrophil 

### 24
influx since they are an important source of several pro

- inflammatory cytokines and since 

### 25
their influx preceded that of neutrophils in human subjects exercising at a moderate level 

### 26
and exposed for 2 hours to 200 ppb O 3 ( Stenfors et al., 2002 ; Blomb erg et al., 1999 )

. 

### 27
Further, a study using mast cell

- deficient mice demonstrated decreased neutrophilic 

### 28
inflammation in response to O 3 (1.75 ppm, 3 hours) compared with wild type mice 

### 29

( Kleeberger et al., 1993 ) . Influx of these inflammatory cell types in the lung is indicative 

### 30

of O 3 - mediated activation of innate immunity as will be discu ssed in Section 5.3.6

. 

### 31

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

### 32
responses to O 3 exposure ( U.S. EPA, 2006b ) . Eicosanoids are one class of secondary 

### 33
oxidation products that may be formed rapidly following O 3 exposure and that may 

### 34

mediate inflammation. Eicosanoids are m etabolites of arachidonic acid

— a 20

- carbon 

### 35

PUFA

— that are released from membrane phospholipids by phospholipase A2

- mediated 

### 36
catalysis. Activation of phospho lipase A2 occurs by several cell signaling pathways and 

### 37
may be triggered by O 3 - mediated lipid peroxidation of cellular membranes ( Rashba

- Step 

### 38

### Draft

– Do Not Cite or Quote 5

- 36 June 2012 et al., 1997 ) . Additionally, cellular phospho lipases A2, C and D may be activated by lipid 

### 1
ozonation products ( Kafoury et al., 1998 ) . While the conversion of arachidonic acid to 

### 2
prostaglandins, leukotrienes and other eicosanoi d products is generally catalyzed by 

### 3
cyclooxygenases and lipoxygenases, non

- enzymatic reactions also occur during oxidative 

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

### 5
species. Further, the release of arachidonic a cid from phospholipids is accompanied by 

### 6
the formation of lysophospholipids that are precursors for platelet activating factors 

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

### 8

O 3 - mediated lipid peroxidation. 

### 9

In additio n, secondary reaction products may stimulate macrophages to produce 

### 10
cytokines such as IL

- 1, IL

- 6 and TNF

- α that in turn activate IL

- 8 production by epithelial 

### 11
cells. Although IL

- 8 has been proposed to play a role in neutrophil chemotaxis, 

### 12

measurements of I L

- 8 in BALF from humans exposed to O 3 found increases that were 

### 13

too late to account for this effect ( Mudway and Kelly, 2000 ) . The time

- course profiles of 

### 14

PGE 2 and IL

- 6 responses suggest that they may play a role in neutrophil chemotaxis in 

### 15

humans ( Mudway and Kelly, 2000 ) . However, pretreatment with ibuprofen attenuated 

### 16

O 3 - induced increases in BALF PGE 2 levels, but had no effect on neutrophilia in human 

### 17
subjects exercising at a heavy level and exposed for 2 hour to 400 ppb O 3 ( Hazucha et al., 

### 18

1996 )

. 

### 19

One set of studies in humans focused on the earliest phase of airways infl ammation 

### 20

(1

- 2 hours following exposure). Human subjects, exercising at a moderate level, were 

### 21
exposed to 200 ppb O 3 for 2 hours and bronchial biopsy tissues were obtained 1.5 and 

### 22

6 hours after exposure ( Bosson et al., 2009 ; Bosson et al., 2003 ; Stenfors et al., 2002 ; 

### 23
Blomberg et al., 1999 ) . Results demonstrated upregulation of vascular endothelial 

### 24

adhesion molecules P

- selectin and ICAM

- 1 at both 1.5 and 6 hours ( Stenfors et al., 2002 ; 

### 25
Blomberg et al., 1999 ) . Submucosal mast cell numbers were increased at 1.5 hours in the 

### 26
biopsy samples without an accompanying increase in neutrophil number ( Blomberg et al., 

### 27

1999 ) . Pronounced neutrophil infiltration was observed at 6 hours in the bronchial 

### 28

mucosa ( Stenfors et al., 2002 ) . Surprisingly, suppression of the NF

- κB and AP

- 1 

### 29
pathways at 1.5 hours and a lack of increased IL

- 8 at 1.5 or 6 hours in bronchial 

### 30
epithelium were observed ( Bosson et al., 2009 ) . The authors suggested that vascular 

### 31
endothelial adhesion molecules, rather than redox sensitive transcription factors, play key 

### 32
roles in early neutrophil recruitment in response to O 3
. 

### 33

Increases in markers of inflammation occurred to a comparable degree in human subjects 

### 34
with mild (least sensitive) and more remarkable (more sensitive) spirometric responses to 

### 35

O 3 (200 ppb, 4 hours, moderate exercise) ( Balmes et al., 1996 ) . Two other studies 

### 36

(200 ppb for 4 hours with moderate exercise and 300 ppb for 1 hour wi th heavy exercise) 

### 37

### Draft

– Do Not Cite or Quote 5

- 37 June 2012 found that acute spirometric changes were not positively correlated with cellular and 

### 1

biochemical indicators of inflammation ( Aris et al., 1993 ; Schelegle et al., 1991 )

. 

### 2
However inflammation was correlated with changes in sRaw ( Balmes et al., 1996 ) . In 

### 3
another study, pretreatment with ibuprofen had no effect on neutrophilia although it 

### 4
blunted the spirometric response in human subjects exercising at heavy level an d exposed 

### 5

for 2 hours to 400 ppb O 3 ( Hazucha et al., 1996 ) . Taken together, results from th ese 

### 6
studies indicate different mechanisms underlying the spirometric and inflammatory 

### 7
responses to O 3
. 

### 8

A common mechanism underlying both inflammation and impaired pulmonary function 

### 9
was suggested by Krishna et al. ( 1997 ) . This study, conducted in human subjects 

### 10
exercising at a light level and exposed to 200 ppb O 3 for 2 hours, demonstrated a 

### 11
correlation between loss of SP immunoreactivity from neurons in the bronchial mucosa 

### 12
and numbers of neutrophils and epitheli al cells (shed epithelial cells are an index of 

### 13
injury) in the BALF 6

- hours postexposure. Furthermore, the loss of SP immunoreactivity 

### 14
was correlated with the observed changes in FEV 1 . Another study found that SP was 

### 15
increased in lavage fluid of exercising human subjects immediately after O 3 challenge 

### 16

(250 ppb, 1 hour, heavy exercise) ( Hazbun et al., 1993 ) . SP is a neuropeptide released by 

### 17
sensory nerves which mediates neurogenic edema and bronchoconstriction ( Krishna et 

### 18

al., 1997 ) . Collectively, these findings suggest that O 3 - mediated stimulation of sensory 

### 19

nerves that leads to activation of central and local axon reflexes is a common effector 

### 20
pathway leading to impaired pulmonary function and inflammation. 

### 21

Studies in animal models have confirmed many of these findings and provided evidence 

### 22

for additional mechanisms involved in O 3 - induced inflammation. A study in mice (2 ppm 

### 23

O 3 , 3 hours) demonstrated that PAF may be important in neutrophil chemotaxis 

### 24

( Longphre et al., 1999 ) , while ICAM

- 1 and macrophage inflammatory protein

- 2 (MIP

- 2), 

### 25

the rodent IL

- 8 homologue, have been implicated in a rat model (1 ppm O 3 , 3 hours) 

### 26

( Bhalla a nd Gupta, 2000 ) . Key roles for CXCR2, a receptor for keratinocyte

- derived 

### 27

chemokine (KC) and MIP

- 2, and for IL

- 6 in O 3 - mediated neutrophil influx were 

### 28

demonstrated in mice (1 ppm O 3 , 3 hours) ( Johnston et al., 2005a ; Johnston et al., 2005b )

. 

### 29
Activation of JNK and p38 pathways and cathepsin

- S were also found to be important in 

### 30
this response (3 ppm O 3 , 3 hours) ( Williams et al., 2009a ; Williams et al., 2008a ; 

### 31

Williams et al., 2007a ) . Matrix metalloproteinase

- 9 (MMP

- 9) appeared to con fer 

### 32
protection against O 3 - induced airways inflammation and injury in mice (0.3 ppm O 3 , 

### 33

6

- 72 hours) ( Yoon et al., 2007 ) . Interleukin

- 10 (IL

- 10) also appeared to be protective 

### 34

since IL

- 10 deficient mice responded to O 3 exposure (0.3 ppm, 24

- 72 hours) with 

### 35
enhanced numbers of BAL neutrophils, enhanced NF

- κB activation and MIP

- 2 levels 

### 36
compared with IL

- 10 sufficient mice ( Backus et al., 2010 )

. 

### 37

### Draft

– Do Not Cite or Quote 5

- 38 June 2012 In addition, lung epithelial cells may release ATP in response to O 3 exposure ( Ahmad et 

### 1

al., 2005 ) . ATP and its metabolites (catalyzed by ecto

- enzymes) can bind to cellular 

### 2
purinergic receptors resulting in activation of cell signaling pathways ( Picher et al., 

### 3

2004 ) . One such metabolite, adenine, is capable of undergoing oxidation leading to the 

### 4
formation of UA which, if present in high concentrations, could ac tivate inflammasomes 

### 5
and result in caspase 1 activation and the maturation and secretion of IL

- 1β and IL

- 18 

### 6

( Dostert et al., 2008 ) . A recent study in human subjects exercising at a moderate level and 

### 7
exposed for 2 hours to 400 ppb O 3 demonstrated a correlation between ATP metabolites 

### 8
and inflammatory markers ( Esther et al., 2011 ) , which provides some support for this 

### 9

mechanism. 

### 10

Several recent studies have focused on the role of Toll

- like receptor (TLR) and its related 

### 11

a daptor protein MyD88 in mediating O 3 - induced neutrophilia. Hollingsworth et a l. (2004 ) 

### 12
demonstrated airways neutrophilia that was TLR4

- independent following acute (2 ppm, 

### 13

3 hours) and subchronic (0.3 ppm, 72 hours) O 3 exposure in a mouse model. However, 

### 14

Williams et al. (2007b ) found that MyD88 was important in mediating O 3 - induced 

### 15
neutrophilia in mice (3 ppm, 3 hours), with TLR4 and TLR2 contributing to the speed of 

### 16
the response. Moreover, MyD88, TLR2 and TLR4 contributed to inflammatory g ene 

### 17
expression in this model and O 3 upregulated MyD88, TLR4 and TLR4 gene expression 

### 18

( Williams et al., 2007a ) . Neutrophilic inflammation was also found to be partially 

### 19
dependent on MyD88 in mice exposed to 1 ppm O 3 for 3 hours ( Li et al., 2011 )

. 

### 20

Hyaluronan was found to mediate a later phase (24 hours) of O 3 - induced inflammation in 

### 21

mice ( Garantziotis et al., 2010 ; Garantziotis et al., 2009 ) . Hyaluronan is an extracellular 

### 22

m atrix component that is normally found in the ELF as a large polymer. Exposure to 

### 23

2 ppm O 3 for 3 hours resulted in elevated levels of soluble low molecular weight 

### 24
hyaluronan in the BALF 24

- hours postexposure ( Garantziotis et al., 2010 ; Garantziotis et 

### 25

al., 2009 ) . Similar results were found in response to 3 hour exposure to 1 ppm O 3 ( Li et 

### 26

al., 2011 ) . Ozone may have caused the depolymerization of hyaluronan to soluble 

### 27
fragments that are known to be endogenous ligands of the CD44 receptor and TLR4 in 

### 28
the macrophage ( Jiang et al., 2005 ) . Binding of hyaluronan fragments to the CD44 

### 29
receptor activ ates hyaluronan clearance, while binding to TLR4 results in signaling 

### 30
through MyD88 to produce chemokines that stimulate the influx of inflammatory cells 

### 31

( Jiang et al., 2005 ) . Activation of NF

- κB occurred in both airway epithelia and alveolar 

### 32
macrophages 24

- hours postexposure to O 3 . Increases in BALF pro

- inflammatory factors 

### 33

KC, IL

- 1β, MCP

- 1, TNF

- α and IL

- 6 observed 24 hours following O 3 exposure were 

### 34
found to be partially dependent on TLR4 ( Garantziotis et al., 2010 ) while increases in 

### 35
BAL inflammatory cells, which consisted mainly of macrophages, were dependent on 

### 36

CD44 ( Garantziotis et al., 2009 ) . BAL inflammatory cells number and injury markers 

### 37

### Draft

– Do Not Cite or Quote 5

- 39 June 2012 following O 3 exposure were similar in wild

- type and TLR4

- deficient animals 

### 1

( Garantziotis et al., 2010 )

. 

### 2

Since exposure to O 3 leads to airways inflammation characterized by neutrophilia, and 

### 3
since neutrophil

- derived oxidants often consume ELF antioxidants, concentrations of 

### 4

ELF antioxidants have been examined during airways neutrophilia ( Long et al., 2001 ; 

### 5

Gunnison and Hatch, 1999 ; Mudway et al., 1999b ) . In human subjects exercising at a 

### 6
moderate level and exposed to 200 ppb O 3 for 2 hours, UA, GSH and α

- TOH levels 

### 7
remained unchanged in BALF 6

- hours postexposure while AH2 was decreased 

### 8
significa ntly in both BALF and plasma ( Mudway et al., 1999b ) . A second study 

### 9
involving the same protocol reported a loss of AH2 from bronchial wash f luid and BALF, 

### 10
representing proximal and distal airway ELF respectively, as well as an increase in 

### 11
oxidized GSH in both compartments ( Mudway et al., 2001 ) . No change was observed in 

### 12
ELF UA levels in response to O 3 ( Mudway et al., 2001 ) . Further, O 3 exposure (0.8 ppm, 

### 13

4 hour s) in female rats resulted in a 50% decrease in BALF AH2 immediately 

### 14

postexposure ( Gunnison and Hatch, 1999 ) . These studies suggested a role for AH2 and 

### 15
GSH in protecting against the oxidative stress associated with inflammation. 

### 16

The relationship between inflammation, antioxidant status and O 3 dose has also b een 

### 17
investigated. The degree of inflammation in rats has been correlated with 18 O

- labeled O 3 

### 18
dose markers in the lower lung. In female rats exposed to 0.8 ppm O 3 for 4 hours, BAL 

### 19
neutrophil number and 18 O reaction product were directly proportional ( Gunnison and 

### 20

Hatch, 1999 )

. Kari et al. (1997 ) observed that a 3

- week caloric restriction (75%) in rats 

### 21
abrogated the toxicity of O 3 (2 ppm, 2 hours), measured as BALF increases in protein, 

### 22
fibronectin and neutrophils, that was seen in normally fed rats. Accompanying this 

### 23

resistance to O 3 toxicity was a reduction (30%) in the accumulation of 18 O reactio n 

### 24
product in the lungs. These investigations also demonstrated an inverse relationship 

### 25

between AH2 levels and O 3 dose and provided evidence for AH2 playing a protective 

### 26
role following O 3 exposure in these studies. Pregnant and lactating rats had lower AH2 

### 27
content in BALF and exhibited a greater increase in accumulation of 18 O reaction 

### 28
products compared with pre

- pregnant rats in response to O 3 exposure ( Gunnison and 

### 29

Hatch, 1999 ) . In the calorie restricted model, a 30% higher basal BALF AH2 

### 30

co ncentration and a rapid accumulation of AH2 into the lungs to levels 60% above 

### 31

normal occurred as result of O 3 exposure ( Kari et al., 1997 ) . However, this relationship 

### 32

between AH2 levels and O 3 dose did not hold up in every study. Aging rats (9 and 

### 33

24 months old) had 49% and 64% lower AH2 in lung tissue com pared with month

- old 

### 34
rats but the aging

- induced AH2 loss did not increase the accumulation of 18 O reaction 

### 35
products following O 3 exposure (0.4

- 0.8 ppm, 2

- 6 hours) ( Vincent et al., 1996b )

. 

### 36

### Draft

– Do Not Cite or Quote 5

- 40 June 2012 A few studies have examined the dose

- or concentration

- responsiveness of airways 

### 1
neutrophilia in O 3 - exposed humans ( Holz et al., 1999 ; Devlin et al., 1991 ) . No 

### 2

concentration

- responsiveness was observed in healthy human subjects exposed for 1 hour 

### 3

to 125

- 250 ppb O 3 while exercising at a light level and a statistically significa nt increase 

### 4
in sputum neutrophilia was observed only at the higher concentration ( Holz et al., 1999 )

. 

### 5

However, concentration

- dependent and statistically significa nt increases in BAL 

### 6
neutrophils and the inflammatory mediator IL

- 6 were reported following exposure to 80 

### 7

and 100 ppb O 3 for 6.6 hours in human subjects exercising at a moderate level ( Devlin et 

### 8

al., 1991 ) . Additional evidence is provided by a meta

- analysis of the O 3 

### 9

dose

- inflammatory response in controlled human exposure studies involving exposure to 

### 10

80

- 600 ppb O 3 for 60

- 396 minutes and exercise levels ranging from light to heavy 

### 11

( Mudway and Kelly, 2004b ) . Results demonstrated a linear relationship between inhaled 

### 12

O 3 dose (determined as the product of concentration, vent ilation and time) and BAL 

### 13
neutrophils at 0

- 6 hours and 18

- 24 hours following O 3 exposure ( Mudway and Kelly, 

### 14

2004b )

. 

### 15

5.3.4 Alteration of Epithelial Barrier Function

Following O 3 exposure, injury and inflammation can lead to alt ered airway barrier 

### 16
function. Histologic analysis has demonstrated damage to tight junctions between 

### 17
epithelial cells, suggesting an increase in epithelial permeability. In addition, the presence 

### 18
of shed epithelial cells in the BALF and increased epithelia l permeability, which is 

### 19
measured as the flux of small solutes, have been observed and are indicative of epithelial 

### 20
injury. This could potentially lead to the loss of ELF solutes that could diffuse down their 

### 21
concentration gradient from the lung to the blo od. Increases in vascular permeability, as 

### 22
measured by BALF protein and albumin, have also been demonstrated ( Costa et al., 

### 23

1985 ; Hu et al., 1982 )

. 

### 24

An early study in sheep measured changes in airway permeability as the flux of inhaled 

### 25
radiolabeled histamine into the plasma ( Abraham et al., 1984 ) . Exposure of sheep to 

### 26

0.5 ppm O 3 for 2 hours via an endotracheal tube resulted in an increased rate of histamine 

### 27
appearance in the plasma at 1 day postexposure. Subsequently, numerous studies have 

### 28
measured epithelial permeability as the flux of the small solute 99m Tc

- DTPA that was 

### 29
introduced into the air spaces in different regions of the RT. Increased pulmonary 

### 30
epithelial permeabili ty, measured as the clearance of 99m Tc

- DTPA from lung to blood, 

### 31

was demonstrated in humans 1

- 2 hours following a 2

- hour exposure to 400 ppb O 3 while 

### 32
exercising at a heavy level ( Kehrl et al., 1987 ) . Another study in human subjects found 

### 33
increased epithelial permeability 19

- hours postexposure to 240 ppb O 3 for 130 minutes 

### 34
while exercising at moderate level ( Foster and Stetkiewicz, 1996 ) . Increased bronchial 

### 35

### Draft

– Do Not Cite or Quote 5

- 41 June 2012 permeability was also observed in dogs 1

- day postexposure (0.4 ppm O 3 by endotracheal 

### 1

tube for 6 hours) and did not resolve for several days ( Foster and Freed, 1999 )

. 

### 2

A role for tachykinins in mediating airway epithelial injury and decreased barrier 

### 3
function has been suggested. Nishiyama et al. (1998 ) demonstrated that capsaicin, which 

### 4
depletes nerve fibers of substance P, blocked the O 3 - induced increase in permeability of 

### 5
guinea pig tracheal mucosa (0.5

- 3 ppm O 3 , 0.5 hours). Pretreatment with propranolol or 

### 6
atropine failed to inhibit this response, suggesting that adrenergic and cholinergic 

### 7
pathways were not involved. In another study, tachykinins working through NK

- 1 and 

### 8
CGRP receptors were found to contribute to airway epithelial injury in O 3 - exposed rats 

### 9

(1 ppm, 8 hours) ( Oslund et al., 2009

, 2008 )

. 

### 10

Kleeberger et al. (2000 ) evaluated genetic susceptibility to O 3 - induced altered barr ier 

### 11
function in recombinant inbred strains of mice. Lung hyperpermeability, measured as 

### 12
BALF protein, was evaluated 72 hours after exposure to 0.3 ppm O 3 and found to be 

### 13
associated with a functioning Tlr 4 gene. This study concluded that Tlr4 was a strong 

### 14

c andidate gene for susceptibility to hyperpermeability in response to O 3 ( Kleeberger et 

### 15

al., 2000 ) . A subsequent study by these same investigators found that Tlr4 modulated 

### 16

mRNA levels of the Nos2 genes and suggested that the protein product of Nos2 , iNOS, 

### 17
plays an important role in O 2 - induced lung hyperpermeability (0.3 ppm, 72 hours) 

### 18

( Kleeberger et al., 2001 ) . More recently, HSP70 was identified as part of the TLR4 

### 19
signaling pathway (0.3 ppm, 6

- 72 hours) ( Bauer et al., 2011 )

. 

### 20

Antioxidants have been shown to confer resistance to O 3 - induced injury. In a recent 

### 21
study, lung hyperpermeability in response to O 3 (0.3 ppm, 48 hours) was unexpectedly 

### 22
reduced in mice deficient in the glutamate

- cysteine ligase modifier subunit gene 

### 23
compared with sufficient mice ( Johansson et al., 2010 ) . Since the lungs of these mice 

### 24
exhibited 70% glutathione depletion, protection ag ainst O 3 - induced injury was 

### 25
unexpected ( Johansson et al., 2010 ) . However it was found that several other antioxidant 

### 26
defenses, including meta llothionein, were upregulated in response to O 3 to a greater 

### 27
degree in the glutathione

- deficient mice compared with sufficient mice ( Johansson e t al., 

### 28

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

### 29
compensatory augmentation of antioxidant defenses ( J ohansson et al., 2010 ) . Antioxidant 

### 30
effects have also been attributed to Clara cell secretory protein (CCSP) and surfactant 

### 31
protein A (SP

- A). CCSP was found to modulate the susceptibility of airway epithelium to 

### 32
injury in mice exposed to O 3 (0.2 or 1 pp m for 8 hours) by an unknown mechanism 

### 33

( Plopper et al., 2006 ) . SP

- A appeared to confer protection against O 3 - induc ed airways 

### 34
inflammation and injury in mice (2 ppm, 3 hours) ( Haque et al., 2007 )

. 

### 35

Increased epithelial permeability has been proposed to play a role in allergic sensitization 

### 36

( Matsumura, 1970 ) , in activation of neural reflexes and in stimulation of smooth muscle 

### 37

### Draft

– Do Not Cite or Quote 5

- 42 June 2012 receptors ( Dimeo et al., 1981 )

. Abraham et al. (1984 ) reported a correlation between 

### 1
airway permeability and airways hyperresponsiveness (AHR) in O 3 - exposed sheep. 

### 2
However a recent study in human subjects exposed to 220 ppb O 3 for 135 minutes whil e 

### 3
exercising at a light to moderate level did not find a relationship between O 3 - induced 

### 4
changes in airway permeability and AHR ( Que et al., 2011 )

. 

### 5

5.3.5 Sensitization of Bronchial Smooth Muscle

Bronchial reactivity is generally determined in terms of a response to a challeng e agent. 

### 6

Non

- specific bronchial reactivity in humans is assessed by measuring the effect of 

### 7
inhaling increasing concentrations of a bronchoconstrictive drug on lung mechanics 

### 8

(sRaw or FEV 1 ). Methacholine is most commonly employed but histamine and other 

### 9

ag ents are also used. Specific bronchial reactivity is assessed by measuring effects in 

### 10
response to an inhaled allergen in individuals (or animals) already sensitized to that 

### 11
allergen. An increase in sRaw in response to non

- specific or specific challenge age nts 

### 12

indicates AHR. 

### 13

In addition to causing mild airway s obstruction as discussed above, acute O 3 exposure 

### 14
results in reversible increases in bronchial reactivity by mechanisms that are not well 

### 15
understood. In one study, bronchial reactivity of healthy subje cts was significa ntly 

### 16

increased 19

- hours postexposure to O 3 (120

- 240 ppb O 3 for 2 hours with moderate 

### 17

exercise) ( Foster et al., 2000 ) . These effects may be more considerable in human subjects 

### 18
with already compromised airways (Section 5.4.2.2 ). 

### 19

Ozone may sensitize bronchial smooth muscle to stimulation through an exposure

- related 

### 20
effect on smooth muscle or through effects on the sensory nerves in the epithelium or on 

### 21
the motor nerves innervating the smooth muscle ( O'Byrne et al., 1984 ; O'Byrne et al., 

### 22

1983 ; Holtzman et al., 1979 ) . It is also recognized that increased bronchial reactivity can 

### 23
be both a rapidly occurring and a persistent response to O 3 ( Foster and Freed, 1999 )

. 

### 24
Tachykinins and secondary oxidation products of O 3 have been proposed as mediators of 

### 25
the early response and inflammation

- derived products have been pro posed as mediators 

### 26
of the later response ( Foster and Fr eed, 1999 ) . Furthermore, bronchial reactivity may be 

### 27

increased as a result of O 3 - mediated generation of ROS. 

### 28

Ozone

- induced increases in epithelial permeability, which could improve access of 

### 29
agonist to smooth muscle receptors, may be one mechanism of s ensitization through a 

### 30

direct effect on bronchial smooth muscle ( Holtzman et al., 1979 ) . As noted above, a 

### 31
correlation between airway permeability and AHR has been reported in O 3 - exposed sheep 

### 32

( Abraham et al., 1984 ) but not in O 3 - exposed human subjects ( Que et al., 2011 )

. 

### 33

### Draft

– Do Not Cite or Quote 5

- 43 June 2012 Neurally

- mediated sensitization has been demonstrated. In human subjects exposed for 

### 1

2 hours to 600 ppb O 3 while exercising at a light level, pretreatment with atropine 

### 2

inhibited O 3 - induced AHR, suggesting the involvement of cholinergic postganglionic 

### 3
pathways ( Holtzman et al., 1979 ) . Animal studies have demonstrated that O 3 - induced 

### 4
AHR involved vagally

- mediated respo nses (rabbits, 0.2 ppm O 3 , 72 hours) ( Freed et al., 

### 5

1996 ) and local axon reflex responses through bronchopulmonary C

- fiber

- mediated 

### 6
release of SP (guinea pigs, 0.8 ppm O 3 , 2 hours) ( Joad et al., 1996 ) . Further, p retreatment 

### 7
with capsaicin to deplete nerve fibers of SP blocked O 3 - mediated AHR (guinea pigs, 

### 8

1

- 2 ppm O 3 , 2

- 2.25 hours) ( Tepper et al., 1993 ) . Other investigators demonstrated that SP 

### 9
released from airway nociceptive n eurons in ferrets contributed to O 3 - induced AHR 

### 10

(2 ppm O 3 , 3 hours) ( Wu et al., 2008c ; Wu et al., 2003 )

. 

### 11

Some evidence suggests the involvement of arachidonic acid metabolites and neutrophils 

### 12
in mediating O 3 - induced AHR ( Seltzer et al., 1986 ; Fabbri et al., 1985 ) . Increased BAL 

### 13
neutrophils and cyclooxygenase products were foun d in one study demonstrating AHR in 

### 14
human subjects exercising at a heavy level immediately postexposure to 600 ppb O 3 for 

### 15

2 hours ( Seltzer et al., 1986 ) . Another study found that ibuprofen pretreatment had no 

### 16

effect on AHR in h uman subjects exercising at a heavy level following exposure to 

### 17

400 ppb O 3 for 2 hours, although spirometric responses were blunted ( Hazucha et al., 

### 18

1996 ) . This study measured arachidonic acid metabolites and provided evide nce that that 

### 19
the arachidonic acid metabolites whose generation was blocked by ibuprofen, 

### 20

(i.e., prostaglandins, thromboxanes and some leukotrienes) did not play a role in AHR. 

### 21
Experiments in dogs exposed for 2 hours to 2.1 ppm O 3 demonstrated a close corr elation 

### 22

between O 3 - induced AHR and airways neutrophilic inflammation measured in tissue 

### 23
biopsies ( Holtzman et al., 1983 )

. Furthermore, the increased AHR observed in dogs 

### 24
following O 3 exposure (3 ppm, 2 hours) was inhibited by neutrophil depletion ( O' Byrne 

### 25

et al., 1983 ) and by pre

- treatment with inhibitors of arachidonic acid metabolism. In one 

### 26
of these studies, indomethacin pre

- treatment did not prevent airways neutrophilia in 

### 27
response to O 3 (3 ppm, 2 hours) providing evidence that the subset of ar achidonic acid 

### 28
metabolites whose generation was inhibitable by the cyclooxygenase inhibitor 

### 29

indomethacin (i.e., prostaglandins and thromboxanes) was not responsible for neutrophil 

### 30

influx ( O'Byrne et al., 1984 )

. It should be noted that these studies did not measure 

### 31
whether the degree to which the inhibitor blocked arachidonic acid metabolism and thus 

### 32
their results should be interpreted with c aution. Taken together, these findings suggest tha t 

### 33

arachidonic acid metabolites may be involved in the AHR response following O 3 

### 34
exposure in dogs. S tudies probing the role of neutrophils in mediating the AHR response 

### 35
have provided inconsistent results ( Al

- Hegelan et al., 2011 )

. 

### 36

Evidence for cytokine and chemokine involvement in the AHR response to O 3 has been 

### 37
described. Some studies have suggested a rol e for TNF

- α (mice, 0.5 and 2 ppm O 3 , 

### 38

### Draft

– Do Not Cite or Quote 5

- 44 June 2012 3 hours) ( Cho et al., 2001 ; Shore et al., 2001 ) and IL

- 1 (mice and ferrets, 2 ppm O 3 , 

### 1

3 hours) ( Wu et al., 2008c ; Park et al., 2004 ) . The latter study found that SP expression in 

### 2
airway neurons was upregulated by IL

- 1 that was released in response to O 3 . Other 

### 3

studies in mice have demonstra ted a key role for CXCR2, the chemokine receptor for the 

### 4
neutrophil chemokines KC and MIP

- 2, but not for IL

- 6 in O 3 - mediated AHR (1 ppm O 3 , 

### 5

3 hours) ( Johnston et al., 2005a ; Johnston et al., 2005b ) . In contrast, CXCR2 and IL

- 6 

### 6
were both required for neutrophil influx in this model ( Johnston et al., 2005a ; Johnst on et 

### 7

al., 2005b ) , as discussed above. Williams et al. (2008b ) demonstrated that the Th2 

### 8
cytokine IL

- 13 contributed to AHR, as well as to airways neutrophilia, in mice (3 ppm 

### 9

O 3 , 3 hours). 

### 10

Other studies have focused on the role of TLR4. Hollingswor th et al. (2004 ) measured 

### 11
AHR, as well as airways neutrophilia, in mice 6 and 24 hours following acute (2 ppm O 3 

### 12

for 3 hours) and subchronic (0.3 ppm for 3 days) exposure to O 3 . TLR4 is a key 

### 13
component of the innate immune system and is responsible for the immediate 

### 14
inflammatory response seen following challenge with endotoxin and other pathogen



### 15
associated substances. In this study, a functioning TLR4 was required for the full AHR 

### 16
response following O 3 exposure but not for airways neutrophilia ( Hollingsworth et al., 

### 17

2004 ) . These findi ngs are complemented by an earlier study demonstrating th at O 3 effects 

### 18
on lung hyperpermeability required a functioning TLR4 (mice, 0.3 ppm O 3 , 72 hours) 

### 19

( Kleeberger et al., 2000 )

. Williams et al. (2007b ) found that TLR2, TLR4 and the TLR 

### 20
adaptor protein MyD88 contributed to AHR in mice (3 ppm O 3 , 3 hours). Ozone wa s also 

### 21
found to upregulate MyD88, TLR4 and TLR4 gene expression in this model ( Williams et 

### 22

al., 2007b ) . Furthermore, a recent study reported O 3 - induced AHR that required TLR4 

### 23

and MyD88 in mice exposed to 1 ppm O 3 for 3 hours ( Li et al., 2011 )

. 

### 24

A newly recognized mechanistic basis for O 3 - induced AHR is provided by studies 

### 25

f ocusing on the role of hyaluronan following O 3 exposure in mice ( Garantziotis et al., 

### 26

2010 ; Garantziotis et al., 2009 ) . Hyaluronan is an extracellular matrix component that is 

### 27
normally found in the ELF as a large polymer. Briefly, TLR4 and CD44 were found to 

### 28
mediate AHR in response to O 3 and hyaluronan. Exposure to 2 ppm O 3 for 3 hour s 

### 29
resulted in enhanced AHR and elevated levels of soluble low molecular weight 

### 30
hyaluronan in the BALF 24

- hours postexposure ( Garantziotis et al., 2010 ; Garantziotis et 

### 31

al., 2009 ) . Ozone may have caused the depolymerization of hyaluronan to soluble 

### 32
fragments that are known to be endogenous ligands of the CD44 receptor and TLR4 in 

### 33
the macrophage ( Jiang et al., 2005 ) . In the two recent studies, O 3 - induced AHR was 

### 34

attenuated in CD44 and TL R4

- deficient mice ( Gara ntziotis et al., 2010 ; Garantziotis et 

### 35

al., 2009 ) . Hyalur onan fragment

- mediated stimulation of AHR was found to require 

### 36
functioning CD44 receptor and TLR4 ( Garantziotis et al., 2010 ; Garantziotis et al., 2009 )

. 

### 37
In contrast, high

- molecular

- weight hyaluronan blocked AHR in response to O 3 

### 38

### Draft

– Do Not Cite or Quote 5

- 45 June 2012 ( Garantziotis et al., 2009 ) . In another study high

- molecular

- weight hyaluronan enhanced 

### 1
repair of epithelial injury ( Jiang et al., 2005 ) . These studies provide a link between innate 

### 2
immunity and the development of AHR following O 3 exposure, and indicate a role for 

### 3
TLR4 in increasing airways responsiveness. While TLR4

- d ependent responses usually 

### 4

involve activation of NF

- κB and the upregulation of proinflammatory factors, the precise 

### 5
mechanisms leading to AHR are unknown ( Al

- Hegelan et al., 2011 )

. 

### 6

In guinea pigs, AHR was found to be mediated by different pathways at 1

- and 3

- days 

### 7
postexposure to a single exposure of O 3 (2 ppm for 4 hours) ( Verhein et al., 2011 ; Y ost et 

### 8

al., 2005 ) . At 1 day, AHR was due to activation of airway parasympathetic nerves rather 

### 9
than to an exposure

- related effect on smooth mus cle ( Yost et al., 2005 ) . This effect 

### 10

occurred as a result of O 3 - stimulated relea se of major basic protein from eosinophils 

### 11

( Yost et al., 2005 ) . Major basic protein is known to block inhibitory M2 muscar inic 

### 12
receptors that normally dampen acetylcholine release from parasympathetic nerves ( Yost 

### 13

et al., 2005 ) . The resulting i ncrease in acetylcholine release caused an increase in smooth 

### 14
muscle contraction following O 3 exposure ( Yost et al., 2005 ) . Eosinophils played a 

### 15

different role 3

- days postexposure to O 3 in guinea pigs ( Yost et al., 2005 ) . Ozone



### 16

mediated influx of eosinophils into lung airways resulted in a different population of cells 

### 17
present 3

- days postexposure compared to those present at 1 day ( Yost et al., 2005 ) . At 

### 18
this time point, eosinophil

- derived major basic protein increased smooth muscle 

### 19
responsiveness to acetylcholine which also contributed to AHR ( Yost et al., 2005 )

. 

### 20
However, the major effect of eosinophils was to protect against vagal hyperreactivity 

### 21

( Yost et al., 2005 ) . The authors suggested that these beneficial effects were due to the 

### 22
production of nerve growth factor ( Yost et al., 2005 ) . Further work by these investigators 

### 23
demonstrated a key role for IL

- 1β in mediating AHR 3

- days postexposure to O 3 ( Verhein 

### 24

et al., 2011 ) . In this study, IL

- 1β increased nerve growth factor and SP that acted through 

### 25
the NK1 receptor to cause vagally

- mediated bronchoconstriction ( Verhein et al., 2011 )

. 

### 26
The mechanism by which SP caused acetylcholine release from parasympathetic nerves 

### 27

followin g O 3 exposure was not determined ( Verhein et al., 2011 ) . Taken together, the 

### 28
above study results indicate that mechanisms involved in O 3 - mediated AHR can vary 

### 29
over time postexposure and that eosinophils and SP can play a role. Results of this anima l 

### 30
model may provide some insight into allergic airways disease in humans that is 

### 31
characterized by eosinophilia (Section 5.4.2.2 ). 

### 32

5.3.6 Modification of I nnate/Adaptive Immune System Responses

Host defense depends on effective barrier function and on innate immunity and adaptive 

### 33
immunity ( Al

- Hegelan et al., 2011 )

. The effect s of O 3 on barrier function in the airways 

### 34

was discussed above (Section 5.3.4 ). This section focuses on the mechanisms by which 

### 35

### Draft

– Do Not Cite or Quote 5

- 46 June 2012 O 3 impacts innate and adaptive immunity. Both tissue damage and foreign pathogens are 

### 1
triggers for the activation of the innate immune system . This results in the influx of 

### 2
inflammatory cells such as neutrophils, mast cells, basophils, eosinophils, monocytes and 

### 3
dendritic cells and the generation of cytokines such as TNF

- α, IL

- 1, IL

- 6, KC and IL

- 17. 

### 4
Further, innate immunity encompasses the acti ons of complement and collections, and 

### 5
the phagocytic functions of macrophages, neutrophils and dendritic cells. Airway 

### 6
epithelium also contributes to innate immune responses. Innate immunity is highly 

### 7
dependent on cell signaling networks involving TLR4. A daptive immunity provides 

### 8
immunologic memory through the actions of B and T

- cells. Important links between the 

### 9
two systems are provided by dendritic cells and antigen presentation. Recent studies 

### 10
demonstrate that exposure to O 3 modifies cells and processes which are required for 

### 11
innate immunity, contributes to innate

- adaptive immune system interaction and primes 

### 12
pulmonary immune responses to endotoxin. 

### 13

Ozone exposure of human subjects resulted in recruitment of activated innate immune 

### 14
cells to the airways. Healthy individuals were exposed to 80 ppb O 3 for 6.6 hours while 

### 15
exercising at a moderate level and airways inflammation was characterized in induced 

### 16
sputum 18

- hours postexposure ( Alexis et al., 2010 ) . Previous studies demonstrated that 

### 17
induced sputum contains liquid and cellular constituents of the ELF from central 

### 18
conducting airways ( Alexis et al., 2001b ) and also identified these airways as a si te of 

### 19
preferential O 3 absorption during exercise ( Hu et al., 1994 ) . Ozone exposure resulted in 

### 20
increased numbers of neutrophils, airway monocytes and dendritic

- like cells in sputum 

### 21

( Alexis et al., 20 10 ) . In addition, increased expression of cell surface markers 

### 22
characteristic of innate immunity and antigen presentation (i.e., CD

- 14 and HLA

- DR) 

### 23
was demonstrated on airway monocytes ( Alexis et al., 2010 ) . Enhanced antigen 

### 24
presentation contributes to exaggerated T

- cell responses and promotes Th2 inflammation 

### 25
and an allergic phenotype ( Lay et al., 2007 ) . Upregulation of pro

- inflammatory cytokines 

### 26
was also demonstrated in sputum of O 3 - exposed subjects ( Alexis et al., 2010 ) . One of 

### 27
these cytokines, IL

- 12p70, correlated with numbers of dendritic

- like cells in the sputum, 

### 28

and is an indicator of dendritic cell activation ( Alexis et al., 2010 ) . These authors have 

### 29
previ ously reported that exposure of human subjects exercising at a light to moderate 

### 30

level to 400 ppb O 3 for 2 hours resulted in activation of monocytes and macrophages 

### 31

( Lay et al., 2007 ) , which co uld play a role in exacerbating existing asthma by activating 

### 32
allergen

- specific memory T

- cells. The current study confirms these findings and extends 

### 33
them by suggesting a potential mechanism whereby O 3 - activated dendritic cells could 

### 34

stimulate naïve T

- cell s to promote the development of asthma ( Alexis et al., 2010 ) . A 

### 35
companion study by these same investigators (described in detail in Section 5.4.2.1 ) 

### 36
provides evidence of dendritic cell activation, measured as increased expression of HLA



### 37
DR, in a subset of the human subjects (GSTM1 null) exposed t o 400 ppb O 3 for 2 hours 

### 38
while exercising at a light to moderate level ( Alexis et al., 2009 ) . Since dendritic cells are 

### 39

### Draft

– Do Not Cite or Quote 5

- 47 June 2012 a link between innate and adaptive immunity, these studies provide evidence for an 

### 1

O 3 - mediated interaction between the innate and adaptive immune systems. 

### 2

Anot her recent study linked O 3 - mediated activation of the innate immune system to the 

### 3
development of non

- specific AHR in a mouse model ( Pichavant et al., 2008 ) . Repeated 

### 4
exposure to 1 ppm O 3 for 3 hours (3 days over a 5 day period) induced non

- specific AHR 

### 5

measured 24 hours following the last exposure ( Pichavant et al., 2008 ) . This response 

### 6
was found to require NKT

- cells, whic h are effector lymphocytes of innate immunity, as 

### 7

well as IL

- 17 and airways neutrophilia ( Pichavant et al., 2008 ) . Since glycolipids such as 

### 8
galactosyl ceramide are ligands for the invariant CD1 receptor on NKT

- cells and serve as 

### 9
endogenous activators of NKT

- cells, a role for O 3 - oxidized lipids in activating NKT

- cells 

### 10

was pro posed ( Pichavant et al., 2008 ) . The authors contrasted this innate immu nity 

### 11
pathway with that of allergen

- provoked specific AHR which involves adaptive immunity, 

### 12
the cytokines IL

- 4, IL

- 13, IL

- 17, and airways eosinophilia ( Pichavant et al., 2008 )

. 

### 13
Interestingly, NKT

- cells were required for both the specific AHR provoked by allergen 

### 14

and the non

- specific AHR provoked by O 3 ( Pichavant et al., 2008 ) . Different cytokine 

### 15
profiles of the NKT

- cells from allergen and O 3 - exp osed mice were proposed to account 

### 16
for the different pathways ( Pichavant e t al., 2008 ) . More recently, NKT

- cells have been 

### 17
found to function in both innate and adaptive immunity ( Vivier et al., 2011 )

. 

### 18

An interaction between allergen and O 3 in the induction of nonspecific AHR was shown 

### 19
in another animal study ( Larsen et al., 2010 ) . Mice were sensitized with the aerosolized 

### 20
allergen OVA on 10 consecutive days followed by exposure to O 3 (0.1

- 0.5 ppm for 

### 21

3 hours) ( Larsen et al., 2010 ) . While allergen sensitization alone did not alter airways 

### 22
responsiveness to a nonspecific challeng e, O 3 exposure of sensitized mice resulted in 

### 23
nonspecific AHR at 6

- and 24

- hours postexposure ( Larsen et al., 2010 ) . The effects of O 3 

### 24
on AHR were independent of airways eosinophilia and neutrophilia ( Larsen et al., 2010 )

. 

### 25
However, OVA pretreatment led to goblet cell metaplasia which was enhanced by O 3 

### 26

exposure ( Larsen et al., 2010 ) . It should be noted that OVA sensitization using only 

### 27
aerosolized antigen in this study is less common than the usual procedure for OVA 

### 28
sensitization achieved by one or more initial systemic injections of OVA and adjuvant 

### 29
followed by repeated inhalation exposure to OVA. This study also points to an interaction 

### 30
between innate and adaptive immune systems in the development of the AHR response. 

### 31

Furthermore, O 3 was found to act as an adjuvant for allergic sensitization ( Hollingsworth 

### 32

et al., 2010 ) . Oropharyngeal aspiration of OVA on day 0 and day 6 failed to lead to 

### 33

all ergic sensitization unless mice were first exposed to 1 ppm O 3 for 2 hours 

### 34

( Hollingsworth et al., 2010 ) . The O 3 - mediated response involved Th2 ( IL

- 4, IL

- 5 and 

### 35

IL

- 9) and Th17 cytokines (IL

- 17) and was dependent on a functioning TLR4 

### 36

( Hollingsworth et al., 2010 ) . Ozone exposure also activated OVA

- bearing dendritic cells 

### 37

### Draft

– Do Not Cite or Quote 5

- 48 June 2012 in the thoracic lymph nodes, as measured by the presence of the CD86 surface marker, 

### 1
which suggests naïve T

- cell stimulation and the involvement of Th2 pathways 

### 2

( Hollingsworth et al., 2010 ) . Thus the adjuvant effects of O 3 may be due to activation of 

### 3
both innate and adaptive immunity. 

### 4

Priming of the innate immune system by O 3 was reporte d by Hollingsworth et al. (2007 )

. 

### 5
In this study, exposure of mice to 2 ppm O 3 for 3 hours led to nonspecific AHR at 24

- 

### 6

and 48

- hours postexp osure, an effect which subsided by 72 hours ( Hollingsworth et al., 

### 7

2007 ) . However, in mice treated with aerosolized endotoxin immediately following O 3 

### 8

exp osure, AHR was greatly enhanced at 48

- and 72

- hours postexposure ( Hollingsworth et 

### 9

al., 2007 ) . In addition, O 3 pre

- exposure was found to reduce the number of inflammatory 

### 10
cells in the BALF, to increase cytokine production and total protein in the BALF and to 

### 11
increase systemic IL

- 6 following exposure to endotoxin ( Hollingsworth et al., 2007 )

. 

### 12

Furthermore, O 3 stimulated the apoptosis of alveolar macrophages 24

- hours 

### 13
postexposure, an effect which was greatly enhanced by endotoxin treatment. Apoptosis of 

### 14
circulating blood monocytes was also observed in response t o the combined exposures 

### 15

( Hollingsworth et al., 2007 ) . Ozone pre

- exposure enhanced the response of lung 

### 16
macrophages to endotoxin ( Hollingsworth et al., 2007 ) . Taken together, these findings 

### 17

demonstrated that O 3 exposure increased innate immune responsiveness to endotoxin. 

### 18
The authors attributed these effects to the increased surface expression of TLR4 and 

### 19
increased signaling in macrophages observed in the study ( Hollingsworth et al., 2007 ) . It 

### 20
was proposed that the resulting decrease in airway inflammatory cells could account for 

### 21

O 3 - mediated decreased clearance of bacterial pathogens observed in numerous animal 

### 22

m odels ( Hollingsworth et al., 2007 )

. 

### 23

More recently, these authors demonstrated that hyaluronan contributed to the O 3 - primed 

### 24
response to endotoxin ( Li et al., 2010 ) . In this study, exposure of mice to 1 ppm O 3 for 

### 25

3 hours resulted in enhanced response s to endotoxin, which was mimicked by 

### 26
intratracheal instillation of hyaluronan fragments ( Li et al. , 2010 ) . Hyaluronan, like O 3 , 

### 27
was also found to induce TLR4 receptor peripheralization in the macrophage membrane 

### 28

( Li et al., 2010 ; Hollingsworth et al., 2007 ) , an effect which is associated with enhanced 

### 29

responses to e ndotoxin. This study and previous ones by the same investigators showed 

### 30
elevation of BALF hyaluronan in response to O 3 exposure ( Garantziotis et al., 2010 ; Li et 

### 31

al., 2010 ; Garantziotis et al., 2009 ) , providing evidence that the effects of O 3 on innate 

### 32
immunity are at least in part mediated by hyaluronan fragments. The authors note that 

### 33
excessive TLR4 signaling can lead to lung injury and suggest that O 3 may be responsible 

### 34
for an exaggerated innate immune response which may underlie lung inju ry and 

### 35

decreased host defense ( Li et al., 2010 )

. 

### 36

### Draft

– Do Not Cite or Quote 5

- 49 June 2012 Activation or upregulation of the immune syste m has not been reported in all studies. 

### 1
Impaired antigen

- specific immunity was demonstrated following subacute O 3 exposure 

### 2

(0.6 ppm, 10 h/day for 15 days) in mice ( Feng et al., 2006 ) . Specifically, O 3 exposure 

### 3
altered the lymphocyte subset and cytokine profile and impacted thymocyte early 

### 4
development leading to immune dysfunction. Further, recent studies demonstrat ed SP

- A 

### 5
oxidation in mice exposed for 3

- 6 hours to 2 ppm O 3 . SP

- A is an important innate 

### 6
immune protein which plays a number of roles in host defense including acting as 

### 7
opsonin for the recognition of some pathogens ( Haque et al., 2009 ) . These investigations 

### 8

found that O 3 - mediated carbonylation of purified SP

- A was associated with impaired 

### 9
macrophage phagocytosis in vi tro ( Mikerov et al., 2008c ) . In addition, O 3 exposure 

### 10

(2 ppm for 3 hours) in mice was found to increase susceptibility to pneumonia infection 

### 11
in mice through an impairment of SP

- A dependent phagocytosis ( Mikerov et al., 2008b ; 

### 12

Mikerov et al., 2008a ) . Furthermore, early life ex posure to O 3 in infant monkeys followed 

### 13
by a recovery period led to hyporesponsiveness to endotoxin ( Maniar

- Hew et al., 2011 )

, 

### 14

as discussed below and in Section 5.4.2.4 and Section 7.2.3.1

. 

### 15

Taken together, results of recent studies provide evidence that O 3 alters host 

### 16
immunologic response and leads to immune system dysfunction through its effects on 

### 17
innate and adaptive immunity. 

### 18

5.3.7 Airways Remodeling

The nasal airways, conducting airways and distal airways (i.e., respiratory bronchioles or 

### 19
CAR depending on the species) have all been identified as sites of O 3 - mediated injury 

### 20

and inflammation ( Mudway and Kelly, 2000 ) . At all levels of the RT, loss of sensitive 

### 21
epithelial cells, degranulation of secretory cells, proliferation of resistant epithelial cells 

### 22
and neutrophilic influx have been observed as a result of O 3 exposure ( Mu dway and 

### 23
Kelly, 2000 ; Cho et al., 1999 ) . An important study ( Plopper et al., 1998 ) conducted in 

### 24
adult rhesus monkeys (0.4 and 1.0 ppm O 3 for 2 hours at rest) found that 1 ppm O 3 

### 25
resulted in the greatest epithelial i njury in the respiratory bronchioles immediately 

### 26
postexposure although injury was observed at all of the RT sites studied except for the 

### 27
lung parenchyma. Exposure to 0.4 ppm O 3 resulted in epithelial injury only in the 

### 28
respiratory bronchioles. Initial cell ular injury correlated with site

- specific O 3 dose since 

### 29
the respiratory bronchioles received the greatest O 3 dose ( 18 O mass/lung weight) and 

### 30
sustained the greatest initial cellular injury. The respiratory bronchioles were also the site 

### 31

of statistically sig nifica nt GSH reduction. In addition, a study in isolated perfused rat 

### 32
lungs found greater injury in conducting airways downstream of bifurcations where local 

### 33

doses of O 3 were higher ( Postlethwait et al., 2000 )

. 

### 34

### Draft

– Do Not Cite or Quote 5

- 50 June 2012 In addition to the degree of initial injury, the degree of airways inflammation due to O 3 

### 1
may have important long

- term consequences since airways inflammation may lead to 

### 2
tissue injury ( Balmes et al., 1996 )

. P ersistent inflammation and injury, observed in animal 

### 3
models of chronic and intermittent exposure to O 3 , are associated with airways 

### 4
remodeling, including mucous cell metaplasia of nasal transitional epithelium ( Harkema 

### 5

et al., 1999 ; Hotchkiss et al., 1991 ) and bronchiolar met aplasia of alveolar ducts 

### 6

( Mudway and Kelly, 2000 ) . Fibrotic changes such as deposition of collagen in the 

### 7
airways a nd sustained lung function decrements especially in small airways have also 

### 8
been demonstrated as a response to chronic O 3 exposure ( Mudway and Kelly, 2000 ; 

### 9
Chang et al., 1992 ) . These effects, described in detail in Section 7.2.3.1 , have been 

### 10
demonstrated in rats exposed to levels of O 3 as low as 0.25 ppm. Mechanisms responsible 

### 11
for the resolution of inflammation and the repair of injury remain to be clarified and there 

### 12
is only a limited understanding of the biological processes underlying long

- term 

### 13
morphological changes. However, a recent study in mice demonstrated a key role for the 

### 14

TGF

- β signaling pathway in the deposition of collagen in the airways wall following 

### 15

chr onic intermittent exposure to 0.5 ppm O 3 ( Katre et al., 2011 ) . Studies in infant 

### 16
monkeys have also documented effects of chronic intermittent exposure to 0.5 ppm O 3 on 

### 17
the developing lung and immune system. Extensive discussion of this topic is found in 

### 18

Section 5.4.2.4 (Lifestage) and in Section 7.2.3.1

. 

### 19

It should be noted that repeated exposure to O 3 results in attenuation of some O 3 - induced 

### 20
responses, including those associated with the activation of neural reflexes 

### 21
(e.g., decrements in pulmonary function), as discussed in Section 5.3.2 . However, 

### 22
numerous studies demonstrate that some markers of injury and inflammation remain 

### 23
increased during multi

- day exposures to O 3 . Mechanisms responsible for atten uation, or 

### 24
the lack thereof, are incompletely understood. 

### 25

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 

### 26
been proposed that lipid oxidation products resulting from reaction of O 3 with lipids in 

### 27
the ELF are responsible for systemic effects, however it is not known whether they gain 

### 28
access to the vascular space ( Chuang et al., 2009 ) . Alternatively, extrapulmonary relea se 

### 29
of diffusible mediators may initiate or propagate inflammatory responses in the vascular 

### 30

or systemic compartments ( Cole and Freeman, 2009 ) . A role for O 3 in modulating 

### 31
endothelin, a potent vasoconstrictor, has also been proposed. Studies in rats found that 

### 32
exposure to 0.4 and 0.8 ppm O 3 induced endothelin system genes in the lung and 

### 33
increased circulating levels of endothelin ( Thomson et al., 2006 ; Thomson et al., 2005 )

. 

### 34
Systemic oxidative stress may be suggested by studies in humans which reported 

### 35

### Draft

– Do Not Cite or Quote 5

- 51 June 2012 associations between O 3 exposure and l evels of plasma 8

- isoprostanes and the presence 

### 1
of peripheral blood lymphocyte micronuclei ( Chen et al., 2007a ; Chen et al., 2006a )

. 

### 2
However, plasma isoprostanes are not a direct measure of systemic oxidative stress since 

### 3
they are stable and can be generated in any compartment before diffusion into the 

### 4
vascular space. Evidence of O 3 - mediated systemic oxidative stress is better provided by 

### 5

new animal studies described below. 

### 6

Ozone

- induced perturbations of the cardiovascular system were recently investigated in 

### 7
young mice and mo nkeys ( Chuang et al., 2009 ) and in rats ( Kodavanti et al., 2011 ; 

### 8
Perepu et al., 2010 ) (see Section 6.3.3 and Section 7.3.1.2 ). These are the first studies to 

### 9
suggest that systemic oxidative stress and inflammation play a mechanistic role in 

### 10

O 3 - induced effects on the systemic vascular and heart. Exposure to 0.5 ppm O 3 for 5 days 

### 11
resulted in oxidative/nitrosative stress, vascular dysfunction and mitoc hondrial DNA 

### 12
damage in the aorta ( Chuang et al., 2009 ) . Chronic exposure to 0.8 ppm O 3 resulted in an 

### 13
enhancement of inflammation and lipid peroxidation in the heart following an ischemia



### 14

rep erfusion challenge ( Perepu et al., 2010 ) . In addition, chronic intermittent exposure to 

### 15

0.4 ppm O 3 increased aortic levels of mRNA for biomarkers of oxidative stress, 

### 16
thrombosis, vasoconstriction and proteolysis and aortic lectin

- like oxidized

- low density 

### 17
lipoprotein receptor

- 1(LOX

- 1) mRNA and protein levels ( Kodavanti et al., 2011 ) . The 

### 18
latter study suggests a role for circulating oxidized lipids in mediating the effects of O 3
. 

### 19

Systemic inflammation and oxidative/nitrosative stress may similarly affect other organ 

### 20
systems as well as the plasma compartment. Circulating cytokines have the potential to 

### 21
enter the brain through diffusion and active transport and to contribute to 

### 22

neuroinflammation, neurotox icity, cerebrovascular damage and a break

- down of the 

### 23

blood brain barrier ( Block and Calderón

- Garcidueñas, 2009 ) (see Section 6.4 and 

### 24

Section 7.5 ). They can also activate neuronal afferents ( Block and Calderón

- Garcidueñas, 

### 25

2009 ) . Vagal afferent pathways originating in the RT may also be responsible for 

### 26

O 3 - mediated activation of nucleus tractus solitarius neurons which resulted in neuronal 

### 27

activation in stress

- responsive regions of the CNS in rats (0.5 or 2 ppm O 3 for 

### 28

1.5

- 120 hours) ( Gackière et al., 2011 ) . Recent studies have demonstrated O 3 - induced 

### 29
brain lipid peroxidation, cytokine production in the brain and upregulated expression of 

### 30

VEGF in rats (0.5 ppm O 3 , 3 hours or 0.25

- 0.5 ppm O 3 , 4 h/day, 15

- 60 days) ( Guevara



### 31

Guzmán et al., 2009 ; Araneda et al., 2008 ; Pereyra

- Muñoz et al., 2006 ) . Further, 

### 32

O 3 - induced oxidative stress resulted in increased plasma lipid peroxides (0.25 ppm, 

### 33
4h/day, 15

- 60 days) ( Santiago

- López et al., 2010 ) , which was correlated with damage to 

### 34
specific brain regions ( Pereyra

- Muñoz et al., 2006 )

. 

### 35

Oxidative stress is one mechanism by which testicular and sperm function may be 

### 36
disrupted (see Section 7.4.1 ). Studies in Leydig cells in vitro have demonstrated that 

### 37

### Draft

– Do Not Cite or Quote 5

- 52 June 2012 stero idogenesis is blocked by oxidative stress ( Diemer et al., 2003 ) . It has been proposed 

### 1
that lipid pero xidation of sperm plasma membrane may lead to impaired sperm mobility 

### 2
and decreased sperm quality ( Agarwal et al., 2003 ) . Further, it has been proposed that 

### 3
oxidative stress may damage DNA in the sperm nucleus and lead to apoptosis and a 

### 4
decline in sperm counts ( Agarwal et al., 2003 ) . One study reported an association 

### 5

between O 3 exposure and semen qual ity and suggested oxidative stress as an underlying 

### 6

mechanism ( Sokol et al., 2006 ) . Additional evidence is required to substantiate this link. 

### 7

A role for plasma antioxidants in modulating O 3 - induced respiratory effects was 

### 8
suggested by a recent study ( Aibo et al., 2010 ) . In this study, pretreatment of rats with a 

### 9
high dose of acetaminophen resulted in increased levels of plasma cytokines and the 

### 10
influx of inflammatory cells into the lung following O 3 exposure (0.25

- 0.5 ppm, 6 hours) 

### 11

( Aibo et al., 2010 ) . These effects were not observed in response to O 3 alone. 

### 12

Furthermore, ace taminophen

- induced liver injury was exacerbated by O 3 exposure. A 

### 13
greater increase in hepatic neutrophil accumulation and greater alteration in gene 

### 14
expression profiles was observed in mice exposed to O 3 and acetaminophen compared 

### 15
with either exposure alon e ( Aibo et al., 2010 ) . Although not measured in this study, 

### 16
glutathione depletion in the liver is known to occur in acetaminophen toxicity. Since liver 

### 17
glutathione is the source of plasma glutathione, acetaminophen treatment may have 

### 18
lowered plasma glutathione levels a nd altered the redox balance in the vascular 

### 19
compartment. These findings indicate interdependence between RT, plasma and liver 

### 20
responses to O 3 , possibly related to glutathione status. 

### 21

5.3.9 Impaired Alveolar

- Arterial Oxygen Transfer

O 3 may impair alveolar

- arte rial oxygen transfer and reduce the supply of arterial oxygen 

### 22
to the myocardium. This may have a greater impact in individuals with compromised 

### 23
cardiopulmonary systems. Gong et al. (1998 ) provided evidence of a small decrease in 

### 24
arterial oxygen saturation in human subj ects exposed for 3 hours to 300 ppb O 3 while 

### 25
exercising at a light to moderate level. In addition, Delaunois et al. (1998 ) demonstrated 

### 26
pulmonary vasoconstriction in O 3 - exposed rabbits (0.4 ppm, 4 hours). Although of 

### 27
interest, the contribution of this pathway to O 3 - induced cardiovascular effects remains 

### 28

uncertain. 

### 29

5.3.10 Summary

This section summarizes the modes of action and toxicity pathways resulting f rom O 3 

### 30

inhalation ( Figure 5

- 8 ). These pathways provide a mechanistic basis for the health effects 

### 31

### Draft

– Do Not Cite or Quote 5

- 53 June 2012 which are described in detail in Chapters 6 and 7. Th ree distinct short

- term responses 

### 1

have been well

- characterized in humans challenged with O 3 : decreased pulmonary 

### 2
function, airways inflammation, and increased bronchial reactivity. In addition, O 3 

### 3
exposure exacerbates, and possibly also causes, asthma and allergic airways disease in 

### 4
humans. Animal studies have demonstrated airways remodeling and fibrotic changes in 

### 5
response to chronic and intermittent O 3 exposures and a wide range of other responses. 

### 6
While the RT is the primary target tissue, cardiovascular and other organ effects occur 

### 7
following short

- and long

- term exposures of animals to O 3
. 

### 8

Figure 5

- 8 The modes of action/possible pathways underlying the health effects resulting from inhalation exposure to ozone

.

The initial key event in the toxicity pathway of O 3 is the formation of secondary oxidation 

### 9
products in the RT. This mainly involves direct rea ct ions with components of the ELF

. 

### 10
The resulting secondary oxidation products transmit signals to the epithelium, 

### 11
nociceptive sensory nerve fibers and, if present, dendritic cells, mast cells and 

### 12
eosinophils. Thus, the effects of O 3 are mediated by compone nts of ELF and by the 

### 13 

## Mode of Action/Possible Pathways

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 changes Altered lung development

### Draft

– Do Not Cite or Quote 5

- 54 June 2012 multiple cell types found in the RT. Further, oxidative stress is an implicit part of this 

### 1
initial key event. 

### 2

Another key event in the toxicity pathway of O 3 is the activation of neural reflexes which 

### 3
lead to decrements in pulmonary function (see Section 6.2.1 ). Evidence is accumulating 

### 4
that secondary oxidation products are responsible for this effect. Eicosanoids have been 

### 5
imp licated in humans, while both eicosanoids and aldehydes are effective in animal 

### 6
models. Different receptors on bronchial C

- fibers have been shown to mediate separate 

### 7

effects of O 3 on pulmonary function. Nociceptive sensory nerves are involved in t he 

### 8

involu ntary truncation of ins piration which results in decreases in FVC, FEV 1 , tidal 

### 9
volume and pain upon deep inspiration. Opioids block these responses while atropine has 

### 10
only a minimal effect. New evidence in an animal model suggests that TRPA1 receptors 

### 11

on b ronchial C

- fibers mediate this pathway. Ozone exposure also results in activation of 

### 12
vagal sensory nerves and a mild increase in airway obstruction measured as increased 

### 13
sRaw. Atropine and β

- adrenergic agonists greatly inhibit this response in humans 

### 14

indic ating that the airway s obstruction is due to bronchoconstriction. Other studies in 

### 15
humans implicated SP release from bronchial C

- fibers resulting in airway narrowing due 

### 16
to either neurogenic edema or bronchoconstriction. New evidence in an animal model 

### 17

sug gests that the SP

- NK receptor pathway caused bronchoconstriction following O 3 

### 18
exposure. Activation of neural reflexes also results in extrapulmonary effects such as 

### 19
bradycardia. 

### 20

Initiation of inflammation is also a key event in the toxicity pathway of O 3
. Secondary 

### 21
oxidation products, as well as chemokines and cytokines elaborated by airway epithelial 

### 22
cells and macrophages, have been implicated in the initiation of inflammation. Vascular 

### 23
endothelial adhesion molecules may also play a role. W ork from severa l laboratories 

### 24
using human subjects and animal models suggest that O 3 triggers the release of 

### 25
tachykinins such as SP from airway sensory nerves which could contribute to 

### 26
downstream effects including inflammation (see Section 6.2.3 and Section 7.2.4 ). 

### 27
Airways neutrophilia has been demonstrated in BALF, mucosal biopsy and induced 

### 28
sputum samples. Influx of mast cells, monocytes and macrophages also occur. 

### 29

Inflammation further contributes t o O 3 - mediated oxidative stress. Recent investigations 

### 30

show that O 3 exposure leads to the generation of hyaluronan fragments from high 

### 31
molecular weight polymers of hyaluronan normally found in the ELF in mice. 

### 32
Hyaluronan activates TLR4 and CD44

- dependent si gnaling pathways in macrophages, 

### 33
and results in an increased number of macrophages in the BALF. Activation of these 

### 34
pathways occurs later than the acute neutrophilic response suggesting that they may 

### 35
contribute to longer

- term effects of O 3 . The mechanisms involved in clearing 

### 36

O 3 - provoked inflammation remain to be clarified. It should be noted that inflammation, 

### 37

### Draft

– Do Not Cite or Quote 5

- 55 June 2012 as measured by airways neutrophilia, is not correlated with decrements in pulmonary 

### 1
function as measured by spirometry. 

### 2

A fourth key event in the t oxicity pathway of O 3 is alteration of epithelial barrier 

### 3
function. Increased permeability occurs as a result of damage to tight junctions between 

### 4
epithelial cells subsequent to O 3 - induced injury and inflammation. It may play a role in 

### 5
allergic sensitizati on and in AHR (see Section 6.2.2 , Section 6.2.6 , and Section 7.2.5 ). 

### 6
Tachykinins mediate this response while antioxidants may confer protection. Genetic 

### 7
susceptibility has been associate d with a functioning Tlr4 and Nos2 genes. 

### 8

A fifth key event in the toxicity pathway of O 3 is the sensitization of bronchial smooth 

### 9
muscle. Increased bronchial reactivity can be both a rapidly occurring and a persistent 

### 10
response. The mechanisms responsible for early and later AHR are not well

- understood 

### 11

(see Section 6.2.2 ). One proposed mechanism of sensitization, O 3 - induced increases in 

### 12
epithelial permeability, would improve access of agonist to smooth muscle receptors. The 

### 13
evidence for this mechanism is not consistent. Another proposed mechanism, for which 

### 14
there is greater evidence, is neurally

- mediated sensitization. In humans exposed to O 3 , 

### 15

atrop ine blocked the early AHR response indicating the involvement of cholinergic 

### 16
postganglionic pathways. Animal studies demonstrated that O 3 - induced AHR involved 

### 17
vagally

- mediated responses and local axon reflex responses through bronchopulmonary 

### 18

C

- fiber

- media ted release of SP. Later phases of increased bronchial reactivity may 

### 19

involve the induction of IL

- 1β which in turn upregulates SP production. In guinea pigs, 

### 20
eosinophil

- derived major basic protein contributed to the stimulation of cholinergic 

### 21
postganglioni c pathways. A novel role for hyaluronan in mediating the later phase effects 

### 22

O 3 - induced AHR has recently been demonstrated. Hyaluronan fragments stimulated AHR 

### 23

in a TLR4

- and CD44 receptor

- dependent manner. Tachykinins and secondary oxidation 

### 24
products of O 3 have been proposed as mediators of the early response and inflammation



### 25
derived products have been proposed as mediators of the later response. Inhibition of 

### 26
arachidonic acid metabolism was ineffective in blocking O 3 - induced AHR in humans 

### 27

while in animal models mixed results were found. Other cytokines and chemokines have 

### 28
been implicated in the AHR response to O 3 in animal models. 

### 29

A sixth key event in the toxicity pathway of O 3 is the modification of innate/adaptive 

### 30
immunity. While the majority of evidence for this key event comes from animal studies, 

### 31
there are several studies suggesting that this pathway may also be relevant in humans. 

### 32
Ozone exposure of human subjects resulted in recruitment of activated innate immune 

### 33
cells to the airways. This included macrophages and monocytes with increased 

### 34
expression of cell surface markers characteristic of innate immunity and antigen 

### 35
presentation, the latter of which could contribute to ex aggerated T

- cell responses and the 

### 36
promotion of an allergic phenotype. Evidence of dendritic cell activation was observed in 

### 37

### Draft

– Do Not Cite or Quote 5

- 56 June 2012 GSTM1 null human subjects exposed to O 3 , suggesting O 3 - mediated interaction between 

### 1

the innate and adaptive immune systems. Animal studies further linked O 3 - mediated 

### 2
activation of the innate immune system to the development of nonspecific AHR, 

### 3
demonstrated an interaction between allergen and O 3 in the induction of nonspecific 

### 4

AHR, and found that O 3 acted as an adjuvant for allergic se nsitization through the 

### 5
activation of both innate and adaptive immunity. Priming of the innate immune system by 

### 6

O 3 was reported in mice. This resulted in an exaggerated response to endotoxin which 

### 7
included enhanced TLR4 signaling in macrophages. Ozone

- medi ated impairment of the 

### 8

function of SP

- A, an innate immune protein, has also been demonstrated. Taken together 

### 9
these studies provide evidence that O 3 can alter host immunologic response and lead to 

### 10
immune system dysfunction. These mechanisms may underlie th e exacerbation and 

### 11

induction of asthma (see Section 6.2.6 and Section 7.2.1 ), as well as decreases in host 

### 12

defense (see Section 6.2.5 and Section 7.2.6 ). 

### 13

Another key event in the toxicity pathway of O 3 is airways remodeling. Persistent 

### 14
inflammation and injury, which are observed in animal models of chronic and 

### 15
intermittent exposure to O 3 , are associated with morphologic changes such as mucous 

### 16
cell metaplasia of nasal epithelium, bronchiolar metaplasia of alveolar ducts and fibrotic 

### 17
changes in small airways (see Section 7.2.3 ). Mechanisms responsible for these responses 

### 18

are not well

- understood. However a recent study in mice demonstrated a key role for the 

### 19

TGF

- β signaling pathway in the deposition of collagen in t he airway wall following 

### 20
chronic intermittent exposure to O 3 . Chronic intermittent exposure to O 3 has also been 

### 21
shown to result in effects on the developing lung and immune system. 

### 22

Systemic inflammation and vascular oxidative/nitrosative stress are also k ey events in the 

### 23
toxicity pathway of O 3 . Extrapulmonary effects of O 3 occur in numerous organ systems, 

### 24
including the cardiovascular, central nervous, reproductive and hepatic systems (see 

### 25

Section 6.3 to Section 6.5 and Section 7.3 to Section 7.5 ). It has been proposed that lipid 

### 26
oxidation products resulting from reaction of O 3 with lipids and/or cellular membranes in 

### 27
the ELF are responsible for systemic responses, however it is not known whether they 

### 28
gain access to the vascular space. Alternatively, release of diffusible mediators from the 

### 29
lung into the circulation may initiate or p ropagate inflammatory responses in the vascular 

### 30
or in systemic compartments. 

### 31



## 5.4 Interindividual Variability in Response

Responses to O 3 exposure are variable within the population ( Mudway and Kelly, 2000 )

. 

### 32
Some studies have shown a large range of pulmonary function responses to O 3 among 

### 33
healthy young adults (i.e., 4 hours to 200 ppb O 3 or for 1.5 hours to 420 ppb O 3 while 

### 34

### Draft

– Do Not Cite or Quote 5

- 57 June 2012 exercising at a moderate level) ( Hazucha et al., 2003 ; Balmes et al., 1996 ) . Since 

### 1
individual responses were relatively consistent across time in these studies, it was thought 

### 2
that responsiveness reflected an intrinsic characteristic of the subject ( Mudway and Kelly, 

### 3

2000 ) . Other studies have shown that age and body mass index may influence 

### 4
responsiveness to O 3. In h uman subjects exercising moderately and exposed to 420 ppb 

### 5

O 3 for 1.5 hours, older adults were generally not responsive to O 3 ( Hazucha et al., 2003 )

, 

### 6
while obese young women appeared to be more responsive than lean young women 

### 7

( Bennett et al., 2007 ) . In another study, the observed lack of spirometric responsiveness 

### 8

in one group of human subjects was not attributable to the presence of endogenous 

### 9
endorphins, which could vary between individuals and which could potentially block C



### 10
fiber stimulation by O 3 (420 ppb, 2 hours, moderate exercise ( Passannante et al., 1998 )

. 

### 11
Other responses to O 3 have also been characterized by a large degree of interindividual 

### 12
variability. For example, interindividual variability in the neutrophilic response has been 

### 13
noted in human subjects ( Holz et al., 1999 ; Devlin et al., 1991 ; Schelegle et al., 1991 )

. 

### 14
One study demonstrated a 3

- fold difference in airways neutrophilia, measured as percent 

### 15
of total cells in proximal BALF, among hum an subjects exposed to 300 ppb O 3 for 1 hour 

### 16
while exercising at a heavy level ( Schelegle et al., 1991 ) . Another study reported a 

### 17

20

- fold difference in BAL neutrophils following exposure to 80

- 100 ppb O 3 for 6.6 hours 

### 18
in human subjects exercising at a moderate level ( Devlin et al., 1991 ) . In contrast, 

### 19
reproducibility of intraindividual responses to 1

- hour exposure to 250 ppb O 3 in human 

### 20
subjec ts exercising at a light level, measured as sputum neutrophilia, was demonstrated 

### 21
by Holz et al. (1999 ) . While the basis for the observed interindividual variability in 

### 22
responsiveness to O 3 is not clear, both dosimetric and mechanistic factors are likely to 

### 23

contrib ute and will be discussed below. 

### 24

5.4.1 Dosimetric Considerations

Two studies have investigated the correlation of O 3 uptake with the pulmonary function 

### 25
responses to O 3 exposure ( Reeser et al., 2005 ; Gerrity et al., 1994 ) . These studies foun d 

### 26
that the large subject

- to

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

### 27
have a dosimetric explanation. Reeser et al. (2005 ) found no significa nt relationship 

### 28

between %ΔFEV 1 and fractional absorption of O 3 using the bolus method. Contrary to 

### 29
previous findings , the percent change in dead space volume of the respiratory tract 

### 30
(%ΔV D ) did not correlate with O 3 uptake, possibly due to the contraction of dead space 

### 31
caused by airway closure. Gerrity et al. (1994 ) found that intersubject vari ability in FEV 1 

### 32
and airway resistance was not related to differences in the O 3 dose delivered to the lower 

### 33
airways, whereas minute ventilation was predictive of FEV 1 decrement. No study has yet 

### 34
demonstrated that subjects show a consistent pattern of O 3 ret ention when re

- exposed 

### 35

### Draft

– Do Not Cite or Quote 5

- 58 June 2012 over weeks of time, as has been shown to be the case for the FEV 1 response, or that 

### 1

within

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

### 2

However, these studies did not control for the differences in cond ucting airway volume 

### 3
between individuals. By controlling for conducting airway volume, it may be possible to 

### 4
estimate how much of the intersubject variation in FEV 1 response at a given O 3 exposure 

### 5

is due to actual inter

- individual variability in dose. 

### 6

5.4.2 Mec hanistic Considerations

This section considers mechanistic factors that may contribute to variability in responses 

### 7
between individuals. It has been proposed that some of the variability may be genetically 

### 8

determined ( Yang et al., 2005a ) . Besides gene

- environment interactions, other factors 

### 9
such as pre

- existing diseases and conditions, nutritional status, lifestage, attenuation, and 

### 10

co

- ex posures may also contribute to inter

- individual variability in responses to O 3 and are 

### 11

discussed below. 

### 12

5.4.2.1 Gene

- environment Interactions

The pronounced interindividual variation in responses to O 3 infers that genetic 

### 13
background may play an important role in responsiveness to O 3 ( Cho and Kleeberger, 

### 14

2007 ; Kleeberger et al., 1997 ) (see also Section 8.4 ). Strains of mice which are prone or 

### 15

resist ant to O 3 - induced effects have been used to systematically identify candidate 

### 16
susceptibility genes. Using these recombinant inbred strains of mice and exposures to 

### 17

0.3 ppm O 3 for up to 72 hours, genome wide linkage analyses (also known as positional 

### 18

clonin g) demonstrated quantitative trait loci for O 3 - induced lung inflammation and 

### 19
hyperpermeability on chromosome 17 ( Kleeberger et al., 1997 ) and chromosome 4 

### 20

( Kleeberger et al., 2000 ) , respectively. More specifically, these studies found that Tnf

, 

### 21
whose protein product is the inflammatory cytokine TNF

- α, and Tlr4 , whose protein 

### 22
product is TLR4, were candidate susceptibility genes ( Kleeberger et al., 2000 ; Kleeberger 

### 23

et al., 1997 ) . Other studies, which used targeted deletion, identified genes encoding iNOS 

### 24

and heat sho ck proteins as TLR4 effector genes ( Bauer et al., 2011 ; Kleeberger et al., 

### 25

2001 ) and f ound that IL

- 10 protects against O 3 - induced pulmonary inflammation ( Backus 

### 26

et al., 2010 ) . Investigations in inbred mouse strains found that differences in expression 

### 27
of certain proteins, such as CCSP (1.8 ppm O 3 for 3 hours) ( Broeckaert et al., 2003 ) and 

### 28

MARCO (0.3 ppm O 3 for up to 48 hours) ( Dahl et al., 2007 ) , were responsible for 

### 29
phenotypic characteristics, such as epithelial permeability and scav enging of oxidized 

### 30
lipids, respectively, which confer sensitivity to O 3
. 

### 31

### Draft

– Do Not Cite or Quote 5

- 59 June 2012 Genetic polymorphisms have received increasing attention as modulators of O 3 - mediated 

### 1
effects. Functionally relevant polymorphisms in candidate susceptibility genes have been 

### 2

studied at the individual and population level in humans, and also in animal models. 

### 3
Genes whose protein products are involved in antioxidant defense/oxidative stress and 

### 4
xenobiotic metabolism, such as glutathione

- S

- transferase M1 (GSTM1) and 

### 5
NADPH:quinone oxidor eductase 1 (NQO1), have also been a major focuses of these 

### 6
efforts. This is because oxidative stress resulting from O 3 exposure is thought to 

### 7
contribute to the pathogenesis of asthma, and because xenobiotic metabolism detoxifies 

### 8
secondary oxidation product s formed by O 3 which contribute to oxidative stress ( Islam et 

### 9

al., 2008 ) . TNF

- α is of interest since it is linked to a candidate O 3 susceptibility gene a nd 

### 10
since it plays a key role in initiating airways inflammation ( Li et al., 2006d )

. 

### 11
Polymorphisms of genes coding for GSTM1, NQO1 and TNF

- α have been associated 

### 12

with altered ri sk of O 3 - mediated effects ( Li et al., 2006d ; Yang et al., 2005a ; Romieu et 

### 13

al., 2004b ; Corradi et al., 2002 ; Bergamaschi et al., 2001 ) . Additional studies have 

### 14
focused on functional variants in other genes involved in antioxidant defense such as 

### 15

catalase ( CAT ), myeloperoxidase, heme oxygenase ( HMOX

- 1 ) and manganese 

### 16
superoxide dismutase ( MnSOD ) ( Wenten et al., 2009 ; Islam et a l., 2008 ) . These studies 

### 17

are discussed below. 

### 18

GSTM1 is a phase II antioxidant enzyme which is transcriptionally regulated by 

### 19

NF

- e2

- related factor 2

- antioxidant response element (Nrf2

- ARE) pathway. A large 

### 20
proportion (40

- 50%) of the general public (across ethnic populations) has the 

### 21

GSTM1

- null genotype, which has been linked to an increased risk of health effects due to 

### 22
exposure to air pollutants ( London, 2007 ) . A role for GSTs in metabolizing electrophil es 

### 23

such as 4

- hydroxynonenal, which is a secondary oxidation product resulting from O 3 

### 24
exposure, has been demonstrated ( Awasthi et al., 2004 ) . A recent study found that the 

### 25
GSTM1 genotype modulated the time course of the neutrophilic inflammatory response 

### 26
following acute O 3 exposure (400 ppb for 2 hours with light to moderate exercise) in 

### 27
healthy adults ( Alexis et al., 2009 ) . In GSTM1

- null and

- sufficient subjects, O 3 - induced 

### 28
sputum neutrophilia was similar at 4 hours. However, neutrophilia r esolved by 24 hours 

### 29
in sufficient subjects but not in GSTM1

- null subjects. In contrast, no differences in 

### 30

24 hour sputum neutrophilia were observed between GSTM1

- null and

- sufficient human 

### 31
subjects exposed to 60 ppb O 3 for 2 hours with moderate exercise ( Kim et al., 2011 ) . It is 

### 32
not known whether the effect seen at the higher exposure level ( Alexis et al., 2009 ) was 

### 33
due to the persistence of pro

- inflammatory stimuli, impaired production of 

### 34
downregulators or impaired neutrophil apoptosis and clearance . However, a subsequent 

### 35
in vitro study by these same investigators found that GSTM1 deficiency in airway 

### 36
epithelial cells enhanced IL

- 8 production in response to 0.4 ppm O 3 for 4 hours ( Wu et 

### 37

al., 2011 ) . Furthermore, NF

- κB activation was required for O 3 - induced IL

- 8 production 

### 38

( Wu et al., 2011 ) . Since IL

- 8 is a poten t neutrophil activator and chemotaxin, this study 

### 39

### Draft

– Do Not Cite or Quote 5

- 60 June 2012 provides additional evidence for the role of GSTM1 as a modulator of inflammatory 

### 1
responses due to O 3 exposure. 

### 2

In addition, O 3 exposure increased the expression of the surface marker CD14 in airway 

### 3

neutro phils of GSTM1

- null subjects to a greater extent than in sufficient subjects ( Alexis 

### 4

et al., 2009 ) . Furthermore, differences in airway macrophages were noted between the 

### 5

GSTM1

- sufficient and

- null subjects. Numbers of airway macrophages were decreased at 

### 6

4 and 24 hours following O 3 exposure in GSTM1

- sufficient subjects ( Alexis et al., 2009 )

. 

### 7
Airway macrophages in GSTM1

- null subjects were greater in number and found to have 

### 8
greater oxidative burst and phagocytic capability than those of sufficient subjects. Airway 

### 9
macrophages and dendritic cells from GSTM1

- null subjects exposed to O 3 expressed 

### 10
higher levels of the surface marker HLA

- DR, suggesting activation of the innate immune 

### 11

system ( Alexis et al., 2009 ) . These differences in inflammatory responses between the 

### 12

GSTM1

- null and

- sufficient subjects may provide biological plau sibility for the 

### 13

differences in O 3 - mediated effects reported in controlled human exposure studies 

### 14

( Corradi et al., 2002 ; Bergamaschi et al., 2001 ) . It should also be noted that GSTM1 

### 15
genotype did not affect the acute pulmonary function (i.e., spirometric) response to O 3 

### 16
which provides additional evidence for separate mechanisms underlying the effects of O 3 

### 17
on pulmona ry function and inflammation in adults ( Alexis et al., 2009 ) . However, 

### 18

GSTM1

- null asthmatic children were previously found to be more at risk of O 3 - induced 

### 19
effects on pulmonary function than GSTM1

- sufficient asthmatic children ( Romieu et al., 

### 20

2004b )

. 

### 21

Another enzyme involved in the metabolism of secondary oxidation products is NQO1. 

### 22
NQO1 catalyzes the 2

- electron reduction by NADPH of quinones to hydroquinones. 

### 23
Depending on the substrat e, it is capable of both protective detoxification reactions and 

### 24
redox cycling reactions resulting in the generation of reactive oxygen species. A recent 

### 25
study using NQO1

- null mice demonstrated that NQO1 contributes to O 3 - induced 

### 26

oxidative stress, AHR and inflammation following a 3

- hour exposure to 1 ppm O 3 

### 27

( Voynow et al., 2009 ) . These experimental results may provide biological plausibility for 

### 28
the increased biomarkers of oxidative stress and increased pulmonary function 

### 29

decrements observed in O 3 - exposed individuals bearing both the wild

- type NQO1 gene 

### 30

and the n ull GSTM1 gene ( Corradi et al., 2002 ; Bergamaschi et al., 2001 )

. 

### 31

Besides enzymatic metabolism , other mechanisms p articipate in the removal of 

### 32
secondary oxidation products formed as a result of O 3 inhalation. One involves 

### 33
scavenging of oxidized lipids via the macrophage receptor with collagenous structure 

### 34
(MARCO) expressed on the cell surface of alveolar macrophages. A recent study 

### 35
demonstrated increased gene expression of MARCO in the lungs of an O 3 - resistant C3H 

### 36

mouse strain (HeJ) but not in an O 3 - sensitive, genetically similar strain (OuJ) ( Dahl et al., 

### 37

### Draft

– Do Not Cite or Quote 5

- 61 June 2012 2007 ) . Upregulation of MARCO occur red in mice exposed to 0.3 ppm O 3 for 

### 1

24

- 48 hours; inhalation exposure for 6 hours at this concentration was insufficient for this 

### 2
response. Animals lacking the MARCO receptor exhibited greater inflammation and 

### 3
injury, as measured by BAL neutrophils, prote in and isoprostanes, following exposure to 

### 4

0.3 ppm O 3 ( Dahl et al., 2007 ) . MARCO also protected against the inflammatory effects 

### 5
of oxidized surfactant lipids ( Dahl et al., 2007 ) . Scavenging of oxidized lipids may limit 

### 6

O 3 - induced injury since ozonized cholesterol species formed in the ELF (mice, 0.5

- 3 ppm 

### 7

O 3 , 3 hours) ( Pulfer et al., 2005 ; Pulfer and Murphy, 2004 ) stimulated apoptosis and 

### 8
cytotoxicity in vitro ( Gao et al., 2009b ; Sathishkumar et al., 2009 ; Sathishkumar et al., 

### 9

2007b ; Sathishkumar et al., 2007a )

. 

### 10

Two studie s reported relationships between TNF promoter variants and O 3 - induced 

### 11
effects in humans. In one study, O 3 - induced change in lung function was significa ntly 

### 12
lower in adult subjects with TNF promoter variants

- 308A/A and

- 308G/A compared with 

### 13
adult subjects with the variant

- 308G/G ( Yang et al., 2005a ) . This response was 

### 14
modulated by a specific polymorphism of LTA ( Yang et al., 2005a ) , a previously 

### 15
identified candidate susceptibility gene whose protein product is lymphotoxin

- α 

### 16

( Kleeberger et al., 1997 ) . In the second study, an association between the TNF promoter 

### 17

variant

- 308G/G and decreased risk of asthma and lifetime wheezing in children was 

### 18

found ( Li et al., 2006d ) . The protective effect on wh eezing was modulated by ambient O 3 

### 19
levels and by GSTM1 and GSTP1 polymorphisms. The authors suggested that the 

### 20

TNF

- 308 G/G genotype may have a protective role in the development of childhood 

### 21

asthma ( Li et al., 2006d )

. 

### 22

Similarly, a promoter variant of the gene HMOX

- 1 , consisting of a smaller number of 

### 23
(GT)n repeats, was associated with a reduced risk for new

- onset asthma in non

- Hispanic 

### 24

white children ( Islam et al., 2008 ) . The number of (GT)
n repeats in this promoter has 

### 25
been shown to be inversely related to the inducibility of HMOX

- 1 . A modulatory effect of 

### 26

O 3 was demonstrated since the beneficial effects of this polymorphism were seen only in 

### 27
children living in low O 3 communities ( Islam et al., 2008 ) . This st udy also identified an 

### 28
association between a polymorphism of the CAT gene and increased risk of new

- onset 

### 29
asthma in Hispanic children; however no modulation by O 3 was seen ( Islam et al., 2008 )

. 

### 30
No association was observed in this study between a MnSOD polymorphism and asthma 

### 31

( Islam et al., 2008 )

. 

### 32

Studies to date indicate that some variability in individual responsiveness to O 3 may be 

### 33
accounted for by functional genetic polymorphisms. Further, the effects of 

### 34

gene

- environment interactions may be different in children and adults. 

### 35

### Draft

– Do Not Cite or Quote 5

- 62 June 2012 5.4.2.2 Pre

- existing Diseases and Con ditions

Pre

- existing diseases and conditions can alter the response to O 3 exposure. For example, 

### 1
responsiveness to O 3 , as measured by spirometry, is decreased in smokers and individuals 

### 2

with COPD ( U.S. EPA, 2006b ) . Asthma and allergic diseases are of major importance in 

### 3

this discussion. In individuals with asthma, th ere is increased responsiveness to 

### 4
bronchoconstrictor challenge. This results from a combination of structural and 

### 5
physiological factors including increased airway inner

- wall thickness, smooth muscle 

### 6
responsiveness and mucus secretion. Although inflammatio n is likely to contribute, its 

### 7
relationship to AHR is not clear ( U.S. EPA, 2006b ) . However, some asthmatics have 

### 8
higher baseline levels of neutrophils, lymphocytes, eosinophils and mast cells in 

### 9
bronchial washes and bronchial biopsy tissue ( Stenfors et al., 2002 ) . It has been proposed 

### 10
that enhanced sensitivity to O 3 is conferred by the presence of greater numbers of resident 

### 11
airway inflammatory cells in disease states such as asthma ( Mudway and Kelly, 2000 )

. 

### 12

In order to determine whether asthmatics exhibit greater responses to O 3 , several earlier 

### 13
studies compared pulmonary function in asthma tic and non

- asthmatic subjects following 

### 14

O 3 exposure. Some also probed mechanisms which could account for enhanced 

### 15
sensitivity. While the majority focused on measurements of FEV 1 and FVC and found no 

### 16
differences between the two groups following exposures o f 2

- 4 hours to 125

- 250 ppb O 3 

### 17

or to a 30

- minute exposure to 120

- 180 ppb O 3 by mouthpiece in human subjects 

### 18
exercising at a light to moderate level ( Stenfors et al., 2002 ; Mudway et al., 2001 ; Holz et 

### 19

al., 1999 ; Scannell et al., 1996 ; Koenig et al., 1987 ; Linn et al., 1978 ) , there were notable 

### 20
exceptions. In one study, greater airway s obstruction in asthmatics compared with non



### 21
asthmatic subjects was observed immedia tely following a 2

- hour exposure to 400 ppb O 3 

### 22
while exercising at a heavy level ( Kreit et al., 1989 ) . These changes were measured as 

### 23
statistically significa nt greater decreases in FEV 1 and in FEF 25

- 75 (but not in FVC) in the 

### 24
absence of a bronchoconstrictor challenge ( Kreit et al., 1989 ) . These results suggest that 

### 25
this group of asthmatics responded to O 3 - exposure with a greater degree of v agally



### 26
mediated bronchoconstriction compared with the non

- asthmatics. A second study 

### 27
demonstrated a statistically significa nt greater decrease in FEV 1 and in FEV 1 /FVC (but 

### 28
not in FVC) in asthmatics compared with non

- asthmatics exposed to 160 ppb O 3 for 

### 29

7.6 hours with light exercise ( Horstman et al., 1995 ) . These responses were accompanied 

### 30
by wheezing and inhaler use in the asthmatics ( Horstman et al., 1995 ) . Aerosol bolus 

### 31
dispersion measurements demonstrated a statistically significa nt greater change in 

### 32
asthmatics compared with non

- asthmatics, which was suggestive of O 3 - induced small 

### 33
airway dysfunction ( Horstman et al., 1995 ) . Furthermore, a statistically significa nt 

### 34
correlation was observed between the degree of base line airway status and the FEV 1 

### 35
response to O 3 in the asthmatic subjects ( Horstman et al., 1995 ) . A third study found 

### 36

similar decreases in FVC and FEV 1 in both asthmatics and non

- asthmatics exposed to 

### 37

### Draft

– Do Not Cite or Quote 5

- 63 June 2012 400 ppb O 3 for 2 hours with light exercise ( Alexis et al., 2000 ) . However, a statistically 

### 1
significa nt decrease in FEF 75 , a measu re of small airway function, was observed in 

### 2

asthmatics but not in non

- asthmatics ( Alexis et al., 2000 ) . Taken together, these latter 

### 3
studies indicate that while the magnitude of restrictive type spirometric decline was 

### 4

similar in asthmatics and non

- asthmatics, that obstructive type changes 

### 5

(i.e., bronchoconstriction) were greater in asthmatics. Further, asthmatics exhibited 

### 6
greater sensitivity to O 3 in terms of small airways funct ion. 

### 7

Since asthma exacerbations occur in response to allergens and/or other triggers, some 

### 8

studies have focused on O 3 - induced changes in AHR following a bronchoconstrictor 

### 9
challenge. No difference in sensitivity to methacholine bronchoprovocation was obse rved 

### 10

between asthmatics and non

- asthmatics exposed to 400 ppb O 3 for 2 hours while 

### 11
exercising at a heavy level ( Kreit et al., 1989 ) . However, increased bronchial reactivity to 

### 12
inhaled allergens was demonstrated in mild allergic asthmatics exposed to 160 ppb for 

### 13

7.6 hours, 250 ppb for 3 hours and 120 ppb for 1 hour while exercising at a light level or 

### 14

at rest ( Kehrl et al., 1999 ; Jorres et al., 1996 ; Molfino et al., 1991 ) and in allergen



### 15
sensitized guinea pigs following O 3 exposure (1 ppm, 1 hour) ( Sun et al., 1997 ) . Similar, 

### 16
but modest, responses were reported for individuals with allergic rhinitis ( Jorres et al., 

### 17

1996 ) . Further, the contractile response of isolated airways from human donor lung 

### 18
tissue, which were sensitized and challenged with allergen, was increased by 

### 19

pre

- exposure to 1 ppm O 3 fo r 20 ( Roux et al., 1999 ) . These studies provide support for 

### 20

O 3 - mediated enhancement of responses to allergens in allergic subjects. 

### 21

In terms of airways neutrophilia, larger responses were observed in asthmatics compared 

### 22

to non

- asthmatics subjects, who were exercising at a light to moderate level and exposed 

### 23

to O 3 , in some ( Balmes et al., 1997 ; Scannell et al., 1996 ; Basha et al., 1994 ) but not all 

### 24

( Mudway et al., 2001 ) of the earlier studies. While each of these studies involved 

### 25
exposure of exercising human subjects to 200 ppb O 3 , the duration of exposure was 

### 26
longer (i.e., 4

- 6 hours) in the former studies than in the latter study (2 hours). Further, 

### 27
statistically significa ntly increases in myeloperoxidase levels (an indicator of neutrophil 

### 28
activation) in bronchial washes was observed in mild asthmatics compared with n on



### 29
asthmatics, despite no difference in O 3 - stimulated neutrophil influx between the 2 groups 

### 30
following exposure to 200 ppb O 3 for 2 hours with moderate exercise ( Stenfors et al., 

### 31

2002 ) . A more recent study found that atopic asthmatic subjects exhibited an enhanced 

### 32
inflammatory response to O 3 (400 ppb, 4 hours, with light to moderate exercise) 

### 33

( Hernandez et al., 2010 ) . This response was characterized by greater numbers of 

### 34
neutrophil s, higher levels of IL

- 6, IL

- 8 and IL

- 1β and greater macrophage cell

- surface 

### 35
expression of TLR4 and IgE receptors in induced sputum compared with healthy 

### 36
subjects. This study also reported a greater increase in hyaluronan in atopic subjects and 

### 37
atopic asth matics compared with healthy subjects following O 3 exposure. Animal studies 

### 38

### Draft

– Do Not Cite or Quote 5

- 64 June 2012 have previously reported that hyaluronic acid activates TLR4 signaling and results in 

### 1

AHR (see Section 5.3.5 ). Furthermore, levels of IL

- 10, a potent anti

- inflammatory 

### 2
cytokine, were greatly reduced in atopic asthmatics compared to healthy subjects. These 

### 3
results provide evidence that innate immune and adaptive responses are d ifferent in 

### 4
asthmatics and healthy subjects exposed to O 3
. 

### 5

Eosinophils may be an important modulator of responses to O 3 in asthma and allergic 

### 6
airways disease. Eosinophils and associated proteins are thought to affect muscarinic 

### 7
cholinergic receptors whic h are involved in vagally

- mediated bronchoconstriction 

### 8

( Mudway and Kelly, 2000 ) . Studies described in Section 5.3.5 which demonstrated a key 

### 9
role of eosinophils in O 3 - mediated AHR may be relevant to human allergic airways 

### 10
disease which is characterized by airways eosinophilia ( Yost et al., 2 005 ) . Furthermore, 

### 11

O 3 exposure sometimes results in airways eosinophilia in allergic subjects or animal 

### 12
models. For example, eosinophilia of the nasal and other airways was observed in 

### 13
individuals with pre

- existing allergic disease following O 3 inhalati on (160 ppb, 7.6 hours 

### 14
with light exercise and 270 ppb, 2 hours with moderate exercise) ( Vagaggini et al., 2002 ; 

### 15

Peden et al., 1997 ) . Further, O 3 exposure (0.5 ppm, 8 hours/day for 1

- 3 days) increased 

### 16
allergic responses, such as eosinophilia and augmented intraepithelial mucosubstances, i n 

### 17
the nasal airways of ovalbumin (OVA)

- sensitized rats ( Wagner et al., 2002 ) . In contrast, 

### 18

Stenfors et al. (2002 ) found no stimulation of eosinophil influx measured in bronchia l 

### 19
washes and BALF of mild asthmatics following exposure to a lower concentration 

### 20

(200 ppb, 2 hours, with moderate exercise) of O 3
. 

### 21

The role of mast cells in O 3 - mediated asthma exacerbations has been investigated. Mast 

### 22
cells are thought to play a key role in O 3 - induced airways inflammation, since airways 

### 23
neutrophilia was decreased in mast cell

- deficient mice exposed to O 3 ( Kleeberger et al., 

### 24

1993 ) . However, another study found that mast cells were not involved in the 

### 25
development of increased bronchial reactivity in O 3 - exposed mice ( Noviski et al., 1999 )

. 

### 26

Nonethele ss, mast cells release a wide variety of important inflammatory mediators 

### 27
which may lead to asthma exacerbations ( Stenfors et al., 2002 ) . A large increase in mast 

### 28

cell number in bronchial submucosa was observed in non

- asthmatics and a significa nt 

### 29
decrease in mast cell number in bronchial epithelium was observed in mild asthmatics 

### 30

6 hours following exposure to 200 ppb O 3 for 2 hours during mild exercise ( Stenfors et 

### 31

al., 2002 ) . While these results point to an O 3 - mediated flux in bronchial mast cell 

### 32
populations which differed between the non

- asthmatics and mild asthmatics, 

### 33
interpretation of these findings is difficult. Furthermore, mast cell number did not change 

### 34
in airway lavages in either group in response to O 3 ( Stenfors et al., 2002 ) 

### 35

Cytokine profi les in the airways have been investigated as an indicator of O 3 sensitivity. 

### 36
Differences in epithelial cytokine expression were observed in bronchial biopsy samples 

### 37

### Draft

– Do Not Cite or Quote 5

- 65 June 2012 in non

- asthmatic and asthmatic subjects both at baseline and 6

- hours postexposure to 

### 1

200 pp b O 3 for 2 hours with moderate exercise ( Bosson et al., 2003 ) . The asthmatic 

### 2
subjects had a higher baseline expression of IL

- 4 and IL

- 5 compared to non

- asthmatics. 

### 3
In addition, expression of IL

- 5, IL

- 8, GM

- CSF, and ENA

- 78 in asthmatics was increased 

### 4
significa ntly following O 3 exposure c ompared to non

- asthmatics ( Bosson et al., 2003 )

. 

### 5

Some of these (IL

- 4, IL

- 5 and GM

- CSF) are Th2

- related cytokines or neutrophil 

### 6
chemoattractants, and play a role in IgE production, airways eosinophilia and suppression 

### 7

of Th1

- cytokine production ( Bosson et al., 2003 ) . These findings suggest a link between 

### 8
adaptive immunity and enhanced responses of asthmatics to O 3
. 

### 9

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

### 10

such as asthma ( Mudway and Kelly, 2000 ) . This could possibly be due to ongoing 

### 11
inflammation which causes antioxidant depletion or to abnormal antioxidant transport or 

### 12
synthesis ( Mudway and Kelly, 2000 ) . For example, basal levels of AH2 were 

### 13
significa ntly lower and basal levels of oxidized GSH and UA were significa ntly higher in 

### 14

bronchial wash fluid and BALF of mild asthmatics compared with healthy control 

### 15
subjects ( Mudway et al., 2001 ) . Differences in ELF antioxidant content have also been 

### 16
noted between species. These observations have led to the suggestion that the amount and 

### 17
composition of ELF antiox idants, the capacity to replenish antioxidants in the ELF or the 

### 18
balance between beneficial and injurious interactions between antioxidants and O 3 may 

### 19

contribute to O 3 sensitivity, which varies between individuals and species ( Mudway et 

### 20

al., 2006 ; Mudway and Kelly, 2000 ; Mudway et al., 1999a ) . The complexity of these 

### 21
interactions was demonstrated by a study in which a 2

- hour exposure to 200 ppb O 3 , 

### 22
while exercising at a moderate level, resulted in similar increases in airway neutrophils 

### 23
and decreases in pulmonary function in both mild asthmatics and healthy controls, 

### 24
despite differences in ELF antioxidant concentrations prior to O 3 exposure ( Mudway et 

### 25

al., 2001 ) . Further, the O 3 - induced increase in oxid ized GSH and decrease in AH2 

### 26
observed in ELF of healthy controls was not observed in mild asthmatics ( Mudway et al., 

### 27

2001 ) . While the authors concluded that basal AH2 and oxidized GSH concentrations 

### 28
were not predictive of responsiveness to O 3 , they also suggested t hat the increased basal 

### 29
UA concentrations in the mild asthmatics may have played a protective role ( Mudway et 

### 30

al., 2001 ) . Thus compensatory mechanisms resulting in enhanced total antioxidant 

### 31
capacity may play a role in modulating responses to O 3
. 

### 32

Collectively thes e older and more recent studies provide insight into mechanisms which 

### 33
may contribute to enhanced responses of asthmatic and atopic individuals following O 3 

### 34
exposure. Greater airways inflammation and/or greater bronchial reactivity have been 

### 35

demonstrated in asthmatics compared to non

- asthmatics. This pre

- existing inflammation 

### 36
and altered baseline bronchial reactivity may contribute to the enhanced 

### 37
bronchoconstriction seen in asthmatics exposed to O 3 . Furthermore, O 3 - induced 

### 38

### Draft

– Do Not Cite or Quote 5

- 66 June 2012 inflammation may contribute to O 3 - mediated AHR. An enhanced neutrophilic response to 

### 1

O 3 has been demonstrated in some asthmatics. A recent study in humans provided 

### 2
evidence for differences in innate immune responses related to TLR4 signaling between 

### 3
asthmatics and healthy subjects. Animal studies have demonstrated a role for eosinophil



### 4
derived proteins in mediating the effects of O 3 . Since airways eosinophilia occurs in both 

### 5
allergic humans and allergic animal models, this pathway may underlie the exacerbation 

### 6
of allergic asthma by O 3 . In a ddition, differences have been noted in epithelial cytokine 

### 7
expression in bronchial biopsy samples of healthy and asthmatic subjects. A Th2 

### 8
phenotype, indicative of adaptive immune system activation and enhanced allergic 

### 9
responses, was observed before O 3 e xposure and was increased by O 3 exposure in 

### 10
asthmatics. These findings support links between innate and adaptive immunity and 

### 11
sensitivity to O 3 - mediated effects in asthmatics and allergic airways disease. 

### 12

In addition to asthma and allergic diseases, obesi ty may alter responses to O 3 . While O 3 is 

### 13
a trigger for asthma, obesity is a known risk factor for asthma ( Shore, 2007 ) . The 

### 14
relationship between obesity and asthma is not well understood but recent investi gations 

### 15
have focused on alterations in endocrine function of adipose tissue in obesity. It is 

### 16
thought that the increases in serum levels of factors produced by adipocytes 

### 17

(i.e., adipokines)

, such as cytokines, chemokines, soluble cytokine receptors and ene rgy 

### 18
regulating hormones, may contribute to the relationship between obesity and asthma. 

### 19
Some of these same mechanisms may be relevant to insulin resistant states such as 

### 20
metabolic syndrome. 

### 21

In a re analysis of the data of Hazucha et al. (2003 ) , increasing body mass index in 

### 22
young women was associated with increased O 3 responsiveness, as measured by 

### 23
spirometry following a 1.5

- hour exposure to 420 ppb O 3 while exercising at a moderate 

### 24

level ( Bennett et al., 2007 ) . In several mouse models of obesity, airways were found to be 

### 25
innately more hyperresponsive and responded more vigorously to acute O 3 exposure than 

### 26

lean controls ( Shore, 2007 ) . Pulmonary inflammatory and injury in response to O 3 were 

### 27

also enhanced ( S hore, 2007 ) . It was postulated that oxidative stress resulting from 

### 28
obesity

- related hyperglycemia could account for these effects ( Shore, 2007 ) . However, 

### 29
responses to O 3 in the different mouse models are somewhat variable and depend on 

### 30
whether exposures are acute or subacute. For example, diet

- induced obesity augmented 

### 31
inflammation and injury, as measured by BALF markers, and enhanced AHR in mice 

### 32
exposed acutely to O 3 (2 ppm, 3 hours) ( Joh nston et al., 2008 ) . In contrast, the 

### 33
inflammatory response following sub

- acute exposure to O 3 was dampened by obesity in a 

### 34

different mouse model (0.3 ppm, 72 hours) ( Shore et al., 2009 ) . It is not known whether 

### 35
differences in responsiveness to O 3 are due to differences in lung development in 

### 36
genetically

- modified animals which result in smaller lungs and thus to differences in 

### 37
inhaled dose because of the altered body mass to lung size ratio. 

### 38

### Draft

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- 67 June 2012 5.4.2.3 Nutritional Status

A further consideration is the compromised status of ELF antioxidants in nutritional 

### 1
deficiencies. Thus, many investigations have focused on antioxidant deficiency and 

### 2
supplementation as modulators of O 3 - mediated eff ects. One study in mice found that 

### 3
vitamin A deficiency enhanced lung injury induced by exposure to 0.3 ppm O 3 for 

### 4

72 hours ( Paquette et al., 1996 ) . Ascorbate deficiency was shown to increase the effects 

### 5

of acute (0.5

- 1 ppm for 4 hours), but not subacute (0.2

- 0.8 ppm for 7 days), O 3 exposure 

### 6
in guinea pigs ( Kodavanti et al., 1995 ; Slade et al., 1989 ) . Supplementation with AH2 

### 7

and α

- TOH was protective in healthy adults who were on an AH2

- deficient diet and 

### 8
exposed to 400 ppb O 3 for 2 hours while exercising at a moderate le vel ( Samet et al., 

### 9

2001 ) . In this study, the protective effect consisted of a smaller reduction in FEV 1 

### 10
following O 3 exposure ( Samet et al., 2001 ) . However the inflammatory response (influx 

### 11

of neu trophils and levels of IL

- 6) measured in BALF 1 hour after O 3 exposure was not 

### 12
different between supplemented and non

- supplemented subjects ( Samet et al., 2001 )

. 

### 13
Other investigators found that AH2 and α

- TOH supplementation fai led to ameliorate the 

### 14
pulmonary function decrements or airways neutrophilia observed in humans exposed to 

### 15

200 ppb O 3 for 2 hours while exercising at a moderate level ( Mudway et al., 2006 ) . It was 

### 16
suggested that supplementation may be ineffective in the absence of antioxidant 

### 17
deficiency ( Mudway et al., 2006 )

. 

### 18

In asthmatic adults, these same dietary antioxidants reduced O 3 - induced bronchial 

### 19
hyperresponsiveness (120 ppb, 45 min, light exercise) ( Trenga et al., 2001 ) . Furthermore, 

### 20
supplementation with AH2 and α

- tocopherol protected against pulmonary function 

### 21
decrements and nasal inflammatory responses which were associated with high levels of 

### 22

ambient O 3 in asthmatic children l iving in Mexico City ( Sienra

- Monge et al., 2004 ; 

### 23

Romieu et al., 2002 ) . Similarl y, supplementation with ascorbate, α

- tocopherol and 

### 24
β

- carotene improved pulmonary function in Mexico City street workers ( Romieu et al., 

### 25

1998b )

. 

### 26

Protective effects of supplementation with α

- tocopherol alone have not been observed in 

### 27
humans experimentally exposed to O 3 ( Mudway and Ke lly, 2000 ) . Alpha

- TOH 

### 28
supplementation also failed to protect against O 3 - induced effects in animal models of 

### 29
allergic rhinosinusitis and lower airways allergic inflammation (rats, 1 ppm O 3 for 

### 30

2 days) ( Wagner et al., 2007 ) . However, protection in these same animal models was 

### 31
reported using γ

- TOH supplementation ( Wagner et al., 2009 ; Wagner et al., 2007 )

. 

### 32

Whether or not this effect was due to enhanced antioxidant status or to a ct ivated signaling 

### 33
pathways is not known. Other investigators found that α

- TOH deficiency led to an 

### 34
increase in liver lipid peroxidation (rats, 0.3 ppm 3 hours/day for 7 months) ( Sato et al., 

### 35

1980 ) and a drop in liver α

- TOH levels following O 3 exposure (mice, 0.5 ppm, 

### 36

### Draft

– Do Not Cite or Quote 5

- 68 June 2012 6 hours/day for 3 days) ( Vasu et al., 2010 ) . A recent study used α

- TOH transfer protein 

### 1

null mice a s a model of α

- TOH deficiency and demonstrated an altered adaptive response 

### 2
of the lung genome to O 3 exposure ( Vasu et al., 2010 ) . Taken together, these studies 

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

### 4

5.4.2.4 Lifestage

Responses to O 3 are modulated by factors associated with lifestage. On one end of the 

### 5
lifestage spectrum is aging. The spirometric response to O 3 appears to be lost in humans 

### 6
as they age, as was demonstrated in two studies inv olving exposures of human subjects 

### 7
exercising at levels ranging from light to heavy to 420

- 450 ppb O 3 for 1.5

- 2 hours 

### 8

( Hazucha et al., 2003 ; Drechsler

- Parks, 1995 ) . In mice, physiological responses to O 3 

### 9

(600 ppb, 2 hours) were diminished with age ( Hamade et al., 2010 ) . Mechanisms 

### 10
accounting for this effect have not been well

- studied but could includ e altered number 

### 11
and sensitivity of receptors, altered signaling pathways involved in neural reflexes or 

### 12
compromised status of ELF antioxidants. 

### 13

On the other side of the lifestage spectrum is pre/postnatal development. Critical 

### 14
windows of development duri ng the pre/postnatal period are associated with an enhanced 

### 15
sensitivity to environmental toxicants. Adverse birth outcomes and developmental 

### 16
disorders may occur as a result (Section 7.4 ). 

### 17

Adverse birth outcomes may result from stressors which impact transplacental oxygen 

### 18
and nutrient transport by a variety of mechanisms including oxidative stress, placental 

### 19
inflammation and placental vascular dysfun ction ( Kannan et al., 2006 ) . These 

### 20

mechanisms may be linked since oxidative/ nitrosative stress is reported to cause vascular 

### 21
dy sfunction in the placenta ( Myatt et al., 2000 ) . As described earlier in this chapter and in 

### 22

Section 7.4 , systemic inflammation and oxidative/nitrosative stress and modification of 

### 23
innate and adaptive immunity are key events underlying the health effects of O 3 and as 

### 24
such they may contribute to adverse birth outcomes. An animal toxicology study showing 

### 25
that exposure to 2 ppm O 3 led to anorexia ( Kavlock et al., 1979 ) (see Section 7.4.2 ) in 

### 26
exposed rat dams provide an additional mechanism by which O 3 exposure could lead to 

### 27
diminished transplacental nutrient transport. Disturbances of the pituitary

- adrenocortico



### 28
placental system ( Ritz et al., 2000 ) may also impact normal intrauterine growth and 

### 29
development. Further, restricted fetal growth may result from pro

- inflammatory 

### 30
cytokines which limit trophoblast invasion during the early stages of pregnancy ( Hansen 

### 31

et al., 2008 ) . Direct effects on mat ernal health, such as risk of infection, and on fetal 

### 32
health, such as DNA damage, have also been proposed as mechanisms underlying 

### 33

adverse birth outcomes ( Ritz et al., 2000 ) . In addition to restricted fetal growth, preterm 

### 34

### Draft

– Do Not Cite or Quote 5

- 69 June 2012 birth may contribute to adverse birth outcomes. Preterm birth may result from the 

### 1
development of premat ure contractions and/or premature rupture of membranes as well 

### 2
as from disrupted implantation and placentation which results in suboptimal placental 

### 3

function ( Darrow et al., 2009 ; Ritz et al., 2000 ) . Genetic mutations are thought to be an 

### 4
important cause of placental abnormalities in the first trimester, while vascular alterations 

### 5
may be the main cause of placental abnormalities in later trimesters ( Jalaludin et al., 

### 6

2007 ) . Ozone

- mediated systemic inflammation and oxidative stress/nitrosative stress may 

### 7
possibly be related to these effects although there is no firm evidence. 

### 8

Enhanced sensitivity to environmental toxicants during critical windows of development 

### 9
may also result in developmental disorders. For example, normal migration and 

### 10
differentiation of neural crest cells are important for heart development and are 

### 11
particularly sensi tive to toxic insults ( Ritz et al., 2002 ) . Further, immune dysregulation 

### 12
and related pathologies are known to be associated with pre/postnatal environmental 

### 13

exposures ( Dietert et al., 2010 ) . Ozone exposure is associated with developmenta l effects 

### 14
in several organ systems. These include the lung and immune system (see below) and 

### 15
neurobehavioral changes which could reflect the effect of O 3 on CNS plasticity or the 

### 16
hypothalamic

- pituitary axis ( Auten and Foster, 2011 ) (see Section 7.4.9 ). 

### 17

The majority of developmental effects due to O 3 have been described for the respiratory 

### 18
system (see Section 7.2.3 and 7.4.8 ). Since its growth and development take place during 

### 19
both the prenatal and early postnatal periods, both prenatal and postnatal exposures to O 3 

### 20
have been studied. Maternal exposure to 0.4

- 1.2 ppm O 3 during gestati on resulted in 

### 21
developmental health effects in the RT of mice ( Sharkhuu et al., 2011 ) . Recent studies 

### 22
involving postnatal exposure to O 3 have focused on differences between developing and 

### 23
adult animals in antioxidant defenses, respiratory physiology and sensit ivity to cellular 

### 24
injury, and on mechanisms, such as lung structural changes, antigen sensitization, 

### 25
interaction with nitric oxide signaling, altered airway afferent innervation and loss of 

### 26
alveolar repair capacity, by which early O 3 exposure could lead to asthma pathogenesis or 

### 27

exacerbations in later life ( Auten and Foster, 2011 )

. 

### 28

An interesting set of studies conducted over the last 10 years in the infant rhesus monkey 

### 29

has identified numerous O 3 - mediated perturbations in the developing lung and immune 

### 30

system ( Plopper et al., 2007 ) . These investigations were prompted by the dramatic rise in 

### 31

t he incidence of childhood asthma and focused on the possible interaction of O 3 and 

### 32
allergens in promoting remodeling of the epithelial

- mesenchymal trophic unit during 

### 33
postnatal development of the tracheobronchial airway wall. In humans, airways growth 

### 34

duri ng the 8

- 12 year period of postnatal development is not well understood. Rhesus 

### 35
monkeys were used in these studies because the branching pattern and distribution of 

### 36
airways in this model are more similar to humans than those of rodents are to humans. In 

### 37

### Draft

– Do Not Cite or Quote 5

- 70 June 2012 ad dition, a model of allergic airways disease, which exhibits the main features of human 

### 1
asthma, had already been established in the adult rhesus monkey. Studies in infant 

### 2
monkeys were designed to determine whether repeated exposure to O 3 altered postnatal 

### 3

g rowth and development, and if so, whether such effects were reversible. In addition, 

### 4
exposure to O 3 was evaluated for its potential to increase the development of allergic 

### 5
airways disease. Exposures were to cyclic episodic O 3 over 5 months which involved 5 

### 6
biweekly cycles of alternating filtered air and O 3 - 9 consecutive days of filtered air and 5 

### 7
consecutive days of 0.5 ppm O 3 , 8 h/day

– and to house dust mite allergen (HDMA) for 

### 8

2 hours per day for 3 days on the last 3 days of O 3 exposure followed by 11 days of 

### 9

filtered air. 

### 10

Key findings were numerous. First, baseline airway resistance and AHR in the infant 

### 11
monkeys were dramatically increased by combined exposure to both HDMA and O 3 

### 12

( Joad et al., 2006 ; Schelegle et al., 2003 ) . Secondly, O 3 exposure led to a large increase in 

### 13
BAL eosinophils ( Schelegle et al., 2003 ) while HDMA exposure led to a large increase of 

### 14
eosinophils in airways tissue ( Joad et al., 2006 ; Schelegle et al., 2003 ) . Thirdly, the 

### 15
growth pattern of distal airways was changed to a large extent by exposu re to O 3 alone 

### 16
and in combination with HDMA. More specifically, longer and narrower airways resulted 

### 17
and the number of conducting airway generations between the trachea and the gas 

### 18
exchange area was decreased ( Fanucchi et al., 2006 ) . This latter effect was not 

### 19
ameliorated by a recovery period of 6 months. Fourthly, exposure to both HDMA and O 3 

### 20
altered the abundance and distribution of CD25+ lymphocytes in the airways ( Mi ller et 

### 21

al., 2009 ) . Lastly, several effects were seen at the level of the epithelial mesenchymal 

### 22
trophic unit in response to O 3 . These include altered organization of the airways 

### 23
epithelium ( Schelegle et al., 2003 ) , increased abundance of mucous goblet cells 

### 24

( Schelegle et al., 2003 ) , disruption of the basement membrane zone ( Evans et al., 2003 )

, 

### 25

reduced innervation ( Larson et al., 2004 ) , increased neuroendocrine

- like cells ( Joad et al., 

### 26

2006 ) , and altered orientation and abundance of smooth muscle bundles ( Plopper et al., 

### 27

2007 ; Tran et al., 2004 ) . Six months of recovery in filtered air led to reversal of some but 

### 28

not all of these effects ( Kajekar et al., 2007 ; Plopper et al., 2007 ; Evans et al., 2004 ) . The 

### 29
authors concluded that cyclic challenge of infant rhesus monkeys to allergen and O 3 

### 30
during the postnatal period compromised airway growth and development and resulted in 

### 31
changes which favor allergic airways responses and persistent effects on the immune 

### 32

system ( Plopper et al., 2007 ) . A more recent study in this same model reported that early 

### 33
life exposure to O 3 resulted in decreased total peripheral blood leukocyte numbers and 

### 34
increased blood eosinophils as well as persistent effects on pulmonary and systemic 

### 35
innate immunity in the infant rhesus monkey model ( Maniar

- Hew et al., 2011 )

. 

### 36

Furthermore, the effect of cyclic episodic O 3 exposure on nasal airways was studied in 

### 37
the infant rhesus monkey model. The three

- dimensional detail of the nasal passages was 

### 38

### Draft

– Do Not Cite or Quote 5

- 71 June 2012 analyzed for developing predictive dosimetry models and exposure

- dose

- response 

### 1
relationships ( Carey et al., 2007 ) . The authors reported that the relative amounts of the 

### 2
five epithelial cell types in the nasal airways of monkeys remained consistent between 

### 3
infancy and adulthood [comparing to ( Gross, 1987 ; Gross, 1982 ) ]. Cyclic episodic O 3 

### 4
exposure (as described in the previous paragraphs) resulted in 50

- 80% decreases in 

### 5
epithelial thickness and epithelial cell volume of the ciliated respiratory and transitional 

### 6
epithelium, confirming that these cell types in the nasal cavity were the most sensitive to 

### 7

O 3 exposure. The character and location of nasal lesions resulting from O 3 exposure were 

### 8
similar in the infant monkeys and adult monkeys similarly expo sed. However, the nasal 

### 9
epithelium of infant monkeys did not undergo nasal airway epithelial remodeling or 

### 10
adaptation which occurs in adult animals following O 3 - mediated injury and which may 

### 11
protect against subsequent O 3 challenge. The authors suggested t hat infant monkeys may 

### 12
be prone to developing persistent necrotizing rhinitis following episodic longer

- term 

### 13

exposures. 

### 14

5.4.2.5 Attenuation of Responses

Repeated daily exposure to O 3 often results in a reduction in the degree of a response, 

### 15

i.e., an attenuation of response. This phenomenon may reflect compensatory mechanisms 

### 16
and is not necessarily beneficial. Furthermore, there is variability among the different 

### 17

O 3 - related endpoints in terms of response attenuation, as will be described below. As a 

### 18

result, attenu ation of some responses occurs concomitantly with the exacerbation of 

### 19

others. 

### 20

In responsive individuals, a striking degree of attenuation of the FEV 1 response occurred 

### 21
following repeated daily exposures to O 3 . Generally, the young O 3 responder was no 

### 22
longe r responsive on the fourth or fifth day of consecutive daily O 3 exposure 

### 23

(200

- 500 ppb O 3 for 2

- 4 hours with light to heavy levels of exercise) and required days to 

### 24

weeks of non

- exposure in order for the subject to regain O 3 responsiveness ( Christian et 

### 25

al., 1998 ; Devlin et al., 1997 ; Linn et al., 1982b ; Horvath et al., 1981 ; Hackney et al., 

### 26

1977b ) . This phenomena has been reported for both lung function and symptoms such as 

### 27
upper airway irritation, nonproductive cough, substernal discomfort and pain upon deep 

### 28
inspiration ( Linn et al., 1982b ; Horvath et al., 1981 ; Hackney et al., 1977b ) . Repeated 

### 29
daily exposures also led to an attenuation of the sRaw response in moderately exercising 

### 30
human subjects exposed for 4 hours to 200 ppb O 3 ( Christian et al., 1998 ) and to a 

### 31
dampened AHR response compared with a single day exposu re in light exercising human 

### 32
subjects exposed for 2 hours to 400 ppb O 3 ( Dimeo et al., 1981 ) . However, one group 

### 33
reported persistent small airway dysfunction despite attenuation of the FEV 1 response on 

### 34

### Draft

– Do Not Cite or Quote 5

- 72 June 2012 the third day of consecutive O 3 exposure (250 ppb, 2 hours, with moderate exercise) 

### 1

( Frank et al., 2001 )

. 

### 2

Studies in rodents also indicated an attenuation of the physiologic response measured by 

### 3
breathing patterns and tidal volume following fi ve consecutive days of exposure to 

### 4

0.35

- 1 ppm O 3 for 2.25 hours ( Tep per et al., 1989 ) . Attenuation of O 3 - induced 

### 5
bradycardic responses, which also result from activation of neural reflexes, has been 

### 6
reported in rodents (0.5

- 0.6 ppm O 3 , 2

- 6 h/day, 3

- 5 days ( Hamade and Tankersl ey, 2009 ; 

### 7

Watkinson et al., 2001 )

. 

### 8

Multi

- day exposure to O 3 has been found to decrease some markers of inflammation 

### 9
compared with a single day exposure ( Christian et al., 1998 ; Devlin et al., 1997 ) . For 

### 10
example, in human subjects exposed for 4 hours to 200 ppb O 3 during moderate exercise, 

### 11
decreased numbers of BAL neutrophils and decreased levels of BALF fibronectin and 

### 12

IL

- 6 were observed after 4 days of consecutive exposure compared with responses after 

### 13

1 day ( Christian et al., 199 8 ) . Results indicated an attenuation of the inflammatory 

### 14
response in both proximal airways and distal lung. However markers of injury, such as 

### 15
lactate dehydrogenase (LDH) and protein in the BALF, were not attenuated in this study 

### 16

( Christian et al., 1998 ) . Other investigators found that repeated O 3 exposure (200 ppb O 3 

### 17

for 4 hours on 4 consecutive days with light exercise) resulted in increased numbers of 

### 18
neutrophils in bronchial mucosal biopsies despite decreased BAL neutrophilia ( Jorres et 

### 19

al., 2000 ) . Other markers of inflammation, including BALF protein and IL

- 6 remained 

### 20
elevated following the multi

- day ex posure ( Jorres et al., 2000 )

. 

### 21

In rats, th e increases in BALF levels of proteins, fibronectin, IL

- 6 and inflammatory cells 

### 22
observed after one day of exposure to 0.4 ppm O 3 for 12 hours were no longer observed 

### 23
after 5 consecutive days of exposure ( Van Bree et al., 2002 ) . A separate study in rats 

### 24
exposed to 0.35

- 1 ppm O 3 for 2.25 hours for 5 consecut ive days demonstrated a lack of 

### 25
attenuation of the increase in BALF protein, persistence of macrophages in the 

### 26
centriacinar region and histological evidence of progressive tissue injury ( Tepper et al., 

### 27

1989 ) . Findings that injury, measured by BALF markers or by histopathology, persist in 

### 28
the absence of BAL neutrophila o r pulmonary function decrements suggested that 

### 29
repeated exposure to O 3 may have serious long

- term consequences such as airway 

### 30
remodeling. In particular, the small airways were identified as a site where cumulative 

### 31
injury may occur ( Frank et al., 2001 )

. 

### 32

Some studies examined the recovery of responses which were attenuated by repeated O 3 

### 33
exposure. In a study of humans undergoing heavy exercise who were exposed for 2 hours 

### 34

to 400 ppb O 3 for five consecutive days ( Devlin et al., 1997 ) , recovery of t he 

### 35
inflammatory responses which were diminished by repeated exposure required 

### 36

10

- 20 days following the exposure ( Devlin et al., 1997 ) . In an animal study conducted in 

### 37

### Draft

– Do Not Cite or Quote 5

- 73 June 2012 parallel ( Van Bree et al., 2002 ) , full susceptibility to O 3 challenge following exp osure to 

### 1

O 3 for five consecutive days required 15

- 20 days recovery. 

### 2

Several mechanisms have been postulated to explain the attenuation of some responses 

### 3
observed in human subjects and animal models following repeated exposure to O 3 . First, 

### 4
the upregulatio n of antioxidant defenses (or conversely, a decrease in critical O 3 - reactive 

### 5
substrates) may protect against O 3 - mediated effects. Increases in antioxidant content of 

### 6
the BALF have been demonstrated in rats exposed to 0.25 and 0.5 ppm O 3 for 

### 7

several hours on consecutive days ( Devlin et al., 1997 ; Wiester et al., 1996b ; Tepper et 

### 8

al., 1989 ) . Second, IL

- 6 was demonstrated to be an important mediator of attenuation in 

### 9
rats exposed to 0.5 ppm for 4 hours on two consecutive days ( Mckinney et al., 1998 )

. 

### 10
Third, a protective role for increases in mucus producing cells and mucus concentrations 

### 11

in the air ways has been proposed ( Devlin et al., 1997 ) . Fourth, epithelial hyperplasia or 

### 12
metaplasia may decrease further effects due to subsequent O 3 challenge ( Carey et al., 

### 13

2007 ; Harkema et al., 1987a ; Harkema et al., 1987b ) . These morphologic changes have 

### 14
been observed in nasal and lower airways in monkeys exposed chronically to 

### 15

0.15

- 0.5 ppm O 3 and reflect a persistent change in epithelial architecture which may lead 

### 16
to other long

- term sequelae. Although there is some evidence to support these 

### 17
possibilities, there is no consensus on mechanisms underlying response attenuation. 

### 18

Rec ent studies demonstrating that O 3 activates TRP receptors suggest that modulation of 

### 19
TRP receptor number or sensitivity by repeated O 3 exposures may also contribute to the 

### 20
attenuation of responses. 

### 21

In summary, the attenuation of pulmonary function response s by repeated exposure to O 3 

### 22

has been linked to exacerbation of O 3 - mediated injury. Enhanced exposure to O 3 due to a 

### 23
dampening of the O 3 - mediated truncation of inspiration may be one factor which 

### 24
contributes to this relationship. 

### 25

5.4.2.6 Co

- exposures with Particu late Matter

Numerous studies have investigated the effects of co

- exposure to O 3 and PM because of 

### 26
the prevalence of these pollutants in ambient air. Results are highly variable and depend 

### 27
on whether exposures are simultaneous or sequential, the type of PM employed and the 

### 28
endpoint examined. Additive and interactive effects have been demonstrated. For 

### 29
example, simultaneous exposure to O 3 (120 ppb for 2 hours at rest) and concentrated 

### 30
ambient particles (CAPs) in human subjects resulted in a diminished systemi c IL

- 6 

### 31
response compared with exposure to CAPs alone ( Urch et al., 2010 ) . However, exposure 

### 32

to O 3 alone did not alter blood IL

- 6 levels ( Urch et al., 2010 ) . The authors provided 

### 33

evidence that O 3 mediated a switch to shallow breathing which may have accounted for 

### 34

### Draft

– Do Not Cite or Quote 5

- 74 June 2012 the observed antagonism ( Urch et al., 2010 ) . Further, simultaneous exposure to O 3 

### 1

(114 ppb for 2 hours at rest) and CAPs but not exposure to either alone, resulted in 

### 2

increased diastolic blood pressure in human subjects ( Fakhri et al., 2009 ) . Mechanisms 

### 3
underlying this potentiation of response were not explored. In some strains of mice, 

### 4

pre

- exposure to O 3 (0.5 ppm for 2 hou rs) modulated the effects of carbon black PM on 

### 5
heart rate, HRV and breathing patterns ( Hamade and Tankersley, 2009 ) . Another recent 

### 6
study in mice demonstrated that treatment with carbon nanotubes followed 12 hours later 

### 7
by O 3 exposure (0.5 ppm for 3 hours) resulted in a dampening of some of the pulmonary 

### 8

effects of carbon nanotubes measured as markers of inflammation and injury in the 

### 9

BALF ( Han et al., 2008 ) . Further, Harkema and Wagner (2005 ) found that epithelial a nd 

### 10
inflammatory responses in the airways of rats were enhanced by co

- exposure to O 3 

### 11

(0.5 ppm for 3 days) and LPS (used as a model of biogenic PM) or to O 3 (1 ppm for 

### 12

2 days) and OVA (used as a mod el of an aeroallergen). Lastly , a recent study 

### 13

demonstrated that maternal exposure to particulate matter (PM) resulted in augmented 

### 14
lung inflammation, airway epithelial mucous metaplasia and AHR in young mice 

### 15
exposed chronically and intermittently to 1 ppm O 3 ( Auten et al., 2009 )

. 

### 16

In summary, many of the demonstrated responses to co

- exposure were more than 

### 17
additive. These findings are hard to interpret but demonstrate the complexity of responses 

### 18
following combined exposure to PM and O 3
. 

### 19

5.4.2.7 Summary

Collectively, these earlier and more recent studies provide some evidence for 

### 20
mechanisms that may underlie the variability in responsiveness seen among individuals 

### 21

( Figure 5

- 9 ). Certain functional genetic polymorphisms, pre

- existing conditions and 

### 22
diseases, nutritional status, lifestage and co

- exposures contribute to altered risk of 

### 23

O 3 - induced effects. Attenuation of responses may also be important, but it is 

### 24
incompletely understood, both in terms of the pathways involved and the resulting 

### 25

consequences. 

### 26

### Draft

– Do Not Cite or Quote 5

- 75 June 2012 Figure 5

- 9 Some factors, illustrated in yellow, that likely contribute to the interindividual variability in responses resulting from inhalation of

ozone.



## 5.5 Species Homology and Interspecies Sensitivity

The previous O 3 AQCDs discussed the suitability of animal models f or 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 r elationships 

### 4
between exposure and health outcome that would be impossible to collect in human 

### 5
studies. Furthermore, animal studies add to a better understanding of the full range of 

### 6
potential O 3 - mediated effects. 

### 7

Still, care must be taken when comparing q uantitative dose

- response relationships in 

### 8
animal models to humans due to obvious interspecies differences. This section will 

### 9
qualitatively describe basic concepts in species homology concerning both dose and 

### 10
response to O 3 exposure. Overall, there have be en few new publications examining 

### 11
interspecies differences in dosimetry and response to O 3 since the last AQCD. These 

### 12
studies do not overtly change the conclusions discussed in the previous document. 

### 13 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
factors

### Draft

– Do Not Cite or Quote 5

- 76 June 2012 5.5.1 Interspecies Dosimetry

As discussed in Section 5.2.1 , O 3 uptake depends on complex interactions between RT 

### 1
morphology, breathing route, rate, and depth, physicochemical properties of the gas, 

### 2
physical p rocesses 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

t oxicology studies. 

### 6

Physiological and anatomical differences exist between experimental species. The 

### 7
structure of the URT is vastly different between rodents and humans but scales according 

### 8
to body mass. The difference in the cross

- sectional shape and size of the nasal passages 

### 9
affects bulk airflow patterns, particularly the shape of major airflow streams. The nasal 

### 10
epithelium is lined by squamous, respiratory, or olfactory cells, depending on location. 

### 11
The differences in airflow patterns in the URT mean th at 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, sheep, and pigs. The 

### 17
branching structure of the ciliated bronchi and bronc hioles 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 mammals. In addition, 

### 20

rodents have fewer terminal bronchioles due to a sma ller lung size compared to humans 

### 21

or canines ( McBride, 1992 ) . The cellular composition i n 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, considerable 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 o n 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 affect the amount of O 3 uptake. 

### 29

As O 3 absorption and re activity relies on ELF antioxidant substances (see Section 5.2.3 ), 

### 30
variability in antioxidant concentrations and metabolism between species may affe ct dose 

### 31

and O 3 - induced health outcomes. The thickness of the ELF in the TB airways varies 

### 32
among species. Mercer et al. (1992 ) found that the human ELF thickness in bronchi and 

### 33

bronchioles wa s 6.9 and 1.8 μm, respectively, compared to 2.6 and 1.9 μm for the same 

### 34
locations in the rat. Guinea pigs and mice have a lower basal activity of GSH transferase 

### 35
and GSH peroxidase, and lower α

- TOH levels in the lung compared to rats ( Ichinose et 

### 36

### Draft

– Do Not Cite or Quote 5

- 77 June 2012 al., 1988 ; Sagai et al., 1987 ) . Nasal lavage fluid analysis shows that human s have a higher 

### 1
proportion of their nasal antioxidants as UA and low levels of AH 2 whereas mice, rats, or 

### 2
guinea pigs have high levels of AH 2 and undetectable levels of UA. GSH is not detected 

### 3
in the nasal lavage of most of these species, but is present in monkey nasal lavage. 

### 4
Guinea pigs and rats have a higher antioxidant to protein ratio in nasal lavage and BALF 

### 5

than humans ( Hatch, 1992 ) . The BALF profile differs from the nasal lavage. Humans 

### 6
have a higher proportion o f GSH and less AH 2 making up their BALF content compared 

### 7
to the guinea pigs and rats ( Slade et al., 1993 ; Hatch, 1992 ) . Similar to the nose, rats have 

### 8

t he highest antioxidant to protein mass ratio found in BALF ( Slade et al., 1993 )

. 

### 9
Antioxidant defenses also vary with age ( Servais et al., 2005 ) and exposure history ( Duan 

### 10

et al., 1996 )

. Duan et al. (1996 ); Duan et al. (1993 ) reported that differences in 

### 11
antioxidant levels between species and lung regions did not appear to be the primary 

### 12

factor in O 3 induced tissue inju ry. However, a close correlation between site

- specific O 3 

### 13
dose, the degree of epithelial in jury, and reduced glutathione depletion was observed in 

### 14
monkeys ( Plopper et al., 1998 )

. 

### 15

Even with these differences h umans and animals ar e similar in the pattern of regional O 3 

### 16

dose distribution. As discussed for humans in Section 5.2.2 , O 3 flux to the air

- liquid 

### 17

interface of the ELF slowly decreases distally in the TB region and then rapidly decreases 

### 18
distally in the alveolar region ( Miller et al., 1985 ) . Modeled tissue dose in the human RT, 

### 19
representing O 3 flux to the liq uid

- tissue interface, is very low in the trachea, increases to 

### 20
a maximum in the CAR, and then rapidly decreases distally in the alveolar region 

### 21

( Figure 5

- 10 ). Similar patterns of O 3 tissue dose profiles normalized to inhaled O 3 

### 22
concentration were predicted for rat, guinea pig, and rabbit ( Miller et al., 1988 ; Overton 

### 23

et al., 1987 ) ( Figure 5

- 10 a). Overton et al. (1987 ) modeled rat and guinea pig O 3 dose 

### 24
distribution and found that after comparing two diff erent morphometrically based 

### 25
anatomical models for each species, considerable difference in predicted percent RT and 

### 26
alveolar region uptakes were observed. This was due to the variability between the two 

### 27
anatomical models in airway path distance to the fir st alveolated duct. As a result, the 

### 28
overall dose profile was similar between species however the O 3 uptake efficiency varied 

### 29
due to RT size and path length (Section 5.2.2 ). A similar pattern of O 3 dose distribution 

### 30
was measured in monkeys exposed to 0.4 and 1.0 ppm 18 O 3 ( Plopper e t al., 1998 ) 

### 31

( Figure 5

- 10 b ). Less 18 O was measured in the trachea, proximal bronchus, and distal 

### 32
bronchus than was observed in the respiratory bron chioles. Again indicating the highest 

### 33

concentration of O 3 tissue dose is localized to the CAR, which are the respiratory 

### 34
bronchioles in nonhuman primates. In addition, the lowest 18 O detected in the RT was in 

### 35
the parenchyma (i.e., alveolar region), mimicki ng the rapid decrease in tissue O 3 dose 

### 36
predicted by models for the alveolar regions of humans and other animals. 

### 37

### Draft

– Do Not Cite or Quote 5

- 78 June 2012 a

.

Note: 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 region,
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 concentrations in the same region.
Source: Panel (a) U.S. EPA (1996a ) (b) Plopper et al. (1998 )



### Figure 5

- 10 Humans and animals are similar in the regional pattern of ozone tissue dose distribution. 0.4 ppm 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. A

### Draft

– Do Not Cite or Quote 5

- 79 June 2012 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 times higher concentrations of O 3 reaction product 

### 3
in BAL cells, surfactant and protein fractions compared to resting r ats similarly exposed 

### 4

(400 ppb) ( Figure 5

- 11 ). The use of 18 O was specifically employed in an attempt to 

### 5
accurately measure O 3 dose to BALF fractions and lung tissue and was normalized to the 

### 6
dried mass of lavaged constituents. It was necessary to expose resting rats to 2 ppm O 3 to 

### 7

achieve the same BALF accumulation of 18 O reaction product that was observed in 

### 8

humans exposed to 400 ppb with intermittent heavy exercise ( V ̇ E ~60 L/min). The 

### 9

concentration of 18 O reaction product in BALF paralleled the accumulation of BALF 

### 10
protein and cellular effects of the O 3 exposure observed such that these responses to 

### 11

2.0 ppm O 3 were similar to those of the 400 ppb O 3 i n exercising humans. This suggests 

### 12
that animal data obtained in resting conditions would underestimate the reaction of O 3 

### 13
with cells in the RT and presumably the resultant risk of effect for humans. However 

### 14
these results should be interpreted with caution given an important limitation in the 18 O 

### 15
labeling technique when used for interspecies comparisons. The reaction between O 3 and 

### 16

some r eactants such as ascorbate produce 18 O

- labeled products that are lost during sample 

### 17
processing. When levels of ascorbate or other such reactants vary between species, this 

### 18
lost portion of the total 18 O

- reaction products will also vary, leading to uncertainty in 

### 19

inter species comparisons. 

### 20

### Draft

– Do Not Cite or Quote 5

- 80 June 2012 No te: 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.

Source: Hatch et al. (1994 )



### Figure 5

- 11 Oxygen

- 18 incorporation into different fractions of BALF from humans and rats exposed to 0.4 and 2.0 ppm 18 

### O 3 

### .

Recently, a quantitative comparison of O 3 transport in the airways of rats, dogs, and 

### 1
humans was conducted using a three

- compartment airways model, based on upper and 

### 2
lower airway casts and mathematical calculation for alveolar parameters ( Tsujino et al., 

### 3

2005 ) . This one

- dimensional gas transport model examined how interspecies anatomical 

### 4
and physiological differences affect intra

- airway O 3 concentrations and the amount of gas 

### 5
absorbed. The morphol ogical model consisted of cylindrical tubes with constant volume 

### 6
and no airway branching patterns. 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 huma ns. The amount of O 3 absorbed 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

### Draft

– Do Not Cite or Quote 5

- 81 June 2012 surface area had a statistically significa nt impact on model results. The model is limited 

### 1
in that it did not account for chemical reactions in the ELF or consider gas diffusion as a 

### 2
driving force for O 3 transport. Also, the model was run at a respiratory rate of 16/min 

### 3
simulating a rest ing individual, however exercise may cause a further deviation from 

### 4

animal models as was seen in Hatch et al. (1994 )

. 

### 5

Overall, animal models exhibit qualitatively similar patterns of O 3 net and tissue dose 

### 6
distribution with the largest tissue dose delivered to the CAR. However, due to 

### 7

anatomical and biochemical RT differences the absolute values of O 3 dose delivered 

### 8
differs. Past results suggest that animal data obtained in resting conditions would 

### 9

underesti mate the O 3 reactions with cells in the BALF and presumably the resultant risk 

### 10
of effect for humans, especially for humans during exercise. 

### 11

5.5.2 Interspecies Homology of Response

Biological response to O 3 exposure broadly shows commonalities in many species. 

### 12
Among rodents, non

- human primates, and humans, for example, ample data suggest that 

### 13

O 3 induces oxidative stress, cell injury, upregulation of cytokines/chemokines, 

### 14
inflammation, alterations in lung function, and disruption of normal lung growth and 

### 15
development (See Chapters 6 and 7). 

### 16

The effects related to early life exposures can differ a ppreciably across species due to the 

### 17
maturation stage of the lung and immune systems at birth. Evidence from non

- human 

### 18
primate studies shows that early life O 3 exposure disrupts lung development producing 

### 19
physiologic perturbations that are similar to those observed in children exposed to urban 

### 20
air pollution ( Fanucchi et al., 2006 ; Joad et al., 2006 ) . Studies of O 3 effects on lung 

### 21

s urface chemistry also show some degree of homology. Lipid oxidation products specific 

### 22

to O 3 reactions with unsaturated fatty acids have been reported, for example, in lavage 

### 23

fluids from both rodents and humans ( Frampton et al., 1999 ; Pryor et al., 1996 ) . In 

### 24
humans, the extent to which systemic effects occur is less well studied; plasma indices of 

### 25
lipid oxidation such as isoprostanes unfortunately do not pinpoint the compartment(s) 

### 26
where oxidative stress has transpired. That oxidative stress occurs systemically in both 

### 27

rodents and non

- human primates ( Chuang et al., 2009 ) , nevertheless, suggests that it 

### 28
likely also occurs in humans. 

### 29

Despite the overall similarities in responses to O 3 among species, studies have reported 

### 30
variability in the responsiveness to O 3 between and within species, as we ll as between 

### 31
endpoints. Rodents appear to have a slightly higher tachypneic response to O 3 and are 

### 32
less sensitive to changes in pulmonary function responses than humans ( U.S. EPA, 

### 33

1996a ) . However, rats experience attenuation of pulmonary function and tachypneic 

### 34

### Draft

– Do Not Cite or Quote 5

- 82 June 2012 ventilatory responses, similar to humans ( Wiester et al., 1996b )

. Hatch et al. (1986 ) 

### 1
reported that guinea pigs were the most responsive to O 3 - induced inflammatory cell and 

### 2
protein influx. Rabbits were the least responsive and rats, hamsters, and mice were 

### 3
intermediate responders. Further analysis of this study by Miller et al. (1988 ) found that 

### 4
the protein levels in BALF from guinea pigs increased more rapidly with predicted 

### 5
pulmonary tissue dose than in rats and rabbits. Alveolar macrophages isolated from 

### 6
guinea pigs and humans mounted similar qualitative and quantitative cytokine responses 

### 7

to in vitro O 3 (0.1

- 1.0 ppm for 60 minutes) exposure ( Arsalane et al., 1995 )

. 

### 8

Also, because of their higher body surface to volume ratio, rodents can rapidly lower 

### 9
body temperature during exposure leading to lowered O 3 dose and toxicity ( Watkinson et 

### 10

al., 2003 ; Iwasaki et al., 1998 ; Slade et al., 1997 ) . In addition to lowering the O 3 dose to 

### 11
the lungs, this hypothermic response may cause: (1) lower metabolic rate, (2) altered 

### 12
enzyme kinetics, and (3) altered membrane function. The thermoregulatory mechanisms 

### 13
also may affect disruption of heart rate that may lead to: (1) decreased card iac output, (2) 

### 14
lowered blood pressure, and (3) decreased tissue perfusion ( Watkinson et al., 2003 )

. 

### 15
These responses have not been observed in humans except at very high exposures, thus 

### 16
further compl icating extrapolation of effects from animals to humans. 

### 17

The degree to which O 3 induces injury and inflammation responses appears to be variable 

### 18
between species. However, the majority of those studies did not normalize the response 

### 19

to the dose received to account for species differences in O 3 absorption. For example, 

### 20

Dormans et al. (1999 ) found that rats, mice, and guinea pigs all exhibited O 3 - induced (0.2 

### 21

- 0.4 ppm for 3

- 56 days) inflammation; however, guinea pigs were the most responsive 

### 22
with respect to alveolar macrophage elicitation and pulmonary cell density i n the 

### 23
centriacinar region. Mice were the most responsive in terms of bronchiolar epithelial 

### 24
hypertrophy and biochemical changes (e.g., LDH, glutathione reductase, glucose



### 25

6

- phosphate dehydrogenase activity), and had the slowest recovery from O 3 exposure. A ll 

### 26
species displayed increased collagen in the ductal septa and large lamellar bodies in Type 

### 27
II pneumocytes at the longest exposure and highest concentration; whereas this response 

### 28
occurred in the rat and guinea pig at lower O 3 levels (0.2 ppm) as well. O verall, the 

### 29

author s rated mice as most responsive , followed by guinea pigs, then rats ( Dormans et al., 

### 30

1999 ) . Rats were also less responsive in terms of epithelial necrosis and inflammatory 

### 31
responses as a result of O 3 exposure (1.0 ppm for 8 hours) compared with monkeys and 

### 32
ferrets, which manifested a similar response ( Sterner

- Kock et al., 2000 ) . Results of this 

### 33
study should be interpreted with caution since no dose metric was used to norma lize the 

### 34
total inhaled dose or local organ dose between species. 

### 35

To further understand the genetic basis for age

- dependent differential response to O 3 , 

### 36

adult (15 week old) and neonatal (15

- 16 day old) mice from 8 genetically diverse strains 

### 37

### Draft

– Do Not Cite or Quote 5

- 83 June 2012 were examined for O 3 - induced (0.8 ppm for 5 hours) pulmonary injury and lung 

### 1

inflammation ( Vancza et al., 2009 ) . Ozone exposure increased polymorphonuclear 

### 2
leukocytes (PMN) influx in all strains of neonatal mice tested, but significa ntly greater 

### 3
PMNs occurred in neonatal compared to adult mice for only some sensitive strains, 

### 4
suggesting a genetic background eff ect. This strain difference was not due to differences 

### 5

in delivered dose of O 3 to the lung, evidenced by 18 O lung enrichment. The sensitivity of 

### 6

strains for O 3 - induced increases in BALF protein and PMNs was different for different 

### 7
strains of mice suggestin g that genetic factors contributed to heightened responses. 

### 8
Interestingly, adult mice accumulated more than twice the levels of 18 O reaction product 

### 9

of O 3 than corresponding strain neonates. Thus, it appeared that the infant mice showed a 

### 10

2

- fold

- to 3

- fold higher response than the adults when expressed relative to the 

### 11

accumulated O 3 reaction product in their lungs. The apparent decrease in delivered O 3 

### 12
dose in neonates could be a result of a more rapid loss of body temperature in infant 

### 13

rodents incident to maternal separation and chamber air flow. 

### 14

In animal studies, inhaled O 3 concentration and exposure history rarely reflect actual 

### 15
human environmental exposures. Generally, very high exposure concentrations are used 

### 16

to induce murine AHR, which in some human subjects is observed at far more relevant 

### 17
concentrations. This calls into question whether the differences in airway reactivity are 

### 18
simply a function of differential nasopharyngeal scrubbing or whether the complexities 

### 19
encompassing a variety of contributor y biological pathways show species divergence. 

### 20

Furthermore, in non

- human primates exposed during early life, eosinophil trafficking 

### 21

occurs, which has not been observed in rodents (unless sensitized) ( Maniar

- Hew et al., 

### 22

2011 ) . This response has been shown to be persistent when O 3 challenges are 

### 23

administered after a recovery period of ≥ 9 months during which no exposure transpired

. 

### 24

Quantitative extrapolation is challenging due to a number of uncertainties. Unfortunately, 

### 25
many input parameters needed to conduct quantitative extrapolations across species have 

### 26
not been obtained or currently remain undefined. It is not clear whether c haracterization 

### 27
of the ELF provides the information needed to compute a profile of reaction products or 

### 28
whether environmentally relevant exposure has altered the physicochemical interactions 

### 29
that occur within the RT surface compartment (e.g., O 3 diffusion through regions where 

### 30
the ELF is thin). That systemic effects have been documented in both rodents and non



### 31
human primates leads to the question of whether reaction products, 

### 32
cytokines/chemokines, or both enter the nasopharyngeal or bronchial circulation, b oth of 

### 33
which show species

- dependent differences ( Chuang et al., 2009 ; Cole and Freeman, 

### 34

2009 )

. 

### 35

In addition, the response to O 3 insult across species and more recent health effects such as 

### 36
immune system development are uncertain. Non

- human primat e studies have shown 

### 37

### Draft

– Do Not Cite or Quote 5

- 84 June 2012 hypo

- responsiveness to endotoxin challenge as a consequence of exposure; whether this 

### 1
occurs in rodents and humans is largely unknown ( Maniar

- Hew et al., 2011 ) . In addition, 

### 2
structural changes (e.g., airway s remodeling, fibrogenesis) might differ appreciably 

### 3
across species. Moreover, whether the upper airways differentially contribute to either 

### 4

distal lung or systemic impacts has not been explored. 

### 5

Some outcomes (e.g., inflammation) support the conclusion of homologous responses 

### 6
across species. However, factors such as age, exposure history, diet, endogenous 

### 7
substrate generation and homeostatic regulat ion, the cellular machinery that regulates 

### 8
inflammatory cell trafficking, responses to other environmental challenges, and the 

### 9
precise chemical species (whether ELF or cell membrane

- derived) that account for 

### 10

exposure

- related initiation of pathophysiologic sequelae might differ across species, but 

### 11
the extent of species

- specific contributing factors remains unknown. Consequently, some 

### 12
level of uncertainty cannot be dismissed. Nonetheless, if experimental animals show 

### 13
pathophysiological consequences of exposur e, assuming that qualitatively similar human 

### 14
health impacts could occur is not unreasonable. 

### 15

5.5.3 Summary

In summary, biological response to O 3 exposure broadly shows commonalities in many 

### 16
species and thus supports the use of animal studies in determining mecha nistic and cause



### 17
effect relationships and as supporting evidence that similar effects could occur in humans 

### 18

if O 3 exposure is sufficient. However, there is uncertainty regarding the similarity of 

### 19
response to ozone across species for some recently described endpoints. Differences exist 

### 20
between species in a number of factors that influence O 3 dosimetry and responses, such 

### 21
as RT anatomy, breathing patterns, and ELF antioxidant concentrations and chemical 

### 22
species. While humans and animals are similar in the pat tern of regional O 3 dose 

### 23
distribution, these differences will likely result in differences in the absolute values of 

### 24

O 3 dose delivered throughout the RT. These considerations limit quantitative comparison 

### 25
between species. 

### 26



## 5.6 Chapter Summary

Ozone is a highly reactive gas and a powerful oxidant with a short half

- life. Both O 3 

### 27
uptake and responses are dependent upon the formation of secondary reaction products in 

### 28
the ELF; however more complex interactions occur. Uptake in humans at rest is 80

- 95% 

### 29

efficient and i t is influenced by a number of factors including RT morphology, breathing 

### 30
route, frequency, and volume, physicochemical properties of the gas, physical processes 

### 31

### Draft

– Do Not Cite or Quote 5

- 85 June 2012 of gas transport, as well as the physical and chemical properties of the ELF and tissue 

### 1
layers . In fact, even though the average LRT dose may be at a level where health effects 

### 2
would not be predicted, local regions of the RT may receive considerab ly higher than 

### 3
average doses due to RT inhomogeneity and differences in the pathlengths, and therefore 

### 4
be at greater risk of effects. The primary uptake site of O 3 delivery to the lung epithelium 

### 5
is believed to be the CAR, however changes in a number of factors (e.g., physical 

### 6
activity) can alter the distribution of O 3 uptake in the RT. Ozone uptake is chem ical 

### 7

reaction

- dependent and the substances present in the ELF appear in most cases to limit 

### 8

interaction of O 3 with underlying tissues and to prevent penetration of O 3 distally into the 

### 9

RT. Still, reactions of O 3 with soluble ELF components or possibly plas ma membranes 

### 10
result in distinct products, some of which are highly reactive and can injure and/or 

### 11
transmit signals to RT

- cells. 

### 12

Thus, in addition to contributing to the driving force for O 3 uptake, formation of 

### 13
secondary oxidation products initiates pathwa ys that provide the mechanistic basis for 

### 14
health effects that are described in detail in Chapters 6 and 7 and that involve the RT as 

### 15
well as extrapulmonary systems. These pathways include activation of neural reflexes, 

### 16

initiation of inflammation, alteratio n of epithelial barrier function, sensitization of 

### 17
bronchial smooth muscle, modification of innate and adaptive immunity, airways 

### 18
remodeling, and systemic inflammation and oxidative/nitrosative stress. With the 

### 19
exception of airways remodeling, these pathwa ys have been demonstrated in both 

### 20
animals and human subjects in response to the inhalation of O 3
. 

### 21

Both dosimetric and mechanistic factors contribute to the understanding of 

### 22
interindividual variability in responses to O 3 . This variability is influenced by differences 

### 23
in RT volume and surface area, certain genetic polymorphisms, pre

- existing conditions 

### 24
and disease, nutritional status, lifestages, attenuation, and co

- exposures. Some of these 

### 25

factors also underlie differences in species homology and sensitivit y. Qualitatively, 

### 26
animal models exhibit similar patterns of O 3 net and tissue dose distribution with the 

### 27
largest tissue dose of O 3 delivered to the CAR. However, due to anatomical and 

### 28

biochemical RT differences, the absolute value of delivered O 3 dose diff ers, with animal 

### 29
data obtained in resting conditions underestimating the dose to the RT and presumably 

### 30
the resultant risk of effect for humans, especially humans during exercise. Even though 

### 31
interspecies differences limit quantitative comparison between sp ecies, many short

- term 

### 32
responses of laboratory animals to O 3 appear qualitatively homologous to those of the 

### 33
human. Furthermore, animal studies add to a better understanding of the full range of 

### 34
potential O 3 - mediated effects. Given the commonalities in man y responses across 

### 35
species, animal studies that observe O 3 - induced effects may be used as supporting 

### 36

evidence that similar effects could occur in humans or in de termining mechanistic and 

### 37

cause

- effect relationships if O 3 exposure is sufficient. 

### 38

### Draft

– Do Not Cite or Quote 5

- 86 June 2012 

### References

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- 843. Aharonson, EF; Menkes, H; Gurtner, G; Swift, DL; Proctor, DF. (1974). Effect of respiratory airflow rate on
removal of soluble vapors by the nose. J Appl Physiol 37: 654

- 657. Ahmad, S; Ahmad, A; McConville, G; Schneider, BK; Allen, CB; Manzer, R; Mason, RJ; White, CW. (2005). Lung ep ithelial cells release ATP during ozone exposure: Signaling for cell survival. Free Radic Biol
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- 226. http://dx.doi.org/10.1016/j.freeradbiomed.2005.03.009 Aibo, DI; Birmingham, NP; Lewandowski, R; Maddox, JF; Roth, RA; Ganey, PE; Wagner, JG; Harkema, JR. (2010). Acute exposure to ozone exacerbates acetaminophen

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- Hegelan, M; Tighe, RM; Castillo, C; Hollingsworth, JW. (2011). Ambient ozone and pulmonary innate immunity. Immunol Res 49: 173

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- z Alexis, N; Soukup, J; Nierkens, S; Becker, S. (2001b). Association between airway hyperreactivity and bronchial macrophage dysfunction in individuals with mild asthma. A m J Physiol Lung Cell Mol Physiol 280: L369

- L375. Alexis, N; Urch, B; Tarlo, S; Corey, P; Pengelly, D; O'Byrne, P; Silverman, F. (2000). Cyclooxygenase metabolites play a different role in ozone

- induced pulmonary function decline in asthmatics compared
to normals. Inhal Toxicol 12: 1205

- 1224. Alexis, NE; Lay, JC; Hazucha, M; Harris, B; Hernandez, ML; Bromberg, PA; Kehrl, H; Diaz

- Sanchez, D; Kim, C; Devlin, RB; Peden, DB. (2010). Low

- level ozone exposure induces airways inflammation and modifies cell surface phenotypes in healthy humans. Inhal Toxicol 22: 593

- 600. http://dx.doi.org/10.3109/08958371003596587 Alexis, NE; Zhou, H; Lay, JC; Ha rris, B; Hernandez, ML; Lu, TS; Bromberg, PA; Diaz

- Sanchez, D; Devlin, RB; Kleeberger, SR; Peden, DB. (2009). The glutathione

- S

- transferase Mu 1 null genotype modulates

ozone

- induced airway inflammation in human subjects. J Allergy Clin Immunol 124: 122 2

- 1228. http://dx.doi.org/10.1016/j.jaci.2009.07.036 Alfaro, MF; Putney, L; Tarkington, BK; Hatch, GE; H yde, DM; Schelegle, ES. (2004). Effect of rapid shallow breathing on the distribution of 18O

- labeled ozone reaction product in the respiratory tract of the rat.
Inhal Toxicol 16: 77

- 85. Alfaro, MF; Walby, WF; Adams, WC; Schelegle, ES. (2007). Breath condensate levels of 8

- isoprostane and leukotriene B4 after ozone inhalation are greater in sensitive versus nonsensitive subjects. Exp Lung
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- 133. Araneda, S; Commin, L; Atlagich, M; Kitahama, K; Parraguez, VH; Pequignot, JM; Dalmaz, Y. (2008). VEGF overexpression in the astroglial cells of rat brainstem fo llowing ozone exposure. Neurotoxicology 29:
920

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Aris , RM; Christian, D; Hearne, PQ; Kerr, K; Finkbeiner, WE; Balmes, JR. (1993). Ozone

- induced airway inflammation in human subjects as determined by airway lavage and biopsy. Am J Respir Crit Care Med
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- 1372. Arito, H; Uchiyama, I; Arakawa, H; Yokoyama, E. (1990). Ozone

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