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i xii

 

ROLE OF GLUTATHIONE IN OZONE-INDUCED EPITHELIAL HYPERPLASIA IN
THE NASAL AIRWAYS OF INFANT MONKEYS AND RATS

By

STEPHAN A. CAREY

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Comparative Medicine and Integrative Biology

2009

ABSTRACT

ROLE OF GLUTATHIONE IN OZONE-INDUCED EPITHELIAL HYPERPLASIA IN
THE NASAL AIRWAYS OF INFANT MONKEYS AND RATS

By
Stephan A. Carey

Ozone, the major oxidant pollutant in photochemical smog, causes toxic epithelial
injury in the nasal and tracheobronchial airways of laboratory animals and people.
Epidemiologic studies suggest that children may be more susceptible to the respiratory
health effects of ozone exposure than adults. The majority of children in the United
States live in areas in which the atmospheric ozone concentration exceeds the current
National Ambient Air Quality Standard for this pollutant. Repeated exposure to high
ambient levels of ozone induces site-specific rhinitis, mucous cell metaplasia, and
epithelial hyperplasia in the nasal airways of adult, laboratory monkeys and rats. These
remodeling events in the nasal airways of adult monkeys and rats are associated with an
altered response to subsequent ozone challenge, and thus may serve as a protective
adaptation. Low molecular weight antioxidants, such as the tripeptide glutathione (GSH),
in the nasal epithelium and the overlying epithelial lining fluid (ELF) are regarded as the
first line of defense against these and other inhaled oxidant pollutants. Recent studies
have also implicated GSH in the development of pulmonary tolerance to ozone-induced
injury, and in the pathogenesis of oxidant-mediated cell proliferation. Few studies have
examined the effects of ozone exposure on the developing nasal airways of immature
animal models. The overall goal of these experiments was to demonstrate that the site-
specificity and temporal pattern of ozone-induced nasal epithelial remodeling in

immature rats and infant monkeys is related to the local regulation of intracellular GSH.

Infant, male rhesus macaques were exposed episodically to repeated cycles of ozone (0.5
ppm) and filtered air (0 ppm) for two or five months. The time-course of morphologic
and immunohistochemical events comprising this ozone-induced nasal epithelial injury
and repair were determined, and these were compared to site-matched changes in the
steady-state levels of GSH in the nasal mucosa. In addition, a whole-animal inhalation
study was conducted using an immature rodent model to determine the role of GSH in
ozone-induced epithelial hyperplasia in the developing nasal airways of rats. Immature
male F344 rats were exposed to three consecutive days of ozone (0.8 ppm) or filtered air.
Infant monkeys episodically exposed to ozone for 5 months developed acute rhinitis,
epithelial hyperplasia, and squamous metaplasia in the anterior nasal cavity. These
animals also exhibited a significant increase in intracellular GSH concentration in the
nasal mucosa corresponding to the site of remodeling. GSH concentration was positively
correlated with the epithelial numeric cell density following filtered air or ozone
exposure. In immature rats, ozone-induced epithelial hyperplasia was also associated with
a local increase in GSH concentration. However, GSH depletion had no effect on ozone-
induced cell proliferation. In conclusion, ozone-induced epithelial hyperplasia in the
nasal airways is temporally correlated with local increases in mucosal GSH
concentrations. However, ozone-induced hyperplasia in the nasal airways of rats is not a
GSH-dependent phenomenon. Furthermore, since GSH upregulation is not unique to
sites of injury in the nasal airways, other factors (e.g., interactions with other nasal
antioxidants) must also contribute to the site-specificity of ozone-related nasal injury and

repair.

To my bride, Regina.
Your love, your sacrifice, and your smile

make this and all things possible.

iv

ACKNOWLEDGEMENTS

I would like to thank the many people whose time, wisdom, and encouragement
contributed to my personal and professional development during this process. I will be
forever indebted to my mentor, Dr. Jack Harkema. His guidance, generosity with his
knowledge and resources, experience, trust, and patience provided me with the
opportunity to develop a relevant research project of which I am truly proud. His
enthusiasm for teaching and passion for research provided a nurturing, welcoming
environment for my graduate training. I am very grateful to my committee members, Dr.
James Wagner, Dr. Susan Ewart, and Dr. Ed Robinson, for their guidance, advice, and
willingness to invest their time and effort into my graduate program and my professional
development. I have great respect for each of them, and it gives me great honor to now
be considered their colleagues. I am also grateful for the guidance, wisdom, and support
of Dr. Vilma Yuzbasiyan-Gurkan, who, despite her many responsibilities, always made
me feel as if I was her first priority.

Through my work with Dr. Harkema, I have also had the unique privilege of
learning from expert collaborators across the country. l owe many thanks to Dr. Charles
Plopper, Dr. Julia Kimbell, Dr. Richard Corley, Dr. Carol Ballinger, and Dr. Kevin
Minard, for sharing their knowledge and expertise. I would especially like to thank Dr.
Edward Postlethwait for being so generously giving of his time, resources, and expertise,
for the benefit of my training. My research project would not have been possible without

their effort and generosity.

My research was made both possible and enjoyable through the efforts of the
members of our lab, Lori Bramble, Ryan Lewandowski, Dr. Daher Ibrahim-Aibo, Dr.
Daven Jackson, Kara Corps, and Dr. Neil Birmingham. My research efforts were also
made possible by the invaluable contributions of Amy Porter, Kathy Joseph, and Rick
Rosebury in the Clinical Center Histology Lab, Ralph Common in the Electron
Microscopy Lab, and Sarah Davis, Brian Tarkington, Louise Olsen, Dr. Ruth McDonald,
Dr. Lisa Miller, Dr. Kristina Abel, and Dr. Veronique de Silva from the California
National Primate Research Center.

None of this would have been possible without generous financial support from
Pfizer Animal Health. I would particularly like to thank Dr. Michael Spensley and Dr.
Michael Kennedy of Pfizer for their support of my work and development. I would also
like to thank Dr. Robert Roth, Dr. Norbert Kaminski, the Center for Integrative
Toxicology, and NIH (N IEHS) P01 E80] 1617 for funding my research program.

Finally, I need to thank my parents for their unconditional love and support, and
for instilling in me the work ethic necessary to complete this milestone. I would like to
thank my precious children, Daria, Sophia, and Harrison, for their resilience during my
long hours away, and for being such wonderful inspirations for me. It is with the deepest
gratitude that I thank my bride, Regina, whose sacrifice was the greatest during this
process. She is the most important and most difficult to thank, as my words fail to firlly
express how essential her effort was to the achievement of this and all of my goals. With
her love, support, care, patience, I have become the husband, father, and scientist that I

am today.

vi

TABLE OF CONTENTS

LIST OF TABLES ..................................................................................................... xi
LIST OF FIGURES ................................................................................................... xii
LIST OF ABBREVIATIONS .................................................................................... xvi
CHAPTER 1

INTRODUCTION ............................................................................................... 1
Ozone-Induced Nasal Airway Injury ............................................................. 2

Human Health Significance ..................................................................... 2

Non-Human Primate Models ................................................................... 3

Fischer 344 Rat Model ............................................................................. 4
Site-Specificity of Ozone-Induced Injury in the Respiratory Tract ............... 6
Airway Antioxidants and Ozone Interactions ................................................ 7
Potential Role of Glutathione in Epithelial Adaptation and Remodeling ...... 10
Overall Goal ................................................................................................... 13
Governing Hypothesis ................................................................................... 13
Specific Aims of Thesis ................................................................................. 14
Chapter 1 References ..................................................................................... 17

CHAPTER 2

LOCAL ANTIOXIDANT PROFILE OF THE RAT NASAL CAVITY:

A REVIEW OF THE EXPERIMENTAL LITERATURE .................................. 22
Abstract .......................................................................................................... 23
Introduction .................................................................................................... 24
Sources of Biologically Relevant Environmental Oxidants .......................... 28

Ozone and Reactive Oxygen Species ...................................................... 28
Nitrogen Oxides (N Ox) and Reactive Nitrogen Species .......................... 30
Antioxidant Enzyme Systems of the Rat Nasal Epithelium .......................... 31
Superoxide Dismutase ............................................................................. 31
Catalase .................................................................................................... 33
NAD(P)H:Quinone Oxidoreductase (N QOl) .......................................... 34
Glutathione Peroxidase and Glutathione Reductase ................................ 35
Peroxiredoxin ........................................................................................... 37
Low Molecular Weight Antioxidants ............................................................ 38
Glutathione ............................................................................................... 38
Ascorbic Acid (Vitamin C) ...................................................................... 39
oc-Tocopherol (Vitamin E) ....................................................................... 40
Discussion ...................................................................................................... 40
Chapter 2 References ..................................................................................... 43

vii

CHAPTER 3
THREE-DIMENSIONAL MAPPING OF OZONE-INDUCED INJURY,
ANTIOXIDANT ALTERATIONS, AND PREDICTED OZONE FLUX,
IN THE NASAL AIRWAYS OF MONKEYS USING MAGNETIC

RESONANCE IMAGING AND MORPHOMETRIC TECHNIQUES .............. 51
Abstract .......................................................................................................... 52
Introduction .................................................................................................... 54
Materials and Methods ................................................................................... 56

Animals and Exposure Regimens ............................................................ 56
Necropsy and Tissue Preservation ........................................................... 59
Magnetic Resonance Imaging of Nasal Airways ..................................... 62
Image Analysis and Airway Segmentation .............................................. 63
Tissue Processing for Light Microscopy and Morphometric Analysis ...66
Three-Dimensional Mapping of Epithelial Distribution .......................... 68
Morphometric Quantitation of Ozone-Induced Nasal Lesions ................ 75
Three-Dimensional Digital Mapping of Ozone-Induced Nasal Injury ....78
Computational Fluid Dynamics Simulations ........................................... 81
Determination of Intracellular Antioxidant Concentrations
In Nasal Mucosa ...................................................................................... 81
Results ............................................................................................................ 82
Nasal Cavity Surface Area and Volume Calculations ............................. 82
Digital Epithelial Mapping and Surface Area Calculations ..................... 85
Ozone-Induced Nasal Epithelial Injury and Morphometry ..................... 85
Computational Fluid Dynamics Simulations ........................................... 9O
Intracellular Concentrations of Low Molecular Weight
Nasal Antioxidants ................................................................................... 92
Discussion ...................................................................................................... 94
Summary ........................................................................................................ 102
Chapter 3 References ..................................................................................... 105
CHAPTER 4

CORRELATION BETWEEN OZONE-INDUCED SQUAMOUS
METAPLASIA AND GLUTATHIONE UPREGULATION IN THE

NASAL AIRWAYS OF INFANT RHESUS MONKEYS .................................. 109
Abstract .......................................................................................................... 110
Introduction .................................................................................................... 1 11
Materials and Methods ................................................................................... 114

Animals and Ozone Exposure .................................................................. 114
Necropsy and Tissue Preservation ........................................................... 115

Tissue Processing for Light Microscopy and Morphometric Analysis ...118
Morphometry of Neutrophilic Inflammation and

Epithelial Numeric Cell Density .............................................................. 118
Morphometry of Epithelial Hyperplasia .................................................. 119
Morphometry of Stored Intraepithelial Mucosubstances ......................... 121

viii

Determination of Intracellular Low Molecular Weight

Antioxidant Concentrations in Nasal Mucosa ......................................... 121
Analysis for GCL-C and GCL-M mRNA in Nasal Tissues .................... 122
Statistical Analyses .................................................................................. 123
Results ............................................................................................................ 123
Nasal Histopathology ............................................................................... 123
Morphometry of Nasal Epithelium .......................................................... 128
Quantitation of Intraepithelial Mucosubstances ...................................... 128
Morphometry of Neutrophilic Inflammation ........................................... 132
Intracellular Concentrations of Low Molecular Weight
Nasal Antioxidants ................................................................................... 134
GCL-C and GCL-M mRNA Expression in the
Anterior Maxilloturbinate ........................................................................ 136
Correlation Analysis for Epithelial Cell Numeric Density and
Antioxidant Concentration Along the Anterior Maxilloturbinate ........... 136
Discussion ...................................................................................................... 139
Chapter 4 References ..................................................................................... 148
CHAPTER 5

ROLE OF GLUTATHIONE IN THE PATHOGENESIS OF
OZONE-INDUCED EPITHELIAL HYPERPLASIA IN RAT

NASAL AIRWAYS ............................................................................................ 155
Introduction .................................................................................................... 156
Study 1: Early Cellular and Molecular Events Preceeding Ozone-Induced
Epithelial Hyperplasia in the Nasal Airways of Immature Rats .......................... 159
Materials and Methods ................................................................................... 159
Animals, Ozone Exposure, and BrdU Treatment .................................... 159
Necropsy and Tissue Preparation for Morphometric Analyses ............... 162
Morphometry of Cellular Injury and Cell Proliferation ........................... 165
Tissue Preparation for mRNA Analysis .................................................. 165
Total RNA Isolation from Nasal Tissues ................................................. 166
Real-time Reverse Transcription Polymerase Chain Reaction ................ 166
Statistical Analyses .................................................................................. 167
Results ............................................................................................................ 168
Ozone-Induced BrdU Incorporation in Immature Rat Nasal Airways ....168
Ozone-Induced Oxidative Stress Gene Expression ................................. 170
Ozone-Induced Pro-Inflammatory Gene Expression ............................... 170
Ozone-Induced CXC Chemokine Gene Expression ................................ 170
Discussion ...................................................................................................... 176
Study 2: Eflect of GSH Depletion on Ozone-Induced Epithelial Hyperplasia 1 78
Materials and Methods ................................................................................... 178
Animals, Ozone Exposures, and Glutathione Depletion ......................... 178
Necropsy and Tissue Preparation for Morphometric Analyses ............... 179

ix

Morphometry of Neutrophilic Inflammation, Epithelial Numeric

Cell Density, and Cell Proliferation ......................................................... 180
Tissue Preparation for HPLC Antioxidant Analysis ................................ 181
Analysis of Intracellular Concentrations of
Low Molecular Weight Nasal Antioxidants ............................................ 182
Statistical Analyses .................................................................................. 183
Results ............................................................................................................ 183
Determination of Low Molecular Weight _
Nasal Antioxidant Concentrations ........................................................... 183
Nasal Histopathology ............................................................................... 188
Morphometric Quantitation of Epithelial Hyperplasia ............................ 191
Morphometric Quantitation of Neutrophilic Inflammation ..................... 198 '
Discussion ...................................................................................................... 200
Chapter 5 References ..................................................................................... 204
CHAPTER 6
SUMMARY AND CONCLUSIONS .................................................................. 209
Chapter 6 References ..................................................................................... 216

LIST OF TABLES

Table 3-1. Calculated total surface areas and volumes

of the nasal passages from the four 180-day-old

monkeys pictured in Figure 3-2. ..................................................... 84
Table 3-2. Estimated epithelial surface areas for one side

of a 180-day-old rhesus monkey ..................................................... 84

xi

Figure 1-1.

Figure 1-2.

Figure 3-1.

Figure 3-2.

Figure 3-3.

Figure 3-4.

Figure 3-5.

Figure 3-6.

Figure 3-7.

Figure 3-8.

Figure 3-9.

Figure 3-10.

LIST OF FIGURES

Potential fates of inhaled ozone in the nasal cavity. ....................... 9

Potential role of GSH in ozone-induced epithelial hyperplasia
in the nasal airways of rats .............................................................. l6

Diagram of the right lateral wall (A) and the left nasal
cavity (B) of a 90-day-old rhesus monkey following
sagittal sectioning ............................................................................ 61

Computer-assisted 3D isosurface renderings of the nasal
passages from two FA-exposed (A and B) and two 1 cycle

ozone-exposed (C and D) 180-day-old infant monkeys ................. 65
Photograph of the lateral wall of the nasal cavity

of a 90-day-old rhesus monkey ....................................................... 67
Three-dimensional mapping of nasal epithelium ............................ 72

Three-dimensional nasal epithelial map. Medial (A)
and lateral (B) views of a 3D reconstruction of the right
nasal passage of a 180-day-old rhesus monkey .............................. 74

Photomicrograph (A) of an H&E-stained transverse section

through the left nasal airway of a 90-day-old infant monkey.
Z-plane MR image (B) of the nasal cavity of the same monkey,
obtained at the level corresponding to the transverse

section in (A) ................................................................................... 76

Three-dimensional mapping of ozone-induced nasal lesions ......... 80
Light photomicrographs of the nasal mucosa lining the dorsal
surface of the anterior aspect of the maxilloturbinate of

90-day-old monkeys exposed to 0 ppm ozone (filtered air, A),

1 cycle ozone (B) or 5 cycle ozone (C) ........................................... 87

Morphometry of ozone-induced nasal epithelial lesions in
90-day-old rhesus monkeys ............................................................ 89

Computational fluid dynamics simulation of airflow and ozone
uptake in the nasal airways ............................................................. 91

xii

Figure 3-11.

Figure 3-12.

Figure 4-1.

Figure 4-2.

Figure 4-3.

Figure 4-4.

Figure 4-5.

Figure 4-6.

Figure 4-7.

Figure 4-8.

Figure 5-1.

Figure 5-2.

Antioxidant concentrations in the nasal mucosa of infant
Monkeys exposed to 1 cycle and 5 cycle ozone ............................. 93

Overview of the imaging, 3D modeling, and
histopathologic methods ................................................................. 104

Anatomic location of the nasal tissues
selected for morphometric analysis ................................................ 117

Light micrographs of the nasal mucosa lining the

dorsomedial surface of the maxilloturbinate of 180-day-old

monkeys exposed to 0 ppm ozone (filtered air, A),

1 cycle ozone (B), or 11 cycle ozone (C). Tissues were

stained with H&E ............................................................................ 126

Morphometry of ozone-induced nasal epithelial injury
in infant rhesus monkeys ................................................................ 130

Light photomicrographs of maxilloturbinates from monkeys

exposed to FA (A) or 11 cycle ozone (C). Tissues were

stained with AB/PAS. (B,D) Morphometry of

intraepithelial mucus ....................................................................... 131

Effect of ozone exposure on the numeric cell density
of neutrophils (PMNs) in the nasal mucosa along the
anterior maxilloturbinate of infant monkeys ................................... 133

Effect of 1 cycle ozone and 1] cycle ozone exposure on the

intracellular concentrations of GSH (A), GSSG (B), AHz (C),

and UA (D) in the nasal mucosa from the anterior

maxilloturbinate, posterior maxilloturbinate, and anterior
ethmoturbinate of infant monkeys .................................................. 135

Effect of 1 cycle ozone and 11 cycle ozone exposure on
GCL-C (A) and GCL-M (B) gene expression in the
nasal mucosa of the anterior maxilloturbinate ................................ 137

Correlations between epithelial hyperplasia and the intracellular

concentrations of GSH (A), AH2 (B), and UA (C) in the
nasal mucosa lining the anterior maxilloturbinate .......................... 138

Anatomic location of rat nasal tissues selected for
morphometric analyses ................................................................... 164

Effect of ozone exposure on BrdU labeling index (A) and
BrdU numeric cell density (B) in the NTE ..................................... 169

xiii

Figure 5-3.

Figure 5-4.

Figure 5-5.

Figure 5-6.

Figure 5-7.

Figure 5-8.

Figure 5-9.

Figure 5-10.

Figure 5-11.

Figure 5-12.

Figure 5-13.

Figure 5-14.

Effect of ozone exposure on expression of HO-l mRNA (A)
and iNOS mRNA (B) in rat nasal mucosa ...................................... 172

Effect of ozone exposure on expression of GCL-C mRNA (A)
and GCL-M mRNA (B) in rat nasal mucosa .................................. 173

Effect of ozone exposure on expression of TNF-a mRN A (A)
and IL-6 mRNA (B) in rat nasal mucosa ........................................ 174

Effect of ozone exposure on expression of MIP-2 mRNA (A)
and CINC mRNA (B) in rat nasal mucosa ...................................... 175

Effect of ozone exposure +/- BSO treatment on intracellular
GSH concentrations in rat nasal mucosa ........................................ 185

Effect of ozone exposure +/- BSO treatment on intracellular
GS SG concentrations in rat nasal mucosa ...................................... 186

Effect of ozone exposure +/- BSO treatment on intracellular
AH; concentrations in rat nasal mucosa ......................................... 187

Light photomicrographs of the dorsal aspect of maxilloturbinates
from rats treated with saline (A, B, C) or BSO (D, E, F) and

exposed to 0 ppm ozone (A and D), exposed to 0.8 ppm ozone

for 2 days (B and D), or exposed to 0.8 ppm ozone for 3 days

and killed 48 hours post-exposure (C and F). Tissues were

stained with H&E ............................................................................ 190

Light photomicrographs of maxilloturbinates from rats

treated with saline and exposed to 0 ppm ozone

(filtered air, A), and from rats treated with saline (B) or

BSO (C) and exposed to 0.8 ppm ozone for 2 days.

Tissues were immunohistochemically stained with anti-PCNA
antibody, and counterstained with hematoxylin ............................. 194

Effect of ozone exposure +/- BSO treatment on PCNA labeling
index in the NTE lining the anterior maxilloturbinate .................... 195

Effect of ozone exposure +/- BSO treatment on the numeric I
density of cells expressing PCNA in the NTE lining the
anterior maxilloturbinate ................................................................. 196

Effect of ozone exposure +/- BSO treatment on the numeric

density of epithelial cells in the NTE lining the anterior
maxilloturbinate .............................................................................. 1 97

xiv

Figure 5-15. Effect of ozone exposure +/- BSO treatment on the numeric
density of intraepithelial neutrophils in the NTE lining
the anterior maxilloturbinate ........................................................... 199

Images in this dissertation are presented in color.

XV

 

3D

AB/PAS
AHz

ANOVA
AP-l
BALF
BALT
BrdU
BSO
CAD
cDNA
CFD
CINC
CSC
CT
DDV
DNA
EH
ELF
ET

F344

LIST OF ABBREVIATIONS

three-dimensional

alcian blue/periodic acid Schiff
ascorbate

analysis of variance

activator protein-1

bronchoalveolar lavage fluid
bronchus-associated lymphoid tissue
bromo-deoxyuridine
L-buthionine-[S,R]-sulfoximine

computer aided design

copy deoxyribonucleic acid

computational fluid dynamics
cytokine-induced neutrophil chemoattractant

cigarette smoke condensate
cycle threshold

digital data viewer
deoxyribonucleic acid
epithelial hyperplasia
epithelial lining fluid
ethmoturbinate

Fischer 344 rat

xvi

FA

FI

GALT

GAPDH

GCL

GCL-C

GCL-M

GGT

GPx

GR

GS'

GSH

GSSG

G6PDH

H&E

HO-l

HP

HPLC

IL

iNOS

LE

MCM

MIP-2

filtered air

fold-increase

gut-associated lymphoid tissue
glyceraldehyde-3 -phosphate dehydrogenase
glutamate cysteine ligase

glutamate cysteine ligase, catalytic subunit
glutamate cysteine ligase, modulatory subunit
y-glutamyltranspeptidase

glutathione peroxidase

glutathione reductase

glutathionyl radical

glutathione

glutathione disulfide

glucose-6-phosphate dehydrogenase
hematoxylin and eosin

heme oxygenase-1

hard palate

high performance liquid chromatography
interleukin

inducible nitric oxide synthase
lymphoepithelium

mucous cell metaplasia

macrophage inflammatory protein-2

xvii

MPO

mRNA
MT
NAAQS
NAC
NADH
NADPH
NALT
NP
NQ01
Nrf2

NTE

OE
PCNA
PCR

PMN

P'Pm

myeloperoxidase

magnetic resonance

messenger ribonucleic acid
maxilloturbinate

national ambient air quality standard
N-acetylcysteine

nicotinamide adenine dinucleotide
nicotinamide adenine dinucleotide phosphate
nasal-associated lymphoid tissue
nasopharynx

NADPHzQuinone oxidoreductase
nuclear factor-erythroid 2 related factor

non-ciliated transitional epithelium
ozone

olfactory epithelium
proliferating cell nuclear antigen
polymerase chain reaction
polymorphonuclear leukocyte
parts per million

respiratory epithelium
ribonucleic acid

reactive nitrogen species

reactive oxygen species

xviii

RT

SE

SEM

SOD

STL

UA

VOC

reverse transcriptase
squamous epithelium
standard error of the mean
superoxide dismutase
stereolithography

tumor necrosis factor

uric acid

volatile organic compound

xix

CHAPTER 1

INTRODUCTION

Tropospheric ozone, the principal oxidant pollutant in photochemical smog, is recognized
as one of the most important environmental pollutants due largely to its adverse health
effects on the respiratory system. The nasal cavity is an important target of inhaled
airborne pollutants such as ozone, due in part to its proximal location in the respiratory
tract. Specialized epithelial functions including absorption of reactive gases (Brain
1970), filtration and removal of particulates and gases (Proctor and Anderson 1982), and
metabolism of inhaled xenobiotics (Dahl and Hadley 1991), equip the nasal cavity to
serve a protective role as a “scrubbing tower,” limiting access of inhaled chemical agents
to the more delicate pulmonary parenchyma and conducting airways (Brain 1970). These
same functions also expose the mucosa of the nasal airways to higher concentrations of
these inhaled xenobiotics, making the nose more susceptible to injury from certain highly

reactive inhaled oxidant pollutants, including ozone.

Ozone-Induced Nasal Airway Injury—Human Health Sggn’ ificance. Residents of

Mexico City, a population chronically exposed to high ambient levels of ozone,
experienced loss of cilia, basal cell hyperplasia, and squamous metaplasia in the nasal
airways (Calderon-Garciduenas et a1. 1992). Controlled ozone experiments using human
subjects indicated that ozone induces similar profiles of nasal and pulmonary airway
inflammation (Graham and Koren 1990; Koren et a1. 1990). Inflammatory markers
(percent neutrophils, IL-6, fibronectin) in bronchoalveolar lavage fluid (BALF) were
decreased in subjects exposed daily to ozone (0.2 ppm) for four consecutive days,
Compared to subjects exposed for a single day (Devlin 1993). A similar attenuation of

BALF inflammation was observed, despite evidence of ongoing airway epithelial injury,

in response to repeated ozone exposures in human volunteers (Christian et al. 1998).
These experiments, which characterize the responses to repeated exposures, are of
particular human health importance, as repeated or episodic exposure likely mimics the

pattern of exposure experienced by most people (Srarn et a1. 1996).

Children may be at increased risk of airway injury induced by oxidant pollutants (Kim
2004). Factors supporting this age-related heterogeneity in airway injury include
differences in airway geometry and delivered ozone dose, differences in the distribution
of susceptible epithelial populations; and differences in intrinsic epithelial defenses
against oxidative stress (antioxidants, mucus, epithelial repair capacity) in the respiratory
tract between children and adults. The respiratory tract undergoes significant post-natal
development in children. Ozone exposures early in life are associated with long-term
consequences in the pulmonary airways. Pollutant exposure during the critical period of
post-natal lung grth in children impairs lung development, which results in lung
function deficits in adulthood (Gauderman et a1. 2004). Childhood ozone exposure also
causes inflammation, epithelial necrosis, ciliary loss, and epithelial hyperplasia in the
nasal airways (Koltai 1994; Calderon-Garciduenas ct al. 1995; Kopp et al. 1999).

However, the long-term ramifications of this early exposure have not been investigated.

Ozone-Induced Nasal Airway Iniury—Non-Human Primate Models. Short- and
lOrig-term (daily) ozone exposures cause significant nasal airway injury in experimental
animal models. Most of these studies during the past 20 years were conducted in adult

IOdent and macaque monkey models, and have been the subject of a recent review

(Nikasinovic et a1. 2003). The macaque monkey serves as an important animal model of
upper airway toxicology because their nasal airways resemble, at a gross and microscopic
level, the nasal airways of humans (Harkema 1991; Yeh et a1. 1997). The first of these
studies used an adult bonnet monkey (Macaca radiata) model exposed to 0.15 or 0.30
ppm ozone for 6 or 90 days. In these studies, ozone-induced lesions were confined to the
anterior nasal airways (Harkema et a1. 1987; Harkema et a1. 1987). The principal lesions
observed in this model included neutrophilic rhinitis at 6 days, and ciliated cell necrosis
with loss of cilia, epithelial hyperplasia, and mucous cell metaplasia (MCM) at both 6
and 90 days. The epithelial remodeling events identified at 90 days were associated with
attenuation of the inflammatory cell infiltration compared to 6-day exposure. These
lesions specifically involved the non-ciliated transitional epithelium (NTE) and the
adjacent ciliated respiratory epithelium (RE). Similar hyperplastic lesions (cuboidal
bronchiolar cell hyperplasia) were also identified in the terminal bronchiolar epithelium
of the lungs in ozone-exposed monkeys (Harkema et a1. 1993). Thus, assessment of nasal
airway injury in monkeys may also serve as a sentinel for injury to the more delicate
pulmonary airways. Furthermore, these reports document that the nasal airways of
monkeys, which are morphologically similar to human nasal airways, are susceptible to
injury at levels near the National Ambient Air Quality Standard ozone concentration of

0.075 ppm.

onne-Induced Nasal Airway hing—Fischer 344 Rat Model. Rats exposed to 0.8

mm ozone, 6h/day for 7 days develop MCM and epithelial hyperplasia in the non-

ciliated transitional epithelium lining the maxilloturbinates, nasoturbinates, and lateral
wall of the proximal aspect of the nasal airways (Harkema et al. 1989). Ozone-induced
MCM persists, and in some regions progresses, for at least seven days post-exposure
(Harkema et al. 1989). The ozone-induced lesions in Fischer 344 (F 344) rats exposed to
0.8 ppm for 7 days histologically resemble those induced in macaque monkeys exposed
to 0.3 ppm ozone for 6 days (Harkema et al. 1987). No nasal lesions were identified in
rats exposed to 0.12 ppm ozone for 7 days. This is in contrast to the MCM induced in the
nasal airways of macaque monkeys exposed to 0.15 ppm ozone, 8h/day for 6 days
(Harkema et al. 1987). This differential, species-dependent response suggests that the
rat NTE may be more resistant to ozone-induced injury than the NTE in the monkey, and
supports the use of higher ozone concentrations for comparative toxicology studies in rat

models.

The temporal relationships among neutrophil influx, epithelial necrosis, and epithelial
DNA synthesis, following a single exposure to 0.5 ppm ozone in rats are well
characterized (Hotchkiss et al. 1997). Epithelial cell loss (due to necrosis) appears within
8 hours after the end of the exposure, and DNA synthesis begins within 12 hours, and
peaks within 24 hours, after the end of a single 8-hour ozone exposure. Cho et al. (Cho et
al. 1999) investigated the time course of cellular events preceding epithelial hyperplasia
and MCM during the course of a three-day ozone exposure in rats (Sh/day, 0.5ppm).
I)uring a three-day exposure, DNA synthesis peaked on day two of exposure, three days
Prior to the peak of the epithelial hyperplastic response (Cho et al. 1999). These findings

indicate that the cellular events underlying the ozone-induced phenotypic alterations in

the nasal epithelium begin within the first eight hours of exposure, and that the cellular

commitment to cell proliferation occurs within the first two days of ozone exposure.

Meeificitv of OWN—WM The epithelial
responses to ozone exposure vary markedly by site within the respiratory tract. In rhesus
monkeys, the nature and magnitude of ozone-induced cellular injury vary significantly by
location in both the nasal and pulmonary airways. In the lungs, ozone exposure targets
the ci liated respiratory epithelium of the trachea and type I pneumocytes lining the
terminal bronchioles (Castleman et a1. 1980; Wilson et al. 1984; Nikula et al. 1988). In
the nasal airways, ozone-induced injury is confined to the anterior nasal cavity,
specifically targeting the NTE and ciliated RE (Harkema et al. 1987; Harkema et al.
1987; Carey et al. 2007). The location of ozone-induced injury in the respiratory tract is
due to a combination of the local site-specific dose of ozone and tissue susceptibility
(Morgan and Monticello 1990). In the nasal airways, regional differences in ozone dose
are related to the ambient concentration of ozone, physicochemical properties of the
i“killed gas mixture, and nasal airflow patterns. The intranasal differences in
Susceptibility to ozone-induced injury can be due to many factors, including structural
differfinces in epithelial populations (e.g. ciliated, keratinized), local metabolic capacity,
or 100311 differences in innervation or vasculature. The basis for the variations in tissue

Susceptibility may also be related to the nature and magnitude of the tissue response to

OXidant challenge.

Airway Antioxidants and Ozone Interactions. Antioxidant molecules in the epithelial
lining fluid (ELF) of the respiratory system are considered to provide the first line of
defense against cellular stress from inhaled oxidant pollutants (Cross et al. 1994).
Intracellular antioxidant molecules also play a role in the cellular defense against inhaled

oxidants, both within the intracellular compartment as well as in the maintenance of ELF

levels- These include low molecular weight antioxidants such as ascorbate (AHz), uric

acid (UA), a-tocopherol, and glutathione (GSH), and larger protein complex enzymes,
including superoxide dismutase (Stenfors et al.), glutathione peroxidase (GPx), and
glutathione reductase (GR). The low molecular weight antioxidants are of particular
interest, since recent evidence suggests that the tissue concentrations of these substances
decrease with age (Lenton et al. 2000), and in certain disease states (Dibbert et al. 1999).
Several studies have correlated fluctuations in the levels of these low molecular weight
antioxidants with the onset of airway cellular damage in vivo (Guidot and Roman 2002),
or with the appearance of indicators of cellular damage in vitro (Ballinger et al. 2005).
Furthermore, recent reports also support associations between oxidant-mediated
respiratory tract injury and changes in the levels or activity of cellular antioxidants

(Kirschvink et al. 2002; Comhair et a1. 2005).

Interactions between ozone and respiratory tract epithelial cells are governed in large part
by ELF and intracellular antioxidants (Figure 1-1). Inhaled ozone may be effectively
r emOVed, or quenched, in the presence of protective levels of antioxidants. Without
adeq Hate antioxidant protection, however, the respiratory airways become susceptible to

deant-medlated damage. Ozone is an extremely reactive ox1dant gas, and rs relatlvely

insoluble in aqueous solutions. For these reason, it is unlikely that ozone itself is capable
of penetrating the ELF layer and oxidizing epithelial cell membranes (Cross et al. 1998).
A growing body of evidence suggests that ozone itself does not produce cytotoxicity to
airway epithelial cells, but rather does so by generating reactive secondary products
within the epithelial lining fluid (ELF), which subsequently induce cellular damage to the
underlying epithelium (Postlethwait et al. 1998). While antioxidants are largely
considered to confer protection to the airway epithelium during oxidant pollutant
exposure, results from several recent investigations support the concept that the presence

of‘ ELF antioxidants may facilitate oxidant damage by mediating this

 

 

EpithelialSurface: 3-
Compartment Model

 

Intracellular
Space

Fig“ re 1-1. Potential fates of inhaled ozone in the nasal cavity. Diagrammatic
1'epl‘E:Sentation of the epithelial surface lining the anterior nasal cavity. Prior to initiating
mactions with cell surface and intracellular targets, gas-phase ozone (O3) in the airway
hurlen must penetrate the epithelial lining fluid (ELF) layer. Reactions with ELF
antloaiidants, including ascorbate (AH), uric acid (UA), and glutathione (GSH) may
completely quench inhaled ozone, conferring complete protection. It is also possible that
Ozone itself may completely penetrate the ELF intact and directly react with membrane
and intracellular lipids and proteins. However, reactions with ELF antioxidants can also
generate reactive oxygen species (ROS), which may mediate ozone-induced cell
n'lefnbrane protein oxidation and lipid peroxidation via “reactive absorption.” ELF
antloxidant levels are maintained in part by the regulation of intracellular antioxidant
ConCentrations. In the case of GSH, this balance is maintained through a combination of
rail novo synthesis, redox recycling of oxidized glutathione (GSSG) Via glutathione
uCtase (GR), and scavenging of extracellular GSH and GSSG.

“reactive absorption,” and thereby yielding toxic reaction products. It has been shown in

vitro, that the presence of low concentrations of ascorbate or glutathione (GSH) within
the ELF augments membrane oxidation during N02 exposure. NOz-induced membrane

damage is ameliorated by higher ELF concentrations of ascorbate or GSH (V elsor and
Postlethwait 1997). In this same in vitro model system, a similar biphasic response was
observed with ozone exposure, demonstrating enhancement of membrane oxidation at
low ascorbate or GSH concentrations, and protection at higher antioxidant concentrations
(Ballinger et al. 2005). These results indicate that ELF antioxidants confer protection
against oxidant-induced airway injury in a concentration-dependent manner, and further
suggest that the regulation of ELF antioxidant levels is an important factor in both the

mediation of and epithelial response to oxidant-induced airway injury.

Potential Role of Glutathione in Epithelial Adaptation and Remodeling. The concept

0f pulmonary tolerance to ozone exposure has been well described in both human
volunteers and experimental animal models. Prolonged exposure to ozone and other
inhaled pollutants results in biochemical changes that render the epithelium resistant to
I"Uri-her oxidant injury. Among these are increases in the activity of several antioxidants,
inCIuding glutathione (GSH), within the airway epithelium (Plopper et al. 1994; Duan et
al- 1 996). Daily ozone exposure (0.75 ppm, 12h/day for 7 days) results in a sustained
inerease in BALF levels of uric acid and glutathione in calves. BALF antioxidant levels
inerease by day 3 of exposure, remain elevated through day 7, and are accompanied by a
corresponding decrease in the number of neutrophils in BALF (Kirschvink et al. 2002).

Mile these mechanisms have not been extensively investigated in the nasal epithelium,

10

the similarity in epithelial types between the nasal and pulmonary airways suggests that

similar adaptation could occur within the nasal cavity.

Injury to nasal and pulmonary epithelial cells exposed to ozone is highly focal and site-
specific in vivo. Several factors may contribute to this site specificity, including local
ozone dose, susceptibility of the local epithelium, and capacity for antioxidant protection.
Significant variability exists across different subcompartrnents of the monkey and mouse
lung in both the rates of consumption and resynthesis of GSH during ozone challenge in
vitro (Duan et al. 1996). In all regions of mouse lung examined (trachea, minor daughter,
and parenchyma), tissue GSH levels were initially depleted by ozone exposure, and
returned to baseline levels within 2 hours of the end of exposure. Tissue GSH levels in
comparable regions of monkey lung were also similarly depleted by ozone exposure, and
required up to 4 hours for re-synthesis or redox recycling to return GSH to baseline
level 3 - There are also variations in the activities of glutathione-S-transferase and
glutatljione peroxidase, enzymes that require GSH as a substrate, across different regions
0f monkey lung (Duan et al. 1993). In young adult monkeys, the sites of ozone-induced
GSH depletion correspond with sites of exposure-related injury in the pulmonary airways
(Plopper et al. 1998). These results suggest that site- and species-dependent
SuSCeptibility to ozone-induced lower airway injury may be in part mediated by the
caIDaCity of the local epithelium to regulate or replace consumed antioxidants. The
relationship between local GSH regulation and susceptibility to oxidant-mediated injury

i . . . .
n the upper airways has not been extensrvely investlgated.

11

Investigators in several previous reports have suggested that the degree of epithelial
injury may play a role in the progression of epithelial remodeling. Reports of ozone and
formaldehyde inhalation studies indicate that sublethal cellular injury is sufficient, and
perhaps necessary, to trigger toxicant-induced cell proliferation (Monticello et al. 1991;
Henderson et al. 1993). Exposure to low concentrations of cigarette smoke condensate
(CSC), a mixture of toxic compounds including particulate matter, endotoxin, and
oxidant pollutant gases (Stockley et al. 2008), triggers cell proliferation in two lines of
bronchial epithelial cells in vitro. Exposure to high concentrations of CSC inhibits cell
proliferation in these same cell lines. This inhibitory effect of the high concentrations is
due in part to higher levels of cytotoxicity. Administration of the cysteine donor
compound, N-acetylcysteine (NAC) ameliorated the inhibitory effect on cell
proliferation, indicating that this effect is mediated in part by CSC-induced depletion of
non-protein thiol levels (e.g. GSH), and that restoration of non-protein thiol levels via
NAC also restores proliferative capacity in these cells (Luppi et al. 2005). This also
indicates that the magnitude of cellular injury plays a role in the subsequent epithelial
relTlOdeling process; and that glutathione, which exhibits site- and species-dependent
kinetics in response to oxidant challenge, may be an integral factor in site- and species-

dependent, oxidant-induced epithelial remodeling in the nasal airways.

Se‘Veral studies implicate glutathione upregulation as a necessary precursor to the

Still‘lulation of cell proliferation, specifically to entry into S-phase of the cell cycle.

I . . . . . .
chreased 1ntracellular GSH concentration IS a precursor to Gl/S transrtlon followmg

1 togenie stimulation in quiescent (G0) fibroblasts. Furthermore, inhibition of GSH

12

synthesis inhibits DNA synthesis and reduces the percentage of cells capable of entering
S-phase (Shaw and Chou 1986; Kavanagh et al. 1990). The molecular mechanisms
underlying this regulation remain unclear. Previous studies have indicated that
intracellular redox balance is an important regulator of cell cycle progression (Menon et
al. 2003)(Lu et al. 2007). Thus, it is possible that intracellular GSH regulates cell cycle
progression, and ultimately, cell proliferation, via its role in the maintenance of

intracellular redox balance.

Overall Goal: The overall goal of my thesis experiments was to determine the
relationship between the regulation of nasal antioxidants and susceptibility to ozone-
induced nasal airway injury and repair. Specifically, my studies were designed to

deterrnine the association between the local regulation of GSH and the site-specificity of
ozone-induced injury and repair in the nasal airways of infant monkeys and rats, and to
detennine the role of GSH in the pathogenesis of nasal epithelial hyperplasia following
ozone exposure in infant monkeys and immature rats. Based I on previous studies
demonstrating correlations between local GSH regulation and ozone-induced injury in the
Plflmonary airways, we anticipated that GSH would be a determinant in the susceptibility
to Ozone injury in the nasal airways, and that changes in GSH regulation would parallel

the Onset of epithelial hyperplasia.

G OVerning Hypothesis: The spatial distribution and temporal pattern of ozone-induced

epithelial hyperplasia in the nasal airways are dependent on the local regulation of GSH.

l3

Specific Aims:
I: To describe the temporal relationships between ozone exposure and the
development of epithelial hyperplasia in the nasal airways of infant monkeys and
rats. These studies were specifically designed to determine the temporal association
between the ozone exposure and the process of epithelial hyperplasia in the anterior nasal
airways of infant rats and monkeys acutely or episodically exposed to high ambient levels
of ozone. Infant monkeys and immature rats were exposed episodically (monkeys) or
daily (rats) to ozone or filtered air. The morphologic responses of the nasal epithelium
were extensively characterized and quantified during the course of daily and episodic

ozone exposures using standard histopathologic and morphometric techniques.

II: To test the hypothesis that GSH upregulation is spatially and temporally
correlated with sites of ozone-induced injury and repair in the nasal airways of
infant monkeys. These studies were designed to test the hypothesis that the site-
SPeeificity of ozone-induced epithelial hyperplasia in the nasal airways of infant monkeys
and rats is due, in part, to the local regulation of GSH following ozone exposure. The
location and severity of ozone-induced injury (including epithelial hyperplasia) in the left
nasal passages (described in Specific Aim 1) was documented using morphometric
techniques, and mapped on three-dimensional models of the lefi nasal airways of infant
rnoI‘lleeys. The biochemical responses to daily and episodic ozone exposure (changes in
antioxidant concentrations, determination of antioxidant and pro-inflammatory gene
expression) were determined in site-matched mucosal samples from the contralateral

nasal passage. Morphologic and biochemical responses were also evaluated in regions

14

that did not exhibit ozone-induced injury. The relationship between ozone-induced
epithelial hyperplasia and steady-state GSH concentrations was described using

correlation analysis.

111: To test the hypothesis that ozone-induced epithelial hyperplasia in the nasal
airways of immature rats is a GSH-dependent event. These studies were designed to
determine the effect of in vivo GSH depletion, during early ozone exposure, on the

initiation of cell proliferation and the subsequent development of epithelial hyperplasia.
Immature male F 344 rats were exposed daily to ozone or filtered air. Half of the rats
were treated with L-buthionine-[S,R]-sulfoximine (BSO), to cause depletion of
intracellular GSH. B80 is an irreversible inhibitor of glutamate cysteine ligase (GCL),
the rate-limiting enzyme in de novo GSH synthesis. These experiments served to
determine the role of GSH and GSH regulation, during early ozone exposures, in the

development of ozone-induced nasal epithelial hyperplasia in immature rats (Figure 1-2).

15

1);») 2. I)‘\.\

 
   

.‘nlllllr‘h

GSH Depletion l.\ l’llnwl

‘unl I"\lnli.lllnll

l ’.;.\\l .’ .mlzuplllll. ‘1- "'- _ _ l).l_\ 15 l'illlllrllul

II: il.1.x.ln.1l1.»ll “' ,1' j ‘ Il)prl|\ll~l..

 

L
Injury _. Inflammation aRepair/RemodeTing

Figure 1-2. Potential role of GSH in ozone-induced epithelial hyperplasia in the nasal
airways of rats. Daily exposures to ozone cause neutrophilic inflammation, epithelial
necrosis and exfoliation on Day 1, followed by the initiation of DNA synthesis and cell
Proliferation on Day 2. These early cellular events precede the development of ozone-
lnduced epithelial hyperplasia on Days 4-5. We hypothesize that ozone-induced GSH
uPregulation maintains intracellular redox balance, facilitating cell cycle progression and
_epithelial hyperplasia, and that GSH depletion will prevent this cytoprotection, and
1nl'libit the development of epithelial hyperplasia.

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analysis of primate nasal airways using magnetic resonance imaging and nasal
casts." J Aerosol Med 10(4): 319-29.

21

CHAPTER 2

LOCAL ANTIOXIDANT PROFILE OF THE RAT NASAL CAVITY: A REVIEW

OF THE EXPERIMENTAL LITERATURE

22

ABSTRACT
Environmental pollutants are a known cause of lung pathology. Inhaled oxidants
such as ozone, nitrogen oxides, and tobacco smoke are important environmental causes of
pulmonary epithelial damage. Antioxidants in the epithelium lining the respiratory tract,
and those in the overlying epithelial lining fluid (ELF), constitute the initial lines of
defense against these airborne toxicants (Cross et al. 1994). The nasal cavity is an
important target for inhaled oxidant toxicants, and also may serve as a sentinel for
potential lower airway toxicity. The rat is an important animal model for inhalation
toxicologic risk assessment studies, and recent studies have begun to evaluate the
antioxidant profiles of the rat nasal cavity as a potential factor in the susceptibility to
oxidant pollutant-mediated airway injury. Assessment of the levels of constitutive and
inducible antioxidants in this model species, as well as comparisons between animal
models and humans, will aid in the understanding of the mechanisms of oxidant damage

by inhaled toxicants, and may reveal important pathways for intervention.

23

INTRODUCTION

The tissues of the respiratory tract are first-line targets of the effects of
environmental pollutants. Several of these pollutants exert their toxic effects on the
respiratory system by directly inducing oxidative damage to the epithelium or to
components of the epithelial lining fluid (ELF). Ozone, the major oxidant pollutant in
photochemical smog, causes peroxidation of cell membrane fatty acids in pulmonary
epithelial cells of humans (Baskin and Salem 1997; Cross et al. 1998). These peroxides
can trigger chain reactions in adjacent molecules, and initiate a cascade of reactions
yielding equally reactive by-products (Sandstrom 1995). The oxides of nitrogen,
primarily generated during high temperature combustion, are capable of causing similar
epithelial cell membrane damage in the respiratory tract. In addition to directly causing
epithelial damage, inhaled oxidants can also damage membrane and surface fluid
proteins, and the products of this molecular damage, including free radicals, can generate
further epithelial damage and perpetuate membrane chain reactions (Maples et al. 1993).
Constituents of tobacco smoke, diesel exhaust, and other pollutant gaseous and
Particulate mixtures possess oxidant properties capable of inducing cellular damage to the

re Spiratory system, alone and in combination with other pollutants (Sandstrom 1995).
The epithelium of the respiratory system possesses an intrinsic antioxidant system
that serves as the initial line of defense against these environmental toxicants (Cross et al.
I 994). This system is comprised of two major classes of antioxidant compounds. The

first class, the antioxidant protein enzymes, including superoxide dismutase (Stenfors et

a1 ~ 2004), catalase (Harkema et al.), glutathione peroxidase (GPx), and glutathione

r
ed uCtase (GR), primarily provide intracellular defense against endogenous oxidants and

24

the secondary effects of exogenous oxidant pollutants. Some membrane-bound and
extracellular forms of these enzymes also exist (e.g. extracellular SOD). The second

class, low molecular weight antioxidant molecules, including glutathione, oc-tocopherol,
ascorbic acid (AHZ), and uric acid (UA), can be located within epithelial cells as well as

within the ELF. These intrinsic antioxidants, primarily synthesized to neutralize
endogenously generated oxidants during cellular respiration, also provide an important

source of initial protection from inhaled oxidants.
The pulmonary effects of inhaled xenobiotic oxidants, including ozone, as well as
the role of lung tissue antioxidants, have been extensively investigated and well
characterized in humans (Barr et al. 1990; Devlin et al. 1991; Frischer et al. 1993; Cross
et a1. 1994; Sandstrom 1995; Calderon-Garciduenas et al. 2000; Calderon-Garciduenas et
a1. 2000; Mudway et al. 2004) and in experimental animal models (Castleman et a1. 1973;
Dungworth et al. 1975; Frank et al. 1978; Castleman et al. 1980; Slade et al. 1985;
Harkema et al. 1999). People demonstrate significant differences in susceptibility to the
harnl ful effects of ozone, with certain subsets of the population exhibiting heightened
sensitivity to ozone exposure (Devlin 1993). Susceptibility of the lung to the effects of
inhaled oxidants is determined in part by the antioxidant capacity, both constitutive and
inducible, of the pulmonary epithelium (Plopper et al. 1998). The importance of this
intrinsic antioxidant system in the lung is illustrated by the heterogeneity among species
and ages in susceptibility to hyperoxia. Adult rabbits, rats, and mice exhibit higher
SeIlsitivity to hyperoxia-induced lung injury than neonates of these species, which can
more readily upregulate tissue levels of antioxidant enzymes, and are therefore resistant

t
0 1 ung injury from similar insult (Frank et al. 1978).

25

Historically, the nasal cavity has been considered primarily for its role as a
“scrubbing tower” for the pulmonary airways in inhalation toxicological studies.
Functions of the nasal cavity relevant to this purpose include warming and humidifying
inspired air, trapping respirable particles and antigens in its mucus layer, and phase I
xenobiotic metabolism (Thornton Manning and Dahl 1997). Recently, the nasal cavity
has received attention as an important primary target for inhaled oxidants, including
ozone. The nasal epithelial and inflammatory responses to ozone exposure have been the
subject of a recent literature review (Nikasinovic et al. 2003). Nasal injury associated
with ozone exposure has been described in humans (Koltai 1994; Calderon Garciduenas

et al. 1996; Calderon Garciduenas et al. 1997; Kopp et al. 1999; Sienra Monge et al.
2004), non-human primates (Harkema et al. 1987; Harkema et al. 1993), and
CXpeIimental rodent models (Hotchkiss et al. 1989; Cho et al. 1999; Wagner et al. 2003),
and significant heterogeneity exists among the location, character, and severity of the
lesions among the different experimental animal models. Several factors may play a role
in the variability observed in ozone susceptibility across species, including differences in
airV'vay geometry and airflow, and the resultant variations in local ozone dose, specific
epithelial type susceptibility, and differences in the distribution of susceptible epithelial
POPUIations. As with the pulmonary airways, site-dependent differences also exist in the
responses to inhaled ozone challenge among different regions of the nasal airways. It
a1 SO follows that susceptibility to airway damage by ozone exposure in the nasal airways

may also be due in part to local antioxidant capacity.

26

As in the pulmonary airways, the epithelium of the nasal cavity provides an
intrinsic antioxidant array, consisting of antioxidant enzymes and low molecular weight
antioxidant molecules. Each of these compounds possesses different substrate specificity
and tissue activity, and thus, the level of protection provided to the epithelium may vary
according to the type of oxidant challenging the epithelium. The antioxidant array in the
nasal cavity of humans has been characterized in previous investigations (Peden et al.
1993; Mudway et al. 1999; van der Vliet et al. 1999). Several studies have suggested that
low molecular weight antioxidant uric acid is the major antioxidant in the human upper
respiratory tract (Peden et al. 1990; Peden et al. 1993; van der Vliet et al. 1999), with
reduced glutathione, ascorbic acid, SOD species, and catalase also conferring protection

to the nasal cavity in humans.
The rat is an important experimental animal model for inhalation toxicological
Studies. Many of these investigations in rats have focused on the effects of oxidant
xenobiotics or pollutants, such as chlorine (Barrow et al. 1979; Hester et al. 2002), ozone
(Hotchkiss et a1. 1989; Cho et al. 1999; Harkema et al. 1999), and nitrogen dioxide
(Mautz et al. 2001) (Ichinose et al. 1991) on the nasal epithelium. Despite the fact that
the Upper airways are susceptible to injury from inhaled oxidants, investigation of the
antioxidant profile of the rat nasal cavity has only recently garnered scientific attention.
The local nasal biochemical and metabolic differences between humans and experimental
animal models must be considered during interspecies extrapolation of the toxic effects of
inhaled oxidant gases. In light of the heterogeneity demonstrated among different ages
arid Species in the pulmonary response to oxidant challenge (Slade et al. 1985; Hatch et

a1 ‘ 1 994), it seems logical that characterization of the susceptibility profile of the nasal

27

cavity of the relevant animal models to oxidant challenge would aid in the selection of
appropriate models for human risk assessment, and facilitate extrapolation of toxicology
data from animal models to humans.

The purposes of this review are to provide a brief review of the major
environmental oxidant pollutants (ozone and nitrogen oxides) and the biochemistry of the
major antioxidant substances in rat nasal epithelium and nasal ELF, to review the
experimental data regarding nasal antioxidant profiles in rat models, and to discuss the
potential implications of these findings with regard to extrapolation from rat models of

inhalation toxicology for human risk assessment.

SOURCES OF BIOLOGICALLY RELEVANT ENVIRONMENTAL OXIDANTS
Ozone and Reactive Oxygen Species
Ozone (O3), the principal oxidant pollutant in photochemical smog, is a highly
Potent oxidant, capable of inducing nasal and pulmonary epithelial damage in rats at
alribient concentrations (Hotchkiss et a1. 1989). In the stratosphere (the atmosphere 10-

20 miles above the earth’s surface), ultraviolet radiation splits molecular oxygen (02)

into atomic oxygen (0), which rapidly reacts with 02 to produce 03. This reaction

y ields ozone at concentrations of up to 10 ppm. Stratospheric ozone, often referred to as
“good” ozone, or the “ozone layer,” serves to limit the amount of damaging ultraviolet
light that reaches the earth’s surface. In the troposphere (the atmosphere within 5 miles
of the earth’s surface), ozone is primarily produced through actions of ultraviolet
r‘E’lCiia-Sltion on polluted air. Tropospheric ozone, also called “ground level” ozone, is

gel7‘el‘ated predominantly through the reaction of nitrogen oxides with oxygen in the

28

presence of sunlight. Ultraviolet radiation reacts with nitrogen dioxide (N02), which
results in the generation of nitric oxide radical (N O-) and O, the latter of which combines

with O; to form ground level ozone. In clean air, the NO- quickly reacts with 0- (see

“Nitrogen Oxides” below) to regenerate N02, and thus prevents ozone accumulation. In

urban centers, the high motor vehicle use results in the emission of volatile organic
compounds (V OCs) into the atmosphere. These VOCs serve as a source of

hydrocarbons, which can react with O- to yield hydrocarbon radicals. These reactive
hydrocarbon radicals can subsequently oxidize NO- to form N02 (Peden et al. 1995). As

a consequence, ozone is no longer consumed in the scavenging of NO-, and is
subsequently allowed to accumulate in the atmosphere.

In addition to environmental sources, occupational sources can also produce
biologically relevant concentrations of ozone. Exposure to ozone can be an occupational
hazard in workplaces due to welding and other electrical processes (Challen 1974;
Salldstrom 1995). During normal operation, ozone concentrations in airplane cabins may
reach 0.20 ppm (Waters 2002). Ozone exposure is also a potential hazard onboard
Subrriarines (Crawl 2003).

While some environmental pollutants are directly toxic to the epithelium of the
respiratory system, most biologically relevant environmental oxidant pollutants exert at
least some of their toxic effects through the generation of reactive oxygen species (ROS)

Wi thin the biological system. ROS are generated in all aerobic biological systems during
notinal cellular respiration. ROS can be generated by the successive single electron

ed uctlons of oxygen to water, whlch occurs durmg ox1dat1ve phosphorylatlon in

'111 tochondria. These species, including superoxide anion (02‘), hydrogen peroxide

29

(H202), and hydroxyl radical (HO-), are endogenously produced at low levels, and are

typically controlled by endogenous antioxidants (Baskin and Salem 1997). They are also
produced in the respiratory airways as a result of exposure to exogenous oxidants. For

example, exposure of the respiratory tract to ozone leads to epithelial cell injury and
concurrent release of Of (Voter et al. 2001), which likely mediates epithelial membrane

injury following ozone exposure, rather than ozone itself.

Nitrogen Oxides (NO‘) and Reactive Nitrogen Species
The oxides of nitrogen (nitrogen dioxide, N02; nitric oxide, NO) provide another

source of environmental oxidants. In the atmosphere, these compounds are
predominantly derived from internal combustion engines in motor vehicles (Sandstrom
1995 ). However, recent source apportionment studies indicate that combustion from
stationary sources (e. g. electric power plants) also provides a significant proportion of
nitrogen oxides in the environment, particularly in the midwestem United States (Elliott
61 a1- 2007). These anthropogenic sources far exceed the contributions from natural
SOLIl‘ces, such as lightning and wildfires.

The oxygen bound to nitrogen atoms in a these compounds can undergo
re<iuetion, forming a special form of oxygen radical known as a reactive nitrogen species
(Elliott et al.). As is the case with ROS, some reactive nitrogen species (Elliott et al.) are

produced under controlled conditions in vivo (Moncada and Higgs 1991). Of particular
importance is nitric oxide, which serves several important roles in the regulation of blood
pressure, leukocyte killing functions, and as a neurotransmitter (Baskin and Salem 1997).

I 11 .
eXcesswe amounts, however, nltrrc ox1de can react Wlth other radlcals, lncludmg

3O

tyrosine radicals, thus inhibiting their normal biological functions. For example, NO- can
react with the tyrosyl radical at the active site of ribonucleotide reductase, an enzyme
required for the initiation of DNA synthesis, and thereby inhibit cell proliferation

(Lepoivre et al. 1994). RNS are also capable of reacting with ROS, yielding more potent

oxidizing agents. Reaction of NO- with 023 results in the formation of the highly
reactive peroxynitrite anion , (ONOT), a potent oxidant which can initiate electrophilic

attack on both membrane components (lipids, proteins) and low molecular weight ELF
molecules, including antioxidants (e.g. ascorbate), yielding reactive cellular components

and initiating peroxidation chain reactions (Radi et al. 1991).

In addition to ROS and RNS, many other molecules can contribute to oxidative

stress in biological systems. These include organic radicals of amino acid, protein, and
lipid oxidation, metal ions (e.g. iron), and reactive carbon species (e.g. CCl3-) (Bowler

and Crapo 2002). Complex gaseous mixtures, such as the components of the gas phase
0f passive cigarette smoke, provide simultaneous exposure to combinations of potent

R0 S , RNS, and organic radicals (Jaimes et al. 2004).

ANTIOXIDANT ENZYME SYSTEMS OF THE RAT NASAL EPITHELIUM

Superoxide dismutase
Superoxide dismutases are a family of metal ion-containing enzymes that are

present in most aerobic organisms (Stenfors et al. 2004). The major function of these

erlZB’mes is to catalyze the dismutation of the superoxide anion radical, 02', resulting in

the generation of the less reactive oxidants hydrogen peroxide and oxygen gas. Since 02'

does not readily cross cell membranes, it must be detoxified in the cellular compartment

31

in which it was generated. For this reason, SODs evolved into three distinct forms, which
differ in their metal ion content at the active site of the enzyme, and their subcellular
locations. In mammals, manganese-SOD (Mn-SOD) is localized to the mitrochondria,
and the cytosol contains dimeric copper/zinc-SOD (Cu,Zn-SOD). Mammalian tissues
also contain an tetrameric, extracellular form of Cu,Zn-SOD (EC-SOD), that is expressed
primarily in the epithelial lining fluid of the lung (Fridovich 1995).

In the Sprague-Dawley rat, immunohistochemical studies have demonstrated that
expression of Mn-SOD and Cu,Zn-SOD in olfactory receptor neurons of olfactory
epithelium occurs as early as embryonic day 14. While immunoreactivity for Mn-SOD
increases throughout the perinatal period to a maximum at postnatal day 11,

immunoreactivity for dimeric (cytosolic) Cu,Zn-SOD increases primarily after birth,
peaking between postnatal days 11 and 24 (Kulkami Narla et al. 1997). Similarly, in
adult F/344 rats, total SOD activity increases, relative to control, in response to prolonged
hyperoxia (Nikula et al. 1991). These findings are supportive of the concept that SOD
levels are responsive to increases in oxygen tension during the postnatal period, and that
this responsiveness persists into adulthood.

In adult rats, one study using enzyme activity assays demonstrated that total SOD

aCtivity is higher in olfactory epithelium than in nasal respiratory epithelium, and that
aetivity in both types of nasal epithelium exceeded that in lung tissue. This study, using
Wi Star-derived albino rats, also demonstrated, via immunohistochemical localization, that
both Mn-SOD and dimeric Cu,Zn-SOD are found primarily within the olfactory
epithelium of the dorsal, medial nasal cavity (Reed et al. 2003). Interestingly, a similar

d1 Stri bution of activity was also found with the other enzymatic antioxidants evaluated

32

(see below). The same distribution of SOD activity was described in a separate study in
adult male Wistar rats. This latter study also found that despite the higher total activity of
both SOD forms in olfactory epithelium, the ratio of mitochondrial to cytosolic activity
was similar in both regions (Lai et al. 1997). This report also found no SOD

immunoreactivity in goblet cells or the interstitium of the respiratory epithelium,

suggesting that these regions of the nasal mucosa may remain vulnerable to 02-mediated

membrane damage. It is also possible that these areas obtain protection from 02' from

other sources, including intraepithelial and submucosal mucus. In response to hyperoxia,
however, the respiratory epithelium demonstrated greater increases in SOD enzymatic
activity relative to control levels (Nikula et a1. 1991). This discrepancy may be due to
regional differences in the capacity for enzyme upregulation, local differences in the
degree or duration of exposure to inhaled oxidants (respiratory epithelium lies primarily
in the ventral nasal cavity, while olfactory epithelium lies dorsally), or may represent a

point of imbalance in the consumption/repletion kinetics of SOD in these two tissues.

Catalase

Catalase serves a major role in the intrinsic antioxidant cascade in mammalian
systems. Its main function is to catalyze the decomposition of hydrogen peroxide to
water, thus continuing the cascade of superoxide radical decomposition. Catalase is
localized predominantly to peroxisomes, and can be found in this location within

epithelial cells and some inflammatory cells, primarily macrophages, (Baskin and Salem

1997).

33

Within the nasal cavity, catalase activity has been demonstrated in transitional,
respiratory, and olfactory epithelium (Nikula et al. 1991; Peshenko et al. 1998), with
highest activity found in respiratory epithelium. Catalase activity in lung tissue exceeded
both nasal epithelial types (Reed et al. 2003). Catalase activity did not increase in
response to hyperoxic (85%) conditions in any of the nasal epithelial types in adult F/344
rats (Nikula et al. 1991). In contrast to the nasal cavity, lung catalase activity showed a
21% increase in response to hyperoxia (95%) in both adult and neonatal Sprague-Dawley
albino rats (Frank et al. 1978). This higher activity and upregulation may be necessary
in the lung to neutralize the respiratory burst activity from the resident macrophage

population in the alveoli.

NAD(P)H:Quinone oxidoreductase (NQOl)

NQOl, also known as DT-diaphorase is an important cytosolic chemoprotective
oxido-reductase, involved largely in the metabolism of oxidizing xenobiotic quinones. It
is a two-electron quinone reductase, which prevents oxidative damage in cells by
preventing the one-electron reduction of quinones, which results in the formation of
semiquinones, which can spontaneously dismutate and yield damaging superoxide
radicals (Lind et al. 1982). The majority of NQ01 resides in the cytosolic compartment
of most cells, with some activity also found in mitochondria (Emster 1958). In addition

to its intracellular activity against xenobiotic quinone oxidants, NQOl participates in the
reduction of endogenous and exogenous quinines in membranes, including cell

mefl’lbrane-bound vitamin E quinones, thereby regenerating antioxidant forms of these

34

 

molecules. NQOl also possesses scavenging activity against exogenously generated

superoxide radical anion.

NQOl activity has been reported in the respiratory and olfactory epithelium of the
nasal cavity of Wistar rats. The levels detected were highest in respiratory epithelium,
and levels in both respiratory and olfactory were higher than those in lung tissue (Reed et
al. 2003). Immunohistochemically, NQ01 has been detected in respiratory, olfactory,
and transitional epithelium of the nasal cavity. Unlike the nasal distribution of catalase
and SOD, which was primarily dorsal and rostral, NQOl showed regions of intense
detection throughout the nasal cavity of the rat, with the most intense detection in the

caudal and ventral regions of the nasal airways (Reed et al. 2003).

Glutathione peroxidase and glutathione reductase
Glutathione peroxidases are a family of enzymes that can be divided into
selenium-dependent and selenium-independent systems. Both types can catalyze the
reduction of hydrogen peroxide and organic peroxides to water and/or alcohol, but the
former group possesses wider substrate specificity. These enzymes exist in cytosolic
(monomeric) and cell membrane-bound (tetrameric) forms.
Glutathione peroxidases utilize reduced glutathione (GSH) as an electron donor in
the reduction of peroxides, in a mechanism involving glutathione redox cycling. This
fiJIICtion of glutathione is independent of its function as a low molecular weight
antioxidant in epithelial lining fluids (see below). The glutathione peroxidase reaction

results in the formation of the glutathionyl radical (GS-). At high GSH concentrations or

35

 

high rates of GSH consumption, GS- quickly dimerizes to form the oxidized form
glutathione disulfide (GSSG). Reduced glutathione is regenerated from GSSG by the
activity of glutathione reductase, in a reaction requiring reducing potential (NADPH)
provided by glucose-6-phosphate dehydrogenase (G6PDH). In this manner, the activities
of glutathione peroxidase, glutathione reductase, and G6PDH are interrelated.

Glutathione peroxidase antioxidant activity has been documented in transitional,
respiratory (Cassee and Feron 1994), and olfactory epithelium of the rat (Nikula et al.
1991; Reed et al. 2003). Glutathione peroxidase activity in respiratory epithelium is
higher in the resting state than olfactory epithelial activity, with glutathione peroxidase
activity in the lung intermediate to the two. Glutathione reductase activities are
comparable to glutathione peroxidase levels in the nasal transitional and respiratory
epithelium, illustrating the close relationship between the two.

Results of glutathione peroxidase activity were widely divergent in the two
studies that evaluated this activity in respiratory epithelium (~130nmol/min/mg protein,
versus 48.6 umol/min/mg protein) (Cassee and Feron 1994; Reed et al. 2003). There are
several methodological differences between the two studies, however, that may explain
these differences. The discrepancy may be due to differences in the ages of the rats (8
weeks versus adult), peroxide substrate differences between the two assays (tert-butyl
hydroperoxide and cumene hydroperoxide), and differences in standardization methods

(cytosolic versus total protein). A similar discrepancy in the measured levels of
Ellitathione reductase activity (~130nmol/min/mg protein versus 275umol/min/mg

PTOtein), between these studies could also be explained by age and standardization

36

 

differences; however, the same enzyme substrate (oxidized glutathione) was used in both
of these studies (Cassee and Feron 1994; Reed et al. 2003).

Nasal transitional and olfactory epithelium both demonstrate increased levels of
glutathione peroxidase activity in response to hyperoxia (85%), while nasal respiratory
epithelium showed no significant increase following hyperoxia in adult F/344 rats
(N ikula et al. 1991). A separate study found no significant increase in lung glutathione
peroxidase activity in response to 95% hyperoxia in adult Sprague-Dawley rats (P rank et

al. 1978).

Peroxiredoxin (thioredoxin)

Peroxiredoxins are a broad family of antioxidant proteins that are ubiquitously
present in all cells across species. A novel, 28-kDa secretory protein isolated from rat
olfactory epithelium was identified as a thiol-specific antioxidant member of the
peroxiredoxin family (Peshenko et al. 1998). Peroxiredoxins exhibit substrate specificity
similar to that of catalase and glutathione peroxidase, possessing activity against
endogenously generated peroxides including hydrogen peroxide.

The 28-kDa peroxiredoxin initially isolated from Wistar rat olfactory epithelium
has since been documented in trachea and lung, as well as several other non-respiratory
tissues in the rat. Within olfactory epithelium of the Wistar rat, the peroxiredoxin
lmunoreactivity specifically localizes to the apical portions of the sustentacular cell

CJ’Tosol and within mucus, both intracellularly and within the airway lumen (Novoselov et

a1. 1999).

37

 

LOW MOLECULAR WEIGHT ANTIOXIDANTS
Glutathione (GSH)

Glutathione is a non-protein tripeptide of y-glutamate, cysteine, and glycine.
Reduced glutathione is the most abundant thiol present in mammalian tissues (Baskin and
Salem 1997). GSH serves many roles in living systems. Many of these involve redox
functions in biological systems, including serving as an electron donor for reduction of
peroxidases via glutathione peroxidase, non-enzymatic reduction of free radicals,
structural maintenance of sulfhydryl residues of protein and enzymes, and fulfilling a
vital role in the reduction of the a-tocopherol radical and semidehydroascorbate radical,
thereby returning them to their functional states as antioxidants (a-tocopherol and
ascorbate, respectively) (Buettner 1993). In addition to its many known redox functions,
GSH also serves as an important signaling molecule for many cellular processes,
including cell proliferation (Shaw and Chou 1986; Luppi et al. 2005; Markovic et al.
2007), and as a sulfliydryl buffer that maintains cysteinyl residues in their reduced states
(Halliwell and Gutterudge 2007)

Three different studies have reported GSH values for rat nasal epithelium.
Reported concentrations of GSH in two regions of olfactory epithelium (2.32umol/g
tissue in dorsal septal olfactory epithelium, 2.16umol/g tissue in dorsal meatus olfactory

epithelium) in adult F/344 rats are comparable to reported values for respiratory
ePithelium (2.67umol/g tissue) in the same study (Potter et al. 1995). Two of these
Studies reported GSH concentrations relative to total cytosolic protein study. Using a

HPLC quantitation method, one group reported concentrations for GSH in nasal

38

 

respiratory epithelium per unit cytosolic protein (~2.5umol/mg cytosolic protein) in 8-
week-old Wistar rats (Cassee and Feron 1994). A more recent study reported lower GSH
concentrations in the olfactory epithelium (27nmol/mg protein) of Long-Evans rats
(Burman et al. 2003); however, these values were obtained in younger rats (5-7 weeks),
using a different detection and quantitation method (o-phthalaldehyde fluorometric

probe).

Ascorbic Acid (Vitamin C)

Ascorbic acid, an essential vitamin in primates, is a water-soluble compound with
several important metabolic roles. It is a potent and effective scavenger in viva, capable
of reacting with radicals of ROS, RNS, and water-soluble organic radicals, and in this
capacity is one of the most important chain-breaking redox agents. One of its principle
antioxidant roles is the regeneration of a-tocopherol and uric acid from their respective
radicals, (Buettner 1993) which are formed during the metabolism of cell membrane
lipid peroxides. The resulting ascorbyl radicals can be reduced back to ascorbic acid via
enzymatic systems utilizing GSH or NADH as a reducing agent (Kagan et al. 1992).

Two studies have determined values for ascorbic acid concentrations in rat nasal
epithelium, and the reported values are highly variable. Ascorbic acid concentrations in
olfactory epithelium (~500ug/mg protein) are 4-fold higher than those reported in

respiratory epithelium (~125ug/mg protein), and are comparable to concentrations
n3Ported in lung tissue (~400ug/mg protein) of adult Wistar rats (Reed et al. 2003).
Another study, however, found significantly lower concentrations (~90nmol/mg protein,

01' N l 6ug/mg protein) of ascorbic acid in olfactory epithelium of 5-7 week-old Long-

39

 

Evans rats (Burman et al. 2003). The ascorbic acid in each of these studies was harvested
via a phosphoric acid extraction technique; however, the final results were obtained via

different detection methods (525nm spectrophotometry versus HPLC).

a—Tocopherol (vitamin E)

Vitamin E is a group of eight naturally occurring lipid-soluble molecules known
as the tocopherols. The lipid solubility of a-tocopherol facilitates its role as the major
radical (lipid peroxyl) chain-breaking agent in plasma membranes. a-tocopherol is also
reactive against superoxide anion radical and hydroxyl radical. a-tocopherol is an
integtral part of plasma membranes, and is widely distributed in all types of plasma
membranes, including the mitochondrial membranes and the endoplasmic reticulum
(Baskin and Salem 1997).

Concentrations of a-tocopherol have been reported in rat nasal epithelium of
Wistar rats (Reed et al. 2003). Concentrations of a-tocopherol were 1.4-fold higher in
olfactory epithelium than in respiratory epithelium. Pulmonary levels of a-tocopherol

were higher than concentrations found in the nasal epithelium.

DISCUSSION
The antioxidant profile and the geographical distribution of antioxidants
throughout the nasal cavity can have important implications for susceptibility to inhaled
oxidant pollutants and xenobiotics. Olfactory epithelium is enriched with a complement
of metabolic enzyme capacity, including cytochrome P4503, glutathione s-transferases,

and antioxidant enzymes. This metabolic capacity can be protective, or in some cases,

40

can exacerbate epithelial injury by generating toxic metabolites. While some
antioxidants have wide substrate ranges (e.g. glutathione, ascorbic acid), others catalyze
or facilitate specific redox reactions (e.g. SOD, catalase). In health, the complete profile
of antioxidant compounds in a living system works in concert to protect the epithelilun
from oxidant insult. This review illustrates that regional differences exist within species.
While other factors are also important (e.g. concentrations of oxidants delivered, airflow
patterns through the nose, generation of immune responses), regional differences in
antioxidant profiles must be considered in susceptibility studies within species.

This review also raises the question of age differences. While no single study
evaluated antioxidant profile in multiple ages within a single species, examination of data
from different studies and species suggests that antioxidant levels may vary with age. A
similar study has been reported in the lung, which found that the ability to upregulate
antioxidant enzyme expression in response to hyperoxia attenuates with age in rabbits
and mice (Frank et al. 1978). This finding illustrates the importance of model selection
in risk assessment studies, and highlights the need for controlled, comparative studies of
multiple animal models and ages.

Several antioxidants that are not reported in this review are important agents in
humans. Values for uric acid, heme oxygenase isoforms, and vitamin A, for example,
have not been reported in the nasal cavity of the rat. Uric acid is a particularly important
antioxidant in the human respiratory tract, and is the major chain-breaking antioxidant in

the human nasal cavity (Peden et al. 1990; Peden et al. 1993). Also of importance is the
fact that many of the chain-breaking antioxidant molecules, as well as some of the

enzymatic antioxidants, reside in both the intracellular compartment and in the overlying

41

epithelial lining fluid in vivo. The concentrations of antioxidants in the intracellular and
extracellular pools of the respiratory tract can differ significantly, and their levels may
fluctuate independently following perturbation (Buhl et al. 1990; Smith et al. 1992; Testa
et al. 1995). Many human studies consider the antioxidant profile of the ELF in risk
assessment and kinetics studies. Nasal lavage methods have been developed for the rat,
so ELF analysis is a feasible option that should be considered for future studies

employing this model.

42

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Barrow, C. S., R. J. Kociba, L. W. Rarnpy, D. G. Keyes and R. R. Albee (1979). "An
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CHAPTER 3

THREE-DIMENSIONAL MAPPING OF OZONE-INDUCED INJURY,
ANTIOXIDANT ALTERATIONS, AND PREDICTED OZONE FLUX, IN THE
NASAL AIRWAYS OF MONKEYS USING MAGNETIC RESONANCE

IMAGING AND MORPHOMETRIC TECHNIQUES

51

ABSTRACT

My governing hypothesis is that the spatial and temporal distribution of ozone-
induced nasal injury is related to the regulation of intracellular glutathione levels.
However, many other factors are likely to contribute to the location and severity of ozone
lesions in the nasal cavity. Age-related changes in gross, biochemical and microscopic
and structure of the nasal cavity may alter local tissue susceptibility as well as the dose of
inhaled toxicant delivered to susceptible sites. This chapter describes a novel method for
the use of magnetic resonance imaging, 3-dimensional airway modeling, and
morphometric techniques to characterize the distribution and magnitude of ozone-
induced nasal injury and site-matched ozone-induced nasal antioxidant fluctuations,
following acute and episodic exposures in infant monkeys. Using this method, we
generated age-specific, 3-dimensional, epithelial maps of the nasal airways of infant
rhesus macaques. These 3-dimensional models were also utilized to perform
computational fluid dynamics simulations of ozone uptake in the nasal airways. These
simulations were used to determine the influence of airway geometry on local ozone
dose, and to compare sites of predicted uptake with sites of injury. Here, we describe the
histopathologic, imaging, biochemical, and computational biology methods developed to
precisely characterize, localize, quantify, and map these nasal lesions. The principal
nasal lesions observed in this primate model of ozone-induced nasal toxicology were
neutrophilic rhinitis, along with necrosis and exfoliation of the epithelium lining the
anterior maxilloturbinate. These lesions, induced by acute or episodic exposures, were
examined by light microscopy, quantified by morphometric techniques, and mapped on

3-dimensional models of the nasal airways. Nasal mucosal samples from these same sites

52

were processed for intracellular nasal antioxidant content. By combining these
techniques, the location and severity of the nasal epithelial injury were correlated with
epithelial type, nasal airway geometry, and local biochemical changes on an individual
animal basis. These correlations are critical for accurate predictive modeling of
exposure-dose-response relationships in the nasal airways, for the determination of
factors contributing to epithelial susceptibility to exposure-related injury, and for the

extrapolation of nasal findings in animal models to humans for determining risk.

53

INTRODUCTION

Understanding the pathogenesis of ozone-induced airway injury and remodeling
in the upper respiratory tract requires knowledge of the dose delivered to the target
airway tissues and the airway tissue’s response to exposure. Nasal injury caused by
ozone exposure has been shown to be site-specific in both laboratory rodents and
primates (Harkema et al. 1987a). The intranasal location of these ozone-induced lesions
may be attributable to the local dose of ozone reaching the affected area, the site-specific
tissue susceptibility, or a combination of these factors (Morgan and Monticello 1990).
The local dose of ozone is dependent upon age-dependent airway geometry, ambient
ozone concentration, and airflow-driven delivery. Local tissue susceptibility is a
complex factor, dependent upon many components, including the distribution of
susceptible epithelial populations in the nasal airways, and the capacity of the local tissue
to respond to oxidant challenge. To determine the relative importance of each factor, it is
essential to first microscopically identify the ozone-associated lesions in the nasal
passages and precisely map the distribution of these specific lesions relative to airway
geometry and epithelial susceptibility.

In previous reports, this has been accomplished experimentally using a sequential
series of cross-sectional airway profiles covering the entire length of the nasal airways of
each experimental animal. This systematic approach has been used to identify patterns of
nasal toxicity to inhaled chemicals in adult monkeys (Young 1981; Harkema et al. 1987d;
Mery et al. 1994; Kepler 1995), and relies on precise characterization and identification
of the distribution of surface epithelial populations (Gross et al. 1982; Gross et al. 1987;

Harkema et al. 1987a; Harkema 1991). This type of detailed characterization, however,

54

has not been previously reported the developing nasal airways of infant monkeys. Age-
related differences between adult and infant monkeys in nasal airway geometry and
epithelial distribution may cause changes in the exposure-dose-response relationship, and
supports the use of infant monkeys as predictive models for nasal toxicology in children.

The rhesus macaque serves as an important animal model of upper airway
toxicology because their nasal airways resemble, at a gross and microscopic level, the
nasal airways of humans (Harkema 1991; Yeh et al. 1997). Knowledge of the patterns of
ozone-induced airway lesions in the developing nasal passages of immature monkeys
provides insight into the relative roles played by regional dose and local tissue
susceptibility in the toxic responses to ozone in the nose, and ultimately, assists in the
assessment of the potential risk of ozone exposure to the health of children. Using a
combination of magnetic resonance (MR) imaging, 3-dimensional (3D) modeling, and
standard histopathologic and morphometric techniques, we developed a detailed and
flexible approach of nasal analysis to identify, localize, and precisely map normal nasal
airway epithelium and ozone-induced nasal airway lesions. This method also provides a
means to correlate morphologic changes to site-specific biochemical changes in the
airways (e.g. regional antioxidant regulation) and with estimates of local ozone dose.
Identifying these correlations is essential for developing predictive models of dosimetry,
and defining exposure-dose-response relationships in nasal airways of immature non-
human primates that are crucial for determining the mode of action in the nasal toxicity
of ozone and other inhaled toxicants.

Ozone exposure (0.3 ppm, 8h/day, 6 days) causes epithelial necrosis, followed by

epithelial hyperplasia, in the nasal airways of adult monkeys (Harkema et al. 1987c).

55

These epithelial lesions are confined to the anterior part of the nasal cavity. The effects
of ozone exposure on the developing nasal airways of infant monkeys have not been
previously investigated. The experiments described in this chapter were designed to
determine the morphologic and biochemical effects of cyclic (episodic) ozone exposures
on the nasal airways of infant monkeys. These studies were conducted to test the
hypothesis that cyclic ozone exposure would cause epithelial necrosis and subsequent
epithelial hyperplasia in the anterior nasal cavity of infant monkeys, similar to that
described in adult monkeys exposed acutely to ozone (Harkema et al. 19870). The
techniques described here also provide a method to investigate the central hypothesis that
the spatial and temporal distribution of ozone-induced nasal epithelial injury and
subsequent epithelial proliferation in infant monkeys are dependent upon the local
regulation of intracellular glutathione (GSH) concentrations. In addition, these
techniques also provide a method to investigate other possible factors contributing to the
site-specificity of ozone-induced nasal airway injury (e.g., local ozone dose). This
chapter will provide a review of the methods that have been employed for ozone
exposure, nasal airway imaging, nasal tissue processing, nasal antioxidant analysis, and
3D nasal airway mapping in the infant rhesus monkey. Attention will be focused on the
details of these methods that provide an efficient and comprehensive characterization of
airway geometry, nasal histopathology, and nasal antioxidant status, in order to maximize

site-matched information obtained from each individual animal.

MATERIALS AND METHODS

Animals and Exposure Regimens

56

The principal laboratory animal used in our ozone inhalation studies was the
infant, male Rhesus macaque (Macaca mulatta). All monkeys selected for these
studies were born and raised at the California National Primate Research Center. Care
and housing of animals before, during, and afier treatment complied with the provisions
of the Institute of Laboratory Animal Resources and conformed to practices established
by the American Association for Accreditation of Laboratory Animal Care

(AAALAC).

While several rodent models of environmental airway disease have been
previously utilized for human risk assessment, some aspects of the airway anatomy and
physiology of rodents make them less desirable for direct extrapolations to humans.
For example, rodents are obligate nasal breathers, while humans may alternate between
nasal and oronasal breathing. Humans also possess simple nasal turbinates, while
rodents possess a very complex nasal turbinate structure (Harkema et al. 2006).
Furthermore, the relative surface epithelial populations lining the nasal airways of
rodents and humans differ considerably. Approximately 50% of the surface area of the
nasal cavity in F344 rats is lined by olfactory epithelium (Gross et al. 1982), while only i
3% of human nasal airways are dedicated to this sensory neuroepithelium (Sorokin
1988). Each of these factors ultimately changes the dose of inhaled toxicants delivered
to susceptible sites between rodents and humans (Kimbell 2006). A non-human
primate model species was selected because the structural and cellular aspects of the
nasal airways of monkeys most closely represent those in humans (Yeh et al. 1997).

Furthermore, infant rhesus monkeys exposed to ozone represent a more relevant model

57

of oxidant airway disease in children (Evans et al. 2003; Larson et al. 2004; F anucchi et

al. 2006).

Inhalation studies were designed to compare the epithelial responses to acute
ozone exposure to the responses induced by episodic ozone exposure in the nasal airways
of infant rhesus monkeys. Thirteen infant male rhesus monkeys were used for this study.
Acute regimens consisted of 5 consecutive days of exposure (8h/day) to 0.5 ppm ozone
(1 cycle, n=5). Episodic regimens consisted of 5 bi-weekly cycles of alternating filtered
air (FA) and ozone exposure (9 consecutive days of 0 ppm [FA], followed by 5
consecutive days of 0.5 ppm ozone, 8h/day; 5 cycle; n=5). 5 cycle exposures were
designed to mimic intermittent exposure to clean and polluted air, which likely mimics
the nature of exposure experienced by people in high pollution regions (Sram et al. 1996).
Control animals were exposed to FA (n=3). Exposures were scheduled so that each
animal reached its target age (90 days of age) at the end of the exposure regimen. All
exposures were performed at the California National Primate Research Center on the
campus of the University of California at Davis.

Ozone exposures were conducted in 4.2 cubic meter (m3) stainless steel and glass,
whole-body inhalation chambers, similar to those previously described (Hinners et al.
1968). Ozone was produced from vaporized liquid, medical grade oxygen by electric
discharge ozonizers (Model 03Vl-0, Ozone Research-Equipment Company, Akron, OH).
Generated ozone was diluted with filtered air (24°C, 40-60% relative humidity), and
injected into the inlet airflow of the exposure chamber. Filtered air was supplied to the
chamber at a flow rate of 2.1m3 per minute, providing 30 air exchanges per hour.

Chamber ozone concentration was monitored throughout exposures with an ultraviolet

58

ozone analyzer (Model 1003-AH Dasibi Corporation, Glendale, CA). During exposures,
a PC-based data acquisition and control system was used (Dell Computer Corporation,
Round Rock, TX and GE Fanuc Automation North America, Inc., Charlottesville, VA).
In addition to monitoring ozone concentration, this computer system also monitored

chamber airflow, temperature, pressure, and humidity.

Necropsy and Tissue Preservation

At the end of exposure, monkeys were sedated using ketamine hydrochloride
(lOmg/kg 1M), followed by induction of deep anesthesia using propofol (Diprivan, 0.1-
0.2mg/kg/min IV). The trachea, sternum, and abdominal cavity were exposed by a
midline incision. Animals were euthanized by exsanguination via the abdominal aorta.

Immediately afier sacrifice, the head was removed from the carcass, and the lower
jaw, skin, musculature, and brain were removed from the head. The nasal cavity was
exposed by splitting the skull sagittally, l-2mm to the right of midline. This sectioning
provided a complete left nasal cavity, including an intact nasal septum, and a free right
lateral wall, with the ethmoturbinate and maxilloturbinate attached (Figure 1). The left
side of the nasal cavity, including the nasal septum, was flushed with a solution of 1%
paraformaldehyde and 0.1% glutaraldehyde, and was then immersed in ~100 ml of the
same solution for storage at 4°C until preparation for MR imaging.

The right side of the nasal cavity was used to sample nasal mucosal tissues for
biochemical antioxidant analyses. These samples were harvested from three specific
locations along the lateral wall of the right nasal cavity, corresponding to the sites

examined by light microscopy in the left nasal cavity. Mucosa] samples were dissected

59

from the anterior maxilloturbinate (MT), posterior MT, and anterior ethmoturbinate (ET)
(Figure 3-1). The other part from each region was immediately placed into 300ul of
10% metaphosphoric acid containing SOuM desferroxamine, and snap-frozen in liquid
nitrogen. Acidified, frozen nasal mucosal samples were later thawed, homogenized for
30 seconds using a Polytron homogenizer, re-frozen, and stored at —80°C until further

processing for low molecular weight antioxidant analysis via high performance liquid

chromatography (HPLC).

60

 

Figure 3-1. Diagram of the right lateral wall (A) and the left nasal cavity (B) of a 90-
day—old Rhesus monkey following sagittal sectioning, showing the intranasal locations of
the maxilloturbinate (MT), ethmoturbinate (ET), and the nasopharynx (NP). N = naris; T
= 15‘ incisor tooth; HP = hard palate.

61

Magnetic Resonance Imaging of Nasal Airways

MR imaging of formalin-fixed nasal cavities has previously been exploited to
facilitate geometric measurements of airway geometry in cynomologus monkeys (Harris
et a1. 2003). In the present study, a similar approach was employed to rapidly compile
detailed three-dimensional descriptions of airway architecture post-mortem, but prior to
nasal airway sectioning and processing for light microscopic examination.

In preparation for MR imaging, the left nasal cavities were first flushed with a
fixative solution of 1% paraformaldehyde/0.1% glutaraldehyde containing the MR
contrast agent Magnevist® (gadopentetate dimeglumine, Berlex Laboratories, Wayne,
NJ), at a volume concentration of 1 part Magnevist® per 500 parts fixative. Each
specimen was then placed in a small container (60ml volume, 42mm I.D., 59mm length),
and immersed in the same fixative solution. The use of Magnevist® in the fixative
solution ensured that acquired MR images had good signal-to-noise and high contrast
between the nasal tissue and the nasal airspaces; thereby, facilitating confident
segmentation of airway geometry. In preparation for imaging, each specimen was
positioned in the specimen container with the rostral aspect of the nasal cavity facing the
bottom of the container, and the plane of the palate parallel to the long axis of the
container. The specimen was fixed into this position using soaked 2” x 2” gauze sponges
packed around the caudal aspect as needed. Prior to sealing each bottle, the specimen
was then degassed to eliminate any small air bubbles that might otherwise adversely
affect MR image quality.

All MR imaging was performed at the Environmental Molecular Sciences

Laboratory (Richland, WA). High-resolution, 3D MR images of fixed nasal tissue were

62

acquired at 2.0 Tesla using a Varian UnityPlus spectrometer (Palo Alto, CA) and an in-
house fabricated radio frequency (Alderman-Grant) coil with an inner diameter of 5.5 cm.
To visualize complex nasal passages raw image data was collected on a 256x1282 matrix
using a Tz-weighted, 3D spin-echo sequence with a 100 msec echo-time, a 150 msec
repetition time, and 4 averages. Each 3D data set therefore required ~2.7 hrs to collect,
during which, sample temperature was maintained at ~4° C. After Fourier image
reconstruction, all 3D data was stored as a series of 256 2D slices — each showing a
squared 4.5 cm field-of-view. Planar resolution within each slice was 350 microns and

each slice was 200 microns thick.

Image Analysis and Airway Segmentation

All airway segmentation and mesh generation was performed using methods
similar to those already described by Minard et al (2006). Briefly, Digital Data Viewer
(DDV; Computer Geometry Consulting, http://www.compgeomco.com) was used to
identify and digitally segment the airway geometries from the 3D MR image data of each
monkey nasal passage. Segmented airways were subsequently used for the development
of geometry databases and 3D mesh reconstruction. To facilitate airway segmentation,
each set of 256, 2D slices was first read into DDV. The set of images were then viewed
in the x, y, and/or 2 directions, slice by slice. To digitally segment (extract) the nasal
passageway, the area on each of the z-slices associated with the airway was highlighted.
A separate file (“overlay”) was then generated from this extracted data and it was used to
create an isosurface rendering of the 3D segmentation (Figure 3-2). During airway

segmentation, the x-, y-, and/or z-planes were viewed separately or in any combination

63

with the isosurface rendering (Figure 3-2) to facilitate comparisons between the
segmented airways and the raw MR image data. The isosurface generated from the
segmentation files of the MR images, along with the images themselves, could be rotated
and viewed from any direction to ensure optimal segmentation. Once completed, this
made them ideally suited to serve as guides for subsequent histopathologic and

morphometric analyses of nasal passages.

64

 

Figure 3-2. Computer assisted 3D isosurface renderings of the nasal passages from two
F A-exposed (A and B) and two 1 cycle ozone-exposed (C and D) l80-day-old infant
monkeys. The black lines in (A) delineate the regions of the nasal vestibule (V), the main
chamber (MC), the maxillary sinus (S), and the nasopharyngeal meatus (NP).

65

A publicly available mesh generator (N WGrid; http://www.emsl.pnl.gov/nwgrid)
was used to transform the segmented data into a boundary-fitted, volume-filling
tetrahedral mesh that was used for computing surface areas and volumes. The volume
meshes were designed to be suitable as the initial finite-element mesh for computational
fluid dynamics simulations. NWGrid was also used to generate surface area and volume

measurements for each nasal airway specimen.

Tissue Processing for Light Microscopy and Morphometric Analysis

After MR imaging, each left nasal cavity specimen was removed from the fixative
with Magnevist® and decalcified in 13% formic acid for 10-12 days, then rinsed with
distilled water for 2-4 hours. After decalcification, the nasal airways were transversely
sectioned at ten or twelve (based on the size of the specimen) specific anatomical
locations, using gross dental and palatine landmarks (Figure 3-3). This method produced
nasal tissue blocks that had cross sectional profiles similar to those described by Kepler,
et al. (1995), and has been used in prior studies to obtain accurate geometrical
coordinates of nasal airways of non-human primates for computer dosimetry modeling
and nasal airway lesion mapping (Kepler et al. 1998). These locations provided
transverse sections through the preturbinate region, main nasal airway chamber, and
nasopharyngeal region. Each tissue block was embedded in glycol methacrylate (GMA;
Immuno-Bed, Polysciences, Inc., Warrington, PA) for airway epithelial mapping and

histopathologic analysis.

66

' ‘ aw“: ..L

«4‘ "M4 l

4
1111 ll

Figure 3-3. Photograph of the lateral wall of the nasal cavity of a 90-day-old Rhesus
monkey. The location of the twelve transverse tissue blocks (Tl-12) selected for light
microscopic examination and morphometric analyses are indicated. NV = nasal
vestibule; ET = ethmoturbinate; MT = maxilloturbinate; NP = nasopharynx. The yellow
Xs identify the intranasal location of ozone-induced necrotizing rhinitis that is illustrated
in Figure 3-8 below.

 

67

Three-Dimensional Digital Mapping of Epithelial Distribution

It has been reported that ciliated respiratory epithelium (RE) and non-ciliated
transitional epithelium (NTE) in the anterior nasal airways are particularly sensitive to
ozone toxicity in adult monkeys (Harkema et al. 1987b; Harkema et al. 1987c), while
other epithelial types remain relatively unaffected. In order to accurately assess the
morphologic response of the nasal mucosa in infant monkeys, we first had to define the
type and extent of epithelial populations in the normal infant monkey. To determine the
specific distribution of surface epithelial populations lining the nasal airways, and to
provide boundary setting information for computer modeling and site-specific tissue dose
predictions, we developed a method to generate a detailed, 3-dimensional digital
epithelial map of the entire nasal airspace of an infant, rhesus monkey. The details of
this method are described below.

Morphological identification of nasal epithelial types. There are five distinct
epithelial populations in the nasal airways of adult macaque monkeys. These include the
squamous epithelium (SE), ciliated respiratory epithelium (RE), non-ciliated transitional
epithelium (NTE), olfactory epithelium (OE), and lymphoepithelium (LE). The
morphological features used to identify each epithelial type for 3D mapping are described
here. The reader is referred to a recent review article for a more thorough discussion of
the structure and function of these epithelial types and their relevance to nasal toxicologic

pathology (Harkema et al. 2006).

Squamous Epithelium

68

The nasal vestibule is lined with keratinized, stratified squamous epithelium. This
nasal epithelial type is composed of basal cells lining the basal lamina, and several layers
of squamous epithelial cells, which become progressively flatter toward the luminal
surface of the epithelium. The epithelium of the vestibule is contiguous to the epidermis
of the skin, and both serve to protect the underlying tissues from exposure to potentially
harmful agents in ambient air. In adult monkeys, the squamous epithelium of the nasal
vestibule forms an abrupt border with the transitional epithelium that lies immediately

distal to it in the main chamber of the nasal airways (Harkema et al. 2006).

Transitional Epithelium

Adjacent to the stratified squamous epithelium of the nasal vestibule, and
proximal to the ciliated respiratory epithelium within the main nasal chamber, is a narrow
zone of non-ciliated transitional epithelium (NTE). In monkeys, this epithelium is
stratified (4-5 cell layers thick), non-keratinized, and consists primarily of cuboidal and
columnar epithelial cells with a paucity of mucous goblet cells. In adult monkeys, this
epithelium is morphologically distinct from the keratinized squamous epithelium anterior

to it, and the RE posterior to it.

Respiratory Epithelium

Most of the main nasal chamber in adult macaque monkeys is lined with ciliated
respiratory epithelium. This pseudostratified to columnar epithelium is comprised
primarily of ciliated cells, with lesser numbers of narrow non-ciliated (serous) cells,

various numbers of mucous (goblet) cells, and basal cells.

69

Olfactory Epithelium

The olfactory epithelium covers a small region of the dorsal meatus and the dorsal
half of the ethmoturbinate in monkeys. The three principal epithelial types that compose
the olfactory epithelium are the olfactory sensory neurons, sustentacular cells, and basal
cells. The supporting, sustentacular cells form a prominent apical row of nuclei. The
olfactory sensory neurons are bipolar neuronal cells interposed between the sustentacular
cells. The dendritic portions of these neurons extend above the epithelial surface and
terminate into a bulbous olfactory knob, from which extend 10-15 irnmotile cilia per cell.

Basal cells line the basal lamina.

Lymphoepithelium

Lymphoepithelium is a specialized epithelium, found primarily in the walls of the
nasopharynx. This epithelium covers discrete, focal aggregates of nasal-associated
lymphoid tissue (NALT) in the underlying lamina propria. The lymphoepithelium is
composed of cuboidal ciliated cells, few mucous cells, and numerous nonciliated,
cuboidal cells with luminal microvilli, called M-cells. These latter cells are similar in
structure and function to the analogous immune surveillance cells found in gut- and

bronchus-associated lymphoid tissues (GALT and BALT, respectively).
Translation of light microscopic data to 3D epithelial map. One nasal airway

specimen from a filtered air-exposed monkey was selected for complete epithelial

mapping. Initial slides were taken from the anterior face of each tissue block and stained

70

with hematoxylin and eosin (H&E) for routine histopathologic examination. Slides were
also stained with AB/PAS for detection of acidic and neutral intraepithelial
mucosubstances. Additional unstained slides were taken for use in future
immunohistochemical analyses (the analyses of intraepithelial mucosubstances and
immunohistochemistry are not presented in this report). The remainder of each tissue
block was completely sectioned at SO-pm increments, and slides were stained with H&E
for routine histopathologic analysis.

Each z-slice of the MR image file was 200nm thick. Sectioning the block at
50pm increments resulted in 4 H&E slides for each image. The H&E slide that best
corresponded to the shape of the nasal airspace for each image was selected and
examined by light microscopy. Each epithelial type was coded to a color in DDV. The
location of nasal epithelial types (squamous epithelium, non-ciliated transitional
epithelium, respiratory epithelium, olfactory epithelium, lymphoepithelium) in each
section was recorded on the corresponding transverse MR image by drawing a color-
coded line along the air-tissue interface at the corresponding site (Figure 3-4). This
process was repeated in all transverse images. These data were saved as a DDV overlay
file, and were used for generation of a 3-dimensional nasal epithelial map as explained

below.

71

 

Figure 3—4. Three-dimensional mapping of nasal epithelium. (A) Z-plane image of the
nasal cavity of a 180-day-old rhesus monkey. Each MR image was visually matched to
an H&E slide for epithelial mapping. A color-coded line was drawn for each epithelial
type along the air-tissue interface. This process was repeated for each image in the MR
file. (B) The epithelial tracings were converted to 2D epithelial blobs that extended
across the air-tissue interface in each image. The intersection of each epithelial blob with
the original nasal airway segmentation (represented by the green line) was used to
generate the surface map for each epithelial type. Purple = olfactory epithelium; yellow
= respiratory epithelium; MT = maxilloturbinate; ET = ethmoturbinate; MS = maxillary
sinus; S = nasal septum.

72

In computational fluid dynamics (CFD) models, different epithelium-specific gas
uptake properties may be accounted for by setting different boundary conditions for gas
uptake at epithelium-specific locations on the nasal surface. This requires a seamless,
single surface where different epithelial types are defined as regions of the surface. Our
approach for producing this surface was to construct separate 3D reconstructions of each
epithelial type as well as the entire nasal surface in DDV and import these reconstructions
as stereolithography (STL) files into ICEM-CF D (Ansys, Inc., Canonsburg, PA), which is
a computer-aided design (CAD) software used for meshing. An STL file defines a surface
enclosing a given volume. Therefore, when exporting the epithelial reconstructions
created in DDV, we actually exported a volumetric blob that corresponds to the volume
defined by the voxels (3D pixels) drawn in DDV while delimiting each epithelial surface
(Figure 3-4). The surfaces confining each of these volumetric blobs were intersected with
the whole-nose surface in lCEM-CF D to determine the 3-dimensional curves separating
the different epithelia. These curves were then used to divide the surface of the model
into epithelial regions. The final result was a smooth, seamless, single surface of the nasal
airway composed of different parts, each representing 'a particular epithelial type (e. g.
squamous, respiratory) (Figure 3-5). This 3D reconstruction was used to visualize the
locations of the major epithelial types, to estimate epithelium-specific surface areas, and

as the initial finite element mesh for CFD simulations.

73

 

Nasopharynx

 

Nafis
Nasopharynx

Figure 3-5. Three-dimensional nasal epithelial map. Medial (A) and lateral (B) views of
a three-dimensional reconstruction of the right nasal passage of a 180-day-old rhesus
monkey. The locations of the five different epithelial types comprising the nasal airways
are indicated by color. Red = squamous epithelium; green = non-ciliated transitional
epithelium; orange = respiratory epithelium; purple = olfactory epithelium; light blue =
lymphoepithelium.

Morphometric Quantitation of Ozone-Induced Nasal Lesions

Light Microscopic Morphometric Analysis. Nasal airway tissues were
processed, decalcified, and embedded as described above. Slides were prepared from the
anterior face of each of the 10-12 tissue blocks at a 1-2 pm thickness. Slides were
stained with H&E for routine histopathologic examination and morphometric evaluation.
Slides from each section were also taken for AB/PAS staining and
immunohistochemistry as described above.

The location, nature, and extent of the specific mucosal lesions in each transverse
microscopic section were characterized for each animal. The nasal epithelial lesions
identified in the current study were confined to the anterior nasal cavity, and were
primarily associated with the ciliated respiratory epithelium. To quantitate the loss of
respiratory epithelium in this region following ozone exposure, we used standard
morphometric techniques to measure the thickness of the respiratory epithelium lining the

dorsal surface of the anterior maxilloturbinate (Figure 3-6).

75

 

 

Figure 3-6. (A) Photomicrograph of an H&E-stained transverse section (T5) through
the left nasal airway of a 90-day-old infant monkey. The region contained within the
black box represents the dorsal maxilloturbinate. The epithelium lining this region was
selected for morphometric analysis. (B) Z-plane MR image of the nasal cavity of the
same monkey, obtained at the level corresponding to the transverse section in (A). The
overlay of the nasal airspace is highlighted in green. MT = maxilloturbinate; ET =
ethmoturbinate; MS = maxillary sinus; S = nasal septum.

76

Severity of respiratory epithelial cell loss was quantified by measuring the
average height of the epithelium overlying the dorsal surface of the first three transverse
sections of the maxilloturbinate. Thickness of the RE within this region was
morphometrically evaluated as previously described for airway epithelium (Hyde et a1.
1990; Hyde et al. 1991; Plopper et al. 1994). All measurements were obtained at a final
magnification of 1,710x using a light microscope (Olympus BX40; Olympus America,
Inc., Melville, NY) coupled to a 3.3-megapixel digital color camera (Q—Color 3 Camera;
Quantitative Imaging Corporation, Burnaby, British Columbia, Canada), and a personal
computer (Dimension 8200; Dell, Austin TX). The morphometric analyses were
performed using a 135-point cycloid grid overlay with an automated software package for
counting points and intercepts within the grid (Stereology Toolbox; Morphometrix,

Davis, CA) (Hyde et al. 1990; Hyde et al. 1991). The percent voltune density (the

proportion of the total epithelial volume), Vv, of cytoplasm, nuclei, and intraepithelial

mucosubstances, was determined by point counting and calculated using the following

formula:

V, = Pp = Pn/P,, [1]

where Pp is the point fraction of P", the number of test points hitting the structure of

interest (e.g. cytoplasm), divided by P,, the total number of points hitting the reference

space (i.e. respiratory epithelium). The volume of each epithelial component of interest

77

per unit of basal lamina length (5,) was determined by point- (epithelial component) and

intercept- (basal lamina) counting and was calculated using the following formula:

3,, =2 10Ly , [2]

where 10 is the number of cycloid intercepts with the object (epithelial basal lamina), and

L 7 is the length of the test line in the reference volume (epithelium within the test grid).

To determine the thickness of the RE, a volume per unit area of basal lamina (cubic
micrometers per square micrometer) was calculated using the following formula for

arithmetic mean thickness (r):

r = V,/s, . [3]

Statistical Analyses. Statistical analyses for all morphometric parameters were
performed using one-way analysis of variance (ANOVA). Pairwise comparisons were
selected a priori, and were performed using Student-Newman-Keuls multiple

comparisons test (p50.05). Statistical outliers were detected using Grubb’s test (p50.05).

Three-Dimensional Digital Mapping of Ozone-Induced Nasal Injury
We used a combination of standard histopathologic methods, MR imaging, and
computer-assisted, 3D airway modeling to generate subject-specific, 3D maps of ozone-

induced nasal lesions. Using DDV, the frontal profile of each transverse section was

78

visually matched to its corresponding z-plane slice (2D image) in the MRI segmentation
(Figure 3-6). The locations of ozone-induced lesions identified on the nasal tissue
sections were recorded on the corresponding sites on the z-plane MR images by
highlighting the tissue-airspace interface at that site (Figure 3-7). The entire z-plane
slice, including the highlighted lesion site, was subsequently superimposed over the
isosurface rendering of the entire nasal airspace to provide 3-dimensional lesion
localization for that specimen (Figure 3-7). Using the slice plane clip sliders in DDV, the
specific Cartesian (x-, y-, and z-plane) coordinates for epithelial lesions can be recorded.
These coordinates can be read into NWGrid and translated into the corresponding sites on
the surface of the computational volume mesh for that animal. The individual cell faces
of the volume mesh corresponding to the site of injury can be digitally highlighted using
the General Mesh Viewer (GMV, Los Alamos National Laboratory, http://www-

xdiv.lanl.gov/XCM/gmv/GMVHome.html) (Figure 3-7).

79

 

Figure 3-7. Three-dimensional mapping of ozone-induced nasal lesions. (A) Z-plane
(transverse) MR image of the nasal cavity of a 90-day-old Rhesus monkey. The fixative
within the nasal airspace and surrounding the specimen contains Magnevist® MR
contrast agent to provide high contrast between nasal tissue and airspaces. (B) Using
DDV, the geometry of the nasal airspace is selected and highlighted (green) in each 2-
plane image. (C) The locations of specific ozone-induced mucosal lesions are identified
on each transverse nasal tissue section, and the region of each lesion is recorded on the
tissue-airspace interface of the corresponding z-plane image. In this section. a lesion
along the dorsal maxilloturbinate is highlighted. (D) The entire z-plane slice can be
superimposed over the isosurface rendering for that specimen to provide 3D localization
of each lesion. (E) The 3D coordinates generated in DDV can be translated into grid
coordinates using NWGrid, and mapped on the corresponding site along the surface of
the computational volume mesh using GMV, for use in CFD simulations. The area
highlighted in blue represents the focal area of injury along the dorsal maxilloturbinate
identified in (C). NV = nasal vestibule; MS = maxillary sinus; D = duct to the maxillary
sinus; NP = nasopharyngeal meatus.

80

Computational Fluid Dynamics Simulations

The 3D nasal epithelial map described above was used as an initial finite element
mesh for CFD simulations. Airflow simulations were performed for resting breathing
(minute volume = 1.0 L/min) using a commercial CF D package (F idap®, Fluent Inc.,
Lebanon, NH). For the initial simulation, ozone uptake by the epithelium was assumed

to obey the following relationship:

Fluxozone = kcozone [4L

where Fluxozone (kg/mz-s) is the ozone flux from air into tissue, k is a rate constant

(0.023m/s), and cozone is the ambient concentration of ozone at the air-tissue interface

(0.5 ppm). Thus, ozone uptake at the epithelial surface was assumed to be proportional to
the ozone concentration at the wall (Taylor et a1. 2007), and uniform throughout the nasal
airspace. Previous reports suggest that squamous epithelium is resistant to uptake of
inhaled reactive gases, including formaldehyde and ozone (Kimbell 1997). To determine
the potential influence of the distribution of squamous epithelium on overall ozone
uptake in the nasal airways, a second simulation was performed with the same
assumptions; however flux over the area of the mesh representing the squamous

epithelium was set equal to 0.

Determination of Intracellular Antioxidant Concentrations in Nasal Mucosa

81

Nasal tissue homogenates were thawed and centrifuged at 12,500 x g for 4 hours.
Protein pellets were resuspended in 500ul of PBS, neutralized with 25 ul 1N NaOH, and
sonicated for 1 hour at 37°C. The protein content of resuspended pellets was measured
using the Pierce BCA Protein Assay (Pierce Biotechnology, Rockford, IL). Supematants
were diluted three-fold in 10% meta-phosphoric acid, and filtered through a 0.22 pm
syringe filter. Triplicate samples of each Supernatant were fractioned on a Shimadzu LC-
10Ai HPLC (Shimadzu Scientific Instruments, Colmnbia, MD), using a Phenomenex
Luna C18(2) 250mm x 4.6mm, SuM reversed phase column, preceded by a Phenomenex
ODS 4mm x 3mm guard column (Phenomenex, Torrance, CA). The mobile phase
consisted of an isocratic mixture of 50mM phosphate buffer, pH 3.1, containing 50uM
octanesulfonic acid and methanol (95:5). Samples were fractioned at a mobile phase
flow rate of 1.0ml/min. Reduced glutathione (GSH), oxidized glutathione (disulfide,
GSSG), ascorbate (AH;), and uric acid (UA) were simultaneously detected with an 8-
channel ESA CoulArray Model 5600A electrochemical detector (ESA, Chelmsford,
MA). GSH, GSSG, AH;, and UA values were normalized to protein content of

centrifuge pellets.

RESULTS

Nasal cavity surface area and volume calculations. Calculations of total surface
areas and volumes of the left nasal passages of two FA-exposed and two 1 cycle ozone-
exposed 180-day-old rhesus monkeys are presented in Table 3-1. It should be noted here
that the calculations of surface area and volume, as well as the data used in epithelial

mapping, include the nasal vestibule, starting at the first enclosed nasal transverse

82

section, and the nasopharyngeal meatus, caudally to the point at which the left and right
sides join to form the nasopharynx. The volume and area measurements presented here
also include the maxillary sinus. The data presented here show that there was
consistency in the nasal surface areas and volume measurements among the age-matched
infant monkeys. There was no significant effect of ozone exposure on the nasal surface
area and volume measurements obtained in these monkeys. This finding of consistency
in nasal size in infant monkeys is in contrast to the significant inter-individual variability
in results reported previously for adult monkeys (Schreider 1986; Gross et al. 1987;

Kepler 1995).

83

 

 

 

 

 

Monkey
Weight (kg) Volume (cm3) Surface Area (cmz)
(exposure)
A (filtered air) 1.30 0.53 10.3
B (filtered air) 1.50 0.64 13.9
C (1 cycle ozone) 1.55 0.59 13.4
D (1 cycle ozone) 1.32 0.62 13.7

 

 

 

 

 

 

Table 3-1. Calculated total surface areas and volumes of the nasal passages from the
four l80-day-old monkeys pictured in Figure 3-2.

 

 

 

 

 

 

 

 

Epithelial Surface Percent of Total

Region Area (cmz) Surface Area (%)
Squamous 1.81 14.9

Transitional 0.1 7 1 .4

Respiratory 7.92 65.1

Olfactory 1.93 15.9
Lymphoepithelium 0.33 2.7

Total 12.16 100

 

 

 

 

 

Table 3-2. Estimated epithelial surface areas for one side of a l80-day-old rhesus

monkey.

84

Digital epithelial mapping and surface area calculations. Complete epithelial
mapping of one half of the nasal cavity was performed in one 180-day-old, filtered-air
exposed rhesus monkey. Estimated surface areas of the five major epithelial types are
presented in Table 3-2. The majority of the nasal airspace in this monkey (65.1%) was
lined with ciliated respiratory epithelium, including the ventral half of the main nasal
chamber, most of the nasopharyngeal meatus, and the entirety of the maxillary sinus,
including the duct. Our results indicated that approximately 16% of the nasal cavity
surface area in this infant rhesus monkey was lined by olfactory epithelium. This is
consistent with an earlier report estimating that ~14% of the adult rhesus monkey nasal
cavity is lined with olfactory epithelium (Gross et al. 1987). We found that the NTE in
this infant monkey was confined to a narrow band in the preturbinate region, between the
squamous epithelium of the nasal vestibule and the respiratory epithelium of the main
nasal chamber. This localization, as well as the finding that NTE comprised
approximately 1.4% of the total nasal epithelial surface area in this monkey, is also
similar to results obtained from adult rhesus monkeys (Gross et al. 1987). The
lymphoepithelium was primarily localized over two discrete aggregations of NALT
located in the septal aspect of the nasopharynx. A small island of LE was also found in

the ventral aspect of the preturbinate region.

Ozone-induced nasal epithelial injury and morphometry. All monkeys exposed
to 1 cycle ozone had a moderate to marked necrotizing rhinitis that was restricted
principally to the nasal mucosa lining the middle meatus in the main nasal chamber.

There were focal regions of epithelial exfoliation, and these lesions were locally

85

extensive in the respiratory epithelium lining the dorsal surface of the anterior
maxilloturbinate. There was an anterior to posterior decrease in the severity of these
intranasal ozone-induced lesions. In 90-day-old monkeys exposed to 1 cycle ozone, the
most severely affected areas of the nasal mucosa had near full-thickness necrosis and
exfoliation of the surface epithelium with numerous neutrophils, and lesser numbers of
eosinophils, infiltrating the subepithelial lamina propria and the remaining surface
epithelium (Figure 3-8). Ninety-day-old monkeys that were exposed to 5 cycles of ozone
also had ozone-induced rhinitis and epithelial necrosis in relatively the same intranasal

locations as the ozone-induced lesions in the 1 cycle ozone-exposed animals.

86

 

87

Figure 3-8. Light
photomicrographs of the nasal
mucosa lining the dorsal
surface of the anterior aspect of
the maxilloturbinate of 90-day-
old monkeys exposed to 0 ppm
ozone (filtered air; A), 1 cycle
ozone (B), or 5 cycle ozone
(C). No microscopic lesions
are present in (A). Marked
epithelial necrosis and full
thickness necrosis of the
surface epithelium (E) along
with marked neutrophilic
inflammation (*) is present
throughout the nasal mucosa,
including the surface
epithelium and underlying
lamina propria containing
blood vessels (BV) and glands
(G).

Morphometric analyses of these ozone-induced nasal lesions indicated that 90-
day-old monkeys that were exposed to 1 cycle ozone (0.5 ppm; 8h/day for 5 days) had a
65% reduction (compared to filtered air controls) in the mean thickness of the nasal
epithelium lining the dorsal surface of the anterior maxilloturbinate due to toxicant-
induced necrosis and exfoliation of this targeted site of nasal airway epithelium.
Furthermore, this ozone-induced atrophy of the nasal epithelium resulted in an 88%,
64%, and 59% loss of the volume densities of airway cilia, epithelial cytoplasm, and
epithelial nuclei, respectively. Interestingly, the character, severity, and distribution of
the ozone-induced nasal epithelial lesions in 5 cycle 03 exposed monkeys were similar to
those in the 1 cycle ozone-exposed infant monkeys of similar age. The loss of mean
airway epithelial thickness in monkeys exposed to 5 cycle ozone was only slightly less

than in infant monkeys that were exposed to 1 cycle ozone (Figure 3-9).

88

    
  
   

    
     

Epithelial 10 Ciliary Volume
60 Thickness 8 Density
61‘
E40 g 6
3 “E 4
20 :3.
" 2
O
Filtered 1-cycle 5-cycle Filtered 1-cycle 5-cycle
A Air ozone ozone B Air ozone ozone

 

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Cytoplasmic 20
Volume
Density

Nuclear Volume
Density

(um3lum2)
N or
O O

a
(um3lum2)
3

1-cycle 5-cycle
C An ozone ozone D Air ozone ozone

0 0
Filtered 1-cycle 5-cycle Filtered

Figure 3-9. Morphometry of ozone-induced nasal epithelial lesions in 90-day-old rhesus
monkeys. 1 cycle and 5 cycle ozone-induced epithelial necrosis resulted in attenuation of
epithelial thickness (A), and reductions in the volume densities of epithelial cilia (B),
epithelial cytoplasm (C), and epithelial nuclei (D).

89

Computational Fluid Dynamics Simulations. CFD simulations and predictions
of ozone uptake in the nasal airspace model are depicted in Figure 10. At an ambient
concentration of 0.5 ppm, regions of high ozone uptake are predicted along the ventral
aspect of the preturbinate region, and along the septal and lateral walls of the middle and
ventral meatus in the region of the anterior maxilloturbinate. A focal region of high
ozone uptake (hotspot) is predicted at the dorsal aspect of the anterior maxilloturbinate, at
the site of the ozone-induced mucosal injury described earlier. In addition, hotspots are
also predicted in regions of the olfactory epithelium, where no ozone-induced injury is

observed.

90

 

OE

E RE

  

i 1.2 x 1o'° Itgrsm2

o kgrsm’

 

Figure 3-10. Computational fluid dynamics simulations of airflow and ozone uptake in
the nasal airways. Lateral (A) and septal (B) views of CFD predictions of ozone uptake
in the model assuming uniform uptake throughout the nasal airways. Lateral (C) and
septal (D) views of CFD predictions of ozone uptake in the model assuming no uptake by
the squamous epithelium. (E) Transverse section through 3D model at the location of the
anterior maxilloturbinate. High uptake (red) is predicted in the respiratory epithelium
(RE) at the dorsal aspect of the anterior maxilloturbinate where lesions were observed in
inhalation experiments (black arrow). Hotspots are also predicted on the olfactory
epithelium (OE), where no lesions were found (white block arrow).

91

Intracellular concentrations of low molecular weight nasal antioxidants. The
intracellular antioxidant measurements in the nasal mucosa are summarized in Figure 3-
1 1. There were no significant differences across the three regions (anterior MT, posterior
MT, anterior ET) in the baseline antioxidant concentrations of GSH, GSSG, M2, or UA
in FA-exposed animals. In animals exposed to 1 cycle or 5 cycle ozone, there was a
decrease in GSH, GSSH, AH;, and UA concentrations in all three nasal regions,
compared to FA exposed animals. There was a statistically significant decrease in AHz
concentrations in the posterior MT following both 1 cycle (63% decrease) and 5 cycle
(55% decrease) exposure. There was also a statistically significant decrease in GSSG
concentrations in the posterior MT (65% decrease and 82% decrease) and in the anterior
ET (85% decrease with both treatments) following 1 cycle and 5 cycle ozone exposure,

respectively.

92

  

 

   

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D

Figure 3-11. Antioxidant concentrations in the nasal mucosa of infant monkeys exposed
to 1 cycle ozone and 5 cycle ozone. 1 cycle and 5 cycle ozone exposure resulted in
depletion of AH2 (A), GSH (B), UA (C), and GSSG (D), in mucosal sections from the
anterior MT (R2), the posterior MT (R3), and the anterior ET (R4).

93

DISCUSSION

This is the first report to describe in detail the 3-dimensional representation of the
normal epithelial distribution in the nasal airways of an infant, non-human primate
model. This technique was designed to develop age-specific epithelial maps of the nasal
airways in experimental animal models for use in toxicant-induced lesion mapping and
CFD simulations of site-specific tissue dosimetry. Our results in this infant monkey,
when compared to a previous report in adults, suggest that the relative amounts of the
five. epithelial types in the nasal airways of monkeys remained consistent between
infancy and adulthood (Gross et al. 1987). However, because there is considerable
change in the size and shape of the head of the rhesus monkey from the neonatal to the
adult stage, we estimated that there would be significant age-related differences in the
location of susceptible epithelial cell populations. These differences in airway geometry
and epithelial lining may both contribute to differences in the responses to oxidant
pollutant exposure by altering the dose delivered to susceptible epithelial types. This
technique allowed for the determination of the specific distribution of susceptible
epithelial populations (i.e. RE, NTE), and ultimately, will serve to reduce uncertainty in
CFD dosimetry modeling and extrapolation of data from experimental animal models to
humans (Kimbell 2006).

It is also crucial to understand the distribution of the normal epithelial populations
in infant monkeys in order to be able to adequately distinguish age-related epithelial
differences from epithelial alterations induced by toxicant exposure. For example, both
squamous metaplasia and respiratory metaplasia of the olfactory epithelium have been

reported as adaptive sequelae to toxicant exposure (Harkema et al. 2006). Prior

94

knowledge of the normal distribution of these epithelial types in the infant nasal airways
is needed to make this distinction. A clear limitation to the data reported here is that this
map represents the nasal airway from a single, FA-exposed, infant rhesus monkey. In
future studies, we plan to complete additional epithelial maps for 90- and l80-day-old
F A-exposed animals.

The utility of three-dimensional imaging techniques in laboratory animals for
toxicant-induced lesion screening and dosimetry modeling has been previously reviewed
(Menache et al. 1997; Wiethoff et a1. 2001; Reed et al. 2003). These approaches used
semi-quantitative or subjective assessments of airway lesions applied to models of the
nasal airspaces of rodents. The use of morphometric techniques for the quantification of
respiratory structures has also been the subject of a recent review (Fanucchi et al. 2006).
We report the first use of MR imaging, computational biological methods, and standard
morphometric techniques to provide a quantitative and site-specific assessment of the
nasal responses to inhaled respiratory toxicants in a non-human primate model. The
methods described in the current report demonstrate the use of stereologic methods to
estimate volume density (Vs) of the airway epithelium as a quantitative assessment of the
epithelial response to ozone exposure in the nasal airways of infant monkeys. In addition
to the Vs methods described here for measurement of airway epithelium, other
morphometric assessments of the epithelium could be combined with 3D mapping to
provide additional information about the response to toxicant challenge. Morphometric
techniques may be used to measure the numeric cell density of specific epithelial cells per
length of basal lamina (i.e. volume of cells per unit area of basal lamina, um3/um2) as an

assessment of hyperplastic or metaplastic responses. These methods may also be

95

combined with immunohistochemistry to quantitate other responses, including
inflammation (e.g. # of neutrophils per mm basal lamina) and cell proliferation data (e. g.
proliferating cell nuclear antigen, PCNA). By providing additional depth to the
characterization of the tissue response to toxicant exposure, the measurement of these
types of responses is crucial to the establishment of exposure-dose-response relationships
in the nasal airways of infant monkeys. Morphometric quantitation of these epithelial
responses, combined with site-specific 3D mapping, provides objective comparisons of
the responses of monkeys to other species, facilitates comparisons to other toxicants, and
provides an important basis for extrapolation of experimental data to human risk

assessment.

3D mapping of toxicant-induced nasal airway lesions is unique in that the
pathology from each individual animal can be applied directly to a three-dimensional
model of its own nasal airways for use in CFD simulations. The nasal airway surface
area and volume measurements presented in this study, when compared to a previous
characterization in adult monkeys (Gross et al. 1987) demonstrated that significant post-
natal grth occurs in the nasal airways of monkeys from infancy to adulthood (Table 3-
1). This suggests that the nasal deposition patterns of inhaled toxicants would exhibit
age-dependent variation, potentially resulting in exposure of different epithelial types to
peak doses of toxicant. By providing estimates of toxicant dose relative to airway
geometry, CFD simulations provide an effective way to explain the contribution of
airway geometry to site-specificity. However, the effectiveness of CF D simulations is

limited by the accuracy of the initial model geometry. The three-dimensional models

96

generated from the MR image segrnentations provide a logical framework not only for
localizing the airway injury and for correlating injury to susceptible epithelial types, but
also to serve as the basis for the initial finite-element meshes to be used for CFD
simulations. The Cartesian coordinate system relationships used to record the specific
locations of the epithelial lesions in DDV are maintained in the finite element meshes
used for predictive dosimetry modeling. This allows for greater accuracy in the
correlations between experimentally induced and predicted airway injury, and also allows
for more specific testing of hypotheses about transport mechanisms and subsequent
extrapolation of animal data to humans. CFD airflow and site-specific tissue dosimetry

simulations using tissue dosimetry simulations using this model are in progress.

The nasal injury in the 90-day-oldinfant monkeys was particularly severe in the
ciliated respiratory epithelium along the dorsal surface of the maxilloturbinate. This is
consistent with the report that adult monkeys acutely exposed to ozone (0.3 ppm, 8h/day
for 6 days) also experienced necrosis and exfoliation of ciliated cells in the anterior nasal
cavity (Harkema et al. 1987c). Despite differences in the ozone concentration and
duration of exposure, our results indicate that the character and location of the ozone-
induced nasal lesions in the 1 cycle ozone-exposed 90-day-old monkeys reported here are
similar to those previously reported for adult monkeys acutely exposed to ozone.
However, while the character and location of toxicant-induced injury can be compared
with previously described methods, the imaging and 3D modeling techniques described
in the current study also allow for the assessment of the extent of airway lesions. In

future studies, we will section the complete nasal airways of ozone-exposed monkeys, in

97

a manner similar to the sectioning described above for epithelial mapping. This will
allow for the calculation of surface areas for ozone-induced lesions, and facilitate direct

comparisons across age groups and ozone doses.

It is possible that the age-related changes in the surface epithelial cell populations
alter the susceptibility of the nasal epithelium to ozone toxicity. Another possibility is
that age-related differences in airway geometry alter the local delivery of ozone to
susceptible epithelial populations within the nasal cavity. Results from our initial
simulations in infant monkey models predict that the highest ozone flux occurs in the
anterior nasal cavity, in the regions lined by NTE and ciliated RE, and that the presence.
of squamous epithelium at the naris and nasal vestibule may increase the overall dose of
ozone reaching the susceptible epithelial populations (NTE and ciliated RE). These
initial simulations are limited, however, for several reasons. First, these simulations
assume that the ozone flux is uniform throughout the regions of the nasal cavity lined
with mucus. The mucus layer is a dynamic part of the nasal airways, and is locally
regulated by many factors. These include the presence or absence of motile cilia, local
density of the intraepithelial mucosubstances, and the presence and type of submucosal
mucus glands (e. g. Bowman’s glands underlying olfactory epithelium versus submucosal
mucus glands in respiratory epithelial mucosa). Second, these simulations were
performed with an estimate of resting breathing at a constant flow rate. In the living
system, airway flow is initiated by the generation of a pressure difference between the
intrapulmonary airways and ambient air. This results in a variable flow rate throughout

the respiratory cycle. These are both factors that will require consideration for future

98

CFD simulations in order to increase the accuracy of the predictions. Providing more
detailed information to this process will provide valuable information on the relative
importance of airway geometry and tissue morphology, as they relate to the location and
extent of ozone-induced nasal airway injury in infant monkeys, and ultimately, in

children.

We report here that the loss of mean airway epithelial thickness and the volume
densities of airway cilia, epithelial cytoplasm, and epithelial nuclei, were not significantly
different between infant monkeys receiving 1 cycle or 5 cycle ozone exposures (Figure
9). Neither group of ozone-exposed infant monkeys exhibited mucous cell metaplasia or
epithelial hyperplasia in the region of airway injury. We have previously reported that
adult monkeys exposed daily (6 or 90 days) to high ambient concentrations (0.3 ppm) of
ozone undergo epithelial hyperplasia and mucous cell metaplasia (MCM) in the nasal
epithelium. Furthermore, this epithelial remodeling was associated with attenuation of
the epithelial necrosis and inflammation in response to subsequent acute ozone exposure.
These ozone-induced epithelial adaptations in adult monkeys may render the epithelium
less susceptible to tissue injury in response to subsequent ozone challenge (Harkema et
al. 1987b; Harkema et al. 1987c). It is important to note, however, that the long-term
exposure regimens used in adult monkey studies involved chronic (90 days), daily
exposures, while the infant monkeys presented in this report were exposed subchronically
(70 days) and episodically. Nevertheless, these findings suggest that infant monkeys
acutely or episodically exposed to ozone do not undergo nasal airway epithelial

remodeling or adaptation. This also suggests the disturbing possibility that infants may

99

develop a persistent necrotizing rhinitis following episodic exposures without any
protective molecular adaptation or morphologic remodeling of the surface epithelium that
would make the nasal airways less susceptible to ozone toxicity. Further studies are
needed to determine the responses to longer courses of episodic exposure (Chapter 4),
and to investigate the potential long-term consequences of ozone-induced nasal airway

injury in childhood.

In addition to anatomic and histologic factors, age-related biochemical changes
may also contribute to differential susceptibility to ozone toxicity. Antioxidants in the
surface lining fluid and epithelium are considered to provide the first line of defense
against inhaled oxidant challenge (Housley et al. 1995; Mudway and Kelly 1998). Nasal
antioxidants may play a role in the morphologic responses to ozone exposure.
Antioxidant upregulation may contribute to the development of tolerance to oxidant
pollutant challenge (Frank et al. 1978; West et al. 2000; Kirschvink et al. 2002).
Antioxidant upregulation may play a role in the regulation of oxidant-mediant cell
proliferation (Shaw and Chou 1986; Luppi et al. 2005). Ozone exposure also causes site-
specific injury in the pulmonary airways of monkeys, which correlates with sites of
ozone-induced GSH depletion (Plopper et al. 1998). In several in vitro models, the
presence of antioxidants in the surface lining fluid serves to augment oxidant pollutant-
induced cell membrane damage (Velsor and Postlethwait 1997; Ballinger et al. 2005). We
found that all antioxidants measured in the nasal mucosa were depleted at the site of
injury (anterior MT) following 1 cycle and 5 cycle ozone exposure. This exposure-

related antioxidant depletion may, in part, explain the lack of epithelial remodeling

100

following 1 cycle and 5 cycle exposure, and may also be related to the persistent
susceptibility to ozone-induced injury following cyclic ozone exposure. However, it is
important to note that a similar pattern of antioxidant depletion was also observed in the
two nasal regions in which no exposure-related injury was observed. This suggests that
while age-related differences in the regulation of airway antioxidants may contribute to
heterogeneity in the responses of immature models to ozone exposure, other factors are
also likely involved. These may include age-related differences in the regulation of

inflammation, mucus synthesis and secretion, and mechanisms of epithelial repair.

By splitting the nasal cavity and preserving the right side of the airway for
biochemical and molecular analyses, we attained the ability to directly correlate site-
specific airway injury and repair with site-specific indicators of tissue susceptibility to
ozone toxicity on an individual animal basis. Furthermore, this dissection technique
allows for the direct comparison of susceptible and resistant epithelial cell populations
within the same animal (e.g. RE vs. OE). The use of morphometry permits correlation of
quantified structural features with physiological and biochemical data (Tyler et al. 1985).
Using the 3-dimensional mapping technique, this information can be applied to predictive
dosimetry models, increasing the specificity of the results obtained from CFD
simulations. Employing this technique, however, assumes that the toxicant-induced nasal
injury is bilateral and symmetrical, and that it is not significantly influenced by the nasal
cycle, the physiologic alternating of airflow from one nasal passage to the other due to

periodic cycling of mucosal congestion and decongestion (Flanagan and Eccles 1997).

101

SUMMARY

Acute and chronic ozone exposures have been shown to cause epithelial
hyperplasia in the anterior nasal airways of adult monkeys. In the present study, we have
shown that acute and episodic (5 cycle) ozone exposure regimens cause epithelial
necrosis, exfoliation, and atrophy in the anterior nasal airways of infant monkeys, with no
morphologic evidence of epithelial hyperplasia. The magnitude and spatial distribution
of nasal airway injury induced by 1 cycle and 5 cycle exposures were similar. These
ozone-induced epithelial alterations were associated with site-matched consumption of
GSH and AH; in the nasal mucosa. Although these results do not establish a causal
relationship between nasal antioxidants and epithelial remodeling, they support the
possibility that ozone-induced epithelial hyperplasia and adaptation are dependent upon
the regulation of intracellular antioxidants and redox balance. Additional mechanistic
studies are needed to further investigate the mechanisms underlying ozone-induced cell
proliferation, and the possible role of antioxidants in this process.

The site-specificity of ozone-induced nasal injury necessitates that histopathologic
assessment be applied with the same site-specificity. By using the stereologic methods
described here, we obtain reliable measurements of the airway epithelium, its individual
components, and the components of the underlying basal lamina and lamina propria.
These measurements provide a quantifiable assessment of the epithelium and its response
to inhaled pollutants. The use of an infant rhesus monkey, while providing data from
nasal airways that are grossly and microscopically similar to the nasal airways of
children, also presents ethical challenges requiring efficient, robust, and pragmatic

experimental design and methods. Combining morphometric techniques with 3D

102

mapping provides the advantage of applying information about the nature, distribution,
and magnitude of nasal airway injury into a single model (Figure 12). Quantitation of
these epithelial responses facilitates the comparisons of susceptible and resistant
epithelial populations within the same animal. More importantly, this also provides an
important method of providing comparisons between experimental animals, and

facilitates extrapolations between species with different airway geometries.

103

Left Nasal
Cavity
We

h /V"'r #

    

N R
Nasal .
- Imaging
H h I
lstopat o ogy 3D imaging.

histopathology, and
airway modeling

fl“... ,ls, ‘. .. '
Morphometry Mapping

       
   

Nasal Epithelial

   

    

20 Airway
3D Isosurface
Generation
Right Nasal *
Lateral Wall
l\-
:7
/"' (
fl.
. I . 3D Airway ‘ Computational
Slte-spemfic mucosal Mapping Volume Mesh

biochemical and

molecular analysis *
CFD
Imulation

Figure 12. Overview of the imaging, 3D modeling, and histopathologic methods.
Sagittal sectioning of the nasal cavity results in a left nasal cavity with nasal septum
attached (top), and a right lateral wall (bottom left). The left nasal cavity is fixed and
submitted for MR imaging (top right). The resulting stack of 256 MR images is used to
digitally segment the nasal airways. This series of 2D segmentations is combined in
DDV to generate a 3D isosurface rendering of the nasal airways. Segmentation data
generated in DDV are read into NWGrid for translation into a computational volume
mesh, which serves as the initial finite element mesh for use in CFD simulations. After
MR imaging, the fixed left nasal cavity is decalcified, sectioned, and embedded for
histopathologic and morphometric analysis (left). Data obtained from histopathologic
analysis are combined with 2D segmentations to generate 2D nasal epithelial and nasal
airway injury maps. The 2D data from histopathologic and morphometric analyses, along
with site-specific biochemical data from the right nasal specimen (bottom left) can be
combined with existing 3D meshes for use in CFD simulations.

104

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Sram, R. J ., I. Benes, B. Binkova, J. Dejmek, D. Horstrnan, F. Kotesovec, D. Otto, S. D.
Perreault, J. Rubes, S. G. Selevan, I. Skalik, R. K. Stevens and J. Lewtas (1996).
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108

CHAPTER 4

CORRELATION BETWEEN OZONE-INDUCED SQUAMOUS METAPLASIA
AND GLUTATHIONE UPREGULATION IN THE NASAL AIRWAYS OF

INFANT MONKEYS

109

ABSTRACT
Children chronically exposed to high ambient levels of ozone (03), the principal

oxidant pollutant in photochemical smog, develop epithelial hyperplasia (EH) and
squamous metaplasia in the epithelium lining the anterior nasal airways. These nasal

alterations may impair normal physiologic functions (e.g. mucociliary clearance) that
serve to protect the lower airways from inhaled xenobiotics. Reactions between 03 and

endogenous nasal antioxidants may play a role in the induction of exposure-related
cellular injury and repair. The purpose of our study was to determine the effect of cyclic

ozone exposures on the developing nasal airways of infant monkeys, and the association

between 03-induced remodeling and nasal antioxidant levels. Infant (l80-day-old)
rhesus macaques were exposed to 5 consecutive days of O3 (0.5ppm, 8h/day; “l-cycle”)

or filtered air (FA), or 11 bi-weekly cycles of 03 (FA days 1-9; 0.5ppm, 8h/day on days

10-14; “1 l-cycle”). The left nasal passage was processed for light microscopy and

morphometric analysis. Mucosal samples fiom the right passage were processed for
glutathione (GSH), glutathione disulfide (GSSG), ascorbate (AHZ), and uric acid (UA)
concentrations. ll-cycle O3 induced squamous metaplasia and EH along the dorsomedial
surface of the maxilloturbinate, resulting in a 39% increase in the numeric density of
epithelial cells. ll-cycle 03 also induced a 65% increase in GSH concentrations at this
site, but no change in GSSG, AH2, or UA levels. l-cycle 03 did not cause EH,
metaplasia, or changes in antioxidant levels. EH was positively correlated with changes

in GSH (1:0.684; p=0.00694). These findings are consistent with 03-induced remodeling

110

observed in children, and provide new insights regarding the role of GSH in 03-induced

cell proliferation.

INTRODUCTION

Ozone, the principal oxidant air pollutant in photochemical smog, causes toxic
injury to the nasal (Graham, D. et al. 1988; Koren et al. 1990) and pulmonary (Graham,
D. E. and Koren 1990; Devlin et a1. 1991) airways in people. The US. Environmental
Protection Agency estimates that the majority (53%) of children in the United States live
in areas that exceed the NAAQS level for ozone (Woodruff et al. 2007). This is of
particular concern because recent evidence suggests that children may be more
vulnerable to the respiratory health effects of ozone than adults (Kim 2004). Exposure to
ambient concentrations of ozone causes acute inflammation in the nasal airways of
children (Frischer et al. 1993). Children residing in southwestern Mexico City, a region
in which ambient ozone concentrations frequently exceed the NAAQS, experience
epistaxis, nasal obstruction, and sinusitis (Calderon-Garciduenas et al. 2001). Nasal
biopsies from children in this region exhibit ciliated cell necrosis, ciliary dyskinesia, and
squamous metaplasia (Calderon-Garciduenas et al. 2001; Calderon-Garciduenas et al.
2001)

Several studies have characterized ozone-induced nasal airway injury using adult
laboratory animal models. Adult monkeys exposed short-term (6 days) to ambient
concentrations (0.15 ppm) of ozone develop acute rhinitis, epithelial hyperplasia, and
mucous cell metaplasia in the non-ciliated transitional epithelium (NTE) and the ciliated

respiratory epithelium (RE). Adult monkeys exposed to long-term ozone (0.15 or 0.30

111

ppm, 90 days) show an increase in intraepithelial mucus compared to short-term
exposure, with resolution of the inflammation and epithelial hyperplasia (Harkema et al.
1987; Harkema et al. 1987). Adult rats also develop acute inflammation, epithelial
hyperplasia, and mucous cell metaplasia in the nasal airways following short-term ozone
exposure (3 or 7 days, 0.5ppm, 8h/day) (Harkema et al. 1989; Cho et al. 1999). These
lesions were also present in adult rats following chronic exposure (13 weeks), and
persisted for several weeks after the end of exposure (Harkema et al. 1999). We have
recently reported that infant monkeys exposed to cyclic episodes of ozone for two months
(5 cycles) also develop acute rhinitis and nasal epithelial necrosis, but do not develop
epithelial hyperplasia or mucous cell metaplasia (Carey et al. 2007). Additional
controlled studies using immature animal models are needed to better understand the
differential effects of ozone exposure on the developing nasal airways of children.

The respiratory tract demonstrates marked heterogeneity with respect to the
anatomic site of injury following ozone exposure. In rhesus monkeys, the nature and
degree of ozone-induced cellular injury vary significantly by location in both the nasal
and pulmonary airways. In the lungs, ozone exposure targets the ciliated respiratory
epithelium of the trachea and type I pneumocytes lining the terminal bronchioles
(Castleman et al. 1980; Wilson et al. 1984; Nikula et al. 1988). In the nasal airways,
ozone-induced injury is confined to the anterior nasal cavity, specifically targeting the
NTE and ciliated RE (Harkema et al. 1987; Harkema et al. 1987; Carey et al. 2007).

The site-specificity of ozone-induced injury in the respiratory tract is due to a
combination of the local tissue susceptibility and the ozone dose delivered to specific

sites. The basis for the variations in tissue susceptibility may be related to the capacity of

112

that tissue to respond to oxidant challenge. Low molecular weight antioxidants in the
epithelium and overlying epithelial lining fluid of the respiratory tract are considered to
provide the first line of defense against inhaled oxidant challenge (Cross et al. 1994).

The major low molecular weight antioxidants lining the respiratory tract include
glutathione (GSH), ascorbate (AHZ), and uric acid (UA). Recent studies suggest that

reactions between oxidants and low molecular weight antioxidants are critical to the
induction of oxidant-mediated epithelial injury (Ballinger et al. 2005) and repair (Luppi
et al. 2005). In primates, GSH is unique among the low molecular weight antioxidants in
that it can be synthesized de novo and locally regulated by respiratory tract epithelial cells
in response to oxidative stress (Dickinson and Forman 2002). In young adult monkeys,
ozone exposure causes site-specific depletion of intracellular GSH concentrations at sites
of exposure-related injury in the pulmonary airways (Plopper, C. G. et al. 1998). There
are also variations in the activities of glutathione S-transferase and glutathione
peroxidase, enzymes that require GSH as a substrate, across different regions of monkey
lung (Duan et al. 1993).

The association between local antioxidant status and susceptibility to ozone-
induced injury in the nasal airways has not been investigated. The purposes of the
present study were, 1) to determine the effect of cyclic ozone exposures on the
developing nasal airways of infant monkeys, 2) to determine the effect of cyclic ozone
exposure on the steady-state levels of intracellular low molecular weight antioxidants,
and, 3) to determine the association between ozone-induced injury, remodeling, and nasal
antioxidant status. We hypothesized that the site-specificity and temporal pattern of

ozone-induced injury and repair are dependent on the local regulation of intracellular

113

ASE“
Ii

flax-gulch-
_ g ir-

nasal antioxidants. We tested this hypothesis in the nasal airways of infant rhesus
monkeys, an animal model whose nasal airways closely resemble those of children. By
splitting the nasal cavity, we were able to make direct comparisons between morphologic
responses and site-matched cellular and molecular responses to ozone exposure. The
results of this study provide new insights into the susceptibility of children to ozone-
induced nasal injury, and the potential relationships among nasal antioxidants, epithelial

injury, and repair.

MATERIALS AND METHODS
Animals and Ozone Exposure

Fourteen male infant rhesus monkeys (Macaca mulatta) were raised from birth in
a filtered air environment. All monkeys were obtained from the breeding colony at the
California National Primate Research Center at the University of California, Davis. Care
and housing of the animals before, during, and after treatment complied with the
provisions of the Institute of Laboratory Animal Resources, and conformed to practices
established by the American Association for Accreditation of Laboratory Animal Care
(AAALAC). While undergoing exposures, animals were housed in stainless steel open-
mesh cages. Prior to exposure, animals were housed in small social groups within the
exposure chambers in order to allow acclimation to the chamber environment. Five

monkeys were exposed to 5 consecutive days of 0.5 parts per million (ppm) ozone for 8
h/day, ending at 6 months of age (1 cycle O3). Five monkeys were exposed to 9

consecutive days of filtered air (FA) followed by 5 consecutive days of 0.5 ppm ozone,

8h/day, for a total of 11 cycles, beginning at 30 days of age and ending at 6 months of

114

 

age (11 cycle O3). Four control monkeys were exposed to filtered air (FA) only. All
animals were 180 +/-3 days of age at the end of exposure.
Ozone exposures were conducted in 4.2-cubic meter (m3) stainless steel and glass,

whole-body inhalation chambers. Ozone was produced from vaporized liquid, medical
grade oxygen by electronic discharge ozonizers. Generated ozone was diluted with
filtered air (24°C, 40-50% relative humidity) to the appropriate concentration, and
injected into the inlet airflow of the exposure chamber. Filtered air was supplied to the

exposure chambers at a rate of 2.1m3 per minute, providing 30 air exchanges per hour.

Chamber ozone concentration was monitored throughout exposures with an ultraviolet

ozone analyzer (Model 1003-AH, Dasibi Corporation, Glendale, CA).

Necropsy and Tissue Preservation

At the end of exposure, monkeys were sedated using ketamine hydrochloride
(10mg/kg 1M), followed by induction of deep anesthesia using propofol (Diprivan, 0.1-
0.2mg/kg/min IV), with the dose adjusted as necessary by an attending veterinarian. The
trachea, sternum, and abdominal cavity were exposed by a midline incision.
Anesthetized animals were euthanized by exsanguination via the abdominal aorta.
Immediately after sacrifice, the head was removed from the carcass, and the lower jaw,
skin, brain, and musculature were removed from the head. The nasal cavity was exposed
by splitting the skull sagittally, 1-2 mm to the right of midline, yielding a complete left
nasal cavity and intact nasal septum, and a free right lateral wall, as previously described
(Carey et al. 2007). The left nasal cavity was prepared for histopathologic and

morphometric analyses. Briefly, the left nasal cavity was lavaged retrograde through the

115

:— .' flfljfifi

nasopharyngeal duct with 2 ml cold phosphate buffered saline, flushed in the same
manner with a solution of 1% paraformaldehyde and 0.1% glutaraldehyde (0.01M
phosphate buffer, pH 7.4), immersed in ~100 ml of 1% paraformaldehyde and 0.1%
glutaraldehyde, and fixed at 4°C for at least 24 hours.

The right side of the nasal cavity was used to harvest site-specific mucosal
samples for RNA isolation and biochemical antioxidant analyses corresponding to the
sites examined by light microscopy in the left nasal cavity. Mucosal samples were
dissected from the anterior maxilloturbinate (MT), posterior MT, and anterior
ethmoturbinate (ET) (Figure 4-1, A). Each mucosal sample was divided into two equal
parts. One part from each region was stored in 0.5 ml RNALater (Ambion, Austin, TX),
held at room temperature for 24 hours, and then stored at-20°C. The samples from the
anterior MT were further processed for RNA extraction and gene expression analyses
(described below). The other part from each region was immediately placed into 300ul
of 10% metaphosphoric acid, and snap-frozen in liquid nitrogen. Acidified, frozen nasal
mucosal samples were later thawed, homogenized for 30 seconds using a Polytron
homogenizer, re-frozen, and stored at —80°C until further processing for low molecular

weight antioxidant analysis via high performance liquid chromatography (HPLC).

116

“.-

 

Figure 4-1. Anatomic location of nasal tissues selected for morphometric analysis. (A)
Exposed right lateral wall of the nasal cavity of a l80-day-old rhesus monkey. Vertical
lines indicate the locations of the twelve transverse sections processed for light
microscopy. The two black lines indicate the sections through the anterior
maxilloturbinate that were used for morphometric analyses. (B,C) Low magnification
photomicrographs of H&E-stained transverse sections through the anterior
maxilloturbinate of a FA-exposed monkey. The area highlighted in green represents the
dorsomedial surface of the maxilloturbinate. The mucosa underlying this region was
selected for morphometric analysis. . n = naris; i = incisor tooth; mt = maxilloturbinate;
ct = ethmoturbinate; hp = hard palate; np = nasopharyngeal meatus; s = nasal septum.

ll7

Tissue Processing for Light Microscopy and Morphometric Analysis

After fixation, each left nasal cavity specimen was decalcified in 200ml of 13%
formic acid for 14-16 days, and then rinsed with distilled water for 2-4 hours. After
decalcification, each nasal cavity was transversely sectioned at 10-12 specific anatomical
locations in a plane perpendicular to the hard palate and nasal septum, using gross dental
and palatine landmarks, as previously described (Carey et a1 2007). Each tissue block
was embedded in paraffin for light microscopic and morphometric analyses. Slides were
prepared using 4-5um thick sections from the anterior face of each of the 10-12 tissue
blocks. A tissue section from each block was stained with hematoxylin and eosin (H&E)
for routine histopathologic examination. Another tissue section was stained with Alcian
blue (pH 2.5)/periodic acid-Schiff (AB/PAS) for identification of acidic and neutral
mucosubstances.

For each animal, the first two transverse tissue sections that included a complete
profile of the MT (i.e., the anterior MT) were selected for morphometric analyses (Figure
4-1). A tissue section from these blocks was stained with rabbit-anti-human granulocytic
myeloperoxidase (MPO) polyclonal antibody (Lab Vision Corporation, Fremont, CA) for

immunohistochemical identification of neutrophils.

Morphometry of Neutrophilic Inflammation and Epithelial Numeric Cell Density
The epithelium lining the dorsomedial surface of the anterior MT of each animal

was analyzed through standard morphometric techniques and computerized image

analysis (Harkema et al. 1989). Neutrophilic inflammation was assessed on tissue

sections immunohistochemically stained for myeloperoxidase. Neutrophil numeric

118

 

densities were determined by quantitating the number of nuclear profiles of neutrophils
within the surface epithelium, in the lamina propria, and within the subepithelial
vasculature of the mucosa lining the dorsomedial MT, and dividing this number by the
total length of the basal lamina underlying the epithelium in this region. Neutrophils
were identified on the basis of characteristic, multilobed nuclear morphology and
cytoplasmic myeloperoxidase immunoreactivity. The basal lamina length was calculated
from the contour length of the basal lamina on a digitized image, using image analysis
software (Scion Image, Scion Corporation, Frederick, MD). The epithelial cell numeric
density was determined by counting the total number of epithelial cell nuclear profiles in
the epithelium lining the dorsomedial MT, and dividing this number by the length of the
basal lamina. The mucous cell numeric density was determined by counting the total
number of AB/PAS positive epithelial cells with nuclear profiles, and dividing this

number by the basal lamina length.

Morphometry of Epithelial Metaplasia

Metaplastic changes in the epithelium lining the dorsomedial, anterior MT were
quantified using stereologic methods. The volume density of nuclei, cilia, and
intraepithelial mucus within this region was morphometrically evaluated as previously
described for airway epithelium (Hyde et al. 1990; Hyde et al. 1991; Plopper, C. G. et al.
1994; Carey et al. 2007). All measurements were obtained at a final magnification of
1,710x using a light microscope (Olympus BX40; Olympus America, Inc., Melville, NY)
coupled to a 3.3-megapixel digital color camera (Q-Color 3 Camera; Quantitative

Imaging Corporation, Brunaby, British Columbia, Canada), and a personal computer

119

 

(Dimension 8200, Dell Corporation, Austin, TX). The morphometric analyses were
performed using a 135-pointcycloid grid overlay with an automated software package for
counting points and intercepts within the grid (Stereology Toolbox; Morphometrix,

Davis, CA) (Hyde et a1. 1990; Hyde et al. 1991). The percent volume density (the

proportion of the total epithelial volume), Vv, of the epithelial constituents was

determined by point counting and calculated using the following formula:

Vv=Pp=Pn/Pt (1)

where Pp is the point fraction of P”, the number of test points hitting the structure of

interest (e. g., nuclei), divided by P,, the total number of points hitting the reference space

(i.e., epithelium). The volume of each epithelial component of interest per unit basal

lamina length (cubic microns per square micron; 5,.) was determined by point- (epithelial

component) and intercept- (basal lamina) counting and was calculated using the

following formula:

Sv = 21014}! (2)

where 10 is the number of cycloid intercepts with the object (basal lamina), and L7 is the

length of the test line in the reference volume (epithelium within the test grid). To

determine the thickness of the total epithelium, a volume per unit area of basal lamina

120

 

(cubic microns per square micron) was calculated using the following formula for

arithmetic mean thickness (r):

1' = Vv/Sv (3)

Morphometry of Stored Intraepithelial Mucosubstances

The amount of stored mucosubstances in the surface epithelium of the
dorsomedial MT was estimated by quantifying the volume of AB/PAS-stained
mucosubstances per unit basal lamina (volume density, Vs), using computerized image
analysis and standard morphometric techniques. The area of AB/PAS-stained
mucosubstances was calculated by circumscribing the perimeter of the stained material
using the Scion Image program. The length of the basal lamina underlying the surface

epithelium was calculated as described above. The volume density was estimated using a
previously described method (Harkema et al. 1987), and was expressed in nl/mm2 basal

lamina.

Determination of Intracellular Low Molecular Weight Antioxidants Concentrations
in Nasal Mucosa

Nasal tissue homogenates were thawed and centrifuged at 12,500 x g for 4 hours.
Protein pellets were resuspended in 500u1 of PBS, neutralized with 25ul 1N NaOH, and
sonicated for 1 hour at 37°C. The protein content of resuspended pellets was measured
using the Pierce BCA Protein Assay (Pierce Biotechnology, Rockford, IL). Supematants
were filtered through a 0.22 pm syringe filter, and samples were fractioned in triplicate on

a Shimadzu LC-10Ai HPLC (Shimadzu Scientific Instruments, Columbia, MD), using a

121

 

Phenomenex Luna C18(2) 250mm x 4.6mm, SuM reversed phase column, preceded by a
Phenomenex ODS 4mm x 3mm guard column (Phenomenex, Torrance, CA). The
mobile phase consisted of an isocratic mixture of 50mM phosphate buffer, pH 3.1,
containing SOuM octanesulfonic acid and methanol (95:5). Samples were fractioned at a

mobile phase flow rate of 0.5 ml/min. Glutathione (GSH), glutathione disulfide (GSSG),
ascorbate (AH;), and uric acid (UA) were simultaneously detected with an 8-channel
ESA CoulArray Model 5600A electrochemical detector (ESA, Chelmsford, MA). GSH,

GSSG, AH;, and UA values were normalized to protein content of centrifuge pellets.

Analysis for Glutamate-Cysteine Ligase Catalytic Subunit (GCL-C) and Modifying
Subunit (GCL-M) mRNA in Nasal Tissues

Tissue samples for RNA isolation were homogenized using a Polytron
homogenizer. Total cellular RNA was isolated with Trizol (Invitrogen, Carlsbad, CA)
according to the manufacturer’s instructions, from samples stored in RNALater
(Ambion). To avoid DNA contamination, RNA pellets were resuspended in nuclease-
free water, and Dnase treated with DNA-free (Ambion) for l h at 37°C. cDNA was
prepared using random hexamer primers (Amersham-Pharmacia Biotech, Inc.,
Piscataway, NJ) and Moloney murine leukemia virus reverse transcriptase (Invitrogen).

Catalytic and modifying subunit mRNA levels were determined for GCL, the
rate-limiting enzyme in de novo GSH biosynthesis, by real-time PCR as previously
described (Abel et al. 2005; Wang et al. 2005). Briefly, samples were tested in duplicate,
and the PCR for the glyceraldehyde-3-phosphate (GAPDH) housekeeping gene and the

target gene from each sample were run in parallel on the same plate. The reaction was

122

 

carried out in a 96-well optical plate (Applied Biosystems, Foster City, CA) in a 25141
reaction volume containing 5 ul cDNA plus 20p] Mastermix (Applied Biosystems).
Sequences were amplified using the 7900 default amplification program (2 min at 50°C,
10 min at 95°C, followed by 40 to 45 cycles of 15 s at 95°C and 1 min at 60°C). The

results were analyzed with the SDS 7900 system software, version 2.1 (Applied

Biosystems). GCL-C and GCL-M mRNA expression levels were calculated from the

normalized AC T values. Fold increases were calculated using the AACT method.

Statistical Analyses

All data were expressed as mean group value i SEM. The differences among
groups were analyzed by one-way analysis of variance (AN OVA). Pairwise comparisons
were performed a priori using Student-Newman-Keuls multiple comparisons test. The
relationship between epithelial cell numeric density and intracellular antioxidant
concentration was analyzed by using post hoc correlation analysis (Pearson product
moment correlation). The criterion for statistical significance was set to p_<_0.05 for all
analyses. All statistical analyses were performed using a commercial statistical software

package (SigmaStat, SPSS Science, Chicago, IL).

RESULTS
Nasal Histopathology

Our histologic and morphometric investigation focused on the epithelium lining
the anterior nasal cavity. This region of the nasal cavity has been reported to be the

primary site for damage induced by inhaled toxicants in monkeys (Harkema et al. 1987;

123

 

Harkema et al. 1987; Harkema et al. 2006) and people (Calderon-Garciduenas et al. 1992;
Calderon-Garciduenas et al. 1994; Calderon-Garciduenas et al. 1995; Calderon-
Garciduenas et al. 1999; Calderon-Garciduenas et al. 2001; Calderon-Garciduenas et al.
2001)

In FA-exposed (0 ppm) infant monkeys, the preturbinate region of the main
chamber was lined with a combination of squamous epithelium, non-ciliated transitional
epithelium, and respiratory epithelium (Figure 4-2, A). Most of the remainder of the
main chamber of the nasal airways, including the entire surface of maxilloturbinate, was
lined with respiratory epithelium. A small region along the dorsal meatus and the dorsal
1/2 to 1/3 of the ethmoturbinate was lined with olfactory epithelium. This epithelial
distribution is in accordance with previous reports on the nasal airways of monkeys
(Harkema et al. 1987; Harkema et al. 1987; Harkema et al. 2006; Carey et al. 2007).
Occasional intraepithelial and sub-epithelial neutrophils were present in the mucosa of
the NTE and the RE lining the anterior nasal cavity in FA-exposed monkeys. This mild
inflammation was primarily localized near the ducts and luminal openings of submucosal
glands, and near the junctions of two adjacent epithelial populations (e.g., the junction of
respiratory epithelium and olfactory epithelium). No gross or microscopic exposure-
related morphologic alterations were observed in the nasal mucosa of monkeys exposed

to FA.
The principal nasal injury in monkeys exposed to 1 cycle O3 was acute,

multifocal, necrotizing rhinitis. This response was restricted primarily to the NTE and

ciliated RE lining the ventral half of the anterior main chamber. In all animals exposed to

1 cycle 03, there were focal areas of epithelial necrosis and exfoliation, localized

124

 

primarily to sites along the maxilloturbinate and ventral aspect of the nasal septum.
Associated with these areas of necrosis was an influx of neutrophils into the affected
epithelium and lamina propria, and endothelial margination of neutrophils in the

underlying capacitance vessels of the nasal mucosa.

' mini-E?

ll. ,.

125

 

 

 

126

Figure 4-2. Light
photomicrographs of the nasal
mucosa lining the dorsomedial
surface of the maxilloturbinate
of l 80-day-old monkeys
exposed to 0 ppm ozone
(filtered air, A), 1 cycle ozone
(B), or 11 cycle 03 (C). The
epithelial surface in figure A is
lined by an intact layer of cilia
(long arrows). Marked
epithelial necrosis and
exfoliation (arrowheads) of the
surface epithelium (e) are
present throughout the nasal
mucosa following acute ozone
exposure (B). Squamous
metaplasia and epithelial
hyperplasia are present in the
surface epithelium following 11
cycle 03 exposure (C). Note the
loss of surface cilia following 1
cycle (B) and 11 cycle (C)
ozone exposure. g = glands; bv
= blood vessels.

The site of the most severe necrotizing rhinitis in 1 cycle O3-exposed animals was

consistently observed in the respiratory epithelium lining the dorsomedial surface of the
anterior MT. This location was further characterized by a conspicuous attenuation or
complete loss of cilia, and a reduction in the amount of stored intraepithelial
mucosubstances (Figure 4-2, B). No significant histologic abnormalities were noted in

the olfactory epithelium, or in the nasal mucosa of the posterior aspect of the nasal cavity

in 1 cycle O3-exposed animals.

The exposure-related injury in 11 cycle 03 ozone-exposed animals was also
restricted to the ventral part of the anterior main chamber, similar to the distribution
observed in the 1 cycle O3-exposed animals. The principal lesions in animals exposed to
11 cycle 03 were epithelial hyperplasia, squamous metaplasia, and necrotizing rhinitis,
located along the dorsomedial surface of the MT (Figure 4-2, C). This location is
consistent with the site of maximal injury found in the 1 cycle O3-exposed animals.
However, in contrast to the pseudostratified ciliated respiratory epithelium described in 1
cycle 03 animals, the surface epithelium at this site in the 11 cycle O3-exposed animals

was 3-4 cells thick, and consisted primarily of layers of small, polygonal epithelial cells

devoid of intracellular mucosubstances or surface cilia. There wasan overall decrease in
the amount of stored intraepithelial mucosubstances in the 11 cycle O3-exposed animals.

However, this overall loss of intraepithelial mucus was accompanied by the appearance
of occasional clusters of small, mucus-containing epithelial cells forming intraepithelial

mucus glands. Focal areas of acute rhinitis and epithelial necrosis with, and without

127

‘F‘H.

 

exfoliation and loss of cilia, were also present along the ventral septum, ventral meatus,

and immediately adjacent to the area of squamous metaplasia along the MT.

Morphometry of Nasal Epithelium

Results of stereologic analysis of the dorsomedial surface of the anterior MT are
presented in Figure 4-3. 11 cycle 03 ozone exposure caused a 30% increase in mean
epithelial thickness compared to FA-exposed animals. Furthermore, this ozone-induced
epithelial thickening observed in '11 cycle O3-exposed animals was associated with a

significant 2.5-fold-increase in the volume density of epithelial nuclei, compared to FA-

exposed animals. There was no significant difference in the mean epithelial height or

nuclear volume density between 1 cycle 03 and FA-exposed animals. Both 1 cycle 03

and 11 cycle O3 exposure regimens caused a significant reduction in the volume density

of cilia (56% and 91% reductions, respectively) from the surface of the anterior MT,

compared to FA exposure.

Quantitation of Intraepithelial Mucosubstances

The epithelial hyperplasia and squamous metaplasia observed in animals exposed
to 11 cycle 03 ozone was also associated with a significant reduction in the amount of

stored intraepithelial mucosubstances (88% decrease) and in the numeric density of

mucous cells (90% decrease) in the anterior MT, compared to FA exposure (Figure 4-4).
1 cycle O3 exposure also caused a mild, but not statistically significant, decrease in stored

intraepithelial mucosubstances (36% decrease) and mucous cells (7.6% decrease)

128

compared to FA exposure. There was no significant difference observed in the mucous

cell numeric density between FA- and 1 cycle O3-exposed animals.

 

 

129

>
—I
o
o

Epithelial Height

   
  
    

    
   

80
2
2 60
o
E 40
20
o #2322 .» _
Filtered 1 Cycle 11 Cycle
Air Ozone Ozone
B 25 *
l Nuclear Volume
A 20‘ Density
N l
S 15:
i
5' 10-
0 ‘~:-:
Flltered 1 Cycle 11 Cycle
Air Ozone Ozone
C s

Ciliary Volume
Densny

     

(um3/lum2)
A

 

Filtered 1 Cycle 11 Cycle
Air Ozone Ozone

Figure 4-3. Morphometry of ozone-induced nasal epithelial injury in infant rhesus
monkeys. 11 cycle 03 exposure caused epithelial hyperplasia and squamous metaplasia,
resulting in an increase in epithelial height (A) and in the volume density of epithelial cell
nuclei (B). 1 cycle and 11 cycle ozone exposure each resulted in a reduction in the
volume density of cilia (C). Bars represent group mean : SEM. * = significantly
different from respective FA group (p50.05). a = significantly different from respective
acute ozone group (p50.05).

130

Intrapeithelial
Mucus

    
     

(“ma/szl

0
Filtered 1 Cycle 11 Cycle
Alf Ozone Ozone

D
Mucous Cell
a 120 Density
E E
E5 80
2 I
- G
33 4o
.9

0
Filtered 1 Cycle 11 Cycle
Air Ozone Ozone

 

Figure 4-4. (A,C) Light photomicrographs of maxilloturbinates from monkeys exposed
to FA (A) or 11 cycle 03 (C). Tissues were stained with AB/PAS to detect acidic and
neutral mucosubstances. Note the presence of cilia (solid arrow) and intraepithelial
AB/PAS-stained mucosubstances (dashed arrow) in the epithelium of the FA-exposed
monkey, and the loss of these features following 11 cycle 03 exposure (C). Dotted line —
basal lamina between epithelium and lamina propria; e = epithelium; bv = blood vessels;
g = glands. (B,D) Morphometry of intraepithelial mucus. Episodic ozone exposure
caused a decrease in the volume density of intraepithelial mucosubstances (B) and
mucous cell numeric cell density (D). Bars represent group mean 1 SEM. * =
significantly different fi‘om respective FA group. a = significantly difi‘erent from
respective 1 cycle 03 group (p50.05).

131

 

Morphometry of Neutrophilic Inflammation

The numeric cell densities of neutrophils in the mucosa along the dorsomedial
surface of the anterior MT are summarized in Figure 4-5. All ozone-exposed animals (1
cycle or 11 cycle) exhibited neutrophilic rhinitis in the nasal airway mucosa lining the
dorsomedial MT. Both of the ozone-exposed groups had ~4-fold more total neutrophils
in the nasal mucosa (intraepithelial + interstitial + intravascular) than the FA-exposed

group. There was no significant difference in the total or interstitial neutrophil numeric
density between 1 cycle 03 and 11 cycle 03 animals. However, the 11 cycle 03 group
had significantly more intraepithelial neutrophils (by 2.4-fold), and fewer intravascular

neutrophils (75% fewer) along this site than the 1 cycle O3 group.

132

 

I.
i.

 

 

A B

  
  

   

   

 

   

60 12
T0131 PMN * ‘ Intraepithelial PMN *a
2 2 ‘
E _ 40 E '-
E 5 s E.
2 _ 2 _.
3 w 3 l'
o 2 2° 4 a
.n .n
O "
Filtered 1 Cycle 11 Cycle Filtered 1 Cycle 11 Cycle
Air Ozone Ozone Alr Ozone Ozone
C D
40 Interstitial pMN * 16 Intravascular PMN
" 30 1'
E 5 e E 12
E E E E
\ Q a
2 : 2° 7. — a
T: g ,7; s
4: 1° 3 4
0 0
plum 1 Cycle 11 Cycle Filtered 1 Cycle 11 Cycle
Alr Ozone Ozone Air 010“. Ozone

Figure 4-5. Effect of ozone exposure on the numeric cell density of neutrophils (PMNs)
in the nasal mucosa along the anterior MT of infant monkeys. (A) Total PMN numeric
cell density (intraepithelial PMN + interstitial PMN + intravascular PMN); (B)
Intraepithelial PMN; (C) Interstitial PMN; (D) Intravascular PMN. Bars represent group
mean : SEM. * = significantly different from respective FA group (p50.05). a =
significantly different from respective acute ozone group (p_<_0.05).

133

Intracellular Concentrations of Low Molecular Weight Nasal Antioxidants

‘ The baseline antioxidant concentrations of GSH, GSSG, AH2, and UA in FA-
exposed animals were comparable in the anterior MT, posterior MT, and the anterior
ethoturbinate. The mucosal concentrations of GSH, GSSG, AH2, and UA did not
change significantly in any of the three nasal regions examined following 1 cycle 03-
exposure. However, there were slight decreases observed in the concentrations of GSH,
AH2, and UA in all regions following 1 cycle 03 exposure compared with FA-exposed
animals. In contrast, 11 cycle 03 exposure caused a significant increase in mucosal GSH

concentrations in the anterior MT (65% increase) and in the posterior MT (140%

increase) compared to FA exposure (Figure 4-6, A). There was also a moderate increase
in GSH concentration in the anterior ethmoturbinate of the 11 cycle 03 group (75%
increase), however this was not statistically significant. There was no change in GSSG
concentration in the two MT regions following 11 cycle 03 exposure, however, the
GSSG concentration in the anterior ethmoturbinate increased by ~2.4-fold (Figure 4-6,

B). No differences were observed in AHZ or UA concentrations in any of the three nasal

regions examined in 11 cycle 03 ozone animals (Figure 4-6, C and D).

134

ll

A

 

    
        

5003— ‘ "g "" 3
.g ‘ launch ‘ GS H .g GSSG
46 400' L-11Cyt:ie *a ‘6 *
a » ___ ----- a 2
3001 ..
E *a g
s g 1
" o
E e
C c
o E.
0 Anterior Posterior Anterior Anterior Posterior Anterior
MT MT ET MT MT
c 300 0.6
C
'3': AH2 3'3 UA
g o
a. 200 E. 0.4
u or
s s
3 100 g 0.2
.6 .5 2':
E e r»:
= =
0 Anterior Posterior Anterior 0 Anterior Posterior Anterior
MT MT ET MT MT ET

Figure 4-6. Effect of 1 cycle 03 and 11 cycle 03 exposure on the intracellular
concentrations of GSH (A), GSSG (B), AHz (C), and UA (D) in the nasal mucosa from
the anterior MT, posterior MT, and anterior ET of infant monkeys. Note the robust
increase in intracellular GSH concentrations in all regions of the nasal mucosa following
episodic ozone exposure. Bars represent group mean i SEM. * = significantly different
from respective FA group (p50.05). a = significantly different from respective 1 cycle 03
group (p50.05).

135

GCL-C and GCL-M mRNA Expression in the Anterior Maxilloturbinate
ll-cycle ozone exposure induced a significant increase (1.4-fold) in the steady-

state level of GCL-C mRNA in the anterior MT, compared to FA exposure (Figure 4-7,
A). There was no change in GCL-C mRNA expression at this site following 1 cycle 03
exposure. There were no changes in the expression of GCL-M mRNA following either 1

cycle 03 or 11 cycle O3 exposure (Figure 4-7, B).

Correlation Analysis for Epithelial Cell Numeric Density and Antioxidant
Concentration Along the Anterior Maxilloturbinate

Correlation analysis was used to determine the association between epithelial cell
numeric density and intracellular antioxidant concentration. There was a significant
positive correlation between epithelial cell numeric density and intracellular GSH
concentration at the anterior MT (F0684; p=0.00694) across all exposure groups (Figure

4-8, A). There were no significant correlations found between epithelial cell numeric
density and the intracellular concentrations of AH2 (r=0.0215; p=0.942) or UA (r=0.328;

p=0.252) (Figure 4-8, B and C).

136

 

>

 

 

 

 

    
    

 

 

 

 

 

 

2 i
2 .
E 3
o s.
6' <
a; 1 l T / .-
sé’ y
5 2 .
2 4'5 l /
.2 l
0 i fl T-I-Z-Z 'I-Z-
Filtered 1 Cycle 11 Cycle
Air Ozone Ozone

 

 

 

 

 

 

 

2‘.
E i. / T
:5 1 j! T 7 .........
‘ ,l //J ..... ..
Filtered 1 Cycle 11 Cycle
Air Ozone Ozone

Figure 4-7. Effect of 1 cycle O3 and 11 cycle 03 exposure on GCL-C (A) and GCL-M
(B) gene expression in the nasal mucosa of the anterior MT. Bars represent group mean
i SEM. a = significantly different from respective FA group (p50.05). b = significantly
different from respective acute ozone group (p50.05).

137

 

>

631-! (nlnoleelmg protein)

AHz (nmoleslmg protein)

0

UA (nmoleslmg protein)

 

    
 

 

soo-
2so» '
zoo-
150»
100»

norm
,0. p-o.oosu

O
O

o . - . . .
soo soo 700 soo 900 1000

Epithelial Numeric Cell Density
(cellslmm basal lamina)

 

 

 

 

 

250 ~
zoo - ' '
O
150 » °
e . l
o e
100 » ° .
.‘
so . ”.0215
. 33-0342
0 » . . . _1
600 600 700 000 000 1000
Epithelial Numeric Cell Density
(cellslmrn basal lamina)
0.5 -
O
0-4 r-0.328
p-0.252
0.3 '
O
0.2 ‘ e
0.1 l ' . e
I
0.0 . . Q . . ,
500 600 700 800 000 1000
Epithelial Cell Numeric Density
(cells/mm basal lamina)

138

Figure 4-8. Correlations
between epithelial
hyperplasia (numeric cell
density, x-axes) and the
intracellular concentrations
of GSH (A), AH; (B), and
UA (C) (y-axes) in the nasal
mucosa lining the anterior
MT. The associations were
described as the Pearson
product moment correlation
coefficients (r), and were
considered significant if
p50.05.

 

DISCUSSION

One purpose of this study was to determine the nature and distribution of mucosal
injury and repair in the nasal airway of infant monkeys exposed to ozone. The nasal
responses to both acute and chronic, daily ozone exposure in adult monkeys have been
extensively characterized. Acute ozone exposure (0.3 ppm, 8h/day, 6 days) causes
neutrophilic rhinitis, epithelial necrosis, and ciliated cell loss, confined to the NTE and
ciliated RE of the anterior nasal cavity in adult monkeys (Harkema et al. 1987; Harkema

et al. 1987). We found that infant (180-day-old) monkeys exposed acutely to ozone (1
cycle 03) also exhibit epithelial necrosis, ciliated cell loss, and neutrophilic

inflammation, which were confined to distinct focal regions in the anterior nasal cavity.
The distribution of these lesions was also consistent with those previously described in
both younger (90-day-old), infant monkeys (Carey et al. 2007) and in adult monkeys
(Harkema et al. 1987a; Harkema et al. 1987b) similarly exposed to ozone. Furthermore,

as we have shown here, the sites that exhibited necrosis and ciliated cell injury after 1

cycle 03 exposure in infant monkeys subsequently developed epithelial hyperplasia and
squamous metaplasia following 11 cycle O3 exposure. The location and nature of this

proliferative response to 11 cycle 03 exposures in these infants was similar to the

response previously described in adult macaques (Harkema et al. 1987a) following acute
exposure (0.3 ppm, 8h/day for 6 days). These results suggest that the site-specificity of
ozone-induced injury is consistent in infants and adults, despite significant post-natal

growth of the nasal cavity in non-human primates (Kepler 1995; Carey et al. 2007).

139

Mucous cell metaplasia is a frequent morphologic feature of airway epithelium
following inhaled pollutant challenge (Rogers and Jeffery 1986; Harkema and Hotchkiss
1991; Jany et al. 1991; Hotchkiss et al. 1998; Cho et al. 1999), and is considered a
pathologic response following airway epithelial injury (Wagner et al 2001; Puchelle et
al. 2006). Adult monkeys developed mucous cell metaplasia in the anterior nasal
airways following both acute (6 days, 8h/day) and chronic (90 days, 8h/day) daily

exposure to 0.3ppm ozone (Harkema et al. 1987a). In the present study, neither 1 cycle

03 nor ll cycle 03 ozone exposure caused mucous cell hyperplasia or metaplasia in

infant monkeys. In fact, both 1 cycle 03 and 11 cycle O3 exposures caused a decrease in

the amount of stored intraepithelial mucosubstances in the ciliated RE lining the anterior
MT. Our results may be related to age-related differences in the ability of the infant nasal
airways to respond to oxidant pollutant exposure. However, since ozone exposure
induces mucus hypersecretion in respiratory airways, it is also possible that the higher
ozone concentration (0.5 ppm versus 0.3ppm) used for the infant exposures caused a
short-term depletion of stored intraepithelial mucosubstances (Noganri et al. 2000).
Squamous metaplasia and hyperplasia of the nasal epithelium have been reported
as sequelae of chronic exposure to oxidant pollutants in children (Calderon-Garciduenas

et al. 2001; Calderon-Garciduenas et a1. 2001). In the present study, monkeys exposed to
11 cycle O3 exhibited focal areas of epithelial hyperplasia and squamous metaplasia of

the ciliated RE lining the anterior MT. This metaplastic change resulted in an increase in
epithelial thickness, and was also associated with a loss of surface cilia and a reduction in
the amount of stored intraepithelial mucosubstances. These morphologic changes in the

nasal airways result in an epithelium that is locally more resistant to uptake of reactive,

140

1 ..rmm'fl

pollutant gases (Kimbell et a1 1997), and may serve as protective adaptations. However,
these alterations may also serve to disrupt nasal mucociliary clearance and reactive gas
absorption in the anterior nasal airways. This loss of normal nasal function could
potentially result in delivery of toxic pollutants to more distal sites in the respiratory tract,
including the conducting airways and pulmonary parenchyma (Brain 1970; Morgan and
Frank 1977). Furthermore, the loss of functional ciliated RE could lead to increased
transit times for airborne toxicants trapped in the nasal airways, possibly resulting in
prolonged contact with the nasal epithelium and enhanced upper airway toxicity.

We previously reported that infant monkeys exposed episodically to 5 cycles of

ozone (0.5 ppm, 8h/day) exhibited persistent neutrophilic rhinitis, similar to that observed
following 1 cycle O3-exposure (Carey et al. 2007). This is in contrast to adult monkeys

exposed to 0.3 ppm ozone daily for 6 or 90 days. While adult monkeys exposed for 6
days developed acute neutrophilic rhinitis, this inflammatory response was not observed
in animals exposed for 90 days (Harkema et al. 1987a). Another purpose of the present
study was to determine the effect of a longer period of episodic ozone exposure on the
inflammatory and epithelial responses of the infant monkey nasal cavity. For these
experiments, we designed a longer regimen of episodic exposure (1 l-cycles) to a high
ambient concentration of ozone (0.5 ppm, 8h/day). These protocols were designed to
mimic intermittent exposures to clean and polluted air, which represents the nature of

exposure experienced by people in high pollution regions (Sram et al. 1996). In the

present study, we found that infant monkeys exposed to 11 cycle 03 had a similar
distribution and magnitude of neutrophilic rhinitis to that observed in 1 cycle O3-exposed

monkeys. There was no significant difference in neutrophil numeric cell density along

141

 

the anterior MT between 1 cycle 03- and 11 cycle O3-exposed monkeys (Figure 4-5).
The presence of epithelial hyperplasia and squamous metaplasia did not influence ozone-
induced inflammation during 11 cycle O3 exposure. Other reports have also described a

decrease in pulmonary inflammation and pro-inflammatory indicators following multi-
day ozone exposures (Plopper, Charles G. and Paige 1999; Wesselkamper et al. 2001;
Kirschvink et al. 2002). While the mechanisms underlying this acquired tolerance to
chronic ozone exposure remain unclear, several reports have hypothesized that epithelial
remodeling may serve a protective role against subsequent oxidant pollutant challenge.

We did not observe an attenuated inflammatory response in the nasal airways following
11 cycle O3 exposure, despite the development of significant epithelial remodeling at the

site of inflammation. These results indicate that the presence of ozone-induced epithelial
hyperplasia alone does not attenuate the inflammatory response to subsequent ozone
challenge in infant monkeys, and further suggest that the nasal airways in infant monkeys
remain susceptible to oxidant pollutant-induced inflammation following episodic
exposures.

Maintenance of neutrophilia at a site of inflammation can be due to persistent
Chemokine-mediated recruitment of neutrophils from circulation, cytokine-mediated
enhancement of neutrophil survival (Dibbert et al. 1999), or a combination of these
factors. While altered neutrophil survival has been demonstrated to play a role in the
development of pulmonary tolerance to ozone (Fievez et al. 2001), given the duration of

the episodic exposure period in our studies, the persistent rhinitis observed following 11
cycle O3 exposure is most likely the result of continuous or repeated neutrophil
recruitment. It is possible that the higher ozone concentration used in our studies

142

 

 

inhibited the development of tolerance. However, these results may also reflect a
fundamental difference in the nasal epithelial response to repeated cycles of injury and
repair caused by episodic ozone exposure. Future studies are needed to examine the
mechanisms behind this differential response to episodic versus daily ozone exposures,
and the possible implications toward the nature of ozone exposure experienced by
children.

The present study was also designed to test the hypothesis that the site-specificity
of ozone-induced epithelial injury, repair, and remodeling in the developing nasal
airways of infant monkeys is due, in part, to local differences in the steady-state levels of

low molecular weight nasal antioxidants. We compared the baseline and post-exposure
tissue concentrations of AH;, UA, GSH, and GSSG at an intranasal site of ozone-induced

injury and repair (anterior MT), and at two sites at which no exposure related
morphologic changes were observed (posterior MT, anterior ET). The only exposure-

related change in antioxidant levels was an increase in GSH concentration following 11-
cycle 03 exposure. In the lungs, site-specificity of injury and repair induced by inhaled

xenobiotics frequently correlates with local changes in the regulation of GSH. Regional
changes in GSH status have been correlated with toxicant-induced responses in the lungs
of monkeys and rats exposed to ozone (Duan et al. 1996; Plopper, C. G. et al. 1998).
Similar GSH responses have been documented in rat lungs following exposure to
hyperoxia (Kimball et al. 1976), and in mice exposed to naphthalene (West et al. 2000).
A correlation between oxidant pollutant-induced mucosal injury and GSH upregulation
has also been demonstrated in the nasal airways of rats exposed to cigarette smoke

(Maples et al. 1993). In our study, the ozone-induced upregulation in GSH levels was

143

T

I- . n

not limited to the site of ozone-induced injury, but was found in samples taken
throughout the nasal cavity. Furthermore, while no significant differences were
discerned among the three regions, the magnitude of this upregulation in GSH
concentrations was greatest in the posterior MT, a site with no observed exposure-related
morphologic changes. This suggests that the upregulation in mucosal GSH concentration
observed in our study was not a site-specific response to ozone-induced injury, but rather
a widespread nasal mucosal response to ozone exposure itself.

Acute ozone exposure initially causes site-specific GSH depletion in the

respiratory tract (Plopper, C. G. et al. 1998). We hypothesize that the increase in GSH
observed following 11 cycle 03 exposure is an adaptive response to repeated cycles of

GSH depletion. Intracellular GSH concentration is maintained by a balance among GSH
consumption/degradation, GSH redox cycling (via glutathione reductase), efflux of GSH
and GSSG into the ELF, uptake of intact GSH (primarily from the liver), and de novo
GSH synthesis (van Klaveren et al. 1997). Following acute ozone exposure and GSH
consumption, subsequent upregulation of GSH is, at least in part, the result of local de
novo synthesis. We found only a slight increase in the mRNA levels of the catalytic
subunit of GCL, the rate-limiting enzyme in de novo GSH synthesis, in animals exposed

to 11 cycle 03. While the increase in GCL-C expression may explain part of the increase

in GSH, other regulators of GSH were also likely involved. Oxidative stress activates
signal transduction pathways that lead to activation and nuclear translocation of redox-
sensitive transcription factors, including activator protein-1 (AP-1) and nuclear factor-

erythroid 2 related factor 2 (Nrf2). In addition to GCL-C and GCL-M, AP-l and Nrf2

activation controls expression of several other GSH-regulatory genes, including 7-

144

T

glutamyl transpeptidase, glutathione reductase, and glutathione synthase (Chan and
Kwong 2000; Cho et al. 2006). Increased expression of these genes would promote
increases in GSH content via GCL-independent mechanisms (Dickinson and Forman
2002). Changes in cellular redox state can also influence GCL activity in the absence of
a transcriptional component. Additional studies are needed to fully elucidate the
mechanisms behind ozone-induced GSH upregulation in monkeys.

In the regions lined by respiratory epithelium (anterior and posterior MT), the
increase in steady state GSH levels occurred with no significant change in GSSG
concentration. In contrast, there was a significant increase in GSSG concentration in the

anterior ethmoturbinate, a region lined primarily by olfactory epithelium. This regional
difference in glutathione disulfide concentration following 11 cycle 03 exposure may

reflect a local difference in the regulation of GSH and GSSG. Glutathione reductase is
an intracellular NADPH-dependent enzyme that catalyzes the reduction of GSSG to
GSH. In the rat, glutathione reductase activity is lower in olfactory epithelium than in
respiratory epithelium (Reed et al. 2003). Lower glutathione reductase activity could
result in higher intracellular concentrations of GSSG, particularly following oxidant
challenge. However, it is also possible that reductions in GSH and GSSG efflux rates
could contribute to increases in intracellular GSH and GSSG. Future studies that
compare intracellular and ELF GSH and GSSG levels would provide valuable
information regarding ozone-induced GSH turnover. Regional differences in other GSH
regulatory enzymes have not been evaluated for the nasal airways of monkeys.

While the exact role of GSH in cell proliferation remains unclear, a growing body

of evidence supports a role for GSH in cell cycle regulation and cell proliferation.

145

{mg " mm“
_ ' l

Previous reports indicate that upregulation and subcellular localization of GSH are
important in the control of Gl/S transition (Markovic et a1. 2007) and DNA synthesis

(Thelander and Reichard 1979). The importance of GSH in oxidant-induced cell
proliferation has been previously documented in cultured bronchial epithelial cells.
Cigarette smoke condensate causes a dose-dependent increase in 5-bromo-2-deoxyuridine
(BrdU) incorporation in primary bronchial epithelial cells in vitro. This effect is inhibited

by glutathione depletion (Luppi et al. 2005). Another recent investigation examined the
role of GSH in Nrf2-mediated cell proliferation. Using type II pneomocytes fi'om Nrf2'l'
mice, these investigators demonstrate that Nrf2-deficiency leads to impaired cell
proliferation in vitro, and GSH supplementation restores replicative capacity to Nrf2/-

cells (Reddy et al. 2007). We found a positive correlation between the total numeric
density of epithelial cells lining the anterior MT, a measure of epithelial hyperplasia, and
the intracellular concentration of GSH at that site. While these experiments were not
designed to elucidate the mechanisms behind ozone-induced remodeling, they establish a
temporal relationship between increases in the steady state, intracellular levels of GSH
and morphologic evidence of cell proliferation. It is important to note, however, that
positive correlation analysis does not establish causation, and that GSH upregulation and
the onset of epithelial hyperplasia and squamous metaplasia may be coincidental events,
both under the influence of additional factors.

We have shown that exposure to repeated episodes of ozone caused acute
epithelial injury, epithelial hyperplasia, and squamous metaplasia in the nasal airways of
infant monkeys. The nature and distribution of these nasal lesions were consistent with

those observed in the nasal airways of children inhabiting chronically polluted

146

ii

environments. The onset of ozone-induced epithelial remodeling coincides with the
ozone-induced upregulation in the steady-state intracellular levels of GSH in the nasal
airways. These local biochemical changes in the nasal mucosa correlate with ozone-
induced epithelial hyperplasia, and may confer a permissive effect on toxicant-induced
cell proliferation. Despite the presence of these ozone-induced morphologic and
biochemical alterations, the infant nasal cavity appears to remain susceptible to injury
and inflammation induced by repeated ozone exposures. Our results raise concerns about
the nature of the mucosal responses to repeated oxidant pollutant exposures in the nasal

airways of children.

147

 

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£37:

CHAPTER 5

ROLE OF GSH IN THE PATHOGENESIS OF OZONE-INDUCED EPITHELIAL

HYPERPLASIA IN RAT NASAL AIRWAYS

155

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INTRODUCTION

The governing hypothesis in these studies was that ozone-induced epithelial
hyperplasia in the nasal airways of infant monkeys and rats is a glutathione-dependent
process. In our previous studies, we demonstrated that chronic, episodic ozone exposure
causes epithelial hyperplasia and squamous metaplasia in the anterior nasal airways of
infant monkeys, and that this hyperplastic response is correlated with the local
upregulation of intracellular glutathione (GSH) concentrations. Although the results
from these studies demonstrate a temporal and spatial association between ozone-induced
epithelial hyperplasia and local GSH concentrations in the nasal airways, they do not
establish a mechanistic relationship among oxidant exposure, cell survival and
proliferation, and the regulation of steady-state GSH levels.

Oxidant-induced nasal epithelial hyperplasia is a multifactorial process. Early
exposures to ozone cause epithelial necrosis in the nasal airways (Hotchkiss et al. 1997).
The resulting epithelial proliferation is a response of the surviving epithelial cell
population to regenerate and repair (Henderson et al. 1993). The early responses to
ozone exposure must include mechanisms that can enhance cell survival following
oxidant challenge. Ozone exposure leads to the rapid induction of oxidant response
genes in the respiratory airways. Heme-oxygenase 1 (HO-l) and inducible nitric oxide
synthase (iNOS) are both inducible forms of anti-oxidant enzymes that are upregulated
early during oxidant exposure (Foucaud et al. 2006), and are considered common
indicators of oxidative stress in the respiratory airways (Morse and Choi 2005). Both
enzymes confer cytoprotective benefits during early ozone exposures that may provide

protection to surviving epithelial cells during repeated oxidant pollutant exposures (Joshi

156

 

 

et al. 1999; Jin and Choi 2005). Furthermore, both enzymes may be directly related to
control of cell cycle and cell proliferation following oxidant exposure or oxidant-induced
cytokine stimulation (Romanska et al. 2002; Colombrita et al. 2003).

Ozone exposure in humans causes secretion of pro-inflammatory cytokines and
chemokines into the respiratory airways. Many of these, including IL-6, TNF-or, and IL-
8, have been implicated in cellular processes related to ozone-induced epithelial injury
and repair (Koren et al. 1989; Devlin et al. 1991; Devlin 1993). Both TNF-a and IL-6
are important regulators of inflammation following ozone exposure (Pendino et al. 1994;
Leikauf et al. 1995). IL-6 also plays an important role in the regulation of epithelial
repair and cell proliferation in the pulmonary airways following ozone exposure. Yu, et
al., demonstrated that ozone-induced cell proliferation is inhibited in the pulmonary
airways of IL-6 knockout mice and in mice treated with anti-IL-6 neutralizing antibodies
(Yu et al. 2002). IL-8, a member of the CXC Chemokine family, is a potent neutrophil
chemoattractant molecule in humans and non-human primates. Studies conducted in
monkeys (Hyde et al. 1992) and rats (Salmon et a1. 1998) provide support for the
contribution of neutrophil chemotaxis in the pathogenesis of lung injury and repair
following short-term ozone exposure. The specific functions of these cytokines and
chemokines in the pathogenesis of ozone-induced nasal epithelial hyperplasia have not
been determined.

The same factors that contribute to activation of cytoprotective and pro-
inflammatory cell signaling cascades may also lead to alterations in the intracellular
regulation of GSH. One of the effects of oxidative stress is the activation of antioxidant

response transcription factors, including nuclear factor E2-related factor-2 (Nrf2). In

157

 

humans, activation of Nrf2 leads to nuclear translocation and binding to promoter
sequences of several antioxidant response genes. Among these are the genes for several
GSH synthetic enzymes, including the catalytic and modulatory subunits of glutamate
cysteine ligase (GCL-C and GCL-M), glutathione synthase (GS), and y-
glutarnyltranspeptidase (GGT) (Lu 2008). Increased expression of these enzymes is one
possible mechanism for local upregulation in GSH levels observed in the nasal airways of
infant monkeys following ozone exposure (Chapter 4), as well as similar mucosal
responses following oxidant exposure in the nasal airways of rats (Maples et al. 1993)
and pulmonary airways of monkeys (Plopper et al. 1998).

Oxidative (or nitrosative) stress can also cause DNA lesions or inhibit DNA repair

mechanisms in susceptible cell populations. This genotoxicity can lead to oxidant-
induced cell cycle arrest at either of the two major cell cycle checkpoints, Gl/S or Gz/M.
Recent studies provide evidence for the role of intracellular GSH in mediating cell cycle
progression through Gl/S (Lu et al. 2007) or G2/M (Reddy et al. 2008) following
oxidative stress. Previous studies also provide evidence supporting a role for GSH
upregulation in progression through Gl/S transition (Shaw and Chou 1986; Kavanagh et
al. 1990). Results from recent in vitro studies indicate that GSH depletion inhibits
oxidant-induced cell proliferation in cultured human bronchial epithelial cells, and that
the cysteine donor N-acetylcysteine, a precursor to GSH synthesis, restores replicative
capacity (Luppi et al. 2005).

The two studies described in this chapter were designed to test the hypotheses that

(1) early ozone exposures cause oxidative stress, expression of pro-inflammatory

cytokines and chemokines, and expression of GSH synthetic enzymes in the nasal

158

 

airways of immature rats prior to the development of ozone-induced epithelial
hyperplasia, (2) that these early molecular responses would lead to upregulation of
intracellular GSH concentrations in rat nasal tissues, and that (3) inhibition of GSH
synthesis during ozone exposures would inhibit the development of ozone-induced
epithelial hyperplasia in the anterior nasal airways of immature rats. In Study 1, we
examined the changes in the expression levels of indicators of oxidative stress (HO-1,
iNOS), pro-inflammatory cytokines (TNF-or, IL-6), CXC chemokines (MIP-2, CINC),
and GSH synthesis enzymes (GCL-C, GCL-M) in the nasal airways of rats, and
compared these with the timing of BrdU incorporation, an indicator of cell proliferation,
following 1 day and 2 days of daily ozone exposure. In Study 2, we examined the

changes in intracellular concentrations of GSH, glutathione disulfide (GSSG), and
ascorbate (AHZ) during the development of ozone-induced nasal epithelial hyperplasia.

Finally, we examined the effect of potent GSH synthesis inhibition (buthionine
sulfoximine, an inhibitor of GCL) on intracellular GSH concentrations, and on the

development of ozone-induced epithelial hyperplasia.

STUDY 1: EARLY CELLULAR AND MOLECULAR EVENTS PRECEEDING
OZONE-INDUCED EPI T HELML H YPERPIASIA IN THE NASAL AIRWAYS 0F

IMlllA T URE RA TS

MATERIALS AND METHODS

Animals, Ozone Exposure, and BrdU Treatment

159

’T’mml

Eighteen male Fischer 344/N rats (Harlan Sprague-Dawley, Indianapolis, IN), 28-
days-old, were used in Study 1. All animals were free of pathogens, and used in
accordance with guidelines set forth by the All-University Committee on Animal Use and
Care at Michigan State University. Rats were housed, three animals per cage, in
polycarbonate shoebox-type cages with filter tops. Water and food (Tek-Lab 1640,
Harlan Sprague-Dawley) were provided ad libitum. Rats were maintained on a 12-h
light/12-h dark cycle, in a temperature— and humidity-controlled environment (l6-25°C;
40-70% relative humidity).

All inhalation exposures were conducted in whole-body exposure chambers (HC-
1000; Lab Products, Maywood, NJ). The rats were acclimated to the exposure chambers,
supplied with filtered air (FA), for 8 hours on the day prior to the start of inhalation
exposures. The rats were individually housed in rack-mounted stainless steel wire cages
with free access to water during acclimation. The exposure chamber temperature,
relative humidity, and light cycle were maintained as described earlier for animal
housing.

To determine the effect of ozone exposure on the molecular responses of the nasal
airways prior to the development of ozone-induced epithelial hyperplasia, we randomly
assigned 18 rats to three experimental groups (n = 6/ group). One group of rats was
exposed to 0.8 ppm ozone for 8h/day for one day. A second group of rats was exposed to
0.8 ppm ozone for 8h/day for two consecutive days. A control group of rats was exposed
to FA (0 ppm) for 8h/day for one day. In previous chapters, we utilized a concentration
of 0.5 ppm ozone for non-human primate studies. We selected a higher ozone

concentration (0.8 ppm) for the rodent experiments based on comparative dosimetry

160

 

studies which suggest that the respiratory airways of rodents are less susceptible to the
health effects of ozone exposure, and require up to five-fold higher ozone concentrations
to elicit comparable levels of respiratory injury (Hatch et al. 1994). Three animals from
each group were dedicated for nasal histopathology and morphometry. The remaining
three animals from each group were dedicated for nasal RNA isolation and mRNA
analysis. At the end of the last day of exposure, each rat dedicated for nasal
histopathology was injected with 5-bromo, 2-deoxyuridine (50mg/kg, intraperitoneally)
to label cells in the S-phase (DNA synthesis) of the cell cycle. All rats in Study 1 were
euthanized two hours following the end of exposure.

Ozone exposures were conducted nightly, from 11:00 pm. to 7:00 am, while the
animals were most active. All exposures were conducted in the Inhalation Toxicology
Exposure Laboratory of the University Research Containment Facility at Michigan State
University. Food was removed, but animals had free access to water during ozone
exposures. Ozone was generated with an OREC Model O3VI-O ozonizer (Ozone
Research and Equipment, Phoenix, AZ), using compressed air (AirGas, Lansing, MI) as
an oxygen source. Ozone was diluted with filtered room air and delivered to the ozone
chambers through Teflon tubing. The total airflow through the exposure chambers was
maintained at approximately 250 L/min, to provide 15 chamber air changes per hour.
Chamber temperature and relative humidity were maintained at the same levels as during
the chamber acclimation period. The concentration of ozone within the chamber was
controlled by adjusting the voltage of the ultraviolet lamp within the ozonizer. Ozone
concentration was monitored during exposure with ozone monitors (Model 1003-AH;

Dasibi Environmental Corporation, Glendale, CA), and recorded with a strip chart

161

”l

 

recorder (Model 707; Chrono-Log Corporation, Havertown, PA). The probes for
sampling the exposure atmosphere were positioned in the breathing zone of the rats
within the ozone exposure chambers. The chamber ozone concentrations during
exposure to 0.8 ppm ozone were 0.786 1 0.028 ppm (mean j; SD). The chamber ozone

concentrations during exposure to filtered air were maintained at less than 0.050 ppm.

Necropsy and Tissue Preparation for Morphometric Analyses

Two hours following the end of exposure, rats were anesthetized with
pentobarbital (50mg/kg, i.p.), and then killed by exsanguination via the abdominal aorta.
After death, the head of each rat was removed from the carcass, and the eyes, lower jaw,
skin, and musculature was removed. For animals in Study 1, the entire nasal cavity was
processed for histopathology. The nasal cavities were flushed in a retrograde manner
through the nasopharyngeal orifice with 2ml of 10% neutral buffered formalin, and then
immersed in a large volume of the same fixative for a minimum of 24 hours.

The formalin-fixed nasal cavities were decalcified in 13% formic acid for 5 days,
and were then rinsed in reverse osmosis filtered water for 2 hours as previously described
(Harkema et al. 1988). A tissue block was removed fiom the anterior aspect of the left
nasal cavity by making two transverse cuts perpendicular to the hard palate. The first cut
was made immediately posterior to the upper incisor tooth (Figure 5-1). The second cut
was made at the level of the incisive papilla. The tissue blocks were embedded in
paraffin, and 5 uM-thick sections were cut from the anterior face of the block. Tissue
sections from each animal were histochemically stained with hematoxylin and eosin

(H&E) for routine histopathologic examination (results presented and discussed in Study

162

1'1

 

2). Other tissue sections were immunohistochemically stained with anti-BrdU antibody
(Becton Dickinson Immunocytometry Systems, San Jose, CA) to detect BrdU-labeled

nuclei, and counter stained with hematoxylin (Johnson et al. 1990).

163

"L

II

 

 

Figure 5-1. Anatomic location of nasal tissues selected for morphometric analyses. (A)
Exposed right lateral wall of rat nasal cavity. MT = maxilloturbinate; NT =
nasoturbinate; ET = ethmoturbinate; HP = hard palate. Vertical line indicates the anterior
surface of the tissue block sectioned for morphometric analyses. (B) Diagram of the
anterior face of a tissue block from the anterior nasal cavity. S = nasal septum; T = root
of incisor tooth; D = nasolacrimal duct. Red circle illustrates the profile of the anterior
maxilloturbinate. (C) Enlarged photonricrograph of the maxilloturbinate from a FA-
exposed rat, illustrating the major tissue compartments. e = non-ciliated transitional
epithelium (NTE); TB = turbinate bone; LP = lamina propria. The normal NTE is a non-
ciliated cuboidal epithelium consisting of 1-2 cell layers.

 

Morphometry of Cellular Injury and Cell Proliferation

Non-ciliated transitional epithelium (NTE) overlying the dorsal aspect of the
anterior maxilloturbinate (Figure 5-1) of each animal was analyzed using computerized
image analysis and standard morphometric techniques (Hotchkiss, Harkema, Henderson
Exp Lung Res 1991). In Study 1, the BrdU labeling index was calculated as an indicator
of reparative DNA synthesis in response to ozone-induced cell injury (Rajini et a1 1993).
The number of BrdU-labeled epithelial cell nuclei was counted, divided by the total
number of epithelial cell nuclei, and multiplied by 100 to yield the percentage of BrdU-
labeled epithelial cell nuclei. The BrdU+ numeric cell density was calculated by
counting the number of BrdU-labeled nuclei, and dividing this number by the length of
the basal lamina. The basal lamina length was calculated from the contour length of the
basal lamina on a digitized image, using image analysis software (Scion Image, Scion

Corporation, Frederick, MD).

Tissue Preparation for mRNA Analysis

For mRNA analysis, rats exposed to filtered air for 1 day, or exposed to ozone for
l or 2 days, were killed 2 hours following the end of exposure. These time points were
chosen for pro-inflammatory, GSH synthesis, and oxidative stress gene expression
analysis on the basis of previous findings in adult rats that neutrophilic inflammation and
cellular injury reach peak levels within the first 2 days of daily ozone exposure (Cho et
al. 1999). The head of each rat dedicated for mRNA isolation was removed and sagittally
split 1m to the right of midline. This sectioning process yielded a left nasal cavity, with

the septum attached, and a free right nasal lateral wall. The right side of the nasal cavity

165

 

was immersed in 10ml RNALater (Ambion; Austin, TX), and stored at room temperature

for 24 hours, then stored at —20°C until microdissection.

Total RNA Isolation from Nasal Tissues

Maxilloturbinates were microdissected from RNALater-preserved, frozen nasal
cavities of each animal. Total cellular RNA was extracted from the maxilloturbinate
according to the manufacturer’s protocol (RNEasy Mini Kit, Qiagen, Valencia, CA).
Briefly, tissues were homogenized in buffer RLT containing B-mercaptoethanol with a
5mm Rotor-sator homogenizer (PRO Scientific, Oxford, CT), and centrifuged at 12,000 x
g for 3 minutes. RNA was purified from the supernatant using the RNEasy capture
column. Samples were treated with Qiagen RNAse-Free DNAse set on the column for
30 minutes. Eluted RNA was diluted 1:5, and was quantified using a Genequant Pro
spectrophotometer (BioCrom, Cambridge, England).

Reverse transcription was performed using High Capacity cDNA Reverse
Transcription Kit reagents (Applied Biosystems, Foster City, CA). Each RT reaction was
run in a 50-p.l reaction volume containing 5-ug of total RNA and cDNA Master Mix
prepared according to the manufacturer’s protocol. Reverse transcription was performed

in a GeneAmp PCR System 9700 Thermocycler PE (Applied Biosystems) at 25°C for 10

minutes, 37°C for 2 hours, and held at 4°C.

Real-time Reverse Transcription Polymerase Chain Reaction
cDNA samples were diluted to 5ng/p.l and dispensed (in duplicate) into a 384-

well reaction plate using the BioMek 2000 Automated Workstation (Beckman Coulter,

166

 

 

Fullerton, CA). Quantitative mRNA expression analyses were performed with the ABI
PRISM 7900 HT Sequence Detection System using Taqman Gene Expression Assay
reagents for GCL-C, GCL-M, iNos, HO-l, macrophage inflammatory protein 2 (MIP-2,
CXCL2), and cytokine-induced neutrophil chemoattractant 1 (CINC, CXCLl) (Applied
Biosystems). The cycling parameters were 48°C for 2 minutes, 95°C for 10 minutes, and
40 cycles of 95°C for 15 seconds followed by 60°C for 1 minute. All mRNA expression
levels were reported as fold-increase (Fl) of mRNA in experimental samples compared to
a control sample. Real-time PCR amplifications were relatively quantified using the
AACt method normalized to the geometric mean of 3 endogenous controls (188, B-actin,
and GAPDH). This normalization strategy has been utilized for accurate RT-PCR
expression profiling in biological samples with small expression differences
(Vandesompele et al. 2002). The delta Ct (ACt) value for the experimental sample is

subtracted from the ACt value of the corresponding control sample (AACt). The F1 in

experimental samples relative to control samples is then calculated as: FI = 2'AACt.

Statistical Analyses

All data were expressed as mean group value i SEM. The differences among
groups in Study 1 were analyzed by one-way analysis of variance (ANOVA). Pairwise
comparisons were performed a priori using Student-Newman-Keuls multiple
comparisons test. The criterion for statistical significance was set to p50.05 for all
analyses. All statistical analyses were performed using a commercial statistical software

package (SigmaStat, SPSS Science, Chicago, IL).

167

 

RESULTS
Ozone-Induced BrdU Incorporation in Immature Rat Nasal Airways

Ozone exposure induced increases in the BrdU numeric cell density along the
dorsal maxilloturbinate in rats exposed for l-day (6.6-fold increase) and for 2-days (21-
fold increase), compared to rats exposed to filtered air. Ozone exposure also induced
similar increases in the BrdU labeling index following both 1-day and 2-day exposures
(7-fold and 22-fold increases, respectively). The increases in both BrdU numeric cell
density and BrdU labeling index following 2-day exposure were significantly different

from both FA-exposed and l-day ozone-exposed rats (Figure 5-2).

168

 

 

 

 

 

    

 

 

 

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Filtered 1-Day 2-Day
Air Ozone Ozone

Figure 5-2. Effect of ozone exposure on BrdU labeling index (A) and BrdU numeric cell
density in the NTE. Bars represent group mean +_ SEM (n = 3/ group). * = significantly
different from FA—exposed rats (p50.05). a = Significantly different from rats exposed to
l-day ozone (p50.05).

169

Ozone-Induced Oxidative Stress Gene Expression

Ozone exposure induced an increase in HO-l mRNA expression in rat nasal
airways following both l-day and 2-day exposures (2.7-fold and 2.4—fold increases,
respectively) (Figure 5-3). Ozone caused an increase in iNOS mRNA expression in rat
nasal mucosa following 2-day exposure (3.8-fold increase versus FA). The increase in
iNOS expression in 2-day-exposed rats was significantly different from both FA-exposed
and l-day-exposed rats (Figure 5-3). There were no significant effects of ozone exposure
on the expression of GCL-C or GCL-M following either l-day or 2-day exposure (Figure

5-4).

Ozone-Induced Pro-Inflammatory Gene Expression

2-day ozone exposure induced a significant increase in the steady state levels of
TNF-a mRNA (3.4-fold increase) and IL-6 mRNA (30-fold increase) versus FA
exposure. TNF-a and IL-6 mRNA levels were also increased following l-day ozone
exposure (1.7-fold and 8.2-fold, respectively). These changes were not significantly
different from FA-exposed controls, but were significantly different from 2-day ozone

exposed rats (Figure 5-5).

Ozone-Induced CXC Chemokine Gene Expression
Two-day ozone exposure caused significant increases in MIP-2 (IO-fold) and
CINC (13-fold) mRNA expression versus FA. The increases in mRNA expression for

each of these genes following 2-day exposure were also significantly different from l-day

170

 

 

ozone exposure. There were no significant differences versus FA in MIP-2 or CINC

mRNA levels following l-day ozone exposure (Figure 5-6).

 

171

N

 

 

A

\NH.

 

 

 

 

 

 

Filtered 1-Day
Air Ozone

 

 

 

B
5)
iv
0
E
g 3
ll.
5 2
E
8 1
E m
0
Filtered 1-Day
Air Ozone

Figure 5-3. Effect of ozone exposure on expression of HO-l mRNA (A) and iNOS
mRNA (B) in rat nasal mucosa. Bars represent group mean +_ SEM (n = 3/group). * =
significantly different from FA-exposed rats (p50.05). a = Significantly different from
rats exposed to 1-day ozone (p50.05).

172

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2
i
2
0
E
2
8
< 1 '-
z
n:
E
q
.l
o
o
0 “
Filtered 1-Day 2-Day
Air Ozone Ozone
B
2 .
O
0)
§
0
E
E
.2
< 1 T
2
a:
E
5.
.l
o
0
O
Filtered
Air

 

Figure 5-4. Effect of ozone exposure on expression of GCL-C mRNA (A) and GCL-M
mRNA (B) in rat nasal mucosa. Bars represent group mean 1 SEM (n = 3/group).

;_n

73

 

 

 

 

 

 

 

 

 

   

 

 

4 *a
0
§
3 3
E
2
8
s 2 T
m 7
E
ii 1 ~ ~' /
‘2'“
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o //,
Filtered 1-Day
Air Ozone
B
40 . *a
8 i
E 30 ‘
0 i
E
g i
.2 20 1
g i
‘E
"f 10
='
0 I ’
Filtered 1-Day
Air Ozone

 

Figure 5-5. Effect of ozone exposure on expression of TNF-a mRNA (A) and IL-6
mRNA (B) in rat nasal mucosa. Bars represent group mean 1: SEM (n = 3/group). * =
significantly different from FA-exposed rats (p50.05). a = Significantly different from
rats exposed to 1-day ozone (p50.05).

174

MIP-Z mRNA Fold Increase
a

 

*a

 

 

 

1 -Day
Ozone Ozone

*a

  

%

 

 

4 .
2 [_LJ
0
Filtered
Air
B
18
o 16
3
g 14
o
E 12
'u
3 10
IL
5 8
E 6
g 4
U 2.
0 I l
Filtered
Air

1-Day 2-Day
Ozone Ozone

Figure 5-6. Effect of ozone exposure on expression of MIP—2 mRNA (A) and CINC
mRNA (B) in rat nasal mucosa. Bars represent group mean _+_ SEM (n = 3/group). "‘ =
significantly different from FA-exposed rats (p50.05). a = Significantly different from
rats exposed to 1-day ozone (p50.05).

175

DISCUSSION

This study was designed to examine the early molecular events preceding ozone-
induced epithelial hyperplasia in the nasal airways of immature rats. Specifically, we
hypothesized that early ozone exposure causes oxidative stress, pro-inflammatory gene
expression, neutrophil chemotaxis, and upregulation of GSH synthesis genes in the nasal
mucosa of immature rats, and that these changes would precede or coincide with the
onset of ozone-induced DNA synthesis. In the present study, we found that daily
exposure to 0.8 ppm ozone (8h/day) induced epithelial damage and triggered cell
proliferation in the nasal airways of immature rats within 2 days of daily exposure. This
finding is similar to results observed in adult rats during daily exposures to 0.5 ppm
ozone (8h/day for three days), which caused peak increases in BrdU incorporation on day
2 of exposure (Cho et a1. 1999).

Ozone exposure caused a rapid increase in nasal HO-l mRNA expression
following l-day ozone exposure that was maintained following 2-day exposure. Ozone
exposure caused a delayed increase in nasal iNOS mRNA expression, with a significant
increase only observed following 2-day ozone exposure. Both indices of oxidative stress
were elevated by the onset of the ozone-induced increase in S—phase DNA synthesis
(BrdU incorporation) on day 2. Our results support the conclusion that early exposure to
ozone causes upregulation of cytoprotective antioxidant enzyme systems in the nasal
airways. In multi-day ozone exposures, this early upregulation may influence cell
survival in the remaining cell populations in the face of subsequent ozone exposures, and
allow time for the prerequisite mechanisms for cell proliferation to become established.

Interestingly, despite the observed increase in HO-l and iNOS expression, there was no

176

 

 

significant increase observed in the expression of the GSH synthetic enzyme
heterodimers GCL-C and GCL-M. GCL is the rate-limiting enzyme in de novo GSH
synthesis, and its expression is responsive to local decreases in the redox state of the cell
(lles and Liu 2005). We hypothesized that early ozone exposures would cause oxidative
stress in the nasal airways, resulting in GSH consumption and a transient decrease in
local redox state, thus increasing gene expression of GCL. In addition to changes in GCL
mRNA expression, intracellular GSH concentrations are regulated by several other
factors, including changes in GCL mRNA stability, variations in GCL-C activity, and
changes in gene expression of other GSH regulatory enzymes, including GGT and GS. It
is possible that these other factors could maintain intracellular redox balance without
necessitating an increase in GCL expression.

We demonstrated a significant induction of mRNA for the pro-inflammatory
cytokines TNF-a and IL-6 in the nasal airways during early exposure to ozone. We also
demonstrated coordinated increases in the expression of the CXC chemokines MIP-2 and
CINC in the nasal airways. In particular, there was a dramatic increase in IL-6 mRNA
induction (3 O-fold increase versus FA) by day 2 of exposure, which may influence ozone-
induced DNA synthesis and cell proliferation. TNF-a and IL-6 are pleiotropic
cytokines, which may play different roles at different times during the process of airway
injury, inflammation, and epithelial repair. Yu, et al., showed that IL-6 knockout mice
exhibit attenuated lung injury, decreased airway inflammation, and decreased DNA
synthesis following ozone exposure (Yu et al. 2002). In contrast, McKinney, et al.,
showed that pre-treatment with IL-6 attenuated ozone-induced lung inflammation, while

pre-treatment with an anti-IL-6 neutralizing antibody augmented ozone-induced

177

? -e'lb..h '. DE:
A

inflammation (McKinney et a1. 1998). Pre-treatment with TNF-a or IL-1 also attenuated
ozone-induced epithelial injury in the nasal airways of rats (Hotchkiss and Harkema
1992). However, another study demonstrated that pre—treatment with anti-TNF-a
antibodies ameliorated ozone-induced lung injury. In addition, this study found that early
blockade of TNF-a activity inhibited the ozone-induced expression of IL-6 (Bhalla et a1.
2002). It is possible that expression of IL-6 and TNF-a prior to ozone exposure serves to
limit or inhibit inflammation, while their expression during the first few days of exposure
may serve to amplify pro-inflammatory signals. It is also possible that their functions
may be dependent upon the concurrent expression levels of other pro-inflammatory
cytokines (e.g., IL-l). In our study, the timing of IL-6 and TNF-a upregulation, along
with the concurrent increase in the expression of CXC chemokines, support a pro-
inflammatory role for these cytokines in the nasal airways during early ozone exposure.
However, given the complexity of the inflammatory responses to IL-6 and TNF-a
manipulation, further investigation into the early interactions between these two

important cytokines is needed.

STUDY 2: EFFECT OF GSH DEPLETION 0N OZONE-INDUCED EPITHELIAL

H YPERPLASIA.

MATERIALS AND METHODS

Animals, Ozone Exposures, and Glutathione Depletion

178

To determine the effect of ozone exposure on the steady-state intracellular
concentrations of GSH in the nasal airways, and to determine the effect of GSH depletion
on the development of ozone-induced epithelial hyperplasia, we randomly assigned 72
rats to twelve experimental groups (n = 6/group). Two groups of animals were exposed
to FA for 8 hours. Two groups of animals each were exposed to 0.8 ppm ozone, 8h/day,
for 1, 2, or 3 consecutive days, and were euthanized two hours following the end of
exposure. Rats in the remaining four groups were exposed to 0.8 ppm ozone, 8h/day, for
three consecutive days, and then allowed to recover for 24 or 48 hours post-exposure
prior to euthanasia. One hour prior to the start of each daily inhalation exposure, half of
the rats were treated with L-buthionine-[S,R]-sulfoximine (lg/kg, intraperitoneally, i.p.;
BSO, Sigma Chemical Co., St. Louis, MO), an inhibitor of glutathione synthesis (Haddad
2001). The goal of B80 therapy was to cause a ~50% depletion in nasal mucosal GSH
levels. This degree of GSH depletion has been successfully achieved in vitro and in vivo
in previous studies without causing systemic side effects or cellular dysfunction (Canals
et a1. 2001; Heales et al. 1996). The other half of the animals received saline vehicle
intraperitoneally (10ml/kg BWT). Animals that were allowed to recover for 24 or 48
hours post-exposure also received BSO or saline at the same time of day on post-

exposure days.

Necropsy and Tissue Preparation for Morphometric Analyses
Two hours, 1 day, or 2 days following the end of exposure, rats were anesthetized
with pentobarbital (50mg/kg, i.p.), and then killed by exsanguination via the abdominal

aorta. After death, the head of each rat was removed from the carcass, and the eyes,

179

lower jaw, skin, and musculature was removed. For animals in Study 2, the nasal cavity
was sagittally split 1m to the right of the midline, yielding an intact lefi nasal cavity
with septum attached, and a free right lateral nasal wall. The left nasal cavity was further
processed for histopathology as described in Study 1.

One nasal section from each animal was histochemically stained with
hematoxylin and eosin (H&E) for morphologic examination and morphometric analyses.
For animals in Study 2, a tissue section was immunohistochemically stained with
monoclonal mouse anti-proliferating cell nuclear antigen, clone PClO (PCNA; Dako

North America, Carpinteria, CA).

Morphometry of Neutrophilic Inflammation, Epithelial Numeric Cell Density, and
Cell Proliferation

The non-ciliated transitional epithelium (NTE) overlying the dorsal aspect of the
anterior maxilloturbinate of each animal (Figure 5-1) was analyzed using computerized
image analysis and standard morphometric techniques (Hotchkiss et aL 1991).
Neutrophilic inflammation was quantified by counting the number of nuclear profiles of
neutrophils in the surface epithelium of the dorsal maxilloturbinate, and dividing this
number by the total length of the basal lamina underlying this epithelium (i.e.,
intraepithelial neutrophils per mm basal lamina). Neutrophils were identified on H&E-
stained slides by morphologic characteristics, including a multi-lobed nucleus and clear
cytoplasm with non-staining granules. The basal lamina length was calculated from the
contour length of the basal lamina on a digitized image, using image analysis software

(Scion Image, Scion Corporation, Frederick, MD). The epithelial cell numeric density

180

 

 

was determined by counting the total number of epithelial cell nuclear profiles in the
epithelium lining the dorsal maxilloturbinate, and dividing this number by the length of
the basal lamina.

The expression of PCNA was used as an indicator of cell injury and proliferation
(Muskhelishvili et al. 2003). PCNA is an auxiliary protein of the DNA polymerase-8
complex that is expressed in the nuclear compartment of proliferating cells. For Study 2,
PCNA was used as a marker of cell proliferation instead of BrdU, in order to avoid
possible inconsistencies in BrdU absorption following repeated intraperitoneal BSO
injections. The PCNA labeling index was determined by counting the number of PCNA-
labeled epithelial cell nuclei, dividing this number by the total number of epithelial cell
nuclei, and multiplying by 100 to yield the percentage of PCNA-labeled epithelial cell
nuclei in the surface epithelium. The PCNA numeric cell density (unit-length labeling
index) was calculated by counting the number of PCNA-labeled nuclei, and dividing this

number by the length of the basal lamina.

Tissue Preparation for HPLC Antioxidant Analysis

The right half of the nasal cavity was processed for HPLC antioxidant analysis.
The head was dissected and split as previously described. The maxilloturbinate from
each animal was microdissected from the right lateral wall, and was immediately placed
into 300p] of 10% metaphosphoric acid, and snap-frozen in liquid nitrogen. Acidified,
frozen nasal mucosal samples were later thawed, homogenized for 30 seconds using a

Polytron homogenizer, re-frozen, and stored at —80°C until further processing for low

181

 

 

molecular weight antioxidant analysis via high performance liquid chromatography

(HPLC).

Analysis of Intracellular Concentrations of Low Molecular Weight Nasal
Antioxidants

Nasal tissue homogenates were thawed and centrifuged at 12,500 x g for 4 hours.
Protein pellets were resuspended in 500p] of PBS, neutralized with 25 pl 1N NaOH, and
sonicated for 1 hour at 37°C. The protein content of resuspended pellets was measured
using the Pierce BCA Protein Assay (Pierce Biotechnology, Rockford, IL). Supematants
were diluted three-fold in 10% meta-phosphoric acid, and filtered through a 0.22um
syringe filter. Triplicate samples of each supernatant were fractioned on a Shimadzu LC-
IOAi HPLC (Shimadzu Scientific Instruments, Columbia, MD), using a Phenomenex
Luna C18(2) 250mm x 4.6mm, SuM reversed phase column, preceded by a Phenomenex
ODS 4mm x 3mm guard column (Phenomenex, Torrance, CA). The mobile phase
consisted of an isocratic mixture of 50mM phosphate buffer, pH 3.1, containing SOuM
octanesulfonic acid and methanol (95:5). Samples were fiactioned at a mobile phase

flow rate of 1.0ml/min. Reduced glutathione (GSH), oxidized glutathione (GSSG), and

ascorbate (AH2), were simultaneously detected with an 8-channel ESA CoulArray Model

5600A electrochemical detector (ESA, Chelmsford, MA). GSH, GSSG, and AH; values

were normalized to protein content of centrifuge pellets.

182

Statistical Analyses

All data were expressed as mean group value i SEM. The differences among
groups in Study 2 were analyzed by two-way AN OVA with interactions. Pairwise
comparisons were performed a priori using Student-Newman—Keuls multiple
comparisons test. The criterion for statistical significance was set to p _<_ 0.05 for all
analyses. All statistical analyses were performed using a commercial statistical software

package (SigmaStat, SPSS Science, Chicago, IL).

RESULTS
Determination of Low Molecular Weight Nasal Antioxidant Concentrations

There was no significant effect of ozone exposure observed in the steady-state
nasal mucosal concentrations of GSH, GSSH, or AH2 at any of the exposure or post-

exposure time points. There was a tendency toward increased concentrations of GSH and
GSSG in all saline-treated, ozone-exposure groups compared to their respective FA
exposed controls (all GSH and GSSG values in ozone-exposed groups were higher than
their respective control values); however, none of these differences was statistically
significant.

BSO treatment caused a 45% decrease in the mucosal GSH concentration in F A-
exposed rats. BSO treatment also caused a decrease in nasal mucosal concentrations in
all ozone-exposed rats compared to similarly exposed, saline-treated rats. This decrease
was statistically significant following l-day (57% decrease) and 2-day (65% decrease)
exposures, and following 3-day exposures + 24 hour (71% decrease) and 48 hour (69%

decrease) recovery periods (Figure 5-7).

183

Following 3-day ozone exposure, BSO treatment caused a significant (69%)
decrease in mucosal GSSG concentrations versus saline-treated rats exposed to ozone for
3 days. BSO treatment caused a decrease in the mucosal GSSG concentrations in all

ozone-exposed rats compared to similarly exposed, saline-treated rats (Figure 5-8).

184

300 -

 

- SAL GSH
.14.“ j- 3 880 GSH

 

 

 

 

250 ‘

200 ‘

150 ~

100 —

GSH (nmoleslmg protein)

50-

  

 

FA 1-day 2-day 3-day 3-day 3-day
03 O3 03 + 24h +48h

Figure 5-7. Effect of ozone exposure +/- BSO treatment on intracellular GSH
concentrations in rat nasal mucosa lining the anterior maxilloturbinate. Bars represent
group mean _+_ SEM (n = 6/gr0up). a = significantly different from respective saline-
treated rats.

185

 

- SAL GSSG
E21 830 GSSG

 

 

 

GSSG (nmoleslmg protein)
.5

     

 

 

FA 1-day 2-day 3-day 3-day 3-day
03 03 03 + 24h + 48h

Figure 5-8. Effect of ozone exposure +/- BSO treatment on intracellular GSSG
concentrations in rat nasal mucosa lining the anterior maxilloturbinate. Bars represent
group mean i SEM (n = 6/group). a = significantly different from respective saline-
treated rats.

186

1401

 

 

 

 

- SAL AH2
120 H m BSO AH2
E 100 -
e
a. _
g 80 ‘ f»
g 60 r I
5 I
:l:N 40 " I ,
< I
20 -
o _

 

 

FA 1-day 2-day 3-day 3-day 3-day
03 O3 03 + 24h +48h

Figure 5-9. Effect of ozone exposure +/- BSO treatment on intracellular AHz
concentrations in rat nasal mucosa lining the anterior maxilloturbinate. Bars represent
group mean 1 SEM (n = 6/group).

187

There was no statistically significant effect of BSO treatment on the steady state nasal

mucosal concentrations of AH2 in FA- or ozone-exposed rats. There were trends toward

increases in nasal mucosal AH2 concentrations of BSO-treated rats observed following

F A-exposure and l-day ozone exposure; however, these changes were not statistically

significant (Figure 5-9).

Nasal Histopathology

Light photomicrographs of representative maxilloturbinates that summarize the
time-dependent progression of inflammatory and epithelial responses to ozone exposure
+/- BSO treatment are illustrated in Figure 5-10. No exposure related lesions were
identified in the nasal airways of rats treated with either saline or BSO and exposed to
filtered air (0 ppm). In all ozone-exposed animals, nasal lesions were restricted to the
nasal transitional epithelium (NTE) lining the lateral meatus of the anterior nasal cavity.
There were no significant microscopic differences observed between saline- and BSO-
treated animals in the time-dependent progression of ozone-induced epithelial injury in
the anterior nasal cavity.

In rats treated with saline or BSO and exposed to 1 day of 0.8 ppm ozone, the
principal morphologic alterations observed were epithelial degeneration and necrosis in
the NTE. These lesions were most severe in the NTE lining the dorsal aspect of the
maxilloturbinate, the ventral surface of the nasoturbinate, and the lateral wall adjacent to
the maxilloturbinate. An influx of neutrophils was observed in the epithelium and lamina

propria lining the maxilloturbinate, nasoturbinate, and lateral nasal wall.

188

Figure 5-10. Light photomicrographs of the dorsal aspect of maxilloturbinates from rats
treated with saline (A, B, C) or BSO (D, E, F) and exposed to 0 ppm ozone (A and D),
exposed to 0.8 ppm ozone for 2 days (B and D), or exposed to 0.8 ppm ozone for 3 days
and killed 48 hours post-exposure (C and F). Tissues were stained with H&E. Note the
presence of scattered mitotic figures (black arrows) in the NTE, along with the influx of
neutrophils (white arrows) into the epithelium (e) and underlying lamina propria (1p) in
the maxilloturbinates of rats exposed to 0.8 ppm ozone for 2 days (B and D). TB =
turbinate bone; bv = blood vessel.

189

_

v

..,/.I
:91 min m

9 for 3 day
i. Note If:

s from '4-
(A and D.
16 influx of

 

5%. 3

 

D). TB=

‘pria (in 2

Figure 5-10

m<4—zw

190

After 2 days of ozone exposure, the principal morphologic alterations in the NTE
of saline- and BSO-treated rats were basal cell hyperplasia and progressive neutrophilic
rhinitis. The NTE lining the dorsal maxilloturbinate exhibited cytoplasmic basophilia,
particularly along the basal cell layer, consistent with epithelial regeneration. Occasional
mitotic figures were observed within the NTE. The degeneration of surface epithelial
cells noted following 1-day exposure was also present on day 2. Neutrophil infiltration
into the nasal mucosa was increased from day 1.

At the end of 3 days of ozone exposure, saline- and BSO-treated rats exhibited
epithelial hyperplasia of the NTE. In 3—day ozone animals, the epithelium lining the
dorsal maxilloturbinate was 3-4 cell layers thick, compared to 1-2 cell layers in the NTE
of FA-exposed rats. Concurrent with the increase in epithelial thickness was a decrease
in the influx of neutrophils into the nasal mucosa following 3-day ozone exposure.

Epithelial morphology was similar in saline- and BSO-treated rats exposed to
ozone for 3 days and allowed to recover for either 24 or 48 hours post-exposure. The
principal lesion observed in these animals was marked epithelial hyperplasia in the NTE
lining the maxilloturbinate, and moderate hyperplasia of the epithelium lining the lateral
wall and ventral surface of the nasoturbinate. A mild neutrophilic rhinitis persisted in the

epithelium and lamina propria lining the maxilloturbinates.

Morphometric Quantitation of Epithelial Hyperplasia
PCNA labeling of maxilloturbinates in saline- and BSO-treated, ozone-exposed
rats is illustrated in Figure 5-11. Ozone induced a significant increase in the PCNA

labeling index in both saline- and BSO-treated rats (96-fold and 86-fold, respectively)

191

 

exposed to 0.8 ppm ozone for 2 days, compared to FA-exposed rats (Figure 5-12). There
were no significant differences in the PCNA labeling index observed at any other
exposure time, compared to FA exposure. Interestingly, the labeling index in saline-
treated rats following l-day of exposure was significantly greater (IO-fold higher) than
that observed in BSO-treated rats at the same time point. However, neither was
significantly different from F A-exposed rats. PCNA labeling was similar between saline-
and BSO-treated rats at all other time points.

Accordingly, ozone exposure also induced a significant increase in the PCNA
numeric cell density in both saline- and BSO-treated rats following 2-day exposure (200-
fold and 75-fold increases, respectively), compared to controls. PCNA numeric cell
densities in saline-treated rats were also higher than control levels following 3-day
exposure + 24 hour recovery (160-fold higher) and 48 hour recovery (160-fold). The
PCNA numeric cell density in saline-treated rats exposed to 1-day of ozone was 12-fold
higher than in similarly exposed BSO-treated rats, however, this difference was not

statistically significant (Figure 5-13).

192

Figure 5-11. Light photomicrographs of maxilloturbinates from rats
treated with saline and exposed to 0 ppm ozone (filtered air, A), and from
rats treated with saline (B) or BSO (C) and exposed to 0.8 ppm ozone for
2 days. Tissues were immunohistochemically stained with anti-PCNA
antibody to detect proliferating cells. Note the nuclear uptake of brown
chromagen (black arrows) in the epithelium (e) lining the dorsal
maxilloturbinate and lateral wall (LW) of the anterior nasal cavity in
saline- (B) and BSO-treated (C), ozone exposed rats, indicating PCNA
expression. tb = turbinate bone.

193

mates from at
it. A), and from
1 ppm ozone for
with anti-PCNA
iptake of brow
ting the dorsal
nasal 63111le
idicating PCNA

 

 

 

194

 

Figure 5-1 1

 

 

60 ~ * — SAL
BSO

 

 

 

PCNA Labeling Index
(%PCNA+ cells)

 

 

FA 1-day 2-day 3-day 3-day 3-day
O3 03 03 + 2411 + 48h

Figure 5-12. Effect of ozone exposure +/- BSO treatment on PCNA labeling index in the
NTE lining the anterior maxilloturbinate. Bars represent group mean i SEM (n =
6/group). * = significantly different from respective FA-exposed rats. a = significantly
different from respective saline-treated rats.

195

180 -

 

 

 

 

   

 

A160“ * -SAL
W
@5140-
§§120~
3:
‘3: 100«
':
f§ 60-
E:
83.: 40‘
20-
0.

FA 1-day 2-day 3-day 3-day 3-day
O3 O3 03 + 24h +48h

Figure 5-13. Effect of ozone exposure +/- BSO treatment on the numeric cell density of
cells expressing PCNA in the NTE lining the anterior maxilloturbinate. Bars represent
group mean : SEM (n = 6/group). * = significantly different from respective FA-
exposed rats.

196

 

0'!
O
O

 

— SALlNE ** **
BSO

   
 

 

 

 

.5
O
O

200 -

Epithelial Numeric Cell Density
(cells/mm basal lamina)

100 .

  

' a

FA 1-day 2-day 3-day 3-day 3-day
03 O3 03 + 24h +48h

 

Figure 5-14. Effect of ozone exposure +/- BSO treatment on the numeric cell density of
epithelial cells in the NTE lining the anterior maxilloturbinate. Bars represent group
mean i SEM (n = 6/ group). * = significantly different from respective FA-exposed rats.

197

In both saline- and BSO-treated rats, ozone induced a significant increase in the
total number of epithelial cells lining the dorsal maxilloturbinate following 3-day
exposure (45% and 26% increases, respectively), 3-day exposure + 24 hour recovery
(88% and 72% increases, respectively), and 3-day exposure + 48 hour recovery (88% and
68% increases, respectively). BSO treatment had no effect on the epithelial numeric cell

density at any exposure time point (Figure 5-14).

Morphometric Quantitation of Neutrophilic Inflammation

After 2-day exposure to 0.8 ppm ozone, both saline- and BSO-treated rats had
significantly greater (28-fold and 8-fold, respectively) neutrophil influx into the NTE
lining the dorsal maxilloturbinates than similarly treated FA-exposed rats (Figure 5-15).
This increase in neutrophilic inflammation was also observed in saline- and BSO-treated
rats following 3-day exposure (16-fold and 8-fold, respectively). There was no

significant effect of BSO-treatment on neutrophil influx at any exposure time point.

198

 

 

 

0|
0

 

_ SALINE
:21 BSO *

 

 

 

.5
O

30~

.3
O

Neutrophil Numeric Cell Density
(pmnlmm basal lamina)

    

 

FA 1-day 2-day 3-day 3-day 3-day
03 O3 03 + 24h +48h

Figure 5-15. Effect of ozone exposure +/- BSO treatment on the numeric cell density of
intraepithelial neutrophils in the NTE lining the anterior maxilloturbinate. Bars represent
group mean 1 SEM (n = 6/group). * = significantly different from respective FA-
exposed rats.

199

DISCUSSION

The results of the present study indicate that ozone-induced cell proliferation in
the nasal airways of immature rats is not dependent upon the local upregulation of
intracellular GSH concentration. Daily exposure to 0.8 ppm ozone caused epithelial
hyperplasia in the nasal airways of immature rats. Treatment with BSO, a potent
inhibitor of GSH synthesis, caused marked depletion of intracellular GSH concentrations
prior to and during ozone exposures. There was a conspicuous lack of DNA synthesis
observed in BSO-treated animals following day 1 of ozone-exposure. Furthermore, by
day 1 of exposure, BSO-treated animals had 57% lower concentrations of intracellular
GSH than saline-treated animal undergoing the same ozone exposure. Despite the
marked GSH depletion and delayed cell proliferation observed on day l, the BSO-treated,
ozone-exposed animals exhibited levels of cell proliferation similar to those observed in
saline-treated, ozone-exposed rats by day 2, the day of peak cell proliferation rates in
both groups. This ultimately resulted in similar degrees of epithelial hyperplasia in
saline- and BSO-treated rats following ozone exposure.

In the present study, exposure to 0.8 ppm ozone, 8h/day, for 3 consecutive days,
caused epithelial hyperplasia and transient neutrophilic inflammation in the anterior nasal
airways of immature rats. The time course of cell proliferation and neutrophilic
inflammation observed in our study was consistent with results from similarly exposed
adult rats (Cho et al. 1999). We did not observe a significant difference in the magnitude
or progression of ozone-induced rhinitis between saline- and BSO-treated animals.
Previous studies have provided conflicting results regarding the potential role of

neutrophilic inflammation in the modulation of ozone-induced cell proliferation. Salmon,

200

et al., showed that apocynin, an NADPH oxidase inhibitor, and the corticosteroid
dexamethasone, were each capable of inhibiting ozone-induced cell proliferation in rat
bronchiolar epithelium (Salmon et al. 1998). However, Cho, et al., demonstrated that
neutrophil depletion did not inhibit ozone-induced epithelial hyperplasia in rat nasal
airways (Cho et al. 2000). While our study demonstrates that GSH depletion does not
affect nasal airway inflammation, it neither supports nor refutes the role of neutrophilic
inflammation in oxidant-mediated cell proliferation.

We hypothesized that early ozone exposures would cause oxidative stress in the
nasal airways of rats, increasing the expression of anti-oxidant, pro-inflammatory, and
GSH synthetic genes. Further, we hypothesized that increased expression of GCL
subunits would result in increases in GSH synthesis that would re-establish a favorable
redox balance, and promote cell proliferation (or prevent cell cycle arrest). We did not
observe a significant increase in GSH concentrations in the nasal mucosa of saline-treated
rats during the course of ozone exposure, nor did we observe an increase in mRNA for
either GCL subunit (Study 1 from this chapter). This may be due, in part, to alternative
mechanisms maintaining a favorable (reduced) redox balance during ozone exposures.
The normal GSH/GSSG redox ratio in cells ranges between 30:1 to 100:1 (Hwang et al.
1992). In our study, the GSH/GSSG redox ratio in nasal mucosa was maintained
between ~40:1 to 60:1 following the three days of exposures (see Figures 5-7 and 5-8).
In the absence of increased GCL expression, other factors may contribute to the
maintenance of favorable redox conditions, including glutathione reductase activity,

which can regenerate GSH via GCL-independent mechanisms.

20]

The intracellular concentration of GSH also influences the regulation and

synthesis of ascorbate (AH;), which may also contribute to the overall redox state of a

cell. In a manner similar to GSH and GSSG, AH; exists in a redox couple with
dehydroascorbate (Moison et al. 1997). Dehydroascorbate reductase, the enzyme that

catalyzes the two-electron reduction of dehydroascorbate back to AH;, uses GSH as a

reducing agent. Thus, in humans, regulation and maintenance of AH; is largely GSH-
dependent. Unlike humans (and rhesus monkeys), rats are capable of de novo synthesis
of ascorbate. Furthermore, results from previous studies show that oxidative stress
induced via GSH depletion in mice can be ameliorated by a compensatory increase in
AH; synthesis (Martensson and Meister 1992). In the present study, BSO treatment
caused marked reductions in GSH concentrations that were accompanied by mild
increases in the intracellular concentration of AH; in the nasal mucosa of rats. It is
possible that the potential oxidative stress induced by GSH depletion was reversed by
compensatory increases in AH;, thus promoting cell cycle progression and cell

proliferation.

In Chapters 3 and 4, we reported the effects of acute and episodic ozone exposure
on intracellular antioxidant concentrations in the nasal mucosa of infant monkeys. Acute
ozone exposure (0.5 ppm, 8h/day for 5 days) and 5-cycle episodic ozone exposure (5 bi-
weekly cycles of 9 days FA, followed by 0.5 ppm, 8h/day for 5 days) each caused
widespread reductions in intracellular GSH concentrations. In contrast, 11 cycle
exposure caused widespread GSH upregulation in the nasal mucosa. We hypothesized

that ozone-induced epithelial hyperplasia was dependent upon GSH upregulation, and

202

that GSH upregulation was a response to early GSH consumption. Since ozone-induced
epithelial hyperplasia developed more quickly in the rat nasal cavity, we hypothesized
that ozone-induced GSH upregulation would occur earlier in the rat than in the monkey.
While we did not observe an ozone-related increase in GSH levels in the present study,
we also did not observe GSH consumption in saline-treated rats following ozone
exposures. Several mechanisms may be involved in this differential response to ozone

exposures between infant monkeys and infant rats. As previously discussed, rats are
capable of synthesizing AH; in response to oxidative stress, which may spare
intracellular GSH concentrations during short-term ozone exposure. In contrast,

monkeys lack the enzyme L-gulonolactone oxidase, which catalyzes the final step in AH;

synthesis, and cannot regulate AH; levels via de novo synthesis (Banhegyi et al 1997). It
is also possible that the ozone concentration selected for the rat studies (0.8 ppm) was
insufficient to decrease GSH concentrations, while the lower concentration selected for
the infant monkey exposures (0.5 ppm) was more potent due to the decreased sensitivity
to ozone-induced injury exhibited by rodents (Hatch et a1. 1994).

In summary, GSH depletion during exposure to ozone does not inhibit the
development of epithelial hyperplasia in the rat. Our results do not support a direct role
for GSH in regulating cell proliferation. However, our findings suggest that
compensatory mechanisms, including AH; synthesis and GSH redox recycling, may
ameliorate oxidative stress induced by GSH depletion, thus facilitating cell cycle
progression and cell proliferation. Further studies examining the complex relationships

among AH;, GSH, and oxidant-mediated cellular injury and proliferation are warranted.

203

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208

CHAPTER 6

SUMMARY AND CONCLUSIONS

209

The inflammatory and epithelial responses to acute and chronic ozone exposure
have been well characterized in the nasal airways of monkeys and rats. Acute or chronic
exposure to ozone causes epithelial necrosis and acute inflammation, followed by
epithelial hyperplasia and mucous cell metaplasia, in the nasal transitional epithelium and
ciliated respiratory epithelium of monkeys (Harkema, Plopper et al. 1987; Harkema,
Plopper et al. 1987), and in the nasal transitional epithelium of rats (Harkema, Barr et al.
1997; Harkema, Hotchkiss et al. 1999). While the previous studies were designed to
describe these nasal epithelial proliferative responses to ozone exposure, subsequent
studies from our laboratory using rodent models were designed to investigate the
mechanisms behind these epithelial alterations. Hotchkiss et al., demonstrated that pre-
treatment with the pro-inflammatory cytokines IL-1 and TNF-a inhibits ozone-induced
DNA synthesis and cell proliferation (Hotchkiss and Harkema 1992). Cho et al.,
demonstrated that while ozone-induced mucous cell metaplasia is neutrophil dependent,
ozone-induced epithelial hyperplasia is neutrophil-independent (Cho, Hotchkiss et al.
2000)

Ozone-induced injury and repair exhibits site-specificity in both the nasal and
pulmonary airways. Ozone-induced lesions are confined to the anterior nasal cavity, and
to the tracheobronchial epithelium and terminal bronchiolar epithelium in the lungs. The
site-specificity of ozone-induced injury and repair is correlated to sites of ozone-induced
antioxidant alterations in the lungs of rats (Plopper, Duan et al. 1994) and monkeys
(Plopper, Hatch et al. 1998). The relationship between local antioxidant status and
ozone-induced injury and repair in the nasal airways had not been previously

investigated. We designed the current research to examine the relationship between site-

210

 

specific ozone-induced epithelial hyperplasia and local glutathione (GSH) status. Our
governing hypothesis was that ozone-induced epithelial hyperplasia in the nasal airways
of monkeys and rats is a GSH-dependent process.

With our initial investigations in infant rhesus monkeys, we sought to determine
the temporal and spatial relationships among ozone-induced nasal airway injury and
repair, and local antioxidant regulation. We first identified, quantified, and mapped the
nasal epithelial lesions induced by acute (0.5 ppm, 8h/day for 5 days), episodic (0.5
ppm.8h/day for 5 days + 9 days of filtered air, 5 cycles), and chronic, episodic (0.5
ppm.8h/day for 5 days + 9 days of filtered air, 11 cycles) exposure to ozone. We also
measured intracellular concentrations of GSH, GSSG, AH2, and UA in site-matched
nasal mucosal samples. Acute ozone exposure caused epithelial necrosis and atrophy in
the ciliated respiratory epithelium lining the anterior maxilloturbinate, with no epithelial
hyperplasia at this site. Cyclic exposure for 2 months (episodic) caused nasal lesions
similar in nature, distribution, and magnitude, to those observed following acute
exposure. Both acute and episodic exposure regimens caused GSH depletion in samples
corresponding to the site of ozone-induced injury. In contrast, cyclic exposure for 5
months (11 cycles) caused epithelial hyperplasia and squamous metaplasia along the
same site as the ozone-induced injury following acute and 2-month cyclic exposures.
Furthermore, this hyperplastic and metaplastic response was correlated with a 65%
increase in the local, intracellular GSH concentration. While these results established a
temporal correlation between GSH concentration and epithelial hyperplasia, they did not

establish a causal relationship between GSH regulation and cell proliferation.

211

Based on the results of our studies in rhesus monkeys, we chose to further
investigate the possible relationship between GSH and epithelial hyperplasia. Results
from several recent reports provide evidence that intracellular redox state in cells can
influence cell cycle progression, and that GSH regulation plays an important role in the
maintenance of redox state and cellular protection during oxidative stress (Luppi,
Aarbiou et al. 2005; Lu, Jourd'Heuil et al. 2007). Using a rodent model of ozone-
induced cell proliferation, we tested the hypothesis that ozone exposure causes
intracellular oxidative stress, induction of cytoprotective, pro-inflammatory, and GSH-
synthetic genes, and GSH upregulation prior to the initiation of ozone-induced cell
proliferation. Furthermore, we hypothesized that GSH depletion would diminish this
cytoprotective effect, and inhibit ozone-induced cell proliferation. For this purpose, we
treated rats with BSO, an inhibitor of GCL, to cause GSH depletion in the nasal mucosa.

The results from this study indicated that ozone exposure induced the expression
of the cytoprotective enzymes (HO-1, iNOS), pro-inflammatory cytokines (TNF-or, IL-6),
and neutrophil chemokines (MIP-2, CINC) in the nasal mucosa during the first 2 days of
ozone exposure, and prior to or concurrent with ozone-induced DNA synthesis (BrdU
incorporation). However, ozone exposure did not cause upregulation of GSH
concentrations, nor did it induce the expression of GCL-C or GCL-M, the rate-limiting
enzymes in de novo GSH synthesis, in rat nasal mucosa. Furthermore, we found that
GSH depletion did not inhibit the development of ozone-induced epithelial hyperplasia.
Interestingly, we also found that BSO-induced depletion of GSH was accompanied by a

mild increase in intracellular AH; concentrations. We speculate that the potential

212

 

increases in oxidative stress caused by GSH depletion may be ameliorated by concurrent
increases in AH;.

The results from these experiments also provide the basis for important
comparisons between rodents and monkeys as animal models of human inhalation. In
Chapter 2, we reviewed and summarized the scientific literature relevant to the
antioxidant profile of the rat nasal cavity. This review highlighted important differences
that exist between rats and humans in the relative abundances of specific low molecular
weight nasal antioxidants, and their role in protection against inhaled oxidant challenge.
In Chapter 3, we detailed the similarities between the nasal airways of humans and
monkeys, and the importance of these structural similarities in making accurate
extrapolations of risk data from histopathologic, biochemical, and dosimetry studies fi'om
monkeys to humans. In Chapter 4, we reported that episodic exposure to ozone causes
squamous metaplasia in the nasal airway of infant monkeys. This nasal epithelial
response in monkeys is similar to that observed in the nasal airways of children
chronically exposed to high ambient concentrations of ozone, and illustrates the
importance of the non-human primate studies in the evaluation of human risk. In Chapter
5, we discussed the potential influence of local AH; regulation on ozone-induced GSH
flux in rat nasal mucosa. These findings bring to light important differences between rats
and monkeys, and important similarities between monkeys and humans, in the regulation
and synthesis of low molecular weight antioxidants. While the results of these
experiments lend significant support for the use of non-human primates in inhalation
toxicologic studies, they also bring to light important species differences that must be

considered when designing comparative mechanistic studies.

213

 

In summary, we determined that ozone exposure causes epithelial hyperplasia and
squamous metaplasia in the nasal airways of infant monkeys. The findings in our non-
human primate model are consistent with the nasal injury observed in children
chronically exposed to high ambient levels of ozone (Calderon-Garciduenas, Valencia-
Salazar et al. 2001) (Calderon-Garciduenas, Rodriguez-Alcaraz et al. 2001).
Furthermore, we determined that these alterations are temporally correlated with
increases in the steady-state intracellular concentrations of GSH in the nasal mucosa
following chronic, episodic exposures. Additional studies are needed to fully understand
the durability and long-term consequences of nasal epithelial hyperplasia and squamous
metaplasia. While this metaplastic change may protect the nasal epithelium from
subsequent oxidant exposure, it may also serve to increase toxicant delivery to the
pulmonary airways. These studies may help to better evaluate and predict the risks and
long-term health effects associated with chronic or repeated childhood exposures to air
pollution.

In contrast, while ozone exposure also caused epithelial hyperplasia in the nasal
airways of rats, there was not a concurrent upregulation of intracellular GSH. We also
found that GSH depletion during ozone exposure does not inhibit the development of
nasal epithelial hyperplasia in rats. However, our findings suggest that other antioxidant
mechanisms (e. g. AH;) may also contribute to the maintenance of redox balance during
ozone exposures in rats, thus facilitating cell cycle progression and cell proliferation.
Future studies are needed to more fully investigate the interactions between GSH and
AH;, and their roles in oxidant-mediated cell proliferation in rats as well as primates,

particularly in light of the fact that primates are not capable of de novo AH; synthesis.

214

 

Results of these studies will contribute to our understanding of the specific cellular
mechanisms involved in ozone-induced epithelial hyperplasia, and contribute to our
general understanding of the role of antioxidants in oxidant-mediated cell proliferation in

these two laboratory species used to predict the risk of ozone toxicity in humans.

215

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216

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217

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