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This is to certify that the
dissertation entitled

EFFECTS OF OZONE ON ACETAMINOPHEN-INDUCED
LIVER AND AIRWAY TOXICITY IN MICE

presented by

Daher Ibrahim Aibo

has been accepted towards fulfillment
of the requirements for the

Ph.D. degree in Pathology and Environmental
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5/08 K:IProjIAcc&Pres/ClRC/DaIeDue.indd

EFFECTS OF OZONE ON ACETAMINOPHEN-INDUCED LIVER AND AIRWAY
TOXICITY IN MICE
By

Daher Ibrahim Aibo

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Pathology and Environmental Toxicology

2009

ABSTRACT

EFFECTS OF OZONE ON ACETAMINOPHEN-INDUCED LIVER AND AIRWAY
TOXICITY IN MICE

By
Daher Ibrahim Aibo

Acetaminophen (APAP) is the most frequently used over-the-counter analgesic
and antipyretic in the United States (U.S.). APAP overdose is responsible for half of
cases of acute liver failure in developed countries. At high doses, APAP also causes acute
lung injury in people and laboratory animals. Ozone (03) is the main oxidant air
pollutant in photochemical smog. More than half of the U.S. population is daily exposed
to levels of 03 exceeding the U.S. Environmental Protection Agency (EPA) national
ambient air quality standards (NAAQS). Recently, 03 has been shown to modulate
systemic levels of antioxidant as well as expression of several families of genes in the
liver. The principal purpose of this work was to investigate the effects of combined
APAP and O3 in the liver and pulmonary airways of mice compared to effects of
individual substances. I also explored some of the mechanisms that could help explain the
pathogenesis of APAP and O3 interaction in the liver and lung of these mice. Three
specific aims have been generated. In aims l and 2, I studied the APAP and 03 effects in
the liver and lung, respectively. To do so, I treated mice with saline or 300 mg/kg APAP
intraperitoneally and 2 h later exposed them to filtered air, 0.25 or 0.5 ppm 03 for 6 h.
Mice were euthanized 9 or 32 h after APAP administration. In the liver and pulmonary
airways, APAP and 03 resulted in greater epithelial damage and acute inflammation
compared to either substance alone. In addition, both locations exhibited APAP-induced

increase in epithelial cell proliferation that was inhibited by 03 coexposure. IL-6, an

important mediator of initial phases of hepatocellular regeneration was upregulated at the
gene and protein levels in the liver of APAP-treated animals but not in APAP/O3-
coexposed mice. In aim 3, I hypothesized that the absence of IL-6 induction in the liver
of APAP/O3-coexposed mice is responsible for the impaired hepatocellular regeneration
that might have contributed to the heightened toxicity in this last group. I exposed IL-6
sufficient or deficient mice to the same experimental ”protocol and found that E-6
deficient mice given APAP or APAP and 03 had deficient hepatocellular proliferation.
At the same time, APAP/O3 deficient mice had greater toxicity than APAP-treated
deficient animals suggesting that IL-6 is not involved in the impaired regeneration and
enhanced toxicity detected in the APAP/O3 group. I also found that IL-6 deficient mice
had impaired airway epithelial regeneration in either APAP or APAP/O3-coexposed
groups suggesting that H..-6 had a role in airway epithelial regeneration. Finally, I
detected several antioxidant or hypoxia-related genes or protein differentially expressed
in the APAP/O3 or APAP alone suggesting a contributory role of oxidative stress or

hypoxia in 03 exacerbation of APAP toxicity.

To Laurence, Marine-Ayan and Idil

iv

ACKNOWLEDGMENTS

It is a pleasure and an honor to thank all the people who made this thesis possible.

First and foremost, I am heartily thankful to my mentor Dr. Jack Harkema, for his
scientific guidance, encouragement, financial support and patience from the early stage of
this research as well as giving me extraordinary opportunities to learn and grow as a
scientist throughout this work and other projects. I am indebted to him more than he
probably considers.

I am especially grateful to Drs. Robert Roth, Patricia Ganey and Jane Maddox
who contributed to the financial and scientific realization of this work. I would also like
to thank the rest of my thesis committee members Drs. Jim Wagner, Timothy
Zacharewski and Colleen Hegg for their insightful comments and suggestions. I would
like to thank Dr. John Lapres for letting me work in his Lab.

I owe my deepest gratitude to Lori Bramble and Ryan Lewandowski from our
Lab for all their help and assistance. I would like to thank Ms. Amy Porter, Kathy
Campbell and Rick Rosebury from the MSU Department of Physiology, Laboratory of
Histopathology. Lori, Ryan, Amy, Kathy and Rick, this thesis would not have been
possible without your support.

I would like to show my gratitude to Drs. Willie Reed, Jennifer Thomas and
Laura McCutcheon, former and present Chairpersons of the Department of Pathobiology
and Diagnostic Investigation at MSU, Dr. Susan Ewart, Associate Dean for Research and
Graduate Studies in the College of Veterinary Medicine and Drs. Karen Klomparens and

Tony Nunez, Dean and Associate Dean of the MSU Graduate School, for their support

made available in a number of ways to me and other students including through several
awards.

I thank my former and present colleagues in our Lab; Drs Neil Birmingham, Steve
Carey, Daven Jackson, Ms. Kara Corps, Ms. Xiao Pan and all the undergraduate students
who helped me; Anthony Guzzardo, Ian Hotchkiss, Robert Buhsrobe and Jennifer Stokes.
I also thank colleagues from others Programs with whom I shared expertise and
friendship.

I wish to thank my entire extended family here and abroad, particularly my dad
and mom for providing me a constant understanding. I am indebted to my wife Laurence
and my two daughters Marine-Ayan and Idil for their love, burden-sharing, patience and
understanding.

Lastly, I offer my regards to all of those who supported me in any respect during

the completion of this thesis.

vi

TABLE OF CONTENTS

LIST OF FIGURES ................................................................................. ix
LIST OF ABBREVIATIONS ..................................................................... xii
CHAPTER 1. INTRODUCTION ................................................................... 1
ACETAMINOPHEN HEPATIC INJURY AND HEPATOCELLULAR
REGENERATION .......................................................................................................... 1
Acetaminophen-induced hepatocellular injury and inflammation .............................. 1
Hepatocellular regeneration after hepatectomy or chemical injury ............................. 4
Acetaminophen-induced injury in the lung ................................................................. 7
OZONE LOCAL AND SYSTEMIC EFFECTS AND AIRWAY REGENERATION 8
OS—induced epithelial injury and inflammation in the lung ........................................ 8
03 effects in the liver ................................................................................................ 14
Airway epithelium regeneration after injury ............................................................. 16
OVERALL HYPOTHESIS AND SPECIFIC AIMS ..................................................... 19
REFERENCES .............................................................................................................. 22
CHAPTER 2. EFFECTS OF ACETAMINOPHEN AND ACUTE OZONE
EXPOSURE IN THE LIVER OF MICE ........................................................ 38
ABSTRACT .................................................................................................................. 38
INTRODUCTION ......................................................................................................... 39
MATERIAL AND METHODS ..................................................................................... 41
RESULTS ...................................................................................................................... 52
DISCUSSION ............................................................................................................... 90
REFERENCES .............................................................................................................. 98
CHAPTER 3. EFFECTS OF ACETAMINOPHEN AND ACUTE OZONE
EXPOSURE IN THE AIRWAYS OF MICE ................................................. 103
ABSTRACT ................................................................................................................ 103
INTRODUCTION ....................................................................................................... 104
MATERIAL AND METHODS ................................................................................... 107
RESULTS .................................................................................................................... 119
DISCUSSION ............................................................................................................. 154
REFERENCES ............................................................................................................ 164
CHAPTER 4. ROLE OF INTERLEUKIN-6 IN ACETAMINOPHEN AND OZONE
HEPATIC AND PULMONARY TOXICITY ............................................... 175
ABSTRACT ................................................................................................................ 175
INTRODUCTION ....................................................................................................... 176
MATERIAL AND METHODS ................................................................................... 178
RESULTS .................................................................................................................... 188
DISCUSSION ............................................................................................................. 214

vii

REFERENCES ............................................................................................................ 223

CHAPTER 5. SUMMARY AND CONCLUSIONS......................................... 227
REFERENCES ............................................................................................................ 236

viii

LIST OF FIGURES

Figure 1. Experimental design of APAP and 03 studies in the liver ............................... 43

Figure 2. Inflammatory cell accumulation in the BALF of APAP and O3
exposed mice .......................................................................................................... 54 and 55

Figure 3. IL-6 (A and B) and MCP-l (C and D) protein concentrations
in the BALF of APAP and O3 exposed mice ......................................................... 57 and 58

Figure 4. IL-6 (A and B), MCP-l (C and D) and KC (E and F) protein
concentrations in the plasma of APAP and O3 exposed mice ............................... 60 and 61

Figure 5. Liver damage induced by APAP and 03 exposure
32 h after APAP ..................................................................................................... 64 and 65

Figure 6. Liver neutrophil infiltration in APAP and O3 exposed
mice 32 h after APAP ............................................................................................ 66 and 67

Figure 7. Hepatocellular proliferation in APAP and O3 exposed mice
32 h after APAP ..................................................................................................... 68 and 69

Figure 8. Intracellular glycogen (A) and HIP-1a (B) staining
32 h after APAP ..................................................................................................... 71 and 72

Figure 9. KC (A and B), MIP-2 (C and D) and MCP-l
(E and F) genes expression in livers of APAP and O3 exposed mice ................... 74 and 75

Figure 10. KC (A and B), MCP-l (C and D) and IL-6 (E and F)
protein concentrations in livers of APAP and O3 exposed mice ........................... 76 and 77

Figure 11. IL-6 (A and B) and PAI—l (C and D) genes
expression in livers of APAP and O3 exposed mice ............................................. 79 and 80

Figure 12. P21 (A and B) and SOCS3 (C and D) genes
expression in APAP and 03 exposed mice ............................................................ 82 and 83

Figure 13. MT -1 (A and B), HO-l (C and D) and GCLC (E and F)
genes expression in livers of APAP and O3 exposed mice ................................... 85 and 86

Figure 14. Total (A) or oxidized (B) glutathione and TBARS (C and D)
concentrations in livers of APAP and O3 exposed mice ....................................... 88 and 89

Figure 15. Experimental design of APAP and 03 studies in the lung. ......................... 108

ix

Figure 16. Schematic representation of lung sectioning levels
for histology and morphometry ...................................................................................... 113

Figure 17. Axial airway epithelial damage induced by APAP
and 03 treatment 32 h after APAP ................................................................................. 120

Figure 18. Epithelial numeric cell density in APAP and 03
treated mice 9 and 32 h after APAP in the axial airway (A and C)
and terminal bronchioles (B and D) ................................................................... 122 and 123

Figure 19. Lung neutrophil infiltration in APAP and 03
treated mice 9 and 32 h after APAP in the axial airway (A and B),
terminal bronchioles (C and D) and alveolar septa (E and F) ............................ 125 and 126

Figure 20. Inflammatory cell accumulation in bronchoalveolar
lavage (BALF) in APAP and 03 treated mice ................................................... 128 and 129

Figure 21. KC (A and B), MIP-2 (C and D) and MCP-l (E and F)
genes expression in the lung of APAP and O3 treatedmice ............................. 136 and 137

Figure 22. IL-6 (A and B), and COX-2 (C and D) genes
expression in the lung of APAP and 03 treated mice ....................................... 139 and 140

Figure 23. lL-6 (A and B) and MCP-l (C and D) protein
concentrations in the BALF of APAP and 03 treated mice .............................. 141 and 142

Figure 24. Airway epithelium cell proliferation in APAP and 03
treated mice 32 h after APAP ............................................................................ 144 and 145

Figure 25. CCSP immunolabeling in the axial airway of APAP
and 03 treated mice ........................................................................................................ 146

Figure 26. Clara cell density (A and B) and CCSP volume density
(C and D) in the axial airway (A and C) and terminal bronchioles
(B and D) 32 h after APAP ................................................................................ 148 and 149

Figure 27. CCSP (A and B) and cyclin-dependent kinase inhibitor P21
(C and D) genes expression in the lung of APAP and 03 treated mice ............ 150 and 151

Figure 28. MT-l (A and B) and HO-l (C and D) genes expression
in the lung of APAP and 03 treated mice .......................................................... 152 and 153

Figure 29. GCLC (A and B) gene expression and oxidized/total
glutathione ratio (C) in the lung of APAP and 03 treated mice ........................ 154 and 155

Figure 30. Experimental design of APAP and 03 studies in IL-6
sufficient and deficient mice ........................................................................................... 180

Figure 31. PCR and electrophoresis-based assessment
of lL-6 deletion in the liver ............................................................................................. 187

Figure 32. IL-6 protein concentration in the BALF (A and B)
or plasma (C and D) of APAP and O3 exposed IL-6 sufficient mice ................ 189 and 190

Figure 33. Liver damage induced by APAP and 03
exposure in IL-6 sufficient or deficient mice ..................................................... 192 and 193

Figure 34. Liver neutrophil infiltration in APAP and O3
exposed IL-6 sufficient and deficient mice ..................................................................... 195

Figure 35. Hepatocellular proliferation in APAP and 03
exposed IL-6 sufficient and deficient mice ..................................................................... 197

Figure 36. Epithelial numeric cell density in the axial airway
(A, B) and terminal bronchioles (C, D) of APAP and O3 exposed
IL-6 sufficient and deficient mice ...................................................................... 201 and 202

Figure 37. Neutrophil infiltration in the axial airway (A, B)
and terminal bronchioles (C, D) of APAP and O3 exposed

IL-6 sufficient and deficient mice ...................................................................... 204 and 205
Figure 38. Neutrophil accumulation in alveolar septa of

IL-6 sufficient and deficient mice 9 h after APAP ......................................................... 206
Figure 39. Inflammatory cell accumulation in the BALF

of IL-6 sufficient and deficient mice ................................................................. 207 and 208
Figure 40. Airway epithelium cell proliferation in APAP

and O3 exposed IL-6 sufficient and deficient mice 32 h after APAP ............................ 210
Figure 41. Clara cell density in the axial airway (A, C)

and terminal bronchioles (B, D) of IL-6 sufficient and deficient mice ............. 212 and 213
Figure 42. Summary of results .......................................................................... 234 and 235

Images in this dissertation are presented in color

xi

LIST OF ABBREVIATIONS

MSU: Michigan State University

APAP: n-acetyl-p-aminophenol

O3: ozone

hzhour

ALF: acute liver failure

FDA: Food and Drug Administration

NIH: National Institutes of Health

BrdU: 5-bromo-2-deoxyuridine

PH: partial hepatectomy

IL-6: interleukin-6

TNF-a: tumor necrosis factor—alpha
MIP-lor: macrophage inflammatory protein-1 alpha
MIP-2: macrophage inflammatory protein-2
IFN—y: interferon-gamma

MCP-l: monocyte chemotactic protein-1
IL-12: interleukin-12.

KC: keratinocyte-derived chemokine

IL- 10: interleukin-10

IL-1(B): interleukin- lbeta

NF-KB: nuclear factor-KB

STAT3: signal transducer and activator of transcription 3

xii

GP130: glycoprotein 130

HGF: hepatocyte growth factor

TGF-d: transforming growth factor-alpha
EGF: epidermal growth factor

CCl4: carbon tetrachloride

P21: cyclin dependent kinase inhibitor
NOx: oxides of nitrogen

VOC: volatile organic compounds

EPA: Environmental Protection Agency
NAAQS: National Ambient Air Quality Standards
ELF: epithelium lining fluid

BALF: bronchoalveolar lavage

NO: nitric oxide

ALT: alanine aminotransferase

CGRP: calcitonin gene-related peptide
H&E: hematoxylin and eosin

HIF-lor: hypoxia inducible factor 1 alpha
GSH: reduced glutathione

GSSG: oxidized glutathione

TBARS: thiobarbituric acid-reactives substances
ND: not detected

CV: central vein

SE: standard error (of the mean)

xiii

PAL 1 : plasmino gen activator inhibitor- 1

SOCS3: suppressor of cytokine signaling 3

HO-l: heme oxygenase-1

MT -1 : metallothionein-l

GCLC: catalytic subunit of glutamate-cysteine ligase

G5: transverse lung section at the level of the fifth bifurcation from the axial airway
G11: transverse lung section at the level of the eleventh bifurcation from the axial airway
CCSP: Clara cell secretory protein

Vs: volume density

18S: small subunit of ribosomal RNA

GAPDH: glyceraldehyde 3-phophate dehydrogenase

COX-2: cyclooxygenase-Z

Cpr: NADPH-cytochrome P450 reductase

xiv

CHAPTER 1
INTRODUCTION

I. ACETAMINOPHEN HEPATIC INJURY AND
HEPATOCELLULAR REGENERATION

I — 1. Acetaminophen-induced hepatocellular injury and inflammation

Acetaminophen (N-acetyl-p-amino-phenol or APAP) is a derivative of acetanilide
synthesized by Morse in the 19'h century (Bertolini et al., 2006). It has been introduced
into regular medical practice and marketed since the mid-twentieth century (Bertolini et
al., 2006). By 1980, APAP use outweighed aspirin and phenacetin use (Bertolini et al.,
2006) and became since then the most widely used over-the-counter analgesic and
antipyretic drug in the United States (Larson et al., 2005). Analysis of non-prescription
analgesic data from the third National Health and Nutritional Examination Survey
(NHANES III, 1988—1994) showed that approximately 5% of U.S. adults reported
frequent monthly use (>14 days/month) of APAP (Paulose-Ram et al., 2005; Paulose-
Ram et al., 2003). In the United Kingdom, 3.2 to 3.5 billion tablets of APAP are used
every year which translates to a mean of 55 tablets per person (Jones, 1998).

Prior to 1980, APAP overdose was not a major cause of acute liver failure (ALF)
in the United States (Ritt et al., 1969). By the end of the 20‘h century however, APAP
overdose was responsible for 20% of all the ALF cases (Schiodt et al., 1999). In a
retrospective study between 1998 and 2003, Larson and collaborators (2005) showed that

the incidence of ALF caused by APAP overdose rose from 28% in 1998 to 51% in 2003.

APAP is now the leading cause of acute liver failure (ALF) in developed countries and
accounts for approximately 50% of ALF cases (Lee et al., 2008). Among these cases,
50% of APAP-related ALF were unintentional or non-suicidal which correlates with the
widespread presence of this drug in analgesic over-the-counter multi-molecule
preparations (Fontana, 2008). APAP is present in more than 100 over-the-counter
preparations and numerous prescription drugs (Fontana, 2008). Recently, several studies
suggested that APAP caused elevation of alanine aminotransferase (an indicator of liver
injury) activity above normal limits in people receiving the maximal daily recommended
dose of 4 g (Dart and Bailey, 2007; Watkins et al., 2006). Additionally, the Food and
Drug Administration’s (FDA) Adverse Event Reporting System and the Acute Liver
Failure Study Group showed that APAP doses of 5 to 7.5 mg/day, close to the maximal
recommended daily intake of 4 g can result in liver injury
(http://www.fda.gov/AdvisoryCommittees/Calendar/ucm143083.htm). Because of this
widespread presence of APAP in several composite analgesics and antipyretics and the
narrow therapeutic range, a U.S. FDA working group recommended in 2009 better
education of the general public about the name of this drug and its role as a liver toxin,
clear indication of its name (acetaminophen and not APAP) when it’s present in a multi-
molecule drug and reduction of the maximal daily adult dose
as well as the single adult dose
(http://www.fda. gov/Drugs/DrugSafety/informationbyDrugClass/ucm l 65 107.htm).
APAP is primarily detoxified in the liver by glucuronidation and sulfation
(Bessems and Vermeulen, 2001). At therapeutic doses, only a small fraction is

metabolized by cytochrome P450 isoforrns (Bessems and Vermeulen, 2001). At high

doses however, more and more APAP molecules are processed by the cytochrome P450
enzymes leading to increased formation of the reactive metabolite, N-acetyl-p-
benzoquinone (NAPQI) (Dahlin et al., 1984; Jollow et al., 1973; Mitchell et al., 1973a).
NAPQI reacts and depletes glutathione (Mitchell et al., 1973b) and covalently binds to
proteins leading to disruption of intracellular homeostasis and subsequent hepatocellular
necrosis (Potter et al., 1973). APAP-induced hepatocellular necrosis has always been
associated with protein covalent binding, however, there has been some evidence that
protein covalent binding could occur without cell death (Tarloff et al., 1996). This led to
the proposal of additional mechanisms to support or supplement the covalent
modification of proteins. One of the most prominent hypotheses is the covalent
modification of mitochondrial proteins and mitochondrial membrane permeability
transition resulting in oxidative stress (Haouzi et al., 2002). In addition to oxidative
stress, reactive nitrogen species such as peroxynitrite have also been detected in
hepatocytes and endothelial cells after APAP overdose (Hinson et al., 1998; Hinson et al.,
2004; Knight et al., 2001). Finally, mitochondrial and nuclear DNA damage have been
associated with APAP overdose (Cover et al., 2005; Ray et al., 1990). '

APAP overdose primarily targets the liver in mammals including laboratory mice
and results in centrilobular hepatocellular necrosis (Bessems and Vermeulen, 2001;
Black, 1984; Clark et al., 1973; Davis et al., 1974; Dixon et al., 1975; Dixon et al., 1971;
Hinson et al., 1981; Placke et al., 1987a; Portmann et al., 1975). The preferential location
of liver lesion around the central vein is thought to be associated with its greater regional
content in cytochrome P450 isoforms, particularly CYP2E1 and its smaller oxygen

supply (Hart et al., 1995). Placke and collaborators (1987a) showed that early in the

course of toxicity, ultrastructural changes in mitochondria and plasma membranes were
among the most specific changes induced by APAP administration, in accordance with
the covalent protein binding mechanisms. Neutrophil accumulation in necrotic regions
after APAP overdose has been described since the early mechanistic studies of the APAP
covalent binding hypothesis (Mitchell et al., 1973a). Infiltration of neutrophils in
damaged areas followed the APAP centrilobular lesion in mice suggesting that
neutrophils are recruited as scavengers of necrotic hepatocytes and did not initiate APAP
damage (Bauer et al., 2000; Lawson et al., 2000). Others reported that depletion of
neutrophils protected from APAP-induced liver injury in mice, suggesting that
neutrophils are important at least in APAP lesions progression (Liu and Kaplowitz, 2005;
Liu et al., 2004). At a dose of 300 mg/kg of body weight, the initial APAP-induced injury
was first detected between 2 and 4 h after administration and continued to increase up to
24 h post-injection (Cover et al., 2006; Gujral et al., 2002). At the same dose of APAP,
hepatocellular damage complete-1y resolved by 72 h post-APAP in most strain of mice

(Donahower et al., 2006; James et al., 2003).

I - 2. Hepatocellular repair after hepatectomy or chemical injury

The most frequently used model in liver regeneration is the rodent partial
hepatectomy (PH) model (Martins et al., 2008). In this model, two-thirds of the liver are
surgically removed and hepatocellular regeneration is studied and has been shown to
proceed from remnant hepatocytes (Pahlavan et al., 2006). After partial hepatectomy in

mice, bromodeoxyuridine (BrdU)-labeled hepatocytes (cells in S phase of the cell cycle)

begin to increase around 32 h post-hepatectomy and reach a maximum 48 h after
hepatectomy (Satyanarayana et al., 2004; Wustefeld et al., 2000). At 72 h after
hepatectomy, a very small proportion of hepatocytes were positive for BrdU
immunostaining similar to non-hepatectomized mice.

Using this model, it has been established that the priming phase (first few hours
after PH corresponding to the transition from the interphase or G0 to the first phase G1 of
the cell cycle) of liver regeneration is under the control of cytokines (tumor necrosis
factor-alpha or TNF-a and interleukin-6 or IL-6) (Fausto et al., 2006; Taub, 2004). TNF-
d and IL-6 involvement in priming regeneration was supported by various findings
including increase of these cytokines in the liver and serum after PH, the activation of
several target transcriptions factors (Nuclear Factor-ch or NF-ICB, Signal Transducer and
Activator of Transcription—3 or STAT-3), the inhibition of liver regeneration after
treatment with an anti-TNF-a antibody or in tumor necrosis factor receptor 1 or IL-6 KO
mice and rescue of this inhibition by administration of IL-6 in the last case (Fausto et al.,
2006). The role of TNF-d is to regulate IL-6 responsible for the priming phase
(Michalopoulos and DeFrances, 1997). Subsequent to the initiation/priming phase,
growth factors such hepatocyte growth factor (HGF), transforming growth factor—alpha
(T GF-d) or epidermal growth factor (EGF) lead to the progression of primed hepatocytes
through the remaining phases of the cell cycle (Pahlavan et al., 2006).

Most studies today agree that the role of IL-6 in liver regeneration is pro-
proliferative. Mice deficient in IL-6 exhibited hepatocellular necrosis and degeneration as
well as greater mortality after partial hepatectomy compared to IL-6 sufficient

hepatectomized mice (Cressman et al., 1996). In the same study, IL-6 deficient

hepatectomized mice had 20 to 25% less BrdU-labeled hepatocytes compared to wild
type hepatectomized mice. Administration of exogenous IL-6 in deficient mice rescued
defective tissue regeneration and prevented necrosis. In another study, lL-6 deficient
mice had impaired liver regeneration at 36, 48 and 60 h post-hepatectomy compared to
IL-6 sufficient hepatectomized mice (Sakamoto et al., 1999). Similarly, mice deficient in
IL-6 exhibited a high rate of mortality and IL-6 injection significantly decreased the rate
of mortality in these mice (Blindenbacher et al., 2003). However, in this last case, only
subcutaneous injection of recombinant IL-6 (sustained action of IL-6) rescued deficient
mice while intravenous (short-acting) IL—6 injection did not show this effect. IL-6 has
also been shown to be important in liver regeneration following chemical toxicity
including APAP hepatotoxicity. Acute exposure to carbon tetrachloride (CCl4) resulted
in greater hepatocellular injury, apoptosis and impaired regeneration in IL-6 deficient
mice compared to similarly treated sufficient mice 36 or 48 h post-treatment (Kovalovich
et al., 2000). In this study, IL-6 administration corrected liver regeneration in IL-6
deficient mice given CCl4. Finally, IL-6 deficient mice given 300 mg/kg of APAP
exhibited greater hepatocellular damage but lower hepatic regenerative capacities
relative to IL-6 wild type mice 48 h after APAP administration (James et al., 2003).

In addition to the expression of IL-6 in the liver, others studies showed that high
systemic IL-6 levels could impair proliferation. Wustefeld and collaborators (2000) for
instance used mice overexpressing the human IL—6 receptor in hepatocytes and stimulated
those mice with human recombinant IL-6 3 h before hepatectomy. In these mice, STAT-3
was activated for more than 72 h whereas in unstimulated mice this elevation was limited

to few early hours and liver regeneration was impaired as measured by BrdU

incorporation and Cyclin A and E expression (Wustefeld et al., 2000). Because STAT-3
controls the expression of cyclin—dependent kinase inhibitor P21, a known cell cycle
inhibitor, expression at the transcriptional level, this team investigated the level of P21
and showed that lL-6 stimulation in transgenic mice overexpressing the IL-6 receptor

resulted in increased P21 protein 6 h post-hepatectomy (W ustefeld et al., 2000).

I — 3. APAP-induced injury in the lung

APAP overdose also causes toxicity in other organ systems in laboratory animals
and people including the upper and lower respiratory tract (Amatya et al., 2002;
Baudouin et al., 1995; Dimova et al., 2005; Dimova et al., 2000; Genter et al., 1998;
Jeffery and Haschek, 1988; Khanlou et al., 1999; Neff et al., 2003; Placke et al., 1987b).
The morphologic hallmark of acute APAP overdose is epithelial degeneration and
necrosis regardless of the tissue involved. In vitro, freshly isolated type H pneumocytes
exposed to subtoxic doses of APAP exhibited dose-dependent increase in cytotoxicity
and loss of intracellular glutathione (Dimova et al., 2000). In vivo, mice given APAP
showed pulmonary bronchioles as well as nasal olfactory and respiratory epithelium and
lateral nasal glands necrosis (Gu et al., 2005; Neff et al., 2003; Placke et al., 1987b).

Bartolone and collaborators (1989) showed that APAP-protein adducts were
detected by western blotting only in organs where toxicity was observed (liver, lung and
kidneys). In this study, the severity of tissue damage and the amount of protein adducts
were reduced in those target tissues when mice were pre-treated with a mixed function

oxidase inhibitor (Bartolone et al., 1989). In the lung, Clara cells have the highest level of

cytochrome P4505 (Amatya et al., 2002; Devereux et al., 1989; Massaro et al., 1994) and
are therefore potential sites for APAP bioactivation. In mice deficient in liver-specific
NADPH-cytochrome P450 reductase (cpr), the electron donor of microsomal P4505, the
severity of lung lesions was decreased while liver toxicity was abrogated suggesting that
liver metabolism was only partially involved in APAP-induced airway epithelial damage
(Gu et al., 2005). This result also suggests that local pulmonary APAP bioactivation is
possible as lung toxicity was not completely eliminated in those cpr null mice. In the
lung, expression of two isoforms responsible for hepatic APAP bioactivation, namely
CYP2E1 and CYP1A2, or their activity have been identified (Dey et al., 1999; Forkert et

al., 2001; Stoilov et al., 2006).

II. OZONE LOCAL AND SYSTEMIC EFFECTS AND AIRWAY
REGENERATION

II - 1. Ozone-induced epithelial injury and inflammation in the lung

Ozone (03) is the principal oxidant air pollutant in photochemical smog. It is
formed at ground level by a chemical reaction of oxides of nitrogen (NOx) and volatile
organic compounds (VOC) and oxygen in the presence of sunlight. Motor vehicle
exhaust and industrial emissions, gasoline vapors, and chemical solvents as well as
natural sources are the main sources of NOx and VOC (EPA, 2008). People are
continuously exposed to 03 at levels detected in their geographical location. In 1997, the
U.S. Environmental Protection Agency (U.S. EPA) set an air quality standard of 0.08

ppm for 03 for a 24-hour period. In 2006, this standard was revised based on a growing

number of geographical areas regularly exceeding the national standards and recent
scientific knowledge of 03 effects on public health. This standard has since been lowered
to 0.075 ppm for the same period of time

(EPA, http://www.epa.gov/air/O3pollution/naaqsrev2007.html). Elevated 03 levels
exceeding the National Ambient Air Quality Standards (NAAQS) are commonly reported
in heavily populated areas in Northeastern, Midwestern and Southwestern USA (Graham,
2004). The U.S. EPA reported that 474 counties have been designated as nonattainment
zones (regions that do not meet the air quality standards) with an estimate of 159 million
people living in those areas

(EPA, http://www.epa.gov/air/O3pollution/naaqsrev2007.html).

Pryor in 1992 demonstrated that 03 itself does not cross the airway epithelium
lining fluid (ELF) where its thickness is greater than 0.1 pm. The thickness of this fluid
varies from 20 to 0.1 pm across the airway tree and is patchy, lacking in some areas in
the distal airway compartments (Pryor, 1992). In areas devoid of ELF, Pryor (1992)
showed that 03 reacts with cell membranes and does not exit airway epithelial cells. In
the ELF, 03 first reacts with antioxidants such as glutathione, uric acid, ascorbate and
vitamin E (Mudway and Kelly, 2000). In a series of subsequent papers, Pryor showed
that part of inhaled 03 not neutralized by small molecular weight antioxidants reacts with
ELF or cell membrane lipids and generates secondary less reactive but longer-lasting
derivatives (Pryor, 1994; Pryor et al., 2006; Pryor et al., 1995a, b). In this process,
peroxidation of membrane lipids by 03 generates lipid ozonation products (aldehydes,
hydroxyhydroperoxides and the criegee ozonide), probably the most important

derivatives at the origin of O3-induced epithelial injury (Mudway and Kelly, 2000; Pryor

et al., 1995a). Indeed, these lipid ozonation products lead to the production of
eicosanoids, platelet-activating factors, reactive oxygen species (hydrogen peroxides,
hydroperoxides, etc) and cytokines responsible for O3-induced toxic effects (Pryor et al.,
1995b).

In people, 03 inhalation in both environmental and experimental settings resulted
in lung function decrement, inflammation and epithelial damage and compromised
antioxidant concentrations in the ELF (Avissar et al., 2000; Calderon-Garciduenas et al.,
2000; Holz et al., 1999; Jorres et al., 2000; Koren et al., 1989; Krishna et al., 1998;
Lippmann and Schlesinger, 2000; Mudway et al., 1999; Seltzer et al., 1986). Exposure of
healthy subjects to 0.3 ppm for 1 h during heavy exercise for instance resulted in
neutrophil accumulation in the BALF that started at 1 h post-exposure and was maximal
between 6 and 24 h post-exposure (Schelegle et al., 1991). Similarly, 03 exposure at high
ambient concentrations (0.08 to 0.1 ppm) for 6.6 h was enough to initiate airway
inflammatory responses in healthy subjects (Devlin et al., 1991). The effects of slightly
greater levels of 03 on neutrophil infiltration in bronchial biopsies have subsequently
been confirmed by morphometric evaluation (Aris et al., 1993). O3 is also a health hazard
for people with pre-existing lung conditions (Bell et al., 2004; Cody et al., 1992; Fauroux
et al., 2000; Friedman et al., 2001; Hiltermann et al., 1998; Thurston et al., 1992; Tolbett
et al., 2000; White et al., 1994). Usually, asthmatic people have their symptoms
exacerbated upon 03 inhalation (Blomberg, 2000). Exposure of subjects with mild
allergic asthma to 0.25 ppm 03 for 3 h with intermittent exercise, exacerbated their
bronchial allergen responsiveness 3 h after exposure. Although lower doses of 03 (0.2

ppm) for slightly shorter time (2 h of exposure and evaluation 6 h post-exposure) seemed

10

to contradict this effect (Jorres et al., 1996; Stenfors et al., 2002). Repeated exposure to
03 at similar levels also enhanced functional and inflammatory responses of people with
pre—existing allergic asthma (Holz et al., 2002). Finally increased asthma symptoms and
medication was reported by physician in children during peak 03 concentrations in Los
Angeles, California (Avol et al., 1998).

In non-human primates, exposure to 0.8 ppm of O3 resulted in ciliated cells and
type I cells loss in the centriacinar region (Castleman et al., 1980). Similarly, exposure to
0.96 ppm of 03 for 8 h resulted in tracheal and respiratory bronchiolar epithelial necrosis
as early as 1 h and became maximal between 12 and 24 h post-exposure. Exposure to 1
ppm of 03 for 2 h resulted in increased neutrophils in the bronchoalveolar lavage
(BALF) 2 h post-exposure (Plopper et al., 1998). Lung tissue and BALF neutrophil
accumulation has been shown to be maximal at the time of epithelial necrosis around 12
h post-exposure and continued up to 24 h (Hyde et al., 1992). Plopper and co—workers
(1998) found that 03 dose to tissue concentration was greatest in the terminal part of the
airway tree where epithelial necrosis was maximal. In the same study, 03 dosimetry
inversely correlated with the content in glutathione in the respiratory bronchioles. With
longer duration of exposures (6 days at 0.15 ppm of O3), monkeys exhibited epithelial
respiratory bronchioles hyperplasia along with an influx of macrophages and thickening
of the underlying lamina propria due to an increase in cellular and acellular components
(Harkema et al., 1993). In infant rhesus monkeys, cyclic exposure to 03 with or without
house dust mite resulted in an array of airway changes that could predispose or aggravate
airways to asthma later in life (Plopper et al., 2007) (reduced airway number, hyperplasia

of bronchial epithelial cells, mucus cell hyperplasia, disorientation and change in

11

abundance of smooth muscle cells in distal pulmonary airways, interrupted basement
membrane development, modifications of airway nerve fiber distribution and
reorganization of the airway vascular and immune systems).

In rodents, acute or subacute exposure to 03 caused morphologic changes in the
distal regions of the airway tree, particularly the distal airways and proximal alveolar
ducts (centriacinar region or junction of the bronchioles with alveoli) (Boorman et al.,
1980; Castleman et al., 1980; Dungworth et al., 1975; Mellick et al., 1975). This effect of
O3 in distal pulmonary regions seems to be related to the O3 dose to tissues which has
been found to be greater in terminal bronchioles and proximal alveolar regions as also
described in non-human primate studies (Kimbell and Miller, 1999; Medinsky and Bond,
2001; Miller et al., 1978; Postlethwait et al., 2000). In an earlier study, rats exposed to 0.5
ppm of 03 developed loss of epithelial cells and type I pneumocytes in terminal
bronchioles ciliated as early as after 2 h after exposure (Stephens et al., 1978). 03 effects
in rats have been confirmed in subsequent studies where acute exposures to 0.5 to 1 ppm
caused centriacinar epithelial cells degeneration, necrosis and sloughing off 4 h after the
initiation of exposure (Pino et al., 1992; Stemer-Kock et al., 2000; Vesely et al., 1999). In
those regions, ciliated cells in the terminal bronchioles and type I pneumocytes are the
most susceptible cells to acute 03 exposure in rodents (Pino et al., 1992; Stemer-Kock et
al., 2000). Dormans and colleagues reported that Clara cells were also affected with
subacute O3 exposures (3 days of continuous exposure to 0.4 ppm of O3) (Dormans et
al., 1999; Schwartz et al., 1976). An acute neutrophilic inflammatory infiltration followed
in subacute cases by infiltration of macrophages is usually associated with O3 epithelial

damage (Pino et al., 1992; Vesely et al., 1999). In mice, exposure to 2 ppm of 03 for 3 h

12

led to increased number of neutrophils in the BALF (Savov et al., 2004). In the C57BL/6
mice strain, this number of neutrophils first increased at 6 h and was maximal 24 h post-
exposure. Exposure of mice to 0.3 ppm 03 for 48 continuous hours resulted in
neutrophils infiltration in the BALF at the end of the exposure (Kleeberger et al., 1997).
In these mice, neutrophil infiltration resolved by 48 h after the end of exposure.

In support of O3 role as an inflammagen, its exposure was also associated with an
elevation of various proinflammatory cytokines. Alveolar macrophages were collected
from rats exposed to 2 ppm 03 for 3 h and grown in vitro (Laskin et al., 1994; Pendino et
al., 1994). Culture media from these alveolar macrophages showed greater amounts of
TNF—a and interleukin-1 (IL-1) 48 h after the end of exposure when compared to filtered
air controls. Alveolar macrophages isolated from guinea pigs exposed to 0.3 ppm 03 for
l h had increased IL—6, TNF-a, interleukin (IL—8) and IL-lB (Arsalane et al., 1995). A
lower concentration of 03 (0.1 ppm) caused these macrophages to release only TNF-Ot.
Exposure of 129 mice to 1 ppm 03 for various times resulted in IL-6 mRNA increase in
the lung starting at 2 h and maximal at 4 h (Mango et al., 1998). In C57BL/6J mice,
exposure to 1 ppm 03 for 4 h or 2.5 ppm for 2 h increased the expression of IL-6 mRN A
in the lung (Johnston et al., 1999). In the same study, macrophage inflammatory protein-1
alpha (MIP-lor) and macrophage inflammatory protein-2 (MIP-Z) chemokines expression
in the lung were also increased in mice exposed to 03. In a study by Vincent and
collaborators (1996) using F344 rats and different 03 concentrations (0.4 or 0.8 ppm) and
time points (2 versus 6 h), IL-6 protein level in the BALF fluid was elevated by 03
exposure. The elevation of IL-6 increased with the age of animals (from 2 months to 9

months to 24 months) and with the dose of 03 (Vincent et al., 1996).

13

II - 2. Ozone effects in the liver

O3-induced systemic changes are less well defined compared to their respiratory
effects. Early studies showed that 03 exposure was associated with prolonged
pentobarbital sleeping time in mice, rats and hamsters, this effect being more pronounced
in females compared to males (Graham et al., 1981). Prolongation of sleeping time has
been subsequently shown to be age-dependent and present in 18 months aged females but
not in young mice (2.5 months of age) exposed to 03 (Canada et al., 1986). In a recent
study by Last and collaborators (2005), exposure of C57BL/6 mice to 1 ppm 03, 8 h a
day for 3 days resulted in a 14% decrease in body weight associated with a 42% decrease
in total food consumption. In this study, 03 exposure also resulted in a significant
downregulation of several cytochrome P450 mRNAs expression, including 1A2, 2A4,
2C9, 3A1 1, 8B1, 7B1, 2D11, 2D10 and 3A16 (Last et al., 2005). CYP4A14 was the only
isoforrn whose expression was upregulated in this study. One of the conclusions drawn
from this study was that the pentobarbital-increased sleeping time reported in previous
studies was probably related to lower metabolic levels of cytochromes P4503 enzymes in
the liver. This team considered interferon-gamma (IFN-y) as a candidate for the lung-
liver interaction as several IFN-y-dependent genes were downregulated in the liver of O3-
exposed mice. One of the first reviews published on extrapulmonary effects of 03 was by
Goldstein (1978) several decades ago. It was reported in this study that as 03 itself is too
reactive to reach the bloodstream, the extrapulmonary effects are probably related to
secondary products generated upon the interaction of 03 with ELF or cell membrane

(Goldstein, 1978). Three groups of derivatives were discussed including reactive oxygen

14

species. According to Goldstein, reactive oxygen species are too reactive and short-lived
(e.g. hydroxyl radical and singlet oxygen) or neutralized by enzymatic or non-enzymatic
cell antioxidant cell defense mechanisms (e.g. superoxide and hydrogen peroxide). The
second group of derivatives described was the products of interaction between 03 and
polyunsaturated fatty acids (lipid hydroperoxides, endoperoxides and ozonides). Those
were theoretically too bulky or hydrophobic to propagate 03 effects to some distance in
the hydrophilic ELF or intracellular environment. Goldstein considered the third groups
of derivatives whom he called end products (e. g. carbonyls such as malonaldehyde) to be
the most promising due to their relative hydrophilicity and the absence of specific
detoxification mechanisms in cells. We today know that this is not entirely the case as
several mechanisms for the detoxification of carbonyls have been since described (Yin,
2000) and as the second group called lipid derivatives have been shown to play an
important role in 03 toxicity at least in the respiratory system (Pryor et al., 1995a).

Others have suggested that signaling molecules generated from lung epithelial or
alveolar macrophages and particularly cytokines such as TNF-a could be the mediators of
O3 distant (and particularly liver) effects (Laskin et al., 1998). Laskin and colleagues
(1998) exposed rat to O3 in the range of 0.5 to 2 ppm for 3 h followed by isolation and
culture of hepatocytes 48 h post-exposure. This team showed greater nitric oxide (NO)
production and protein synthesis by these cells when exposed to O3 alone or O3 followed
by additional inflammatory stimuli (interferon-gamma or lipopolysaccharide) compared
to hepatocytes isolated from air-exposed rats. They hypothesized that because some of
the best known stimuli for NO or protein synthesis in hepatocytes are TNF-a and

interleukin-1, those cytokines are the most likely mediators of O3 hepatic effects. In the

15

same study, NO and TNF-a were also produced in excess by alveolar macrophages in
O3-exposed animals. Laskin and collaborators later suggested that at least in the lung,
TNF-a has deleterious effects upon 03 exposure specially through activation of NF-KB

and induction of NO and peroxinitrite (Roberts et al., 2009).
II — 3. Airway epithelium regeneration after injury

O3-induced airway epithelial injury in rodents is followed by epithelial
regeneration as in any other organ. In the nose for instance, epithelial proliferation in rats
exposed to 0.5 ppm for 8 h was first detected in the transitional epithelium at 12 h post-
exposure and was maximal at 20 h (Hotchkiss et al., 1997). At 36 h, BrdU labeling
indices were still above baseline levels in these rats. In the lung, rats exposed to 0.4 ppm
03 during 12 h had a significant elevation of the number of cycling epithelial cells in
terminal bronchioles (van Bree et al., 2002). Similarly, rats exposed to 0.96 ppm 03 for 3
consecutive days exhibited an increase of radiolabeled thymidine indices in upper and
lower parts of the tracheal epithelium at the end of exposure (Nikula et al., 1988). In
C57BL/6 mice, acute (2 ppm for 3 h) or subacute (0.5 ppm for 24 h) 03 exposure caused
significant elevation of BrdU-labeled cells in terminal bronchioles at the end of exposure
(Longphre et al., 1999; Yu et al., 2002). At 10 days no more proliferation was observed
in these airways. The renewal rate of the bronchiolar epithelium in mammals is very low
and increased BrdU labeling usually reflects compensatory epithelial regeneration after

induced cell demise (Evans, 1982; Kauffman, 1980; Stripp and Reynolds, 2008).

16

Murine axial airway are populated by ciliated, Clara and pulmonary
neuroendocrine cells with a small number of basal cells whereas terminal bronchioles are
mainly lined by Clara cells associated with a smaller proportion of ciliated cells (Liu et
al., 2006; Plopper and Hyde, 2008). Most of what is known in cellular regeneration of the
bronchiolar airway epithelium in mice has been done using a naphthalene toxicity model.
This substance targets the Clara cells, particularly those containing the cytochrome P450
2F2 isoform (Plopper et al., 1992). Within 2 to 3 h after administration almost all Clara
cells expressing the cytochrome P450 2F2 were killed and proliferation of variant Clara
cells not expressing the above mentioned isoform began within 2 days and was complete
by 2 weeks (Buckpitt et al., 1995; Stripp et al., 1995; Van Winkle et al., 1995). Basal
cells and Clara cells in the axial airway and Clara cell in the more distal terminal
bronchioles are probably the main regenerative cells within murine airways. Basal cells
have been shown to repopulate naphthalene-injured airways in an inducible Cre-lox mice
model under the control of cytokeratin 14 promoter (marker of basal cells) (Hong et al.,
2004a, b). In the more distal airways, two airway microenvironments or niches populated
by cells expressing Clara cell secretory protein and resistant to naphthalene injury
(because lacking the cytochrome P450 2F2) have been identified and their cell population
are called variant Clara cells. The first niche co-localized with neuroendocrine bodies
(Hong et al., 2001; Reynolds et al., 2000a; Reynolds et al., 2000b) and the second one has
been identified at the bronchoalveolar junction (Giangreco et al., 2002). These variant
Clara cells are regarded as facultative progenitor cells capable of dividing on demand
after an injury and regenerate both ciliated and fully differentiated Clara cells (Evans et

al., 1978; Evans et al., 1976; Giangreco et al., 2009; Stripp and Reynolds, 2008; Zemke et

17

al., 2009). The main difference between these cells and an undifferentiated progenitor
cell is the expression of differentiation markers detected in the former group
(secretoglobin, family 1A, member 1 or SCGBlAl, calcitonin gene-related peptide or
CGRP and pulmonary surfactant-associated protein C or SFTPC) (Rawlins and Hogan,
2006).

At the molecular level, mechanisms behind the induction of basal or Clara cell
differentiation (and maybe “dedifferentiation” in the case of Clara cells) into their
progeny are not well characterized. Numerous factors from different sources (respiratory
epithelial cells, extracellular matrix and particularly the basement membrane and cellular
and acellular components of the underlying subepithelial connective tissue) have been
identified in the induction of epithelial cell regeneration and differentiation and
thoroughly reviewed elsewhere (Coraux et al., 2005; Jetten, 1991). Several growth
factors, their receptors or cytokines have been involved in respiratory epithelial migration
(epidermal growth factor receptor, hepatocyte growth factor, keratinocyte growth factor,
interleukin-1a and B) or proliferation and differentiation (epidermal growth factor, trefoil
factor family 2, heregulin-a, monocyte chemoattractant protein-1 and platelet-derived
growth factor) (Coraux et al., 2005). More recently, mice deficient in the signal
transducer and activator of transduction 3 (STAT3, a transcription factor involved in IL-6
signaling) or glycoprotein 130 or GP130 (a co-receptor for the IL-6 family of cytokines)
were unable to restore their bronchioles epithelial cell shape and number after
naphthalene injury (Kida et al., 2008). The Wnt/B—catenin signaling pathway induction or
inhibition is usually associated with stimulation or arrest of epithelial regeneration (Stripp

and Reynolds, 2008). Recent studies showed that B—catenin is not involved in the

18

maintenance or repair of bronchiolar epithelium in mice exposed to naphthalene (Zemke
et al., 2009). In this study, B-catenin deficient or sufficient mice exhibited similar
regenerative epithelial units in specific niches of the bronchiolar epithelium, similar
mitotic indices and similar restoration of this epithelium after damage. Finally,
pretreatment of mice with an antagonist of CGRP receptor protected from O3-induced
terminal bronchioles injury and at the same time reduced cell proliferation (Oslund et al.,
2009). This team concluded that CGRP contributed to epithelial injury and repair after

03 exposure.
HI. OVERALL HYPOTHESIS AND SPECIFIC AIMS

APAP targets the liver but also affects the lung and particularly Clara cells
responsible for xenobiotic metabolism and protection from oxidative injury. In addition,
these cells have a progenitor potential and are responsible for regeneration of themselves
but also airway ciliated cells after injury. 03 also targets the airway epithelium but the
ciliated cells are the prime targets. 03 inhalation also results in systemic effects and more
and more data are becoming available to strengthen a novel role of O3 in liver
pathobiology. We therefore sought to study the coexposure of APAP and O3 in the liver
and in the lung.

The main hypothesis of my work was that 03 exposure exacerbates APAP-
induced hepatocellular and pulmonary airway epithelial injury. The specific aims are:
;_Ai_n_1_1: to determine the effects of a single exposure of 03 on acute APAP-induced

hepatic toxicity.

19

Agni: to determine the effects of a single exposure of 03 on acute APAP-induced
pulmonary airway toxicity.

Aim; to determine the role of interleukin-6 in the acute APAP and O3 induced hepatic
and pulmonary airway toxicity and repair.

To address aims l and 2, I used C57BL/6 mice as an animal model. Mice were
fasted overnight and then injected saline or 300 mg/kg of APAP intraperitoneally. Two
hours later, I exposed those mice to filtered air or to 0.25 or 0.5 ppm 03 for 6 h. Nine (1
h after 03) or 32 h (24 h after 03) after the APAP or saline administration, mice were
euthanized. Before the latter sacrifice time, mice were injected intraperitoneally
bromodeoxyuridine to label proliferating hepatocellular or airway epithelial cells. I
utilized histopathological, histochemical (intracellular glycogen staining using the
periodic acid Schiff) or immunohistochemical (neutrophils, bromodeoxyuridine for
epithelial cell proliferation, Clara cell secretory protein and hypoxia inducible factor-1)
and morphometric techniques to characterize and quantitate hepatocellular and
pulmonary airway injury. In addition, molecular (real time PCR for gene expression
analysis), biochemical (glutathione and thiobarbituric acid-reactive substances
commercial kits used as indicators of oxidative stress induction) and immunological
(cytokines evaluation by flow cytometry) methods have been used to explore plausible
mechanisms behind 03 potentiation of APAP-induced epithelial injury.

In the liver, 1 found that 03 potentiated APAP-induced hepatocellular injury. I
also observed that APAP and 03 when associated inhibited cell regeneration, a

mechanism that might have contributed to O3 potentiation of APAP hepatic injury. I

observed in aims l and 2 that IL-6 gene expression was elevated in the lung and plasma

20

in mice given APAP and 03 but not changed in the liver of these animals. At the same
time, APAP alone caused increased expression of IL-6 in the liver. As stated previously,
IL-6 is important in liver regeneration and in aim 3, I tested the hypothesis that IL-6
induced by APAP alone treatment but not by APAP and O3 coexposure might be the
main contributor to the impaired regeneration in the latter group. To do so, I used an
experimental design similar to the one described for aims l and 2 and compared the
effects of APAP and 03 in IL-6 deficient and sufficient C57BIJ6 mice. The results of
these studies demonstrate for the first time that a high ambient 03 concentration could
potentiate locally (respiratory tract) but also systemically (liver) the effects of APAP and

may pose a risk to people with pre-existing liver or lung diseases.

21

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37

CHAPTER 2

EFFECTS OF ACETAMINOPHEN AND ACUTE OZONE COEXPOSURE IN
THE LIVER OF MICE

I. ABSTRACT

Ozone (03), an oxidant air pollutant in photochemical smog, principally targets epithelial
cells lining the respiratory tract. However, changes in gene expression have also been
reported in livers of O3-exposed mice. Overdose with acetaminophen (APAP) is the most
common cause of drug-induced liver injury in developed countries. In the present study,

we examined the hepatic effects of acute 03 exposure in mice pretreated with a
hepatotoxic dose of APAP. C57BL/6 male mice were fasted overnight and then given
APAP (300 mg/kg ip) or saline vehicle (0 mg/kg APAP). Two hours (h) later, they were
exposed to 0, 0.25 or 0.5 ppm 03 for 6 h, then were sacrificed 9 h or 32 h after APAP
administration (1 h or 24 h after 03 exposure, respectively). Animals euthanized at 32 h
were given 5-bromo-2-deoxyuridine (BrdU) 2 h before sacrifice to identify hepatocytes
undergoing reparative DNA synthesis. Saline-treated mice exposed to either air or 03 had

no liver injury. All APAP-treated mice developed marked centrilobular hepatocellular
necrosis that increased in severity with time after APAP exposure. 03 exposure increased
the severity of APAP-induced liver injury as indicated by an increase in necrotic hepatic
[1 5 S He and plasma alanine aminotransferase (ALT) activity. O3 also caused an increase in

van trophil accumulation in livers of APAP-treated animals. APAP induced a 10-fold

k- 38

increase in the number of bromodeoxyuridine-labeled hepatocytes that was markedly
attenuated by 03. Gene expression analysis 9 h after APAP revealed differential
expression of genes involved in inflammation, oxidative stress and genes related to
regeneration in mice treated with APAP and 03 compared to APAP or 03 alone, possibly
providing some indications of the mechanisms behind the APAP and O3 synergy. These

results suggest that acute exposure to an oxidant air pollutant exacerbates APAP-induced

liver injury and delays hepatic repair.

11. INTRODUCTION

O3 is the principal oxidant pollutant in photochemical smog. Approximately half
0f the U.S. population lives in areas that persistently exceed the U.S. environmental
protection agency or EPA’s National Ambient Air Quality Standard (NAAQS) for this
highly reactive and irritant gas (EPA, 2008). Short- and long-term exposures to high
ambient concentrations of 03 have been linked to adverse health outcomes that include
increases in both morbidity and mortality from respiratory causes (Bell et al., 2004;
Jerrett et al., 2009; Katsouyanni et al., 1995). Though numerous studies in laboratory
animals and human subjects have documented the toxic effects of inhaled 03 on the lung,
Il'luch less is known about its effects on extrapulmonary organs like the liver (EPA,
2008).

Recently, using global gene expression analyses, investigators found that livers of
C57BU5 mice acutely exposed to inhaled 03 had significant down-regulation of gene

fanfilies rElated to lipid, fatty acid and carbohydrate metabolisms that were consistent

39

 

with systemic cachexic responses to exposure (Last et al., 2005). Transcription of several
mRNAs encoding enzymes of xenobiotic metabolism was also decreased in livers of
these O3-exposed mice. Since several interferon (lFN)-dependent hepatic genes were
down-regulated with 03 exposure, the investigators suggested that IFN may act as the
signaling molecule between the lung and liver. Interestingly, mice exposed to 03 have
prolonged pentobarbital sleeping time (Graham et al., 1981) also suggesting impairment
of hepatic drug metabolism.

To our knowledge, no studies investigating the potential hepatotoxic interactions
of inhaled environmental pollutants and commonly used therapeutic drugs have been
reported, In the present study we investigated the acute effects of inhaled high ambient
COncentrations of 03 on drug-induced liver injury in mice caused by a widely used
antiIDyretic/analgesic agent, APAP. APAP is one of the most commonly used
nonprescription drugs in the world, and although remarkably safe within therapeutic

doses, it has a relatively narrow therapeutic window. Indeed, APAP overdose is a
‘ commonly reported cause of liver failure in the United States (Larson et al., 2005). Like
in humans, mice receiving an overdose of APAP develop acute liver injury that is
characterized pathologically by centrilobular hepatocellular degeneration and necrosis
with elevated blood activity of liver enzymes such as alanine aminotransferase (ALT)
(Jemnitz et al., 2008; Tee et al., 1987).
Commonly reported risk factors for APAP-induced liver injury include chronic
a‘leohol use as well as the concurrent intake of some medicinal agents (e.g., isoniazid,
Iallenytoin, zidovudine) (McClements et al., 1990; Shriner and Goetz, 1992).

IT“"ironmental pollutants have also been recognized as risk factors in pulmonary,

40

cardiovascular and metabolic conditions such as type H diabetes (Gent et al., 2003;
Mon'is et al., 1995; O‘Neill et al., 2005). More recently, it has been reported that
inhalation of ambient air particulates promotes systemic and liver oxidative stress in mice
(Araujo et al., 2008). Though these risk factors are well documented, the potential
interactive effects of inhaled air pollutants, like 03, with APAP have not been
investigated. With the present study, we report for the first time that a single, near
ambient exposure to O3 exacerbates APAP-induced hepatic injury in mice, resulting in

more severe hepatocellular necrosis and attenuation of early hepatic repair mechanisms.
111. MATERIALS AND METHODS
111 ~ 1. Laboratory Animals

Pathogen-free male, C57Bl/6 mice (8-10 weeks of age, the Jackson Laboratory,

3211‘ Harbor, ME) were used in this study. Mice were housed in polycarbonate cages on
1leaf-treated aspen hardwood bedding (Nepco-Northeastem Product Corp, Warrensburg,
NY). Boxes were covered with filter bonnets, and animals were provided free access to
fQQd (Harlan Teklad Laboratory Rodents 22/5 diet, Madison, WI) and water. Mice were
maintained in Michigan State University (MSU) animal housing facilities accredited by
the Association for Assessment and Accreditation of Laboratory Animal Care and
a‘Qeording to the National Institutes of Health guidelines as overseen by the MSU

In

Slliitutional Animal Care and Use Committee. Rooms were maintained at temperatures

41

of 21-24°C and relative humidities of 45-70%, with a 12-hour light/dark cycle starting at

7:30 AM.
III — 2. Experimental protocol: APAP Treatment and O3 Exposures

Mice were randomly divided into twelve groups, each consisting of six animals.
They were given intraperitoneally 0 (saline-vehicle) or 300 mg/kg APAP (Sigma
Chemical Co., St. Louis, MO) in 20 ml/kg saline. Animals were fasted overnight before
the administration of APAP. Two hours after APAP administration, mice were exposed
to 0 (air), 0.25 or 0.5 ppm 03 for 6 h (Figure 1). Mice were killed 9 or 32 h after APAP
(1 or 24 h after 03 exposure, respectively). As no significant differences were detected in
Prelitninary studies, no morphological evaluation was conducted at 0.25 ppm for the 9 h
time point and at 32 h, data analysis in animals given 0.25 [ppm was limited to

IIlol‘phological evaluations (liver necrosis and BrdU immunostaining) and plasma ALT

E‘<:‘:ivity at the later time (32 h) (Figure 1).

42

Mice sacrificed at

 

Day 2: Mice given 9 h (1 h after 03)
Animal arrival rasDtgti tlwivhrnight ip 0 (saline) °' 033: $332137:th
300 mg/kg APAP treatment
I l Izh-l ll
1 week animal .
acclimation T £2195 V3";
Day 2: Inhalation before
Exposure, 0 (air), sacrifice
0.25 or 0.5 ppm 03

Figure 1. Experimental design of APAP and 03 studies in the liver. 8-10 weeks old
C57BL/6 male mice were given 0 (saline) or 300 mg/kg APAP and then exposed to O3 (0
or air, 0.25 or 0.5 ppm) for 6 h. Mice were euthanized 9 or 32 h after APAP injection (1

or 24 h after 03 exposure, respectively).

Mice were housed individually and exposed to O3 in stainless steel wire cages,
Whole-body inhalation exposure chambers (HC-100, Lab Products, Maywood, NJ). 03
was generated with an OREC 03Vl-O ozonizer (03 Research and Equipment Corp, AZ)
I: Sing compressed air as a source of oxygen. Total airflow through the exposure chambers
was 250 L/min (15 chamber air changes/hour). The concentration of 03 within chambers
was monitored during the exposure using Dasibi 1003 AH ambient air 03 monitors

(Dasibi Environmental Corp, Glendale, CA). Two 03 sampling probes were placed in

43

the middle of the ozone chambers, 10-15 cm above cage racks. Airborne concentrations
during the inhalation exposures were 0.26 +/- 0.02 ppm or 0.53 +/- 0.01 ppm (mean +/-

standard error of the mean) for O3 chambers and 0.02 +/- 0.009 ppm for air chambers.
III — 3. Animal Necropsies and Microscopic and Biochemical Analyses

Two hours before sacrifice, mice euthanized at the 32 h time were given 5-bromo-
2-deoxyuridine (BrdU; 50 mg/kg, Fisher Scientific, Fair Lawn, NJ) intraperitoneally. At
the time of necropsy, mice were anesthetized with an intraperitoneal injection of sodium
Pentobarbital (50 mg/kg; Fatal Plus, Vortech Pharmaceuticals, Dearbom, MI), the
abdortninal cavity was opened, and blood was collected from the abdominal vena cava in
heParinized tubes (BD Microtainer, Franklin Lakes, NJ). Animals were then killed by
exsElnguination.

The liver was removed from the abdominal cavity, and the left liver lobe was
fle'ied in 10% neutral buffered formalin (Fisher Scientific, Fair Lawn, NJ) for light
II‘m-icroscopic examination and morphometric analyses. The caudate liver lobe from each
111Ouse was removed and placed in RNAlater (Qiagen, Valencia, CA) at 4°C for 24 h and

then stored at -20°C for gene expression analyses using real time PCR. The remaining

1i Ver lobes were frozen and stored at -80°C for biochemical analysis of inflammatory
Q 3’tokines, glutathione and thiobarbituric acid-reactive substances.

. After collection of liver samples, hernidiaphragms were punctured to allow

Q Qllapse of right and left lung lobes, and the thoracic cavity was opened for the removal

Qf the trachea and heart-lung en bloc. After the trachea was cannulated, the heart-lung

block was excised and the lungs were gently lavaged twice with 0.9 ml of sterile saline.
Approximately 75-90% of the intratracheally instilled saline was recovered as

bronchoalveolar lavage fluid (BALF) from the lavaged lung lobes and immediately

placed on ice until further analysis.
III — 4. Cellular and Biochemical Analyses of Bronchoalveolar Lavage Fluid

Total cell counts in the collected BALF from each mouse were determined using
a hemocytometer. Cytological slides prepared by centrifugation at 600 rpm for 10
minutes using a Shandon cytospin 3 (Shandon Scientific, Sewickley, PA) were stained
With Diff-Quick (Dade Behring, Newark, DE). Differential counts of neutrophils,
e0Sinophils, macrophages and lymphocytes were assessed on a total of 200 cells.
ReIllaining BALF was centrifuged at 1,500 rpm for 15 minutes to collect the supernatant

fraCtion, which was stored at -80°C for later biochemical analysis.
11:1 - 5. Flow Cytometric Analyses for Inflammatory Cytokines

BALF supematants were assayed for inflammatory cytokines that included
i literleukin-lbeta (IL-113), tumor necrosis factor-alpha (TNF-or), interferon-gamma (IFN-
?) ’ interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-l), interleukin-12 (IL-
1 2), keratinocyte-derived chemokine (KC) and interleukin-10 (IL-10). Plasma cytokine
QQIleentrations for KC, TNF-oc, MCP-l and IL-6 were also determined. All cytokine kits

eI‘e purchased as Flex Set reagents or as a preconfigured cytometric bead array kit (BD

45

Biosciences, San Diego, CA). Cytokines analysis was performed using a FACSCalibur
flow cytometer (BD Franklin Lakes, NJ). Briefly, 50 pl of BALF or plasma was added to
the antibody-coated bead complexes and incubation buffer. Phycoerythrin-conjugated
secondary antibodies were added to form sandwich complexes. After acquisition of
sample data using the flow cytometer, cytokine concentrations were calculated based on

standard curve data using FCAP Array software (BD, Franklin Lakes, NJ).

Liver tissues designated for similar cytokine analyses were suspended in
phosphate-buffered saline at 4°C and homogenized on ice. Homogenates were then
centrifuged at 12,700 rpm for 10 minutes at 4°C. Fifty microliters of the resulting

Supernatant were collected and assayed for IL-6, MCP-l, KC, TNF-a and IL-10 by flow

Cytometry as described above.
111 - 6. Plasma Alanine Aminotransferase (ALT) Assay

Blood collected at the time of necropsy was used to evaluate plasma ALT activity

Slbectrophotometrically using Infinity ALT reagents purchased from Thermo Electron

Qorp. (Louisville, CO).
111 — 7. Liver Tissue Processing for Light Microscopy and Immunohistochemistry

Transverse sections from the middle of the left liver lobe were embedded in
Daraffin, cut at a thickness of 5 pm and stained with hematoxylin and eosin (H&E) for

I‘Qlatine histopathological examination and morphometric analyses. Other tissue sections

46

were histochemically stained with periodic acid Schiff staining and counterstained with
hematoxylin (PASH) to identify intracellular glycogen.

Routine immunohistochemical techniques were used for hepatocellular detection
of nuclear BrdU, hepatic infiltration of neutrophils, and hepatocellular expression of
hypoxia inducible factor 1 alpha (HIP-lot). Briefly, liver sections were deparaffinized in
xylene and rehydrated through descending grades of ethanol and immersed in 3%
hydrogen peroxide to block endogenous peroxides. Sections were incubated with normal
sera to inhibit nonspecific proteins (normal horse, rabbit or goat sera for BrdU,
neutrophils or HIF-lor immunostaining, respectively, Vector Laboratories Inc.,
Burlingame, CA) followed by specific dilutions of primary antibodies (1:40, monoclonal
mouse anti-BrdU antibody, BD, Franklin Lakes, NJ; 122500, monoclonal rat anti-
neutrophil antibody, AbD Serotec, Raleigh, NC; 1:200, polyclonal rabbit anti-HIF-la,
Novus Biologicals, Littleton, CO). Tissue sections were subsequently covered with
secondary biotinylated antibodies, and immunostaining was developed with the Vector
RTU Elite ABC kit (BrdU and HIF-lor, Vector Laboratories Inc) or the RTU
Phosphatase-labeled Streptavidin kit (neutrophils, Kirkegaard Perry Labs, Gaithersburg,
MD) and visualized with Vector Red (neutrophils, Vector Laboratories Inc) or DAB
(3,3’-diaminobenzidine) (BrdU or HIF-lor, Sigma Chemicals, St. Louis, MO)
chromogens. Slides were counterstained with Gill 2 hematoxylin (Thermo Fisher,

Pittsburgh, PA).

47

III - 8. Morphometric Analyses of Liver

BrdU-stained and unstained hepatocellular nuclei were counted in 10 medium
power fields (X200) for each animal, starting with a randomly selected field and
evaluating every third field. The hepatocellular labeling index (LI; % of hepatocytes
undergoing DNA synthesis) was determined by counting the number of BrdU-labeled
cells divided by the total number of hepatocytes and multiplying by 100.

Hepatic neutrophil accumulation was assessed by averaging the numbers of
neutrophils in 10 medium power fields (X200) in each slide. Analyzed fields were
selected in an unbiased manner with a random start and counting every third field.
Neutrophils were identified by positive immunohistochemical staining with the
neutrophil-specific antibody and their polymorphologic nuclear profiles.

Hepatocellular degeneration/necrosis in sections from the left liver lobe was
quantified using standard morphometric methods that were similar to those previously
described in detail (Yee et al., 2000). Briefly, H&E-stained liver sections from the left
liver lobe were visualized with an Olympus BX-40 light microscope (Olympus Corp,
Lake Success, NY) coupled with a 3.3-megapixel digital color camera (Qimaging,
Surrey, BC, Canada). Images at a magnification of X200 were evaluated employing a
168-point lattice grid overlaying fields of hepatic parenchyma to determine (1) the total
area of liver analyzed, (2) the area of degenerative/necrotic hepatic parenchyma and (3)
the area of normal parenchyma. The area of each object of interest (e.g. lesion) was

calculated using the following expression (Cruz-Orive, 1982):

48

Afealnterest = 2 POintSInterest X Area/Point

. . 2 . . 2
Area/Point = (Distance between Pomts) lMagnlficatlon

Distance between points was 13 um. Accordingly, the area represented by each
point was 511 um 2. Section from the liver of each mouse was systematically scanned
using adjacent, non-overlapping microscopic fields. The first image field analyzed in
each section was chosen randomly. Thereafter, every third field was evaluated
(approximately 10-14 fields evaluated/section). The measured fields represented
approximately 65% of the total area of each liver section. Percent lesion area was

estimated based on the following formula:

[AreaLesion of Interest/ (Area/1,11 Lesions + AreaNormal Parenchyma)l X 100-

III — 9. Quantitative Real Time RT-PCR for Hepatic Gene Expression

The caudate liver lobe was isolated and placed in RNAlater (Qiagen, Valencia,
CA) and kept at 4°C for at least 24 h then transferred to -20°C until processed. Total
RNA was extracted using RNeasy Mini Kit according to the manufacturer’s instructions
(Qiagen, Valencia, CA). Briefly, tissues were homogenized in RLT buffer containing [3-
mercaptoethanol with a 5 mm Rotor-Sator Homogenizer (PRO Scientific, Oxford, CT)
and centrifuged at 10,700 rpm for 3 minutes. Samples were then treated with Rnase-Free
Dnase, Rnase-free buffer and water on the column for 30 minutes (Qiagen). Eluted RNA
was diluted 1:5 with Rnase free water and quantified using a GeneQuant Pro

spectrophotometer (BioCrom, Cambridge, England).

49

Reverse transcription (RT) reaction was performed using reverse transcription
high capacity cDNA reagents (Applied Biosystems, Foster City, CA) and a GeneAmp
PCR System 9700 Therrnocycler PE (Applied Biosystems). Each RT reaction was run in
5 ill of sample with 20 ul of cDNA Master Mix prepared according to the manufacturer’s
protocol (Applied Biosystems).

Expression analyses of isolated mRNA were performed by quantitative real-time
PCR using individual animals’ cDNA with the ABI PRISM 7900 HT Sequence Detection
System using Taqman® Gene Expression Assay reagents (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. Individual data are reported as fold
change of mRNA in experimental samples compared to the saline/air control group. Real-
time PCR amplifications were quantified using the comparative Ct method normalized to
the mean of two endogenous controls (188 and GAPDH). The cycle number at which
each amplified product crosses the set threshold represented the Ct value. The amount of
target gene normalized to the mean of the endogenous reference genes was calculated by
substracting the endogenous reference Ct from the target gene Ct (ACt). Relative mRNA
expression was calculated by substracting the mean ACt of the treated samples from the
ACt of the control samples (saline-treated, air-exposed) (MO). The absolute values of

the comparative expression level (fold change) was then calculated by using the formula:

' —AACT
Fold change = 2 .

50

III - 10. Glutathione Assay

To determine hepatic concentrations of oxidized glutathione (GSSG) and total
glutathione (reduced plus oxidized; GSH and GSSG, respectively), median lobes of the
liver (preserved at -80°C) were homogenized in cold MES buffer (0.4 M 2-(N-
morpholino)ethanesulfonic acid, 0.1 M phosphate, and 2 mM EDTA, pH = 6).
Homogenates were centrifuged at 9,700 rpm for 15 minutes at 4°C, and the supematants
were collected and deproteinated. The total glutathione concentration was then assayed as
recommended by the manufacturer (Cayman Chemical Co., Ann Arbor, MI). GSSG
concentration was determined after derivatization of GSH with 2-vinylpyridine. Sample
absorbance was determined at 405 nm, and the total or oxidized glutathione
concentrations in liver homogenates was assessed by comparison of absorption to

standard curves.

III — 11. Thiobarbituric Acid-Reactives Substances (TBARS) Assay

Lipid peroxidation in the liver was estimated using a commercially available kit
according to the manufacturer’s recommendations and malonaldehyde as a standard
(TBARS kit, Cayman Chemical Co., Ann Arbor, MI). Liver tissue was homogenized on
ice in RIPA Buffer and Proteases Inhibitor (Thermo Scientific, Rockford, IL).
Homogenates were centrifuged at 3,900 rpm, and the supernatant was collected and used

to detect malonaldehyde and TBARS adducts in acidic conditions and under high

temperature (100°C). Absorbance was measured at 530 nm.

51

1H - 12. Statistical Analyses

Data were reported as mean +/- SE. Differences among groups were analysed by a one-
or two-way AN OVA followed by Student-Newman-Keuls post hoc test. When normality
or variance equality failed, a Kruskal-Wallis ranked test was conducted. All analyses
were performed using SigmaStat software (SigmaStat; Jandel Scientific, San Rafael,

CA). Significance was assigned to p values smaller than or equal to 0.05.

IV. RESULTS

IV - 1. Inflammatory Responses Reflected in BALF

Saline treatment/O3 exposure (03 alone or SAL/O3 group) did not cause changes
in BALF total inflammatory cell number at any time compared to saline treatment/air
exposure (controls or SAL/air group) (Figure 2A, B). As compared to control mice,
animals that were administered APAP and were exposed to either air (APAP/air or APAP
alone) or 03 (APAP/0.5 ppm 03 or APAP and O3-coexposed mice) had a time-
dependent, statistical increase in the number of total inflammatory cells in the BALF at 9
and 32 h after APAP administration (Figure 2A, B). Though not statistically significant,
there was a trend for greater total inflammatory cells in the lungs of the APAP and 0.5
ppm O3-coexposed mice compared to APAP alone (Figure 2A, B). No difference in total
inflammatory cell number was detected between APAP alone and APAP/0.25 ppm 03

groups at 32 h (Figure 2B).

52

Pulmonary inflammatory cell responses, as reflected in the BALF, were due to
increases in alveolar macrophages and/or neutrophils (Figure 2C, D, E, F). 03 alone did
not cause changes in neutrophil or macrophage number in BALF at any time (Figure 2C,
D, E, F). At 9 and 32 h after APAP treatment, mice exposed to APAP alone or with 03
had significant increases of alveolar macrophages (Figure 2E, F). This response was
somewhat greater at 32 compared to 9 h. On the other hand, the number of neutrophils in
BALF was not affected by APAP at 9 h (Figure 2C, D). At 32 h, only APAP/O3-
coexposed mice had marked increases in neutrophil numbers in BALF (Figure 2D),

indicating a synergistic effect.

53

Figure 2. Inflammatory cell accumulation in the BALF of APAP and O3 exposed mice.
Graphs represent total inflammatory cells (A and B), neutrophils (C and D) and
macrophages (E and F) per ml of BALF. Animals were given 0 (saline) or 300 mg/kg
APAP ip and 2 h later exposed to 0 (air), 0.25 (32 h only) or 0.5 ppm 03 for 6 h. 9 or 32
h after APAP administration, animals were euthanized and BALF harvested and analyzed
as described in Materials and Methods. Data are expressed as mean :1: SE (n = 6). a,
significantly different from saline/air group; b, significantly different from saline/0.5 ppm
O3 group; c, significantly different from saline/0.25 ppm 03; (p S 0.05). ND, not
detected.

54

Figure 2

A. BALF total Cells at 9 h

200

    
   
     

=1 Saline/Air
ES! Saline/O3
n APAP/Air
- APAP/0.503

.5
0|
G

Total Cells (x103)/mr
or 8
O O

O

C. BALF neutrophils at 9 h

E10

z E! Saline/Air
":9 8 as: Saline/O3
5 6 n APAP/Air

a _ APAP/0.503
g 4

9

*5 2

g

o

 

E. BALF macrophages at 9 h

200
El Saline/Air

150 Saline/O3 b
m APAP/Air

- APAP/0.503

    

0|
0

Macrophages (X103)lm|
O
O

O

55

B. BALF total Cells at 32 h

' 1:1 Saline/Air
\ . 1:1 Saline/0.2503
”o as: Saline/0.503 a
{£200 - m APAP/Air
v m APAP/0.2503

    
  
   

vvvvv

 

€150 ‘ -APAP/0.503
0100

o , = V
*- Igsx

D. BALF neutrophils at 32 h

  
 
 
  
   

 

   
    

 

_ 50 . a
g :1 Saline/Air
¢«3‘40 a Saline/0.2503
S is: Saline/0.503
530 a APAP/Air
2 cu APAP/0.2503
£20 . - APAP/0.503
2
510 ‘
d!
z 0 _
F. BALF macrophages at 32 h
:1 Saline/Air b
£250 ‘ 1:1 Saline/0.2503
on ‘ 53 Saline/0.503
3 20° W APAP/Air ,,,,,
5.150 , thPAP/O.2503
E100
I: :o:o:o:o:c
a 000...:
e 50 ‘ 0...:
g 1
E o _ .....

BALF was analyzed by flow cytometry for exposure-induced changes in several
inflammatory cytokines (IL-1, TNF-or, IL-10, IFN-y, IL-6, MCP-l, IL-12 and KC, IL-
10). Most of these cytokines were not significantly changed at either examined time (data
not shown), with the exception of IL-6 and MCP-l. APAP or O3 alone did not cause an
increase in IL-6 at 9 h and only a minimal increase occurred at 32 h with the APAP
treatment (Figure 3A, B). APAP/O3 coexposure resulted in a significant increase of IL-6
in BALF compared to either substance alone at 9 h, but not 32 h after APAP
administration (Figure 3A, B). At the early time, MCP-l was undetectable in the BALF
of mice from any of the groups (Figure 3C). 03 exposure caused a slight, but significant,
elevation of MCP-l at 32 h after APAP treatment (Figure 3D). MCP-l in BALF was
similarly and significantly elevated in APAP alone- or APAP/O3-coexposed mice 32 h

after APAP (Figure 3D).

56

Figure 3. IL-6 (A and B) and MCP-l (C and D) protein concentrations in the BALF of
APAP and O3 exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2
h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP administration,
animals were euthanized and BALF harvested and cytokine concentrations evaluated as
described in Materials and Methods. Data are expressed as mean t SE (n = 6). a,
significantly different from saline/0.5 ppm 03 group; b, significantly different from
APAP/air group; c, significantly different from saline/air group; (p S 0.05). ND, not
detected.

57

Figure 3

 

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A. BALF IL-6 at 9 h B. BALF IL-6 at 32 h
a
40 ' r:l Saline/Air b 30 "
ts Saline/O3 25 « g 23:!"913';
=30 - a APAP/Air = a '"e .
g - APAP/0 5 03 E 20 ' “ APAP’A"
g3 ' or - APAP/0.5 03
:20 515
a 3 1o
10 . - l
5 1
o — ND 0 .
C. BALF MCP-l at 9 h D. BALF MCP-l at 32 h
1° ' 1:1 Saline/Air 12° ‘ c: Saline/Arr
=3 . is: Saline/O3 ._..1oo , In Saline/Q3
g I! APAP/Air g 80 ‘ m APAP/Arr c
2 5 . - APAP/0.5 03 2 II APAP/0.5 O3
:4 : 60 ‘ r I I I I I
n'. ‘ o'.
o o 40 ‘ C
2 2 ‘ 2 20 , T V
0 ND ND ND ND 0 r-T—N\\\

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

58

IV — 2. Inflammatory Cytokine Concentrations in Plasma

Exposure-related changes in plasma cytokines were restricted to IL-6, MCP-l and
KC. Changes in plasma IL-6 reflected those in BALF with only APAP/O3 coexposure
inducing significant elevation in IL-6 concentration and only at 9 h after treatment
(Figure 4A, B). 03 alone-exposed mice had no plasma MCP-l change at any time
compared to controls. APAP alone caused significant elevation of plasma MCP-l
concentration 9 h after treatment that was not observed in the APAP/O3 coexposure
group (Figure 4C). At 32 h, APAP alone and APAP/O3-coexposed mice had similar
increases in plasma MCP-l compared to controls (Figure 4D).

03 exposure did not change the plasma concentrations of KC, a neutrophil
chemokine (Figure 4E, F). KC was significantly elevated only in APAP/O3-coexposed
mice at 9 h after APAP treatment (Figure 4E). At the later time, plasma KC was slightly
above saline/O3 levels in APAP/O3-coexposed mice and there was a significant elevation

in KC concentration in the plasma of mice given APAP alone (Figure 4F).

59

Figure 4. IL-6 (A and B), MCP-l (C and D) and KC (E and F) protein concentrations in
the plasma of APAP and O3 exposed mice. Animals were given 0 (saline) or 300 mg/kg
APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized and blood collected and analyzed as described in
Materials and Methods. Data are expressed as mean :1: SE (n = 6). a, significantly
different from saline/0.5 ppm 03 group; b, significantly different from APAP/air group;
c, significantly different from saline/air group; (p S 0.05).

60

Figure 4

A. Plasma IL-6 at 9 h

.5
O

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

:1 Saline/Air a
A 30 , Saline/O3
'g m APAP/Air
a, - APAP/0.5 03
9,- 20 t
‘3
_ 10 ‘ ”I
0
C. Plasma MCP-l at 9 h
50 i
t: Saline/Air 3‘:
=40 . m Saline/O3
E u APAP/Air
3’30 r - APAP/0.5 03
E 20 . a
U
2 1o .
o

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

E.P1asmaKCat9b

  
   
      

20
1:1 Saline/Air
as: Saline/O3
15 m APAP/Air

- APAP/0.5 03

KC (pg/ml)
or 3

O

61

B. Plasma IL-6 at 32 h

40 .
:1 Saline/Air
30 ‘ Saline/O3
a APAP/Air
II APAP/0.5 O3

IL-6 (pg/ml)
N
O

.3
O

 

 

D. Plasma MCP-l at 32 h

    
   
     
       
   

(II
C

:1 Saline/Air

A 40 Saline/O3

E m APAP/Air

‘3.) 3o - APAP/0.5 03
E 20

O

E 10

O

F. Plasma KC at 32 h

N
O

r: Saline/Air
as: Saline/O3

:- 15 m APAP/Air
é - APAP/O3
a:

e 10

O

X

OI

O

IV - 3. Histopathology and Morphometric Assessment of Liver Injury

Saline-treated mice exposed to either air or 03 had no hepatic histopathology at
either time postexposure (Figure 5A, B). All APAP-treated mice developed hepatic
centrilobular necrosis at 9 (data not shown) and 32 h (Figure 5C, D). This drug-induced
liver lesion increased in severity with time after APAP administration. Inhalation
exposures to either 0.25 or 0.5 ppm 03 markedly increased the APAP-induced
centrilobular necrosis at 32 b (Figure 5F), but not 9 h after APAP administration (24 h
and 1 h after 03 exposure, respectively). At the later time, APAP and O3-coexposed
mice had expanded areas of centrilobular necrosis compared to APAP alone-treated mice.
These expanded areas were rimmed by a distinctive layer of enlarged hepatocytes with
highly vacuolated cytoplasm and pyknotic nuclei. This 1-2 cell layer of hepatocytes
undergoing ballooning degeneration separated the conspicuous centrilobular areas of
coagulative necrosis from the normal midzonal and periportal hepatocytes.
Morphometrically, APAP-treated mice exposed to 0.25 or 0.5 ppm 03 had 1.46 or 1.62
time increases, respectively, in hepatocellular necrosis compared to APAP alone-treated
mice (Figure SF).

No changes in plasma ALT activity, a marker of hepatocellular injury in
circulating blood, were detected in 03 alone-exposed mice as compared to control mice
(Figure 5E). As expected, plasma ALT activity was significantly elevated in APAP-
treated mice at 9 (data not shown ) or 32 h after administration (Figure 5E). At the early
time, no significant differences in ALT activity were observed between APAP alone- and

APAP/O3-coexposed mice (data not shown). At 32 h after APAP injection, however,

62

ALT activity was significantly greater in APAP/O3-coexposed mice as compared to
APAP/air-exposed mice (Figure 5E). This finding was reflected in differences in the

extent of the centrilobular lesions between these groups.

63

 

Figure 5. Liver damage induced by APAP and 03 exposure 32 h after APAP. Effects of
APAP and 03 on alanine aminotransferase (ALT) activity (E) in plasma. Morphometric
evaluation of hepatocellular damage (F). Animals were given 0 (saline) or 300 mg/kg
APAP ip and 2 h later exposed to 0 (air), 0.25 or 0.5 ppm 03 for 6 h. Thirty-two hours
after APAP administration, animals were euthanized, blood and liver tissue were
collected and ALT and liver tissue evaluated as described in Materials and Methods. Data
are expressed as mean :1: SE (n = 6). a, significantly different from saline/air group; b,
significantly different from saline/0.25 ppm 03 group; c, significantly different from
saline/0.5 ppm 03; d, significantly different from APAP/air group; (p S 0.05). Solid
arrow, centrilobular necrosis; stippled arrow, vacuolar degeneration; CV, central vein.
ND, not detected.

Figure 5 (cont’d)

E. Plasma ALT at 32 h F. Liver injury at 32 h

1:: Saline/Air . :1 Saline/Air

   
   

     
 

 

 

C m
a Saline/0.25 03 b d E, 5° a Saline/0.25 03 d
4 ‘ ES! Saline/0,5 03 d -= ES! Saline/0.5 03
g are APAP A" g 40 - m APAP/Air
g 3 , rm APAP/0.25 03 e co APAP/0.25 03
E - APAP/0.5 03 g 30 . — APAP/0.5 03
\ 0
22 ‘ “3 20 -
'- 8
2' 1 - r: 10 -
“5
o . ————— e\° 0 -

No neutrophilic accumulation was observed in livers of O3 alone-exposed mice at
either time postexposure compared to controls (Figure 6A, B, E). In APAP-treated mice,
neutrophilic accumulation was observed predominantly within the areas of hepatocellular
degeneration and necrosis (Figure 6C, D). APAP/air and APAP/O3 groups had similar
numbers of neutrophils in the liver as determined by morphometric analyses 9 h post-
treatment (data not shown). At 32 h, the number of neutrophils in the livers of APAP/O3

mice was slightly but not significantly greater compared to APAP/air mice (Figure 6E).

65

A Saline/Arr 7 ,, B. Saline/0.5 ppm 03
. r . ““ ‘. .1 v ' ,ff.’

 
 

h ..
‘HAy‘
o it.) e v 9 w, r
. k c o .
‘ I. t! I a v 8 5 , . l ‘3 -- ‘
,2 I 1:..- O. ' , B ' I ’9‘.
. - .4 ., ~ Nun‘s.
v ' ‘7‘ r g G "O" -1 a e
‘ 't‘ ‘~ wt . . ‘ I - 14'
33} a ,, o ‘ . . ,4 .
t ‘. ' r 43 ' f -: _ It. . ’
r‘n"~:f~ . i=2: .‘ '39 ‘pII‘ Wadi
‘ " ; ‘3 . o 5 ‘ o
1' r .‘ . o , I _ c ’ . I
. . J r _ r- ,
f. a- z x :r 1: ': \A'" 0 J}!
9 r .3 . _ ’ -‘ . ' uI ‘ . a ' — .
, ' ‘ ' ~ , r ‘f z .7 {7 ..
’- .'3. . .1 i i u ‘ — I ~ I. . , 5' 1 -0
o
c. APAP/Arr o. APAP/0.5 ppm 03

Figure 6. Liver neutrophil infiltration in APAP and O3 exposed mice 32 h after APAP.
Morphometric evaluation of neutrophil infiltration in hepatic parenchyma (E). Animals
were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm
03 for 6 h. Animals were euthanized and livers collected and evaluated as described in
Materials and Methods. Data are expressed as mean 1 SE (n = 6). a, significantly
different from saline/air group; b, significantly different from saline/O3 group; (p S 0.05).

Black arrows indicate neutrophils; CV, central vein.

66

Figure 6 (cont’d)

E. Liver neutrophils at 32 h

   
   
      

.3 30° :1 Saline/Air
E to 250 mSalineloa
"- ; IIIIAPAPIAir
g g 200 -APAPIO.5 03 a
g 5 150
a is
e 9: 100
E ° so
2
0

IV - 4. Hepatocellular Regeneration and Hypoxia

BrdU was administered to mice euthanized 32 h after APAP to identify
hepatocytes undergoing DNA synthesis (S phase of the cell cycle). 03 exposure alone
had no effect on hepatocellular BrdU incorporation compared to controls (Figure 7A, B,
E). APAP treatment caused a marked increase of BrdU immun0positive nuclei which
was dose-dependently reduced by coexposure with 03 (Figure 7C, D, E). 0.5 ppm 03

completely blocked the APAP-induced increase in BrdU incorporation in the liver

(Figure 7E).

67

”A. Saline/Air B. Saline/0.5 ppm 03

., PV
[CV . .
’7‘ cv *bv.

;.,y- ~ “ 1' .. .
. , ‘ a. '1‘ ' CV
.2 (y- . ~ .. _ , - . '
. . a: _. ,, ,y- _
c. APAP/Air D. APAP/0.5 ppm 03

Figure 7. Hepatocellular proliferation in APAP and O3 exposed mice 32 h after APAP.
Morphometric evaluation of cycling hepatocytes in S phase (E). Animals were given 0
(saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air), 0.25 or 0.5 ppm 03 for 6
h. Two hours before euthanasia, mice received BrdU administration. Animals were
euthanized and livers collected and evaluated as described in Materials and Methods.
Data are expressed as mean t SE (n = 6). a, significantly different from saline/air group;
b, significantly different from APAP/air group; (p S 0.05). Black arrows indicate BrdU-
labeled hepatocellular nuclei; PV, portal vein; CV, central vein.

68

Figure 7 (con’t)

E. Hepatocellular proliferation at 32 h

1:: Saline/Air

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

3 ' 1:: Saline/0.25 03
ES! Sgline/Ofi 03 _a_
'3 a A AP Arr
3 8 2 an APAP/0.25 03
n 5 - APAP/0.5 03
3 o
= ‘5 b
E 81 I
m :j: b
°‘° “L“ i
o r \

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

At 32 h, no change in glycogen or HIF-la staining was seen in mice exposed to
O3 alone compared to controls (Figure 8A1-2, 8B1-2). At the same time, necrotic areas
in the liver of APAP-treated mice were surrounded by a one to two cell-thick layer of
glycogen-depleted hepatocytes (Figure 8A3). Interestingly, in APAP/O3-coexposed mice,
the ballooning degeneration of hepatocytes were located in this layer of glycogen
depletion and appeared to be the targeted tissue for the O3—induced expansion of APAP-
induced liver injury (Figure 8A4). APAP hepatotoxicity was accompanied by an increase
in hepatocellular HIF-la, a key transcription factor that mediates cellular response to
hypoxia (Pouyssegur et al., 2006; Semenza, 2003). HIF-lor accumulation in APAP-

treated mice was consistently found in the cytoplasm and less frequently in the nucleus of

69

hepatocytes located in glycogen-depleted areas (junction of centrilobular necrotic zone
and healthy parenchyma) (Figure 8B3). In the APAP/O3 group, few hepatocellular
nuclei, located primarily at the periphery of the necrotic zone, had HIF-lor accumulation

(Figure 8B4).

70

Figure 8. Intracellular glycogen (A) and HIF-la (B) staining 32 h after APAP. Light
photomicrographs of liver sections from mice given saline/air (A1, B1), saline/0.5 ppm
03 (A2, B2), APAP/air (A3, B3) and APAP/0.5 ppm 03 (A4, B4). Animals were given 0
(saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h.
Animals were euthanized and livers collected and evaluated as described in Material and
Methods. nH, normal hepatocytes, HH, hypoxic hepatocytes; *, hepatocellular necrosis;
black arrows in A or black arriowheads in B indicate hepatocellular degeneration; black
arrows in B indicate HIF-la hepatocellular cytoplasmic staining, open arrowhead in B
indicate endothelial cell HIF-la staining; stippled arrow in B, HIF-la nuclear staining;
CV, central vein.

71

Figure 8

' j , .salperp.'5pr5m:035,'{_

 

72

IV - 5. Relative Gene Expression and Protein Concentration of Inflammatory

Cytokines in the Liver

03 exposure alone did not cause relative gene expression changes in neutrophil
chemokines KC or MIP-2 in the livers at any time postexposure (Figure 9A-D). This
correlated with the lack of neutrophil liver accumulation in these mice. APAP treatment
or APAP/O3 coexposure caused significant increases in relative expression of KC and
MIP-2 genes 9 h after APAP (Figure 9A, C). KC protein concentration was also elevated
in APAP/air and APAP/O3 groups at both 9 and 32 h after APAP (Figure 10A, B). At 9
h, no differences were observed in expression levels. of KC between APAP/air and
APAP/O3 groups (Figure 9A). At the same time, APAP/O3-coexposed mice had
approximately three times the MIP-2 mRNA expression level of the APAP-alone group
(Figure 9C). Relative gene expression of these chemokines declined to levels similar to
those of controls at 32 h after APAP (Figure 9B, D).

03 exposure alone had minimal effects on relative expression or protein
concentration of MCP-l in the liver at any time (Figure 9E, F and Figure 10C, D). APAP
treatment alone caused a significant increase of MCP-l relative expression or protein
concentration over control levels 9 h after its administration (Figure 9E and Fig 10C). In
comparison, 0.5 ppm 03 exposure caused a more than five-fold reduction in APAP-
induced increase in the mRNA or protein concentration of MCP-l (Figure 9E and Fig
10C). At 32 h, both APAP/air and APAP/O3 groups had significant increases in MCP-l

mRNA expression and protein concentration (Figure 9F and Figure 10D).

73

Figure 9. KC (A and B), MlP-2 (C and D) and MCP-l (E and F) genes expression in
livers of APAP and O3 exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP
ip and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized and liver samples collected in RNALater and
evaluated as described in Materials and Methods. Data are expressed as mean :1: SE (n =
6). a, significantly different from saline/air group; b, significantly different from
saline/0.5 ppm 03 group; c, significantly different from APAP/air group; (p S 0.05). FC,
fold change.

74

Figure 9

 
        

A. Liver KC expression at 9 h B. Liver KC expression at 32 h

is: Saline/O3

J: 25 . _= 4 m APAP/Air

g 15:1 Sallnel03 < - APAP/0.503

g m APAP/Air b E

= 20 — APAP/0.503 E b

a N 2 '0‘o'o'o"'

m m :0000.:.1

O 1 5 o

H 0-0 o r. .

E10 E

2 it‘s

a s a '2

O

u. 0 E -4

C. Liver MIP-2 expression at 9 h D- Liver MIP'Z expression at 32 h
g or: Saline/O3 :50 15:1 Salin /o3
.5200 m APAP/Air 2 m APAPelAir
'5 — APAP/0.503 = 4° — APAP/0 503
a) a '
c,150 "1 3 ‘
a O
o i-l
E100 g 2
m H
g 50 E 1
U I!
u. 0 E

   

 
  
 

  
        

E. Liver MCP-l expression at 9 h F. Liver MCP-l expression at 32 h
_ ES Saline/Q3 ”32100 : Riggs/[ACE
3100 a m APAP/Arr a - APAP/0 503
a — APAP/0.503 .5 30 '
.s 80 . 3
a ‘ "’ b
m 60 2 so .......
3 o
>
3 4° ': 4°
% 20 tir’ 20
n: U
o ...... LL 0 ______
8 -10 a

75

Figure 10. KC (A and B), MCP-l (C and D) and IL-6 (E and F) protein concentrations in
livers of APAP and O3 exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP
ip and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized and liver samples collected and evaluated as
described in Materials and Methods. Data are expressed as mean :1: SE (n = 6). a,
significantly different from saline/air group; b, significantly different from saline/0.5 ppm
03 group; c, significantly different from APAP/O3 group; (p S 0.05).

76

Figure 10

A. Liver KC at 9 h

  

50
r: Saline/Air

 
   
      
  

40 m Saline/O3
E m APAP/Air
Ta 30 I- APAP/0.503
o.
o 20
x

10

0

C. Liver MCP-l at 9 h

:1 Saline/Air

 
   
   
  

         
   

 

    
     
    

           

:- 5! Saline/O3
E 600 m APAP/Air
c» - APAP/0.503
o. :-'~:~:-:~:~:
" 400 :::::::=:::::
r 0.9...O.O.O.I
I o’c’o‘o‘o’o‘r
m .;.:.;.;.;.;.
O O O O O O .
U .0.0 D 0 0.9
5 200 . ..

E. Liver IL-6 at 9 h

1: Saline/Air

6 as: Saline/O3
5 m APAP/Air
... - APAP/0.503
E 4
E 3
3 2
1
o

 

77

B. Liver KC at 32 h

:1 Saline/Air
Saline/O3

m APAP/Air W.,“.
- APAP/0.503

   
   
    

O

  

IL-6 (pg/ml)
N .1;

O

D. Liver MCP-l at 32 h

800 1:1 Saline/Air a
m Saline/O3
600 m APAP/Air

- APAP/0.503

MCP-1 (pg/ml)

 

F. Liver IL-6 at 32 h

r: Saline/Air

   
   
     

1:31 Saline/03
A 6 m APAP/Air
E - APAP/0.503
E 4
3
- 2
0

03 exposure had no effect on IL-6 or plasminogen activator inhibitor 1 (PAI-l)
relative expression at any time (Figure 11A-D). APAP treatment on the other hand
resulted in significantly elevated hepatic IL-6 and PAI-l mRNA 9 or 32 h after its
administration (Figure 11A, B). 03 coexposure tended to reduce the increases of IL-6
and PAL] mRNA caused by APAP, but this reduction reached statistical significance at
the early time only (Figure 11A, B). In agreement with these gene expression data, IL-6
protein was increased in the liver by APAP, but not APAP/O3, at 9 h only after APAP

treatment (Figure 10E, F).

78

Figure 11. IL-6 (A and B) and PAL] (C and D) genes expression in livers of APAP and
O3 exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later
exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP administration, animals
were euthanized and liver samples collected in RNALater and evaluated as described in
Materials and Methods. Data are expressed as mean :I: SE (n = 6). a, significantly
different from saline/air group; b, significantly different from APAP/air group; c,
significantly different from saline/O3 group; (p S 0.05). FC, fold change.

79

Figure 11

A. Liver IL-6 expression at 9 h

a m Saline/O3
m APAP/Air
- APAP/0.503

.3
O

   
 
   
  

FC Relative to Saline/Air
o N :- a: on

C. Liver PAI-l expression at 9 h

  

E 250 a m Saline/O3
g m APAP/Air
% 200 - APAP/0.503
U)

3 150 b

a C
.5100

2

g 50

U

"- o

B. Liver IL-6 expression at 32 h

m Saline/O3
m APAP/Air
- APAP/0.503

4. m

 

 

 

 

C

i

 

 

 

 

 

 

 

 

 

 

 

 

 

one-moo

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FC Relative to Saline/Air

I
N

 

W

D. Liver PAI-l expression at 32 h

m Saline/O3
a a APAP/Air
- APAP/0.503

N
O
O

   

.3
0|
0

FC Relative to Saline/Air
on 3
o c

O

80

IV - 6. Regeneration-Related Gene Expression in Liver Tissue

03 or APAP alone caused an increase in liver mRNA expression of the cyclin
dependent kinase inhibitor P21, at the early time postexposure (Figure 12A). At the same
time, APAP/O3 coexposure resulted in significantly greater expression of P21 mRNA
expression compared to either APAP or 03 (Figure 12A). At 9 h, suppressor of cytokine
signaling 3 (SOCSB) expression was decreased by 03 exposure but increased by APAP
treatment (Figure 12C). APAP/03 group had no statistically significant increase in
expression of SOCS3 as compared to APAP-alone group (Figure 12C). At 32 h, APAP-
alone and APAP/O3 mice had similar increases in the expression of P21 and SOCS3 as

compared to control mice (Figure 12B, D).

81

Figure 12. P21 (A and B) and SOCSB (C and D) genes expression in APAP and 03
exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later
exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP administration, animals
were euthanized and liver samples collected and evaluated as described in Materials and
Methods. Data are expressed as mean :1: SE (n = 6), a, significantly different from
saline/air group; b, significantly different from saline/0.5 ppm 03 group; c, significantly
different from APAP/air group; (p S 0.05). FC, fold change.

82

Figure 12

   
  
   

A. Liver P21 expression at 9 h B. Liver P21 expression at 32 h
1 50 m Saline/O3
m Salinel03 b a n APAP/Air
1 m APAP/Air C 40 - APAP/0.503
1 - APAP/0.503
1

30
20
1 0

FC Relative to SalineIAir
O

O N -h O: on O N :5 65
FC Relative to Saline/Air

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

C. Liver SOCS3 expression at 9 h D. Liver SOCS3 expression at 32 h
.. new
.2 b a ir
< 14 ‘ Saline/O3 '- 4 a
g 12 « m APAP/Air % " APAP'O'503
a 10 . - APAP/0.503 g 3 b
(I) 8 4 8 2
8 6‘ g
g 4 1 a g 1
‘5 2 - '= o
'3 o 2
g .2 . m 33 -1
U
"" ‘4 ‘ LL -2

 

83

IV — 7. Liver Oxidative Damage (Antioxidant Genes, Glutathione and TBARS
Assays)

Heme oxygenase-l (HO-l), metallothionein-l (MT-1) and the catalytic subunit
of glutamate-cysteine ligase (GCLC) were evaluated as markers of oxidative stress. For
all three of these antioxidant genes, mRNA expression was significantly elevated with
APAP treatment whereas only MT-l was increased with 03 exposure 9 h after APAP
(Figure 13A, C, B). At 9 h, APAP/03 coexposure resulted in significant increase of MT-l
expression above APAP or 03 levels (Figure 13A). At the later time (32 h), expression
of these genes declined in APAP/air or APAP/O3 groups as compared to the early time,
and APAP/O3-coexposed mice had less or comparable mRNA expression compared to

APAP/air—treated mice (Figure 138, D, F).

84

Figure 13. MT-l (A and B), HO-l (C and D) and GCLC (E and F) genes expression in
livers of APAP and O3 exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP
ip and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized and liver samples collected in RNALater and
evaluated as described in Materials and Methods. Data are expressed as mean :t SE (n =
6). a, significantly different from saline/air group; b, significantly different from
saline/0.5 ppm 03 group; c, significantly different from APAP/air group; (p S 0.05). FC,
fold change.

85

Figure 13

A. Liver MT-l expression at 9 h

  
   

 

€16 b
314 mSaline/OS °
=12 a APAPIAir

g -APAPIO.503

310

a): 8

:3 6

E 4

o 2

u- o

C. Liver HO-l expression at 9 h

 
     
   

9.- b
5 3° Saline/03

g 25 m APAP/Air

= - APAP/0.503
g 20

3 15 a

g 10

E 5

d)

a: o

if .5

E. Liver GCLC expression at 9 h

‘6 m Saline/O3
14 a APAP/Air

12 II APAP/0.503
10

FC Relative to Saline/Air

ON-‘OO

 

86

B. Liver MT-l expression at 32 h

FC Relative to Saline/Air

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

15 ‘ s: Saline/03
m APAP/Air
1o . .? - APAP/0.503
5 ' b
o \ ...=_—_
as
-5 .

. Liver HO-l expression at 32 h

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

§15 ' m Saline/O3

g m APAP/Air

$10 . - APAP/0.503

‘9 a

E 5 ‘ ”l... b
E t: 5 c
as o '” "

o m

LI.

F. Liver GCLC expression at 32 h
314 . m Saline/O3

312 , m APAP/Air

g - APAP/0.503

m 10 ‘

2 8 -

.‘z’ 6 ‘

E 4 -

O

n: 2 -

g o .W

 

To explore further 03 exacerbation of APAP-induced liver toxicity and the role of
oxidative stress, we evaluated concentrations of GSH and GSSG. At 9 h, total glutathione
concentration was greater in APAP alone and APAP/03 coexposure groups than in
control animals (Figure 14A). At the same time, OS-exposed mice had less total
glutathione concentration in the liver compared to control mice (Figure 14A).
Interestingly, 03 alone and APAP/O3 groups had more GSSG than control and APAP
alone groups, respectively (Figure 14B).

We also evaluated lipid peroxidation levels in livers using TBARS assay. Mice
coexposed to APAP and 03 had greater concentrations of TBARS at the early time
postexposure relative to APAP alone-treated mice. (Figure 14C). 03 alone-exposed mice
had no significant increase in the concentration of TBARS compared to controls (Figure
14C). By 32 h, TBARS concentrations in APAP alone and APAP/03 groups were less

than that of their respective control groups (Figure 14D).

87

Figure 14. Total (A) or oxidized (B) glutathione and TBARS (C and D) concentrations in
livers of APAP and O3 exposed mice. Animals were given 0 (saline) or 300 mg/kg APAP
ip and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized and liver samples collected and evaluated as
described in Materials and Methods. Data are expressed as mean :t SE (n = 6). a,
significantly different from saline/air group; b, significantly different from saline/0.5 ppm
03 group; c, significantly different from APAP/air group; (p _<_ 0.05).

88

Figure 14

A. Liver total glutathione at 9 h B. Liver oxidized glutathione at 9 h

   

g 1:: Saline/Air . _

= 3 m Saline/O3 a :60 D Sa'lne’A"

g m APAP/Air g 50 ‘9 Saline/Q3

2 - APAP/0.503 3 fl APAP/Alf

o 2 a 40 - APAP/0.503

g 2 a

v o 30

a 2...

1 ..

(D

2 § 10

8 o ‘3 o

C. Liver TBARS at 9 h D. Liver TBARS at 32 h
:1 Saline/Air S |' /A'
m Saline/O3 : $3.335;
m APAP/Air B APAP/A'
— APAP/0.503 — APAP/0.IgO3

   

TBARS (nmollg liver)
TBARS (nmollg liver)

89

V. DISCUSSION

To our knowledge, this is the first study to examine the pulmonary and systemic
effects of APAP/O3 coexposure in laboratory animals. Acute exposure to 0.25 or 0.5
ppm 03 alone did not cause pulmonary inflammation in the mice of our study, as
evidenced by BALF analysis. In contrast, APAP treatment alone did cause acute
inflammation of the lung, and this drug-induced pulmonary response was exacerbated by
03 coexposure. The most remarkable finding, however, was that a single 6 h inhalation
exposure to O3 resulted in exacerbation of APAP-induced liver injury. After a
hepatotoxic dose of APAP, exposure of mice to 0.5 ppm 03 resulted in a 60% increase
in hepatocellular necrosis and an 80% decrease in hepatocellular regeneration as
compared to mice treated with APAP alone. How a single acute exposure of 03 caused
such marked enhancement of this drug-induced liver injury in mice is unknown.

It is not likely that 03 caused direct injury to the liver, since it is one of the most
reactive chemicals known and others have demonstrated that when inhaled it reacts
quickly with airway surface lining fluid and is converted to secondary lipid ozonation
products (Miller, 1995; Pryor, 1992; Pryor et al., 1995). These secondary products (e.g.,
aldehydes, hydroxyhydroperoxides etc) are thought to be the principal toxicants
responsible for 03’s toxicity to epithelial cells lining the airway surfaces. Therefore 03
could not have entered the systemic circulation to be transported from the lung to the
liver resulting in direct hepatotoxicity.

Though the present study was not designed to definitively determine how acute

inhalation exposure to 03 caused exacerbation of APAP-induced liver injury, the results

90

of our biochemical and molecular analyses suggest some plausible hypotheses that will
have to be addressed in future studies.

One possibe hypothesis is that the O3—induced enhancement of APAP
hepatotoxicity is due in part to increased oxidative stress in the liver (i.e., more injurious
oxidant free radicals than protective antioxidants). Goldstein et al. (1978) suggested that
extrapulmonary effects of 03 are related to lipid oxidation products, particularly
malonaldehyde released after the interaction of 03 with airway epithelial cell membrane
fatty acids. Interestingly, oxidative stress has also been suggested to be play a key role in
the progression of APAP-induced liver injury, specifically through induction of
mitochondrial permeability transition pore formation (Jaeschke et al., 2003). It is
plausible that the systemic increase in oxidative stress induced by inhaled 03 may have
been partially responsible for the exacerbation of both APAP-induced liver injury
observed in of our study.

In the present study, APAP/O3 coexposure resulted in significant expression of
the oxidative stress-responsive genes, MT-l, HO-l, and GCLC in the liver. We also
found that 03 exposure in either APAP- or saline—treated mice caused significant
increases of hepatic GSSG, another indicator of oxidative stress (i.e., oxidation of GSH).
In addition, lipid peroxidation, an indicator of oxidant-induced cellular injury, was
biochemically evident in the livers of O3-exposed mice.

The greatest measured response in antioxidant gene expression in the liver of
APAP/O3-coexposed mice was MT-l. Metallothioneins, including MT-l, are cysteine-
rich proteins with various protective roles including antioxidant properties (Kang, 2006).

Both APAP and 03 have been shown to induce increases of these proteins in the liver

91

and lung of mice, respectively (Johnston et al., 1999; Wormser and Calp, 1988). In
addition, mice lacking MT-l and MT-2 are more sensitive to APAP or 03 toxicity
compared to their wild-type counterparts (Inoue et al., 2008; Liu et al., 1999). Last et al.
(2005) also reported that mice exposed to O3 exhibited greater hepatic expression of MT -
1 compared to air-exposed animals.

The antioxidant effects of MTs have been ascribed to their abundant cysteine
moieties and direct scavenging properties of phenoxyl or hydroxyl radicals and
superoxide anions as demonstrated by in vitro studies (Schwarz et al., 1994).
Alternatively, the effect of MTs could be due to antioxidant properties of zinc released
from MTs by reactive oxygen species (Powell, 2000; Schwarz et al., 1994). Owing to
these roles, it is probable that the greater induction of MT-l in the combined APAP and
O3 coexposure was in response to enhanced oxidative stress.

With- immunohistochemical analysis, we found that mice given APAP had
conspicuous accumulation of HIP-la in hepatocytes immediately surrounding the
centrilobular areas of necrosis. These same hepatocytes had concurrent loss of
cytoplasmic glycogen as demonstrated by PAS histochemistry. It was these HIF-lot-
overexpressing, glycogen-depleted hepatocytes at the edge of the drug-induced necrotic
regions that appeared microscopically to be further altered (e. g., ballooning degeneration
and necrosis) by acute coexposure to 03. It is known that APAP directly targets
mitochondria inhibiting oxidative phosphorylation and compromising ATP synthesis with
subsequent induction of glycolysis (Kon et al., 2004). APAP also induces HIP-la
accumulation in a hypoxia-unrelated, oxidative stress-dependent fashion (James et al.,

2006). In addition, HIF-lot induction results in decreased oxidative phosphorylation

92

and stimulation of glycolysis in the liver of mice (Denko, 2008). Thus, glycogen
depletion in the peri-necrotic hepatocytes in APAP-treated mice might have been
mediated by HIF—loc in our study.

03 inhalation is also known to compromise cardiopulmonary function by
increasing breathing frequency and pulmonary resistance, and decreasing tidal volume,
forced vital capacity, heart rate, and mean arterial pressure (EPA, 2008). These
alterations in function could decrease the delivery of adequately oxygenated blood to
distant extrapulmonary organs and enhance damage in poorly oxygenated areas like the
centrilobular hepatic zone. Interestingly, other studies in mice have recently demonstrated
that chronic intermittent hypoxia can cause lipid peroxidation in the liver and exacerbate
APAP-induced hepatic injury (Savransky et al., 2007). It is therefore reasonable to
believe that the APAP-induced loss of glycogen and increased HIP-10L in these
hepatocytes might have made these cells more susceptible to O3 toxicity.

In the present study, PAI-l expression was increased in the livers of APAP-
treated mice similar to that reported by others (Bajt et al., 2008; Ganey et al., 2007; Reilly
et al., 2001). In contrast, I APAP/O3-coexposed mice in our study had markedly less
PAI—l liver expression as compared to mice given APAP alone. PAI—linhibits
plasminogen activators involved in the formation of plasmin and as such inhibits
fibrinolysis in mice (Bajt et al., 2008). In mice deficient in PAI-l, APAP caused greater
plasma ALT activity, hepatocellular necrosis and reduced hepatocellular regeneration
(Bajt et al., 2008). Like these PAI-l deficient animals, the APAP/O3-coexposed mice in
our study had greater hepatocellular injury and impaired hepatocellular repair (i.e.,

reduction in BrdU labeling index). One explanation for the role of PAH in APAP/O3 co-

93

toxicity could be that reduced PAI-l led to early fibrinolysis in these animals which then
resulted in an ischemia/reperfusion-like mechanism.

It has been reported that P21 mRNA was increased in APAP-treated, PAI-l
deficient mice (Bajt et al., 2008). In our study, APAP/O3-coexposed mice had reduced
expression of PAH compared to APAP alone and an increase in the expression of P21
mRN A. P21 halts the cell cycle in the G1 phase by inhibiting the activity of cyclinE/cdk2
complexes (Weinberg and Denning, 2002). Therefore it is possible that the enhanced
liver pathology in APAP/O3 -coexposed mice might be due in part to an O3-induced
reduction in PAI—l which in turn caused an increase in P21 that led to impairment of
hepatocellular regeneration.

As mentioned above, the results of our study clearly demonstrated that 03
exposure significantly impaired reparative hepatocellular regeneration in APAP-treated
mice. This suggested to us that the O3 enhancement of APAP-induced liver injury may
be due in part to reduced repair mechanisms. Mehendale and collaborators have
proposed that the severity of acute chemical-induced liver toxicity is strongly dependent
upon tissue repair processes (Soni et al., 1999). They have shown that cotreatment with
small doses of hepatotoxicants (e.g., chlordecone and carbon tetrachloride, CCl4) can
cause synergistic toxicity by inhibition of tissue repair (Soul and Mehendale, 1998). The
differential expression of several genes in the livers of APAP/O3-coexposed mice in our
study suggests some possible mechanisms by which 03 exposure might have
compromised the hepatocellular regeneration after APAP-induced injury. For example,
IL-6 gene and protein expression in the liver of APAP/O3-coexposed mice was

significantly reduced compared to that in mice treated with APAP alone. This correlated

94

with the marked reduction in hepatocellular BrdU-labeling (reduced DNA synthesis).
IL-6 is known to be an essential protein for the initial phases of hepatocellular
regeneration, transitioning cells from the GO to the GI phase of the cell cycle (Fausto et
al., 2006; Taub, 2004).

Cressman and co-workers (1996) have shown that after partial hepatectomy, mice
deficient in IL-6 had greater hepatocellular injury and reduced reparative regeneration as
compared to IL-6 sufficient hepatectomized mice. IL-6 deficient mice treated with CCl4
had greater hepatocellular damage and decreased number of hepatocytes in the S phase of
the cell cycle as compared to IL-6 sufficient mice (Kovalovich et al., 2000). Pretreatment
of IL-6 deficient mice with IL-6 significantly reduced CCl—4-induced liver injury and
restored the reparative induction of DNA synthesis. We found that 03 inhalation caused
impairment of hepatocellular repair following APAP-induced injury. Though the
downregulation of hepatic IL-6 expression may be responsible for impaired liver
regeneration 32 h after APAP, there may be other mechanisms involved.

Other potential candidates responsible for the impaired regeneration in
APAP/O3-coexposed mice are P21 and MCP—l. In APAP/O3-coexposed mice,
expression of P21 was increased relative to mice given APAP or 03 alone. Activation of
P21 after DNA damage is known to delay or arrest the cell cycle (Garner and Raj, 2008).
APAP treatment or 03 exposure cause DNA damage in the liver or lung, respectively
(Bornholdt et al., 2002; Hongslo et al., 1994; Ito et al., 2005; Ray et al., 1990). In the
present study, mice treated with APAP or exposed to O3 alone had greater hepatic P21
expression than saline-treated and air-exposed control mice. APAP/O3-coexposed mice

had even greater P21 expression compared to mice receiving only one of these chemical

95

agents. Though the level of DNA damage in the liver was not measured in this study, the
marked increase in P21 expression in the liver of coexposed mice might have been due to
increased DNA damage which is known to lead to hepatic cell death (Corcoran and Ray,
1992).

Another interesting finding in this study was the effect of APAP and 03 on the
expression of the inflammatory chemokine MCP-l in the liver. APAP treatment caused a
significant increase in the expression of this chemokine, but 03 exposure caused a
marked reduction in MCP-l expression. This reduction was associated with enhanced
liver toxicity and defective hepatocellular regeneration responses. MCP-l has been
shown to be involved in cell regeneration and tissue repair in various tissues after
different types of induced cell injury (Kim et al., 2003; Low et al., 2001; Shireman et al.,
2007). Mice lacking MCP-l or the receptor for MCP-l have been reported to be more
(Hogaboam et al., 2000) or similarly (Dambach et al., 2002) sensitive to APAP-induced
hepatotoxicity compared to their wild-type counterparts. Interestingly, mice lacking the
receptor for MCP-l also had decreased reparative DNA synthesis in a murine model of
arterial injury (Kim et al., 2003). It is not known how a reduction of MCP-l could lead to
impaired cell regeneration, but the role of MCP-l in the O3 enhancement of APAP-
induced hepatotoxicity should be explored in future studies.

In conclusion, we found that a single 6 h inhalation exposure of mice to high
ambient concentrations of 03 caused marked enhancement of APAP-induced
hepatotoxicity in mice. The present study was not designed to determine the underlying
mechanism(s) responsible for this observed sytemic effect caused by this common

oxidant air pollutant. Several biochemical and molecular markers of oxidative stress were

96

elevated in the livers of APAP/O3-coexposed mice compared to mice that received only
APAP or 03 alone. In addition, we found that concurrent with the enhancement of
hepatotoxicity, 03 also caused a marked attenuation of normal increases in DNA
synthesis necessary for hepatocellular regeneration and repair in response to chemical-
induced liver injury. This specific finding suggests a possible role of impaired cellular
regeneration and enhanced toxicity in the liver of coexposed mice. Though it is unclear
how inhalation of this highly reactive gas could enhance chemical-induced liver injury,
several endogenous hepatic proteins such as IL-6, HIP-la, PAI-l, P21, and MCP—l have
been identified as potentially playing important roles.

These results in mice also suggest that exposures to high ambient concentrations
of O3 and other air pollutants (e.g., particulate matter) may pose a risk to people with
pre—existing chemical-induced and other liver diseases. Further studies are needed not
only to understand the biologicallmechanisms underlying this sytemic effect of inhaled
03, but also to determine the smallest doses of O3 and APAP at which these synergistic
responses in the liver occur. In addition, epidemiological studies investigating the
potential interactive effects of pharmaceutical agents and air pollutants on both hepatic
and pulmonary disease appear to be warranted, especially in the light of recent reports
that headaches are commonly reported in association with increased concentrations of air
pollutants (Larrieu et al., 2009) and that APAP and other nonsteroidal, over-the—counter

pain medications, are commonly taken by the general public.

97

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CHAPTER 3

EFFECTS OF ACETAMINOPHEN AND ACUTE OZONE COEXPOSURE IN
THE LUNG OF MICE

1. ABSTRACT

Acetaminophen (APAP) is a safe drug when used within its therapeutic limits.
Overdoses of APAP cause hepatic and pulmonary toxicity in laboratory animals and
people. Ozone (03), the principal oxidant pollutant in photochemical smog is also a
pulmonary toxicant. The purpose of our study was to investigate adverse effects of APAP
and O3 coexposure in the murine lung. C57BL/6 male mice were treated with APAP (0
or 300 mg/kg ip). Two hours later, mice were exposed to 0, 0.25 or 0.5 ppm 03 for 6 h.
They were sacrificed 9 and 32 h after APAP treatment. Bronchoalveolar lavage fluid
(BALF) was collected and lungs were processed for morphometric and biochemical
analyses. At 300 mg/kg, APAP alone induced a bronchiolitis with necrosis and loss of
epithelial cell in both axial airways and terminal bronchioles. 03 alone did not cause
epithelial or inflammatory changes in either location. APAP and O3 coexposure induced
loss of epithelial cell greater than the one elicited by APAP alone, in the axial airway and
terminal bronchioles. Neutrophil numbers were increased in the BALF and airways after
APAP treatment, but were greatest with coexposure. In addition, APAP and 03
coexposure compromised the regenerative capacity of bronchiolar epithelium. Several
oxidant stress responsive genes (MT-l, GCLC) as well as the cyclin dependent kinase

inhibitor P21 were elevated in the combined treatment. Coexposure of these 2 pulmonary

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toxicants pose a greater risk when combined compared to either of these substances. This
might constitutes a health risk in certain populations such as people with preexisting
respiratory conditions or under chronic use of acetaminophen in heavily polluted urban

areas.

II. INTRODUCTION

Ozone (03) is the most pervasive oxidant air pollutant. It is generated upon the
interaction of atmospheric oxygen, ultraviolet solar radiation and anthropogenic and
biogenic pollutants (nitrogen oxides, volatile organic compounds, carbon monoxide, etc)
(EPA, 2008). As of June 2009, more than 100 million people in the United States were
located in areas that do not meet the national ambient air quality standards (NAAQS) for
O3 established by the U.S. Environmental Protection Agency (U.S. EPA)
(httpzllwww.epa.gov/oar/oaqps/greenbk/gnsum.html). Experimental studies have shown
that 03 inhalation cause pulmonary epithelial injury and inflammation and airway
hyperresponsiveness (Bhalla and Gupta, 2000; Carey et al., 2007; Depuydt et al., 1999;
Dormans et al., 1999; Dye et al., 1999; Harkema et al., 1993; Hotchkiss et al., 1997;
Hotchkiss et al., 1989; Pino et al., 1992b). More recently, several studies reported
potential interactions and harmful toxic synergies between 03 and other pollutants or
biological substances on the respiratory tract (Cassee et al., 2002; Churg, 2003;
Goldsmith et al., 2002; Han et al.,. 2008; Harkema and Wagner, 2005; Jakab and
Hemenway, 1994; Kobzik et al., 2001; Last and Pinkerton, 1997; Osebold et al., 1988;

Vincent et al., 1997; Wagner et al., 2003; Yu et al., 2002). Several of these studies

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showed that the harmful potential of these pollutants is greater in people with pre-
existing lung conditions (i.e., asthmatics, etc) and animal models of human lung
diseases compared to healthy subjects (Balmes et al., 1997; Depuydt et al., 2002;
Goldsmith et al., 2002; Last et al., 2004a; Last et al., 2004b; Miller et al., 2009; Wagner
et al., 2007). Very few studies however have investigated the interaction of commonly
used drugs and environmental pollutants. In a model of pulmonary fibrosis for instance,
instillation of bleomycin followed by 03 exposure resulted in enhanced inflammation
and fibrosis (Oyarzun et al., 2005). In addition, coexposure of rat lung fibroblasts to O3
and several antineoplastic agents showed that the combination of 03 with vitamin K3
resulted in greater injury as measured by chromium 51 radioisotope release (W enzel and
Morgan, 1983).

APAP is one of the most commonly used over-the-counter analgesic and
antipyretic and is a remarkably safe drug when used within prescribed therapeutic limits,
although this therapeutic window is narrow (Larson et al., 2005). In cases of
overdosage, APAP targets several organs including the respiratory system (Baudouin et
al., 1995; Placke et al., 1987b). In addition, frequent therapeutic use of APAP has
recently been associated with asthma and allergic rhinitis in adults and children
(Newson et al., 2000; Shaheen et al., 2000). Several studies showed that frequent use of
APAP by women in mid-to-late pregnancy resulted for the offspring in greater risk of
having higher levels of immunoglobulin E, asthma and wheezing later in life (Persky et
al., 2008; Shaheen et al., 2005).

Interestingly one of the most prevalent hypothesis through which APAP might

contribute to asthma is oxidative stress and depletion of glutathione (GSH) (Eneli et al.,

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2005; Fogarty and Davey, 2005; Shaheen et al., 2000). Moreover, both APAP and 03
have been shown to affect airway glutathione concentrations in vivo and in vitro
(Dimova et al., 2005; Micheli et al., 1994; Plopper et al., 1998). APAP or 03 also
targets Clara cells known to produce Clara cell secretory protein (CCSP). CCSP has
been shown to be protective against oxidative lung injury such as hyperoxia or 03
inhalation (Amatya et al., 2002; Stripp et al., 2000). These effects of APAP or 03 on the
airway antioxidant levels (e.g. GSH, CCSP) or Clara cells themselves might potentially
constitute the basis for a toxic synergy between these substances.

In the present study we investigated the acute effects of near ambient
concentrations of 03 on APAP-induced airway epithelial injury and inflammation in
male mice. Our hypothesis was that 03 inhalation would potentiate APAP induced
pulmonary toxic changes. The findings of this study demonstrate for the first time that
03 inhalation exacerbates APAP-induced airway epithelial injury and acute
inflammation and impairs epithelial regeneration. We found that APAP and O3
sequential exposure resulted in more Clara cell damage than either one of these

substances alone and results in greater induction of oxidative stress responsive genes.

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III. MATERIAL AND METHODS

III — 1. Laboratory Animals

Pathogen-free male, C57Bl/6 mice (8-10 weeks of age, the Jackson Laboratory
Bar Harbor, ME) were used in this study. Mice were housed in polycarbonate cages on
heat-treated aspen hardwood bedding (Nepco-Northeastem Product Corp, Warrensburg,
NY). Boxes were covered with filter bonnets, and animals were provided free access to
food (Harlan Tekad laboratory rodents 22/5 diet, Madison, WI) and water. Mice were
maintained in Michigan State University (MSU) animal housing facilities accredited by
the Association for Assessment and Acreditation of Laboratory Animal Care and
according to National Institutes of Health guidelines as overseen by the MSU
Institutional Animal Care and Use Committee. Rooms were maintained at temperatures
of 21-24°C and relative humidities of 45-70%, with a l2-hour light/dark cycle starting at

7:30 AM.

111 - 2. Experimental Protocol

Mice were randomly divided into ten groups, each consisting of six animals. They
were given intraperitoneally 0 (saline-vehicle) or 300 mg/kg APAP (Sigma Chemical
Co., St. Louis, MO) in 20 ml/kg saline. Animals were fasted overnight before the
administration of APAP. Two hours after APAP administration, mice were exposed to 0

(air), 0.25 or 0.5 ppm 03 for 6 h (Figure 1). Mice were killed 9 or 32 h after APAP (1 or

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24 h after 03 exposure, respectively). As no significant differences were detected in
preliminary studies, no morphological evaluation was conducted at 0.25 ppm for the 9 h
time point and at 32 h, data analysis in animals given 0.25 ppm was limited to
morphological evaluations (airway epithelial damage and 5—bromo-2-deoxyuridine

(BrdU) immunostaining) at the later time (32 h) (Figure 15).

 

 

 

 

  

 

 

D M Mice sacrificed
. . ay 2: ice 9 h (1 h after 03)
8-10 weeks Dav1- Mice given or 32 h (24 h after
old C57BL/6 fasted ip saline or 03) after AP AP
male mice overnight 300 mglkg treatment
arrival ApAp
l 2 h l l
1 k ” Inhalation
aim: exposure Mice given
. . 0, 0-25 0' BrdU ip 2 h
acclimation 0.5 ppm 03 before
for 5" sacrifice

 

 

 

Figure 15. Experimental design of APAP and 03 studies in the lung. (A) Eight to ten
weeks old C57BL/6 male mice were given 0 (saline) or 300 mg/kg APAP and then
exposed to ozone (0 or air, 0.25 or 0.5 ppm) for 6 h. Mice were euthanized 9 (1 h after
03 exposure) or 32 h (24 h after 03 exposure) after APAP injection.

Mice were housed individually and exposed to O3 in stainless steel wire cage,
whole-body inhalation exposure chambers (HC-100, Lab Products, Maywood, NJ). 03

was generated with an OREC 03V1-O ozonizer (03 Research and Equipment Corp., AZ)

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using compressed air as a source 'of oxygen. Total airflow through the exposure chambers
was 250 l/min (15 chamber air changes/hour). The concentration of 03 within chambers
was monitored during the exposure using Dasibi 1003 AH ambient air 03 monitors
(Dasibi Environmental Corp., Glendale, CA). Two 03 sampling probes were placed in
the middle of the 03 chambers, 10-15 cm above cage racks. O3 airborne concentrations
during the inhalation exposures were 0.26 +/- 0.02 ppm or 0.53 +/- 0.01 ppm (mean +/-
stande error of the mean) for 03 chambers and 0.01 +/- 0.008 or 0.02 +/- 0.009 ppm for

air chambers.

III - 3. Animal Necropsy, Bronchoalveolar Lavage, and Tissue Selection

for Microscopic and Biochemical Analyses

Two hours prior to scheduled sacrifice, mice were given BrdU intraperitoneally
(50 mg/kg, Fisher Scientific, Fair Lawn, NJ) for nuclear incorporation and
immunohistochemical detection of airway epithelial cells undergoing DNA synthesis
(cycling cells in S phase). At the time of necropsy, mice were anesthetized with an
intraperitoneal injection of sodium pentobarbital (50 mg/kg; Fatal Plus, Vortech
Pharmaceuticals, Dearbom, MI), the abdominal cavity was opened and blood was
collected from the abdominal vena cava in BD Microtainer tubes (Franklin Lakes, NJ).
Animals were then killed by exsanguination.

The thoracic cavity was opened by puncturing the hemidiaphragms to allow
collapse of lung lobes. After the trachea was cannulated, the heart-lung block was excised
and the lung was gently lavaged twice with 0.9 ml of sterile saline. Approximately 75-

90% of the intratracheally instilled saline was recovered as BALF from the lavaged lung

109

lobes and immediately placed on ice until further analysis. The right lung lobes were tied
off at the bronchus level and severed from the left lobe. The left lobe attached to the heart
bloc was gravity-perfusion inflated at a constant pressure of 25 cm of water for at least
1.5 hour using 10% neutral buffered formalin (NBF) (Fisher Scientific, Fair Lawn, NJ)
and then immersed in NBF for light microscopic, morphometric and
immunohistochemical analyses. The right cranial lobe was immersed in RNAlater
(Qiagen, Valencia, CA), kept at 4°C for 24 h and then transferred to a -20°C freezer for
gene expression analyses using real time PCR. The right middle, caudal and accessory
lobes were frozen and stored at -80°C for biochemical analysis of glutathione and

thiobarbituric acid reactive substances.

III - 4. Cellular Analysis of Bronchoalveolar Lavage Fluid

Total cell counts in the collected BALF from each mouse were determined using
a hemocytometer. Cytological slides were prepared using a Shandon cytospin 3 (Shandon
Scientific, Sewickley, PA), centrifuged at 600 rpm for 10 minutes and stained with Diff-
Quick (Dade Behring, Newark, DE). Differential counts of neutrophils, eosinophils,
macrophages and lymphocytes were assessed on a total of 200 cells. Remaining BALF
were centrifuged at 1500 rpm for 15 minutes to collect the supernatant fraction that was

stored at -80°C for later biochemical analyses.

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III - 5. Flow Cytometric Analyses for Inflammatory Cytokines

BALF supematants were assayed for selected inflammatory cytokines that
included interleukin-lbeta (IL-113), tumor necrosis factor-alpha (TNF-a), interferon-
gamma (IFN-y), interleukin—6 (H.-6), monocyte chemoattractant protein-1 (MCP-l),
interleukin-12 (IL-12), keratinocyte-derived chemokine (KC) and interleukin-10 (IL-10).
Plasma cytokines concentrations for KC, TNF-oc, MCP-l and IL-6 were also determined.
All cytokines were purchased as Flex Set reagents or as a preconfigured cytometric bead
array kit (BD Biosciences, San Diego, CA). Cytokines analysis was performed using a
FACSCalibur flow cytometer (BD Franklin Lakes, NJ). Briefly, 50 pl of BALF or
plasma was added to the antibody-coated bead complexes and incubation buffer. Samples
were incubated with the beads. Phycoerythrin (PE)-conjugated secondary antibodies were
then added to form sandwich complexes. Following acquisition of sample data using the

flow cytometer, cytokine concentrations were calculated based on standard curves using

FCAP Array software (BD, Franklin Lakes, NJ).

III — 6. Lung Tissue Processing for Light Microscopy and Immunohistochemistry

The left lung lobe was collected as described previously and 2 sections transverse
to the axial airway were cut at the level of the fifth (G5) or eleventh generation (G11)
from the axial airway (Figure 16). These sections were embedded in paraffin, cut at a
thickness of 5 um and stained with hematoxylin and eosin (H&E) for routine

histopathological evaluation.

111

Routine immunohistochemical techniques were used for detection of airway
epithelial cell nuclear BrdU and cytoplasmic CCSP. Neutrophils accumulation in the
airway and parenchyma was also detected and quantified using immunohistochemistry.
Briefly, lung sections were deparafinized in xylene and rehydrated through descending
grades of ethanol and immersed in 3% hydrogen peroxide to block endogenous
peroxides. Sections were incubated with normal sera to inhibit nonspecific proteins
(normal horse, rabbit or goat sera for BrdU, neutrophils or CCSP immunostaining,
respectively, Vector Laboratories Inc., Burlingame, CA) followed by specific dilutions of
primary antibodies (1:40, monoclonal mouse anti-BrdU antibody, BD, Franklin Lakes,
NJ; 1:2500, monoclonal rat anti-neutrophil antibody, AbD Serotec, Raleigh, NC; 1/1600,
polyclonal rabbit anti-CCSP antibody, Seven Hills Bioreagents, Cincinnati, OH). Tissue
sections were subsequently covered with secondary biotinylated antibodies and
immunostaining was developed with the Vector RTU Elite ABC kit (BrdU and CCSP
Vector Laboratories Inc) or the RTU Phosphatase-labeled Streptavidin kit (neutrophils,
Kirkegaard Perry Labs, Gaithersburg, MD) and visualized with Vector Red (Vector
Laboratories Inc). Slides were counstertained with Gill 2 hematoxylin (T hermo Fisher,

Pittsburgh, PA).

112

lntrapulmonary
Axial Airway Sections

 

Figure 16. Schematic representation of lung sectioning levels for histology and
morphometry. Two transverse sections were taken. One at the level of the fifth generation
(G5) from the central airway for analysis of axial airway morphology and morphometry
and the other one at the eleventh level (G11) for terminal bronchioles evaluation.

III — 7. Lung Morphometric Analyses

Evaluation of epithelial, inflammatory or proliferation changes in the axial airway
or terminal bronchioles in the section taken at at the level of the fifth (G5) and the one
taken eleventh (G11) bifurcation showed no differences. Therefore, morphometric
evaluation of axial airway changes were conducted at G5 while terminal bronchioles

evaluation was done at the level of G11. Evaluation of alveolar septa neutrophils was

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conducted at the level of G5 as no differences were detected between sections at G5 and
G11.

Bromodeoxyuridine stained and unstained airway epithelial cell nuclei were
counted in the axial airway in sections taken at the level of G5 and in all terminal
bronchioles in sections taken at the level of G11. The BrdU labeling index for the axial
airway (AA) or terminal bronchioles (TBS) was determined by dividing the number of
BrdU positive cells by the number of total (stained and unstained) epithelial cells and
multiplying by 100 (Cho et al., 1999). Similarly, the CCSP labeling index was
determined by dividing the CCSP stained cells (cytoplasmic staining) by the total stained
and unstained cells and multiplying by 100. To estimate the amount of the intraepithelial
CCSP in the airway epithelium, the volume density (Vs) of CCSP staining was quantified
using computerized image analysis and standard morphometric techniques. The area of
CCSP staining was calculated from the automatically circumscribed perimeter of stained
intraepithelial material using the public domain NIH Image program (written by Wayne
Rasband, U.S. National Institutes of Health). The length of the basal lamina underlying
the surface epithelium was calculated from the contour length of the digitized image of
the basal lamina. The v01ume of stored CCSP per unit of surface area of epithelial basal

lamina was estimated using the method described in detail by Harkema and collaborators

2
(Harkema et al., 1987). The Vs of CCSP is expressed as nanoliters of CCSP per mm of

basal lamina.
Proximal or distal airway acute inflammatory cell accumulation was assessed by
counting the numbers of immunohistochemically labeled neutrophils (cell membrane

labeling) in the axial airway (G5) or terminal bronchioles (G11) Surface epithelium,

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respectively, divided by the length of the underlying basal lamina (Cho et al., 1999). The
length of the basal lamina underlying the surface epithelium was calculated from the
contour length of the basal lamina, by using a National Institutes of Health (NIH) image
analysis software (NIH Image; written by Wayne Rasband at the U.S. NIH). It is reported
as the number of neutrophils per mm of basal lamina. Alveolar septa neutrophil
accumulation was assessed by averaging the numbers of neutrophils enumerated in 5
medium power fields (X200) in each slide. Analyzed fields were selected in an unbiased
manner with a random start and count of every other field. Fields comprising the axial
airway and major pulmonary vessels were excluded from evaluation.

To quantify APAP and/or O3-induced epithelial injury, morphometric analyses
were conducted on H&E sections. The numeric epithelial cell density (i.e., number of
epithelial cell per mm of basal lamina) was determined by counting the total number of
surface epithelial cell nuclear profiles in transverse airway sections normalized to the
length of the underlying basal lamina (Cho et al., 1999). Numeric cell densities were
evaluated in the axial airway at the level of the fifth generation (Figure 2, G5) and in all
terminal bronchioles in the section taken at the level of the eleventh bifurcation from the
axial airway (Figure 2, GI 1). A terminal bronchiole was identified as any airway portion
blending into an alveolar bed on one side and connecting to another airway (first
junction) on the other side. For the terminal bronchioles, the numeric cell density
presented is the mean of numeric densities for each section. The length of the basal
lamina was measured using the NIH Image program (Wayne Rasband, U.S. National

Institutes of Health).

115

111 — 8. Quantitative Real Time PCR

Total RNA was extracted using RNeasy Mini Kit according to the manufacturer’s
instructions (Qiagen, Valencia, CA). Briefly, lung tissues were homogenized in RLT
buffer containing B-Mercaptoethanol with a 5 mm Rotor-Sator Homogenizer (PRO
Scientific, Oxford, CT) and centrifuged at 10,700 rpm for 3 minutes. Samples were then
treated with Rnase-Free Dnase set on the column for 30 minutes. Eluted RNA was
diluted 1:5 with Rnase free water and quantified using a GeneQuant Pro
spectrophotometer (BioCrom, Cambridge, England).

Reverse transcription (RT) reaction was performed using reverse transcription
high capacity cDNA reagents (Applied Biosystems, Foster City, CA) and a GeneAmp
PCR System 9700 Thermocycler PE (Applied Biosystems). Each RT reaction was run in
5 pl of sample with 20 ul of cDNA Master Mix prepared according to the manufacturer’s
protocol (Applied Biosystems).

Expression analyses of isolated mRNA were performed by quantitative real-time
PCR using individual animals’ cDNA with the ABI PRISM 7900 HT Sequence Detection
System using Taqman® Gene Expression Assay reagents (Applied Biosystems). The
cycling parameters were 48°C for 2 minutes, 95°C forlO minutes, and 40 cycles of 95°C
for 15 seconds followed by 60°C for 1 minute. Individual data are reported as fold
change of mRN A in experimental samples compared to the saline/air control group. Real-
time PCR amplifications were quantified using the comparative Ct method normalized to
the mean of 2 endogenous controls (188 and GAPDH). The cycle number at which each

amplified product crosses the set threshold represents the Ct value. The amount of target

116

genes normalized to the mean of the endogenous reference genes was calculated by
subtracting the endogenous reference Ct from the target gene Ct (ACt). Relative mRNA
expression was calculated by subtracting the mean ACt of the treated samples from the
ACti of the control samples (saline-treated, air-exposed) (AACt). The absolute values of

the comparative expression level (fold change) was then calculated by using the formula:

-AACT
Fold change = 2 .

III — 9. Glutathione Assay

Lung total (reduced or GSH and oxidized or GSSG) and oxidized glutathione
were homogenized in cold MES buffer (0.4 M 2-(N-morpholino)ethanesulfonic acid, 0.1
M phosphate, and 2 mM EDTA, pH = 6). The homogenates were then centrifuged at
9,700 rpm for 15 minutes at 4°C and the supemanant collected and deproteinated. The
total glutathione concentration was assayed on the deproteinated samples as
recommended by the manufacturer (Cayman Chemical Co., Ann Arbor, MI). GSSG
concentration was determined after derivatization of reduced glutathione with
vinylpyridine. Sample absorbance was determined at 405 nm, and the total or oxidized
glutathione concentration in lung homogenates was assessed by comparison of absorption

to standard curves.

117

III - 10. Thiobarbituric Acid-Reactive Substances Assay (TBARS Assay)

Lipid peroxidation in the lung was estimated using a commercially available kit
according to the manufacturer’s recommendations and malonaldehyde as a standard
(TBARS kit, Cayman Chemical Co., Ann Arbor, MD. Lung tissue was homogenized on
ice in RIPA Buffer and Proteases Inhibitor (Thermo Scientific, Rockford, IL). The
homogenates were centrifuged at 3,900 rpm, and the supernatant was collected and used
to detect malonaldehyde and thiobarbituric acid adducts in acidic conditions and under

high temperature (100°C). Absorbance was measured at 530 nm.

111 — 11. Statistical Analysis

Data are reported as mean +/- SE. Differences among groups were analysed by a
one or two-way ANOVA followed by Student-Newman-keuls post hoc test. When
normality or variance equality failed, a Kruskal-Wallis ranked test was conducted. All
analyses were performed using a SigmaStat software (SigmaStat; Jandel Scientific, San

Rafael, CA). Significance was assigned to p values smaller than or equal to 0.05.

118

IV. RESULTS

IV - 1. Lung Histopathology and Morphometric Assessment of Epithelial
Injury and Inflammation

APAP treatment (APAP alone or APAP/air) or 03 exposure (03 alone or
SAUO3) were compared to effects of the combined treatment (APAP/O3 or APAP and
O3-coexposed groups) or controls (SAL/air). 03 exposure did not induce
histopathological changes in the lung airway 9 or 32 h after saline administration (Figure
17A, B). At 9 h, APAP administration caused limited airway epithelial degeneration with
very few epithelial necrosis (data not shown). Epithelial degeneration was characterized
by cell swelling with cytoplasmic clarification and vacuolation. At 32 h, APAP treatment
resulted in epithelial degeneration, necrosis and exfoliation along the entire length of
airway epithelia (Figure 17C). Histopathological evaluation also revealed that both Clara
and ciliated cells were affected by APAP administration although Clara cells seemed to
be predominantly targeted (data not shown). Along the airway tree, APAP-treated
animals apparently had more epithelial damage in the axial airway compared to the
terminal bronchioles (see below, airway epithelium morphometry results). APAP and O3-
coexposed mice had changes similar to the APAP/air group with extension in severity of
both degeneration and necrosis of airway epithelial cells (Figure 17D). Very few
inflammatory cell were detected in H&E slides in the axial airway or terminal
bronchioles epithelium in the combined treatment but not with APAP or 03 alone (data

not shown).

119

A. Saline/Air B. Saline/0.5 ppm 03

 

Figure 17. Axial airway epithelial damage induced by APAP and 03 treatment 32 h after
APAP. Thirty-two hours after APAP administration, animals were euthanized, left lung
lobe was collected and evaluated morphologically. Solid arrow, airway epithelial
degeneration; asterisk, airway epithelial necrosis and exfoliation.

120

Morphometric evaluations showed that 03 alone had no effects on epithelial cell
densities at any time (Figure 18A-D). At 9 h, APAP alone caused a slight nonsignificant
decrease of epithelial cells in the axial airway but not in terminal bronchioles (Figure
18A, C). At the same time, 0.5 ppm 03 exposure enhanced APAP—induced airway
epithelial damage as the APAP/0.5 ppm 03 co-treatment was the only group with
significant loss of epithelial cell in the axial airway (Figure 18A, C). At 32 h, the cell loss
due to APAP alone treatment in the axial airway reached statistical significance, while in
the terminal bronchioles a nonsignificant reduction of the number of epithelial cell was
observed (Figure 18B, D). At 32 h, epithelial loss further increased in the axial airway of
the APAP/O3 co-treated group in an O3 dose-dependent way, while in the terminal
bronchioles APAP and O3 coexposure-induced airway epithelial loss reached statistical

significance (Figure 18B, D).

121

Figure 18. Epithelial numeric cell density in APAP and 03 treated mice 9 and 32 h after
APAP in the axial airway (A and C) and terminal bronchioles (B and D). Animals were
injected ip with 0 (saline) or 300 mg/kg APAP and 2 h later exposed to 0 (air), 0.25 or 0.5
ppm 03 for 6 h. 9 or 32 h after APAP administration, animals were euthanized and left
lung lobe collected, routinely stained and evaluated as described in Material and
Methods. Data are expressed as mean :1: SE, (n = 6). a, significantly different from
saline/0.5 ppm 03 group; b, significantly different from saline/air; c, significantly
different from APAP/air group; d, significantly different from saline/0.25 ppm 03 group;
(p_<_0.05). BL, Basal lamina.

122

Figure 18

   
   

A. Axial airway epithelial B. Axial airway epithelial
density at 9 h density at 32 h
:1 Saline/Air
l: Saline/0.2503
:1 galinelAi; igr§Q3503
m 1' IO Ir
120 E Agx'FEIAir —| 140 _ cm APAP/0.2503
-' - APAP/0.503 '3 - APAP/0.503
5100 a E 120 -
g 80 ' 2 10° ‘
so {323:
_ ID ’0’
g 40 = 40 . 52:
3 20 3 $222525:
'3 'a 20 5555::
Lu 0 I“ 0

 

 

C. Terminal bronchioles D. Terminal bronchioles
epithelial density at 9 h epithelial density at 32 h

[:1 Saline/Air
E Saline/0.2503
m Saline/0.503

1:1 Saline/Air m APAP/Air
ES! Saline/Q3 4140 _ um APAP/0.2503
4160 2£B£3581g03 m - APAP/0.503
m - E 120 -
£100
2
T:
U
.72
3
5
a s

 

   

123

No neutrophil infiltration was seen in the axial airway or terminal bronchioles
epithelium with any treatment regimen at 9 b (Figure 19A, C). Later, at 32 h, 03-
exposure had no effect on neutrophil numbers in the airways while APAP alone caused
neutrophils accumulation that reached statistical significance in the axial airway (Figure
19B, D). 03 at a dose of 0.5 ppm significantly increased APAP-induced neutrophil
accumulation in the epithelium of the axial airway but not terminal bronchioles (Figure
19B, D).

Unlike airways, alveolar septa had significant neutrophil accumulation in APAP
alone or APAP and O3-coexposed mice 9 h after APAP (Figure 19E). At the later time,
neutrophil accumulation progressed in both APAP alone and APAP/0.5 ppm 03 groups

but no significant differences were detected between these groups (Figure 19F).

124

Figure 19. Lung neutrophil infiltration in APAP and 03 treated mice 9 and 32 h after
APAP in the axial airway (A and B), terminal bronchioles (C and D) and alveolar septa
(E and F). Animals were injected ip with 0 (saline) or 300 mg/kg APAP and 2 h later
exposed to 0 (air), 0.25 or 0.5 ppm 03 for 6 h. Lung sections were
immunohistochemically stained and neutrophils evaluated in the airways or alveolar septa
as described in Material and Methods. Data are expressed as mean :1: SE, (n = 6). a,
significantly different from saline/air group; b, significantly different from saline/0.25
ppm 03 group; c, significantly different from saline/0.5 ppm 03 group; d, significantly
different from APAP/air group; (pS0.05). ND, not detected; BL, basal lamina.

125

Figure 19

A. Axial airway

neutrophils at 9 h
ii I: Saline/Air

4 IS! Saline/O3

E m APAP/Air
g - APAP/0.503
i 2
E
‘5
o
2

O

C. Terminal bronchioles

neutrophils at 9 h
_l
m 4 1:1 Saline/Air
E m Saline/O3
E u APAP/Air
.‘L’ - APAP/0.503
E
a 2
2
‘5
a:
2

O

E. Alveolar septa

neutrophils at 9 h

120
g 1:: Saline/Air
“100 in Saline/O3
'L" 80 Iii“APPA PlAir
g - APAP/0. 503
i so
9
*5 40
o
z 20

 

 

 

B. Axial airway

 
   
 
 
    

neutrophils at 32 h

10 - 1:1 Saline/Air
.1 E Saline/0.2503 c
m 8 . Saline/0.503 d
E m APAP/Air
E 6 _ [DJ APAP/0.2503
g - APAP/0.503
.C
3 4 '
a 2 -
z

o l

 

D. Terminal bronchioles

  
  
 
 
  

neutrophils at 32 h

10 . I: Saline/Air c
_, ‘3 Same/0.2503
m Saline/0. 503
E 8 ‘ mAPAP/Ai
E [n APAP/0. 2503
E 6 - - APAP/0. 503
E.
e 4'
3 2 -
z

0

 

 

F. Alveolar septa
neutrophils at 32 h

:1 Saline/Air

=1 Saline/0.2503 C

‘ “ Saline/0i r503

- m APA PIA

_ r11] APAP/0. l2503
- APAP/0. 503

dAA
QON-h
OOOO

Neutrophils/5 FP
a 01
O O

 

N
OD

126

IV — 2. Bronchoalveolar Lavage Fluid Inflammatory Accumulation

03 exposure did not cause changes in BALF total inflammatory cells at any time
when compared to control mice (Figure 20A-F). Compared to controls, APAP alone or
APAP and O3 coexposure caused a time-dependent, statistical increase in the number of
total inflammatory cells in the BALF at 9 and 32 h after APAP administration (Figure
20A, B). Although not significant, there was a trend for greater total inflammatory cells
in the lungs of the APAP and O3-coexposed mice compared to APAP alone (Figure 20A,
B).

Pulmonary inflammatory cell responses as reflected in the BALF were due to
increases in macrophages and/or neutrophils (Figure 20C, D, E, F). 03 alone did not
cause changes in neutrophil or macrophages in BALF at any time (Figure 20C, D, E, F).
At 9 and 32 h, mice exposed to APAP alone or APAP and O3-coexposed mice had a
time—dependent increase of macrophages and neutrophils (Figure 20C, D, E, F). APAP
alone and APAP/03 groups were the only ones with detectable levels of neutrophil
infiltrates in the BALF at 9 h (Figure 20E). At the later time, APAP and O3-coexposed
mice had marked, 03 dose-dependent increase in neutrophil numbers in BALF (Figure

20F), indicating a synergistic neutrophil effect of APAP and O3 coexposure.

127

Figure 20. Inflammatory cell accumulation in bronchoalveolar lavage (BALF) in APAP
and 03 treated mice. Total inflammatory cells (A and B), macrophages (C and D) and
neutrophils (E and F) per ml of BALF 9 and 32 h after APAP administration. Animals
were injected ip with 0 (saline) or 300 mg/kg APAP and 2 hours later exposed to 0 (air),
0.25 or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP administration, animals were
euthanized and BALF harvested and analyzed as described in Material and Methods.
Data are expressed as mean :l: SE, (n = 6). a, significantly different from saline/air group;
b, significantly different from saline/ 0.5 ppm 03; c, significantly different from
saline/0.25 ppm 03; (p S 0.05). ND, not detected.

128

Figure 20

A. BALF total cells at 9 h

  

 

 

      

_ 200

E El Saline/Air
$150 53 Saline/03

2 n APAP/Air

25, - APAP/0.503 a
2 100

8

E 50

o

l-

O

C. BALF macrophages at 9 h

N
O
O

E! Saline/Air
Saline/O3
m APAP/Air
- APAP/0.503

.3
0|
O

    

0|
O

Macrophages (X103)/m|
O
c

O

E. BALF neutrophils at 9 h

E10

> 1:: Saline/Air
"E3 8 m Saline/O3
5 e m APAP/Air

2 _ APAP/0.503
g 4

9

*5 2

g

o

 

B. BALF total cells at 32 h

’ 1:: Saline/Air
. :1 Saline/0.2503

' m APAP/Air
' - APAP/0.503

 
  
  
   

m Saline/0.503 a
m APAP/0.2503 §

 

 

D. BALF macrophages at 32 h

NM
GUI
CO

Macrophages (x103)/mI

‘ 1:: Saline/0.2503
‘ [SS Saline/0.503

150 -

.a
UIO
0°

 

O

:1 Saline/Air

 
  
  
   

m APAP/Air ,,,,,
In APAP/0.2503
- APAP/0.503

.....

F. BALF neutrophils at 32 h

-50-
E

«3‘40 -

 

129

  
 
 
  
   

1:: Saline/Air
I: Saline/0.2503
ES Saline/0.503
a APAP/Air

cu APAP/0.2503
- APAP/0.503

““‘

IV - 3. Relative Genes Expression and Protein Concentrations of
Inflammatory Cytokines in the Lung and BALF

In order to investigate factors responsible for the acute pulmonary inflammation
in the coexposure group, several chemokines involved in neutrophil trafficking were
evaluated in lung tissues. Neutrophils chemoattractants KC and MIP-2 expression were
significantly elevated with 03 but not with APAP at 9 h (Figure 21A, C). At this early
time, APAP and O3-coexposed animals had 3 times more MIP-2 expression than 03-
exposed mice, although significance was not observed (Figure 21A, C). At 32 h post-
APAP, KC and MIP-2 expressions declined in 03 or APAP/O3 groups from expression
levels seen at 9 h and no differences were present between SAL/O3, APAP/air or
APAP/O3 groups (Figure 21B, D).

Macrophages infiltration was not evaluated at the tissue level. However, in the
BALF of APAP alone or APAP and O3-coexposed mice, there was an increase of
macrophages accumulation compared to controls (Figure 20C, D). At 9 h, MCP-l, a
monocyte chemokine, had increased relative expression in APAP or 03 alone groups but
was greatest in the coexposed group compared to either substance alone (Figure 21E). At
32 h, mRNA expression of MCP-l in the APAP and O3-coexposed group slightly

declined but was still above APAP or 03 alone group expression level (Figure 21F).

130

Figure 21. KC (A and B), MIP-2 (C and D) and MCP-l (E and F) genes expression in
the lung of APAP and 03 treated mice. Animals were injected ip with 0 (saline) or 300
mg/kg APAP and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized and lung samples collected in RNAlater. Data
are expressed as mean :t SE, (n = 6). a, significantly different from saline/air group; b,
significantly different from APAP/air group; c, significantly different from saline/O3
group; (p50.05). FC, fold change.

131

II-“l':lllla " ‘l

‘I‘l.ll (I.

 

 

Figure 21

A. Lung KC expression at 9 h

cs: Saline/O3 b
m APAP/Air
- APAP/0.503

    

FC Relative to Saline/Air
O -I N w «5 GI 01

C. Lung MIP-2 expression

at9h

314 mSalineIO3 b
:12 mAPAP/Air
= -APAP/0.503
«:10

0)

g 8

E 6

E 4

d)

n: 2

E o

E. Lung MCP-l expression
at 9 h

m Saline/O3
m APAP/Air
- APAP/0.503

00'

FC Relative to Saline/Air
o .- N w -h 01 a:

 

 

B. Lung KC expression at 32 h

 
     
  

3: 3 Saline/O3
E m APAP/Air

g 2 - APAP/0.503
(U

(D

8 1

.°z’ o

5

Q4

8.2

D. Lung MIP-2 expression
at 32 h

m Saline/O3
m APAP/Air
- APAP/0.503

N

  
    

.3

 
  
 

0 0
90‘0 0 0.0.0
.‘0‘0’0’0'0’0
03.0.0.0.»

FC Relative to Saline/Air
l. o

I
N

F. Lung MCP-l expression
at 32 h

m Saline/O3
m APAP/Air
- APAP/03

  
 
   

a

FC Relative to Saline/Air

 

Two other markers of inflammation were evaluated in the lung tissue. These
include a pro-inflammatory cytokine, IL-6, and an enzyme involved in the generation of
inflammatory mediators, cyclooxygenase 2 (COX-2). At 9 h, IL-6 but not COX-2
expression was elevated in APAP or 03 alone over controls (Figure 22A, C). At the same
time, APAP and O3-coexposed mice had significant elevation of IL-6 and COX-2
expression compared to either APAP or 03 alone (Figure 22A, C). At 32 h, APAP alone,
03 alone or APAP/O3 had similar mRNA expressions of IL-6 or COX-2 (Figure 22B,

D).

133

Figure 22. IL-6 (A and B), and COX-2 (C and D) genes expression in the lung of APAP
and 03 treated rrrice. Animals were injected ip with 0 (saline) or 300 mg/kg APAP and
2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP administration,
animals were euthanized and lung samples collected in RNAlater. Samples were
evaluated as described in Materials and Methods. Data are expressed as mean :1: SE, (n =
6). a, significantly different from saline/air group; b, significantly different from
saline/O3 group; c, significantly different from APAP/air group; (pS0.05). FC, fold
change.

134

Figu

L.<\0P.:rwuw Cu Aux/=3...“ Chm

Figure 22

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A. Lung IL-6 expression at 9 h B. Lung IL-6 expression at 32 h
3 -= 20
3140 m Saline/O3 % i “-9 53""9’03
0 120 ‘ r: u APAP/Air
.5 n APAP/Air = 15 l a — APAP/0 503
«810° , - APAP/0.503 g T -
o 80 r .9. \
2 w E § °
'07- 4 ‘5 a
fig: 2 5* X .1.
‘ a a \ : E

E o E 0 \\ ’

C. Lung COX-2 expression D. Lung COX-2 expression

at 9 h » at 32 h

4 3 .
Es: Saline/O3 b m Saline/O3
' n APAP/Air

n APAPIA" - APAP/0.503

- APAP/0.503

(A)

    

N

.3
A

 

FC Relative to Saline/Air
to
FC Relative to Saline/Air

O .
O

135

IL-6 protein concentration was significantly elevated in the BALF of APAP/O3-
coexposed mice 9 h post-APAP when compared to APAP or 03 alone (Figure 23A). At
32 h, IL-6 protein concentration was slightly above control concentration in APAP alone-
treated mice and no change was detected in other groups compared to SAL/air control
animals (Figure 23B). MCP-l was not detected at the protein level in the BALF of any
group including control mice at 9 b (Figure 23C). At 32 h, APAP or 03 alone and APAP
and O3-coexposed groups had significantly increased concentrations of MCP-l compared
to controls (Figure 23D). Although not significant, APAP alone and APAP/O3 groups
had approximately 3 times the concentration of MCP-l measured in the BALF of 03-

exposed mice (Figure 23D).

136

Figure 23. IL-6 (A and B) and MCP-l (C and D) protein concentrations in the BALF of
APAP and 03 treated mice. Animals were injected ip with O (saline) or 300 mg/kg APAP
and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized, BALF harvested and cytokines evaluated by
flow cytometry as described in Material and Methods. Data are expressed as mean :t SE,
(n = 6). a, significantly different from saline/03 group; b, significantly different from
APAP/air group; c, significantly different from saline/air group; (p S 0.05). ND, not
detected.

137

Figure 23

      

A. Lung IL-6 at 9 h B. Lung IL-6 at 32 h
a
40 b 10 :1 Saline/Air
I: Saline/Air m Saline/O3
E! Saline/O3 8 m APAP/Air
_‘ 30 “ APAP/Air =5? - APAP/0.503
E - APAP/0.503 \ 5
\ a
8’ 20 v
'9 =‘
5 1o 2
0

O

   

C. Lung MCP-l at 9 h D. Lung MCP-l at 32 1]

1° :I Saline/Air 12° 5:13 Saline/Air
:: 8 I: Saline/O3 =100 m aims/I21:
E - figfipia'goa 5 so - APAP/o '503 a c
u: m .
5 6 5 60
n- 4 n-
3 3 4o
2 2 5 20

o o

138

1V - 4. Airway Epithelial Regeneration and Related Genes Expression

Administration of APAP in mice caused an elevation in the number of cycling

airway epithelial cells in the axial airway and terminal bronchioles 32 h after treatment
(FigUre 24C, E, F). Compared to control mice, 03 alone resulted in an increase of BrdU
Positive cells in the terminal bronchioles but did not change the number of cycling
epithelial cell in the axial airway (Figure 24A, B, E, F). In either location, 03 exposure
caused a dose-dependent reduction of APAP-induced epithelial cell proliferation (Figure
24D, E, F). This effect seemed to be more pronounced in the axial airway compared to
the terminal bronchioles and coexposure of APAP and 0.5 ppm 03 almost completely
suppressed the number of cycling epithelial cells (Figure 24E, F).

Regeneration of pulmonary airway epithelia is a very slow process compared to
rapidly cycling other epithelia (Rawlins and Hogan, 2006) and proceeds in mice airways
from Clara cells among other cells (Giangreco et al., 2002; Hong et al., 2001). We
therefore decided to evaluate Clara cells damage within the axial airway and terminal
bronchioles at the time where statistical differences were present (32 h). At this time,
APAP or 03 alone caused reduction of CCSP immunostaining in the axial airway
compared to control animals (Figure 25A, B, C). At the same location, APAP/O3-
coexposed mice had further reduction of CCSP immunostaining compared to animals

given either substance alone (Figure 253, C, D).

139

 

A. Saline/Air

B. Saline/0.5 ppm 03
t ‘3‘ «O - ~ ' \f-g‘.‘ f"
x. Q‘."” ““0. i. b. 1'--.
. O ‘O .5 ‘ I“ d
’ a . s ’
\ “ ’1... v 4 ¢‘_ W ‘ ‘
- .~5g£m. '
o ‘ 'w I
’ a
. X

5 1" J '2'; ’
D. APAP/0.5 ppm 03

- . "' F ._
’6' ‘ ‘. I. ’ " g " '5 ”9-4..r
r. ‘ . a- 3 ‘
‘ ' 'H' - " y’ ,w 3 ~- In
C
‘- t

Figure 24. Airway epithelium cell proliferation in APAP and 03 treated mice 32 h after
APAP- BrdU-labeled epithelial cells were evaluated in the axial airway (A-D and E) and
in terminal bronchioles (F). Animals were injected ip with O (saline) or 300 mg/kg APAP
and? h later exposed to 0 (air), 0.25 or 0.5 ppm 03 for 6 h. Thirty two hours after APAP
admmiStration, animals were euthanized and left lung lobes collected. Lung sections were
immunohistochemically stained and evaluated as described in Materials and Methods.
Data are expressed as mean t SE, (n = 6). a, significantly different from saline/air group;

b, S.‘gtlificantly different from APAP/air group; c, significantly different from saline/O3
groups; (p.<_0.05). Black arrowS indicate cells in S phase.

140

Figure 24 (cont’d)

E. Axial airway cell F. Terminal bronchioles cell
proliferation at 32 h proliferation at 32 h

c: Saline/Air
c: Saline/0.2503
m Saline/0.503

_ :1 Saline/Air B APAP/Air

.h
0

g :SalineI0.2503 a a, 12 IIIAPAPIO.2503
8 mSaline/0.503 i, -APAPIo.503 a
1, 30 - n APAP/Air o 10
2 uzuAPAPIO.2503 g 8.
w -APAP/0.503 7,
fi 2° ‘ ' n 6-
_| N
.1 4.
5’3 10 - g
“3 b I5 2 ‘
°\ 0- — g o-

 

 

   

These morphologic changes were confirmed when Clara cell density (number of
Clara cell per mm of basal lamina) and CCSP volume density (amount in nanoliters of '
intracytoplasmic CCSP in epithelial cells per mm2 of basal lamina) were evaluated in the
airWay surface epithelium. 03 exposure resulted in a slight but significant reduction of
Clara cell number in the terminal bronchioles but not axial airway (Figure 26A, B). In
either location, 03 alone caused a significant reduction of CCSP content in Clara cell
(Figure 26C, D). On the other hand, APAP alone caused significant loss of Clara cell as
well as loss of CCSP content in the axial airway and terminal bronchioles (Figure 26A-
D)- In either location, 03 exposure dose-dependently enhanced APAP-induced loss of

Clara Cell, reaching significance at the high dose of 03 (Figure 26A, B). Coexposure of

141

these substances resulted in a slight nonsignificant trend toward a smaller content of

CCSP in Clara cells as compared to APAP or 03 alone (Figure 26C, D).

A. Saline/Air B. Saline/0.5 ppm 03

   
  

«‘13.“.MI.3!
‘ 5‘." -

o
I

I
O

we? a: 2“
a. 4 1‘ z
i. “‘H '

ll‘ . . A
D. APAP/0.5 ppm 03

 

. 5 Vi "'" T \ ‘i‘k 5’ ’zti‘g
l’«\\r \ ~._
35’” ’25—?» V“ b ’4':
- /' r *\-- ‘
#3 ‘~. ”~24_..""1‘”".\' a.“ . z I ‘ ”A
‘5 i 1"” ‘ ~ \" “ ‘ §‘-"i ‘ u
' A,” I ~‘ 4 D o ‘1 ‘l . ‘1
x) .2711... "a h ' “ .’1’ 0:..." .'

Figure 25. CCSP immunolabeling in the axial airway of APAP and 03 treated mice.
Thirty—two hours after APAP administration, animals were euthanized, left lung lobe was
collected and immunohistochemically stained and evaluated. Red staining represents
CCSP protein immunolocalization in the cytoplasm of airway epithelial cells.

142

CCSP gene expression in the lung tissue was decreased in all treatment groups
and reached statistical significance in the APAP alone or the APAP/O3 group at the 9 h
time point (Figure 27A). At 32 h, mRNA expression of CCSP was still lower than
baseline in APAP and APAP/O3-coexposed groups (Figure 27B). In relation with the
regeneration and repair of airway epithelia, the cell cycle-dependent kinase inhibitor P21
expression was significantly upregulated in APAP or 03 alone 9 h post-APAP (Figure
27C). At the same time, APAP/O3 mice, the group with deficient epithelial proliferation
had greater P21 mRN A expression compared to APAP or 03 alone (Figure 27C). At 32
h, relative P21 expression was still elevated in APAP/air, SAL/O3 and APAP/O3 but no

differences were detected between these groups (Figure 27D).

143

A. Axial airway Clara cell

 

density at 32 h
1:: Saline/Air
=1 Saline/0.2503
is: Saline/0.503
m APAP/Air
ccn APAP/0.2503
£100 + — APAP/0.503
o
g 80 - 1
0
jg so -
3
$ 40 -
o 20 -
0
.\° 0

 

 

 

 

 

B. Terminal bronchioles Clara

cell density at 32 h

% CCSP Labeled Cells

A
thQO
OOOOOO

 

 

1:: Saline/Air
I: Saline/0.2503
as: Saline/0.503
m APAP/Air
Em APAP/0.2503
- APAP/0.503
c b

.....
¢

  

4
O 0
0.0.0.0.:

.

0090

e o e o o

o o o o l
§ '30....”

Figure 26. Clara cell density (A and B) and CCSP volume density (C and D) in the axial
airway (A and C) and terminal bronchioles (B and D) 32 h after APAP. Animals were
injected ip with 0 (saline) or 300 mg/kg APAP and 2 h later exposed to 0 (air), 0.25 or 0.5
Ppm 03 for 6 h. Thirty-two hours after APAP administration, animals were euthanized,
left lung lobe was collected and immunohistochemically stained. Data are expressed as
mean 1 SE, (n = 6). a, significantly different from saline/air group; b, significantly
different from APAP/air group(s); c, significantly different from saline/0.25 ppm 03
group; (I, significantly different from saline/0.5 ppm 03; (p50.05).

Figure 26 (cont’d)

C. Axial airway CCSP
volume density at 32 h

N

1:: Saline/Air

I: Saline/0.2503
l is: Saline/0.503

m APAP/Air
T u:n APAP/0.2503
- APAP/0.503

 

 

 

 

 

 

 

 

 

 

 

0

§ Em

 

 

 

 

 

 

 

 

 

 

Intraepithellal CCSP
(nllmm2 of Basal Lamina)

O

D. Terminal bronchioles CCSP '
volume density at 32 h

r: SalineIAlr
a SalineIO.2503

 

 

 

 

 

 

 

2 . -r issSalineIO.503
Ill APAP/Air
1- :n APAP/0.2503
- APAP/0.503
1 ‘ a c

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

lntraeplthelial CCSP
(nllmm2 of Basal Lamina)

O

 

 

 

 

a

 

 

 

 

 

 

 

 

 

 

 

 

 

 

145

Figure 27. CCSP (A and B) and cyclin-dependent kinase inhibitor P21 (C and D) genes
expression in the lung of APAP and 03 treated mice. Animals were injected ip with O
(Saline) or 300 mg/kg APAP and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or
2 h after APAP administration, animals were euthanized and lung samples collected in

Alater. Samples were evaluated as described in Materials and Methods. Data are
efpressed as mean 1 SE, (n = 6). a, significantly different from saline/air group; b,
Slgmficantly different from saline/O3 groups; c, significantly different from APAP/air
gr CUP; (P5005). FC, fold change.

146

Figure 27

A. Lung CCSP expression
at 9 1.

FC Relative to Saline/Air

F0 Relative to Saline/Air
O A N u A in a

O

l
J

b

 

-3 is: Saline/O3 a
a APAP/Air
- APAP/0.503

l.

C. Lung P21 expression
at 9 h

05'

SS! Saline/O3
m APAP/Air
- APAP/0.503

    

B. Lung CCSP expression
at 32 h

C

I
A

 

FC Relative to Saline/Air

.2 a
as: Saline/03
a APAPIAir

-3 III APAP/0.503

D. Lung P21 expression
at 32 h

as: Saline/O3
n APAP/Air
- APAP/0.503

    

FC Relative to Saline/Air
O A N U «5 0| a:

147

IV - 5- Pulmonary Oxidative Stress
Several oxidative stress responsive genes as well as glutathione concentrations
were eValuated in control and treated mice to assess a potential contributory role of
Oxidative stress in the 03 enhancement of APAP-induced lung airway injury. At 9 h post-
APAP, 03 but not APAP caused greater expression of metallothionein l (MT-1)
compared to controls (Figure 28A). At the same time, APAP/O3 coexposure resulted in
Significantly greater MT-l expression compared to O3 alone (Figure 28A). At 9 h, heme
Oxygenase 1 (HO-1), another oxidative stress responsive gene had significantly elevated
eXpression in APAP alone or APAP/O3-coexposed mice compared to control mice
(Figure 28C). At 32 h, MT-l and HO-l had slightly above or comparable expression in

APAP and /or 03-treated groups compared to controls (Figure 28B, D).

148

Figure 28- MT-l (A and B) and HO—l (C and D) genes expression in the lung of APAP
and03 treated mice. Animals were injected ip with O (saline) or 300 mg/kg APAP and 2
WW? exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP administration,
animals Were euthanized and lung samples collected in RNAlater. Samples were
evaluated as described in Material and Methods. Data are expressed as mean :t SE, (n =
6)" a, Sigt‘nificantly different from saline/air group; b, significantly different from
saline/O3 group; c, significantly different from APAP/air group; (pS0.0S). FC, fold

149

Figure 28

A. Lung MT-l expression

at

d—h

FC Relative to Saline/Air
o N a 05 03 O N

FC Relative to Saline/Air

911

s: Saline/O3
m APAP/Air
- APAP/0.503

C. Lung HO-l expression

as Saline/O3
u APAP/Air
— APAP/0.503

00'

 

 

B. Lung MT-l expression
at 32 h

m Saline/O3
m APAP/Air
— APAP/0.503

 
 
 

A

  

FC Relative to Saline/Air
-l O

I
N

D. Lung HO-l expression

at 32 h
_= Saline/O3
g 2 m APAP/Air

g - APAP/0.503
.. c
E

in

.9

a:

..>.

E

0

m

U

u.

 

150

An additional evidence for a role of oxidative stress in APAP/O3 enhanced
toxicity came from the gene expression analysis of the catalytic subunit of glutamate-
cysteine ligase (GCLC) involved in the synthesis of glutathione. At 9 h, GCLC was
significantly greater in the APAP/O3—coexposed group compared to APAP treatment or
03 exPosure (Figure 29A). No differences in GCLC expression were detected between

APAP alone, 03 alone or APAP/O3 groups 32 h after APAP administration (Figure
29B ) - Evaluation of glutathione in lung tissue 9 h after APAP administration showed that
the ratio of oxidized glutathione (GSSG) to total oxidized and reduced (GSH) glutathione

Was significantly elevated in animals exposed to 03 associated or not to APAP treatment
C S ALJO3 and APAP/03 groups) (Figure 29C).
The thiobarbituric acid-reactive substance assay did not show differences between

the treatment and control groups at any time post—APAP (data not shown).

151

Figure 29- GCLC (A and B) gene expression and Oxidized/total glutathione ratio (C) in
(aching Of APAP and 03 treated mice. Animals were injected ip with O (saline) or 300
milkg APAP and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 and 32 h (B) after
APAP administration, animals were euthanized and lung samples collected in RNAlater.
Data are ex pressed as mean t SE, (n = 6). a, significantly different from saline/air group;

b.5igflificantly different from saline/O3 group; c, significantly different from APAP/air
group, (1330-05). FC, fold change.

152

Figure 29

A- Lung GCLC expression

at 9h

rc Relative to galineIAir

Saline/O3
m APAP/Air

- APAPIO.503

 

C - Lung oxidized to total GSH ratio at 9 h

cssc/(ossc+csu)x1oo

AAA-l
ONhOGON-fifl

  

:1 Saline/Air
m Saline/03

m APAP/Air
- APAPIO.503

b

  

153

B. Lung GCLC expression

 

at 32 h

E 3 as: SalineIO3
3 n APAP/Air
g - APAP/0.503
c3 2

o a

fi

.2

5 1

0

a:

‘u’. o

V. DIS CUSSION

We report in this study that 0.5 ppm 03 did not cause epithelial damage or
inflammatory cell accumulation in the axial airway, terminal bronchioles or BALF at l or
24 h after exposure (9 or 32 h after saline administration, respectively). APAP at a dose
Of 300 mg/kg caused epithelial damage in the axial airway and terminal bronchioles. At
this dose, APAP caused a time-dependent airway epithelial cell loss and inflammatory
cell accumulation composed of macrophages and neutrophils at 9 and 32 h. APAP and
O3 coexposure caused greater airway epithelial damage and neutrophil accumulation as
W61 1 as greater neutrophil exudation in the BALF compared to APAP alone.
In the axial airway and terminal bronchioles, APAP alone increased the number
0f proliferating epithelial cells 32 h after its administration. 03 exposure resulted in
inhibition of APAP-induced epithelial cell proliferation in the axial airway and terminal
bronchioles. Early in the course of toxicity, several oxidative stress responsive genes
(MT— 1, GCLC) and a cyclin-dependent kinase inhibitor (P21) expressions were elevated
in the APAP/O3 group compared to APAP or 03 alone. 03 an hour after the end of the 6
h edicposure period (9 h after saline or APAP administration) resulted in increased
O’Cidized to total glutathione ratio.
APAP at high doses causes airway epithelial damage as shown by several studies
(3 alldouin et al., 1995; Genter et al., 1998; Jeffery and Haschek, 1988; Placke et al.,
1987a; Placke et al., 1987b). In mice, APAP administration resulted in bronchiolar
epithelial degeneration 4 h after injection while necrosis was evident 8 h after
adrrlinistration (Placke et al., 1987b). In our study, we observed degeneration of airway

epitllelial cells 9 h after APAP with very few necrotic cells. Frank necrosis was evident at

154

32 h after APAP injection. Clara cells are present within the entire length of the
pulmonary airway epithelium and are sensitive to the toxic effects of APAP (Amatya et
al., 2002; Jeffery and Haschek, 1988; Pack et al., 1980). We observed APAP-induced
damage in both Clara and ciliated cells in airway epithelia. APAP induced greater total
epithelial cell or Clara cell damage in the axial airway compared to the terminal
bronchioles. The reasons for this gradient of toxicity are not clear but could be related to
Clara cell number or content.

APAP is metabolized in the liver through the cytochrome P450 monooxygenases
(Dahlin et al., 1984; Jollow et al., 1973; Mitchell et al., 1973a; Mitchell et al., 1973b). In
the lung, Clara cells have the highest level of cytochrome P4505 among all resident lung
cells (Devereux et al., 1989; Massaro et al., 1994) and are therefore potential sites for
APAP bioactivation. In mice deficient in liver specific NADPH-cytochrome P450
reductase (cpr), the electron donor of microsomal P4503, the severity of lung lesions was
decreased while liver toxicity was abrogated suggesting that liver metabolism was only
partially involved in APAP-induced airway epithelial damage (Gu et al., 2005). This
result also suggests that APAP bioactivation also occurred in the lung as lung toxicity
was not completely eliminated in those cpr null mice. In the lung, at least two isoforms
responsible for hepatic APAP bioactivation, namely CYP2E1 and CYP1A2, or their
activity are involved in xenobiotics metabolism (Dey et al., 1999; Forkert et al., 2001;
Stoilov et al., 2006). The greater APAP lung toxicity in the axial airway compared to the
terminal bronchioles could be related to site-specific differences in Clara cell functional
properties including bioactivation (pool of cytochrome P4503 expressed and activity, etc)

in the different airway subcompartments. One of the few studies comparing axial airway

155

and terminal bronchioles xenobiotics toxication capabilities showed that mice treated ip
with naphthalene, a Clara cell toxicant, dependent upon specific cytochrome P450
isoforms bioactivation, resulted in more distal damage compared to the proximal airway
epithelium (Plopper et al., 1992a; Plopper et al., 1992b). For APAP itself, isoforms
responsible of its bioactivation in the different lung subcompartments are not known and
could be an important factor in the differential toxicity between the proximal and distal
airway regions.

Clara cell antioxidants (e.g., GSH, CCSP, etc) content is also another important
factor to consider in APAP-induced airway proximal and distal toxicity. Clara cells in the
axial airway epithelium had higher content of GSH and fewer cells with low GSH content
compared to terminal bronchioles (West et al., 2000). The relative abundance of Clara
cells (containing the antioxidant CCSP) in distal areas compared to proximal locations
may have been an important contributor to the lesser toxicity seen in the former areas
(Plopper and Hyde, 2008; Plopper et al., 2006). Our results are showing that the distal
region in the control mice had a slightly greater proportion of Clara cells and CCSP
volume density than the proximal airway. It is possible that this greater content in CCSP
in the distal bronchioles had a protective effect on the smaller number of associated
ciliated cells. From these studies we can conclude that APAP bioactivation and the
abundance of Clara cells as well as their content in antioxidants could be potential factors
responsible for differences in APAP toxicity in the axial airway compared to the terminal
bronchioles.

Acute exposures to 1 ppm 03 caused centriacinar epithelial cell damage and

inflammation as early as 4 h after the initiation of exposure in rodents (Boorman et al.,

156

1980; Dungworth et al., 1975; Mellick et al., 1975; Pino et al., 1992a; Stephens et al.,
1974; Sterner-Kock et al., 2000). In those regions, ciliated cells and type I pneumocytes
are the most susceptible cells although Clara cells are also affected by 03 exposure
(Dormans et al., 1999; Schwartz et al., 1976). In our study, 03 exposure at the dose of 0.5
ppm for 6 h did not cause airways epithelial damage or inflammation. 03, however,
enhanced APAP-induced epithelial cell loss and inflammation in both the axial airway
and terminal bronchioles. Mechanisms behind 03 potentiation of APAP injury are not
clear, however 03 modulation of APAP bioactivation, enhanced neutrophil accumulation
or greater oxidative stress in the coexposure group as well as 03 suppression of APAP-
induced epithelial regeneration might have had a role in this process.

In early reports of O3 systemic effects, 03 inhalation prolonged pentobarbital
sleeping time in mice, rats and hamsters (Graham et al., 1981). These results led to the
hypothesis that 03 could modulate enzymes involved in the metabolism of pentobarbital
(Graham et al., 1981). Recent studies in mice showed that 03 inhalation downregulated
expression of several cytochrome P4505 in the lung (CYP2E1) and in the liver (several
isoforms including CYP2E1 and 3A11) (Gohil et al., 2003; Last et al., 2005) while others
reported an increase or decrease of CYP2E1 in rats exposed to 03 (Watt et al., 1997). In
our study, no change in CYP2E1 gene expression was detected in the lung of 0.5 ppm
O3-exposed mice 1 or 24 h after exposure (data not shown); Moreover, APAP was given
2 h before 03 and APAP metabolism is a fast process almost complete in an hour or so as
suggested by GSH depletion 2 h after administration (Dai et al., 2006; Jollow et al.,
1973). Therefore, it is unlikely that 03 modulation of cytochromes P450 isoforms played

a major role in the APAP/O3 toxic synergy.

157

Neutrophil infiltration of higher magnitude was detected in mice exposed to
APAP and 03 compared to APAP or 03 alone-treated mice 32 h after APAP. It is not
clear whether neutrophil infiltration in animals given both APAP and 03 had a role in the
greater epithelial damage or was rather a consequence associated with the scavenging of
damaged epithelial cells. Neutrophils usually contribute to epithelial injury through
generation and release of reactive oxygen species and specific proteases (Ho et al., 1996;
J aeschke, 2000). Neutrophil infiltration due to bacterial or viral stimuli shifted to the left
the dose-response curve of low non-toxic doses of several drugs including APAP
(Maddox et al., ; Roth et al., 1997; Shaw et al., 2009). Therefore, enhanced neutrophil
accumulation in APAP and O3-coexposed mice might have had a contributory role in the
greater airway epithelial toxicity. Neutrophil accumulation in the alveolar septa in APAP
or APAP/O3 groups 9 h post-APAP are most likely located in the alveolar capillaries that
are sites of neutrophils extravasation into the alveolar spaces (Wagner and Roth, 2000).
This was interpreted as an APAP-related increase in neutrophil trafficking as no injury
was detected in these sites by light microscopy.

APAP and O3 coexposed animals had greater induction of oxidative stress
responsive genes (MT -1 and GCLC) compared to either substance alone. Oxidative stress
plays a role in APAP-induced liver toxicity, particularly through mitochondrial proteins
covalent binding (Jaeschke et al., 2003; James et al., 2003). Furthermore, APAP depletes
antioxidant small molecules in both liver and lung, particularly GSH. APAP-treated mice
(375 mg/kg ip) for instance had their lowest level of GSH 2-4 h after injection in both
liver and lung, liver depletion being of higher magnitude compared to lung depletion

(Chen et al., 1990). 03 on the other hand reacts with epithelial lining fluid and cell

158

membranes to yield secondary reactive hydroxyl and lipid radicals (Pryor, 1994; Pryor et
al., 1995a, b). Subsequently, these secondary products lead to consumption of antioxidant
molecules including GSH and unbalance the pro/antioxidant equilibrium toward an
oxidative state (Kirichenko et al., 1996; Li et al., 1996; Menzel, 1994). We report here
that 03 alone at the high dose caused increased oxidized (GSSG) to total glutathione ratio
(GSH + GSSG). APAP or 03 individually induces oxidative stress in the lung and the
increased toxicity observed in the combination of these substances may have been the
results of elevated consumption of anti-oxidants and greater oxidative damage.
Metallothionein is a cysteine-rich small molecular weight protein which has
radicals scavenging and antioxidant properties (Coyle et al., 2002; Kang, 2006; Kumari et
al., 1998; Sato and Kondoh, 2002). In our Study, MT-l had the greatest level of mRNA
expression in the coexposure group compared to either APAP or 03 alone. Others
reported that APAP treatment upregulated hepatic expression of MT-l in mice (Last et
al., 2005; Liu et al., 1999). 03 exposure (at doses of 0.5, l or 2.5 ppm for 4 h) was also
responsible for increased expression of MT expression in the lung of exposed mice
(Johnston et al., 1999; Mango et al., 1998). In this last study, mice deficient in CCSP had
greater MT mRNA expression by 2 h of exposure. Mice deficient in MT-l and MT -2
exposed to 03 had greater epithelial damage and inflammatory changes than wild type
mice. Metallothioneins deficient mice exposed to 03 also exhibited greater inflammation
and oxidative stress as shown by higher levels of IL-6, HO—l, 8-hydroxy-deoxyguanosine
and nitrotyrosine (Inoue et al., 2008). Overall, these studies demonstrated that
metallothioneins are protective from APAP or 03 oxidative stress, particularly in the

absence of other anti-oxidant molecules (e.g., CCSP). Therefore, APAP/O3-induced

159

greater MT—l expression in our study could be interpreted as a response to an ongoing
greater oxidative damage.

Clara cell secretory protein (CCSP) represents a protein secreted by Clara cells in
airways of mammalian species including mice (Hermans and Bernard, 1999). Various
roles have been ascribed to CCSP including anti-inflammatory and antioxidant activities
and binding of lipophilic xenobiotics and other chemicals (Hermans and Bernard, 1999;
Plopper et al., 2006). In our study, APAP or 03 alone caused a significant reduction of
CCSP in the axial airway and terminal bronchioles. APAP/O3 animals had a slight but
not significant trend toward a reduction of CCSP in either location suggesting that APAP
or O3 alone induced substantial reduction of cytoplasmic CCSP and that further reduction
in coexposed animals probably extended beyond the sensitivity of the detection method.
This loss of CCSP detected in treated animals is probably due to increased secretion of
CCSP into the epithelial lining fluid but also to decreased synthesis as shown by the
CCSP gene expresssion analysis. Exposure to high concentration of oxygen in mice
resulted in decreased Clara cell number and CCSP content of these cells (Johnston et al.,
1998). Exposure of CCSP deficient or sufficient mice to 1 ppm 03 for 8 h resulted in
epithelial injury in both the proximal airway and terminal bronchioles in either genotype
(Plopper et al., 2006). In this study, CCSP deficient mice had increased susceptibility of
ciliated cells and Clara cells to 03 effects and in deficient mice, 03 damage extended to
more proximal airway sites not affected in the wild type CCSP sufficient animals. As
described previously, mice deficient in CCSP had greater expression of MT-l when
CXpOSCd to 03, suggesting that these 2 proteins might act as a back up for each other

(Mango et al., 1998). Taken together, these studies suggest that 03 exacerbation of

160

APAP toxicity could be partially explained by greater oxidative injury due to O3
depletion of CCSP from APAP sensitized Clara cell. In this scenario, a potentiation by
O3-induced inflammatory cell oxidative activity is also plausible.

Exposure of O3 in mice treated with APAP resulted in significant decrease of
airway epithelial cell proliferation induced by APAP. Various populations of adult stem
cell responsible for epithelial regeneration have been identified within different
compartments of the lung. In the axial airway area of rodents, Clara cells and basal cells
are considered having stem cell potential (Boers et al., 1998; Breuer et al., 1990; Liu et
al., 2006; Plopper and Dungworth, 1987). Clara cells have been shown to dedifferentiate
into immature cells upon O3 exposure in rat and then able to generate mature Clara or
ciliated cells (Evans et al., 1976). Those immature cells were also able to produce
undifferentiated Clara cells known as facultative progenitor cells (to distinguish them
from ‘professional’ progenitor) (Evans et al., 1978; Stripp, 2008). A variant of Clara
cells, immunohistochemically positive for CCSP with a resistant phenotype to
naphthalene injury have been shown to be true (‘professional’) stem cell and to localize
in neuroepithelial niches or bronchioloalveolar junction (Giangreco et al., 2002; Hong et
al., 2001). In case of severe airway epithelial damage, both progenitor and true stem cells
participated in epithelial reconstruction while only the progenitor cell population
maintained homeostatic epithelial regeneration or epithelial renewal following less severe
injury (Giangreco et al., 2009). It is therefore possible that the absence of regeneration in
APAP/O3 animals is related to a greater depletion of progenitor-based repair.

APAP and O3 coexposure resulted in greater expression of P21 compared to

either substance alone. The cyclin-dependent kinase inhibitor P21 blocks cells in 61

161

phase to allow repair (Weinberg and Denning, 2002). DNA damage from oxidative stress
or other causes induces P21 which then arrest the cell cycle in GI (Clement et al., 2001).
Indeed, under hyperoxic conditions, lung epithelial cells exhibited upregulation of P21
mRNA and protein concentration, DNA fragmentation and inhibition of epithelial cell
proliferation (Clement et al., 2001; Corroyer et al., 1996; O'Reilly et al., 1998; O'Reilly et
al., 2001). Interestingly, APAP or 03 have both been reported to cause DNA damage.
APAP genotoxic activity at high doses has been related to 3 main mechanisms, namely
inhibition of ribonucleotide reductase, direct DNA damage by the reactive metabolite of
APAP and APAP-induced increase of intracellular calcium (Bergman et al., 1996).
Inhalation of O3 caused oxidative DNA damage in BALF cells, double strand break
through generation of hydroxyl radical or direct base modification (Haney et al., 1999;
Ito et al., 2005). Assessment of DNA damage for strand breaks (Comet assay, 8-oxo-dG
evaluation, etc) or DNA base modifications could therefore shed some light on the
impaired regeneration observed in APAP/O3 animals as compared to APAP alone-treated
or 03 alone-exposed animals.

In neutrophil depleted rats (using a rabbit antirat neutrophil antibody), 03
inhalation resulted in the presence of less BrdU—labeled cells in the terminal bronchioles.
This led to the conclusion that neutrophils had an important role in cleaning up the distal
bronchioles and by doing so contribute to the epithelial repair process (Vesely et al.,
1999). In our study, APAP and 0.5 ppm 03 coexposure resulted in more neutrophils
associated to almost completely nonexistant BrdU-labeled cells which is in contradiction

with the results of Vesely and collaborators (1999). Furthermore, in the nasal cavity of

162

rats exposed to 03, the presence of neutrophils was reported to be important for mucus
cell metaplasia but not for nasal epithelial repair after 03 injury (Cho et al., 2000).

In conclusion, APAP and O3 coexposure in mice resulted in greater airway
epithelial damage in the axial airway and terminal bronchioles as well as greater BALF
and airways neutrophil accumulation. APAP administration alone resulted in greater
number of cycling airway epithelial cells. 03 exposure following APAP treatment
resulted in inhibition of APAP-induced epithelial proliferation. APAP/O3 mice also had
greater loss of Clara cell in the airways and greater expression of the cyclin dependent
kinase inhibitor P21. O3 alone or APAP/O3-coexposed mice had greater lung GSSG to
total glutathione ratio compared to controls or APAP alone. Finally, APAP and O3-
coexposed mice had greater induction of several oxidant responsive genes (MT-1,

GCLC) compared to either substance.

163

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174

CHAPTER 4

ROLE OF INTERLEUKIN-6 IN ACETAMINOPHEN AND OZONE HEPATIC
AND PULMONARY TOXICITY

1. ABSTRACT

In APAP alone-treated mice, Interleukin-6 (IL-6) expression and protein
concentration were elevated 9 h after administration while no changes in gene expression
or protein concentration were detected in APAP and O3 co-treated mice. IL-6 is involved
in initial phases of hepatocellular regeneration and has been shown to release cells from
interphase to enter the cell cycle after injury or hepatectomy. In addition, mice lacking
IL-6 had deficient hepatocellular regeneration and were more susceptible to chemical
injury. We therefore hypothesized that in APAP and O3-coexposed mice, the lack of IL-6
induction was responsible for the impaired hepatocellular regeneration and for the greater
toxicity. IL-6 sufficient and deficient mice were given APAP or 03 alone or sequentially
treated with APAP and then O3. IL-6 sufficient mice recapitulated previous results where
APAP alone induced hepatocellular proliferation but not APAP and O3 coexposure at 32
h after APAP. At the same time, the APAP/ 03 group had greater toxicity compared to
APAP alone. In IL-6 deficient mice, APAP alone or APAP/O3 groups had no changes in
hepatocellular proliferation compared to control mice 32 h after APAP administration.
However, APAP and O3-coexposed IL-6 deficient mice had greater hepatocellular
toxicity compared to APAP-treated deficient mice. These results suggest that H.-6 is

important in hepatocellular regeneration after APAP-related liver injury but not involved

175

in 03 inhibition of APAP-induced hepatocellular proliferation or toxicity. In the same
study, IL-6 knock-out mice given APAP alone or APAP and O3 sequentially also had
deficient lung airway epithelial regeneration suggesting that IL-6 is also involved in
airway epithelial regeneration. At the same time, APAP and O3 coexposure resulted in
greater airway epithelial toxicity compared to APAP alone. This study demonstrates that
IL-6 is important in hepatocellular and pulmonary airway epithelial regeneration after
APAP or APAP and O3 injury but not in 03 inhibition of APAP-induced cell

proliferation nor in the exacerbation by 03 of APAP-induced liver or airway toxicity.

11. INTRODUCTION

In our previous chapters, we reported the effects of APAP and O3 coexposure in
the liver and lung of C57BL/6 male mice 9 or 32 h after APAP administration. In the
liver, APAP/O3-coexposed mice had greater parenchyma] damage but significantly
smaller reparative hepatocellular regeneration compared to APAP alone-treated mice. 03
alone had no effect in the liver of mice. In the liver of APAP alone-treated mice,
interleukin-6 (IL-6) expression and protein concentration were elevated compared to
control mice. At the same time, APAP/O3-coexposed mice with defective regeneration
had levels of IL-6 similar to control animals. We also found that APAP/O3 coexposure
caused greater airway epithelial damage compared to APAP alone. 03 alone did not
cause airway injury at the dose used in these studies. Similar to the liver, airway
regeneration was also impaired in the APAP and O3-coexposed group compared to

APAP alone-treated mice.

176

IL-6 is a cytokine produced by a variety of cells including macrophages, T and B
cells, fibroblasts and endothelial cells among others (Naka et al., 2002). This cytokine has
pleiotropic effects including roles in the immune system (B cell differentiation and
maturation), in the acute phase response and in hematopoiesis where it releases blast cells
from the interphase to divide into functional cell lines (Kishimoto, 1989). In the liver, IL-
6 is mainly derived from non-parenchymal cells including Kupffer and endothelial cells
and has many roles including induction of liver regeneration (Fausto et al., 2006; Klein et
al., 2005; Taub, 2004; Zimmermann, 2004). IL-6 is involved in the initial phases of liver
regeneration where hepatocytes transition from the interphase (G0) to the first phase of
the cell cycle (G1) to replace necrotic hepatocytes (Fausto et al., 2006; Taub, 2004). In
this process, this cytokine is thought to increase the sensitivity of hepatocytes to the
effect of growth factors that lead hepatocytes through subsequent steps of the cell cycle
(Zimmermann, 2004). IL-6 is produced in the liver in response to APAP injury (Bourdi et
al., 2002; James et al., 2003b). Additionally, mice lacking lL-6 had defective
regeneration after partial hepatectomy, chemical injury or ischemia (Camargo et al.,
1997; Cressman et al., 1996; Kovalovich et al., 2000; Sakamoto et al., 1999). For
instance, IL-6 deficient mice with impaired regeneration had increased sensitivity to
CCl4 administration compared to sufficient animals (Katz et al., 1998; Kovalovich et al.,
2000). It has also been shown that defective tissue repair in the liver increases in a dose-
dependent fashion after chemical injury, up to a point where tissue regeneration is
inhibited and results in an unopposed progression of injury (Soni and Mehendale, 1998).

This team for instance coexposed rats to a small non-toxic dose of chlordecone followed

177

by carbon tetrachloride (CCl4) that resulted in inhibition of hepatocellular proliferation
which correlated with greater hepatotoxicity and mortality in mice (Mehendale, 1994).
We therefore hypothesized that the greater liver toxicity in APAP and O3-
coexposed mice is related to the impaired regeneration seen in this group and that IL-6,
an important inducer of liver regeneration, is involved in this process. T 0 support our
hypothesis, we exposed IL-6 sufficient and deficient mice to APAP and/or O3. If IL-6
were involved in liver impaired regeneration in APAP/O3-coexposed mice, this last
group would not exhibit hepatocellular impaired regeneration and would have liver
toxicity similar to the APAP alone deficient group. Because IL-6 deficient mice have
been reported to be protected from O3 airway epithelial injury (Johnston et al., 2005; Yu
et al., 2002), we further hypothesize that APAP/O3-coexposed IL-6 deficient mice will

have airway epithelial injury similar to APAP alone-treated deficient animals.

III. MATERIAL AND METHODS

III - 1. Laboratory Animal

. tmIKopf
Pathogen-free male, IL-6 defiClent (C57BIJ6J—IL-6 , referred to as IL-6 -

/- or deficient animals) or IL-6 sufficient mice (C57BL/6J referred to as IL-6 wild type or
sufficient animals) were purchased from the Jackson Laboratory at the age of 8-10 weeks.
IL-6 gene disruption has been produced by placing a neomycin resistance cassette within

the second exon of the IL-6 gene. Mice heterozygous for the mutation (IL-6+/-) were

178

interbred to produce homozygous mice deficient in both alleles of IL-6 (IL-6 -/-) (Kopf et
al., 1994). Mice were housed in polycarbonate cages on heat-treated aspen hardwood
bedding (Nepco-Northeastem Product Corp, Warrensburg, NY). Boxes were covered
with filter bonnets and animals were provided free access to food (Harlan Tekad
laboratory rodents 22/5 diet, Madison, WI) and water. Mice were maintained in Michigan
State University (MSU) animal housing facilities accredited by the Association for
Assessment and Acreditation of Laboratory Animal Care and according to National
Institutes of Health guidelines as overseen by the MSU Institutional Animal Care and
Use Committee. Rooms were maintained at temperatures of 21-24°C and relative

humidities of 45-70%, with a 12-hour light/dark cycle starting at 7:30 AM.

111 — 2. Experimental Protocol

Mice were randomly divided into 16 groups consisting of 6 animals each. IL-6
sufficient or deficient mice were administered intraperitoneally 0 (saline-vehicle) or
300 mg/kg body weight of APAP (Sigma Chemical Co., St. Louis, MO) in 20 ml/kg
saline. Animals were fasted overnight prior to the administration of APAP. Two hours
after APAP administration, mice were exposed to 0 (air) or 0.5 ppm O3 for 6 h. Mice
were sacrificed 9 or 32 h after APAP (1 or 24 h after O3 exposure, respectively) (Figure
30). Mice were individually housed and exposed to O3 in stainless steel wire cage
whole-body inhalation exposure chambers (HC-lOO, Lab Products, Maywood, NJ). 03
was generated with an OREC 03V1-O ozonizer (O3 Research and Equipment Corp., AZ)

using compressed air as a source of oxygen. Total airflow through the exposure chambers

179

was 220 1/rnin (13 chamber air changes/h). The concentration of 03 within chambers was
monitored during the exposure using Dasibi 1003 AH ambient air O3 monitors (Dasibi
Environmental Corp., Glendale, CA). Two 03 sampling probes were placed in the middle
of the ozone chambers, 10-15 cm above cage racks. Airborne concentrations during the
inhalation exposures were 0.56+l-0.02 ppm (mean +/- standard error of the mean) for

ozone chambers and 0.007+/- 0.002 ppm for filtered air chambers.

8-10 weeks old Mice sacrificed
IL-6 ,4... or Day 1: Mice Day 2: Mice given 9 h (1 h after 03) or
IL-6 _/. male fasted ‘ ip saline or 32 h (24 h after ()3)
mice arrival overnight 300 mg/kg APAP after APAP

treatment

    

 

 

    

 

 
 

‘ a Day 2:
1 week animal Inhalation . -
acclimation exposure $5 a???
0 or 0.5 ppm 03 before
for 6 b

Figure 30. Experimental design of APAP and 03 studies in IL-6 sufficient and deficient
mice. 8-10 weeks old C57BL/6 male mice were given 0 (saline) or 300 mg/kg APAP and
then exposed to O3 (0 or 0.5 ppm) for 6 h. Mice were euthanized 9 or 32 h (l or 24 h
after 03 exposure, respectively) after APAP injection.

180

III — 3. Animal Necropsy, Bronchoalveolar Lavage, and Tissue Selection
for Microscopic Evaluation

Two hours prior to scheduled sacrifice, mice euthanized at the 32 h time point
were given 5-bromo-2-deoxyuridine (BrdU) intraperitoneally (50 mg/kg, Fisher
Scientific, Fair Lawn, NJ) for nuclear incorporation and immunohistochemical detection
of airway epithelial cells undergoing DNA synthesis (cycling cells in S phase). At the
time of necropsy, mice were anesthetized with an intraperitoneal injection of sodium
pentobarbital (50 mg/kg; Fatal Plus, Vortech Pharmaceuticals, Dearbom, M1), the
abdominal cavity was Opened and blood was collected from the abdominal vena cava in
BD Microtainer tubes (Franklin Lakes, NJ). Animals were then killed by exsanguination.

Immediately after death, the liver was removed from the abdominal cavity. The
left liver lobe was fixed in 10% neutral buffered formalin (Fisher Scientific, Fair Lawn,
NJ) for light microscopic examination and morphometric analyses. The caudate liver lobe
from each mouse was removed and placed in RNAlater (Qiagen, Valencia, CA) at 4 °C
for 24 h and then stored at -20°C. The remaining liver lobes were frozen and stored at
-80 °C. The thoracic cavity was then opened by puncturing the diaphragm to allow
collapse of lung lobes. After the trachea was cannulated, the heart-lung block was excised
and the lung was gently lavaged twice with 0.9 ml of sterile saline. Approximately 80%
of the intratracheally instilled saline was recovered as BALF from the lavaged lung lobes
and immediately placed on ice until further analysis. The right lung lobes were tied off at
the bronchus level and severed from the left lobe. The left lobe attached to the heart bloc
was gravity-perfusion inflated at a constant pressure of 25 cm of water for at least 1.5

hour using 10% neutral buffered formalin (NBF) (Fisher Scientific, Fair Lawn, NJ) and

181

then immersed in NBF for light microscopic and morphometric analyses. The right
cranial lobe was immersed in RNAlater (Qiagen, Valencia, CA) at 4°C for 24 h and then
transferred for storage to a -20°C freezer. The right middle and caudal lobes were frozen

and stored at -80°C.

III - 4. Cellular Analysis of Bronchoalveolar Lavage Fluid

Total cell counts in the collected BALF from each mouse were determined using
a hemocytometer. Cytological slides were prepared using a Shandon cytospin 3 (Shandon
Scientific, Sewickley, PA), centrifuged at 600 rpm for 10 minutes and stained with Diff-
Quick (Dade Behring, Newark, DE). Differential counts of neutrophils, eosinophils,
macrophages and lymphocytes were assessed on a total of 200 cells. Remaining BALF
were centrifuged at 1,500 rpm for 15 minutes to collect the supernatant fraction that was

stored at -80°C for IL-6 evaluation by flow cytometry.

1H - 5. Plasma Alanine Aminotransferase (ALT) Assay

Blood collected at the time of necropsy was used to evaluate plasma ALT activity

by spectrophotometry using Infinity ALT reagents purchased form Thermo Electron

Corp. (Louisville, CO).

182

III - 6. Flow Cytometric Analyses for Inflammatory Cytokines

BALF supematants and plasma were assayed for IL-6. IL-6 was purchased as a
Flex Set reagent (BD Biosciences, San Diego, CA). Analysis was performed using a
FACSCalibur flow cytometer (BD Franklin Lakes, NJ). Briefly, 50 ul of BALF or
plasma were added to the antibody-coated bead complex and incubation buffer. Samples
were incubated with the beads. Phycoerythrin (PE)-conjugated secondary antibodies were
then added to form sandwich complexes. Following acquisition of sample data using the
flow cytometer, cytokine concentrations were calculated based on standard curve data

using FCAP Array software (BD, Franklin Lakes, NJ).

111 - 7. Lung and Liver Tissue Processing for Light Microscopy
and Immunohistochemistry

The left lung lobe was collected as described previously and a section transverse
to the axial airway was cut at the level of the fifth (fifth generation, G5) bifurcation from
the axial airway. This section was embedded in paraffin and cut at a thickness of 5 pm.
Transverse sections from the middle of the left liver lobe were embedded in paraffin and
cut at a thickness of 5 pm. Lung and liver sections were stained with hematoxylin and
eosin (H&E) for routine histopathological examination and morphometric analyses.

Routine immunohistochemical techniques were used for detection of
hepatocellular or airway epithelium cell nuclear BrdU incorporation and airway epithelial
cell Clara cell secretory protein (CCSP) detection. Neutrophils infiltration in the airway

and liver parenchyma was also detected using immunohistochemistry. Briefly, tissue

183

sections were deparafinized in xylene and rehydrated through descending grades of
ethanol and immersed in 3% hydrogen peroxide to block endogenous peroxides. Sections
were incubated with normal sera to inhibit nonspecific proteins (normal horse, rabbit or
goat sera for BrdU, neutrophils or CCSP immunostaining, respectively, Vector
Laboratories Inc., Burlingame, CA) followed by specific dilutions of primary antibodies
(1:40, monoclonal mouse anti-BrdU antibody, BD, Franklin Lakes, NJ; 1:2500,
monoclonal rat anti-neutrophil antibody, AbD Serotec, Raleigh, NC; 1/1600, polyclonal
rabbit anti-CCSP antibody, Seven Hills Bioreagents, Cincinnati, OH). Tissue sections
were subsequently covered with secondary biotinylated antibodies and immunostaining
was developed with the Vector RTU Elite ABC kit (BrdU and CCSP Vector Laboratories
Inc) or the RTU Phosphatase-labeled Streptavidin kit (neutrophils, Kirkegaard Perry
Labs, Gaithersburg, MD) and visualized with Vector Red (neutrophils and CCSP, Vector
Laboratories Inc) or DAB (3,3’-diaminobenzidine) (BrdU, Sigma Chemicals, St. Louis,
MO) chromogens. Slides were counterstained with Gill 2 hematoxylin (Thermo Fisher,

Pittsburgh, PA).

III - 8. Lung and Liver Morphometric Analyses

Bromodeoxyuridine stained and unstained airway epithelial cell nuclei were
counted in the axial airway and in all terminal bronchioles in the lung section. The BrdU
labeling index was determined by dividing the number of BrdU positive cells counted in
the axial airway (AA) or terminal bronchioles (TBS) by the number of total (stained and

unstained) epithelial cells and multiplying by 100 (Cho et al., 1999). Similarly, the CCSP

184

labeling index was determined by dividing the CCSP stained cells (cytoplasmic staining)
by the total stained and unstained cells and multiplying by 100. Proximal or distal airway
acute inflammatory infiltration was assessed by counting the numbers of
immunohistochemically labeled neutrophils (cytoplasmic labeling) in the axial airway or
terminal bronchioles surface epithelium, respectively, divided by the length of the
underlying basal lamina (Cho et al., 1999). The length of the basal lamina underlying the
surface epithelium was calculated from the contour length of the basal lamina, by using a
National Institutes of Health (NIH) image analysis software (NIH Image; written by
Wayne Rasband at the U.S. NIH). It is reported as the number of neutrophils per mm of
basal lamina.

BrdU stained and unstained hepatocellular nuclei or neutrophils infiltration were
counted in 10 medium power fields (X200) for each animal starting with a randomly
selected field and evaluating every third field. The hepatocellular BrdU labeling index
(LI) was determined by counting the number of BrdU labeled cells divided by the total
number of hepatocytes (stained and unstained) and multiplying by 100.

To quantify APAP and/or O3-induced airway epithelial injury, morphometric
analyses were conducted on H&E sections. The numeric epithelial cell density (i.e.,
number of epithelial cells per mm of basal lamina) was determined by counting the total
number of surface epithelial cell nuclear profiles in transverse airway sections divided by
the length of the underlying basal lamina (Cho et al., 1999). For the temlinal bronchioles,
the numeric cell density presented is the mean of numeric densities from all terminal
bronchioles present on the section. The length of the basal lamina was measured using

the NIH Image program (Wayne Rasband, U.S. National Institutes of Health).

185

The amount of hepatocellular degeneration/necrosis in sections from the left liver
lobe were morphometrically determined using standard morphometric methods that are
similar to those previously described in details in chapter 2 (Yee et al., 2000). Briefly,
images at a magnification of X200 were evaluated employing a 168-point lattice grid
overlaying fields of hepatic parenchyma. Sections from the liver of each mouse were
systematically scanned using adjacent, non-overlapping microscopic fields. The first
image field analyzed in each section was chosen randomly. Thereafter, every third field

was evaluated (approximately 12 fields evaluated/section).

III - 9. Identification of IL-6 deficient gene by PCR and gel electrophoresis

IL-6 gene silencing in deficient mice was confirmed on liver tissue or BALF and
plasma samples from wild type and deficient mice by PCR and gel electrophoresis or
flow cytometry, respectively. Briefly, 2 PCR primers were used to identify the mutated.
and wild type alleles. The first primer ('ITC CAT CCA GTI‘ GCC TTC TI‘G G )
hybridizes to the 5' upstream region of exon 2 in the wild type gene (the disrupted exon in
the IL-6 gene) and the second primer (CCG GAG AAC CTG CGT GCA ATC C) is a
downstream primer which hybridizes within the neomycin cassette used to disrupt exon
2. As shown in figure 31, the first primer was amplified as a 174 bp fragment while the
mutated (second) primer was amplified as a 380 bp fragment (Hilbert et al., 1995).
Furthermore, IL-6 protein was detected in the BALF or plasma of IL-6 sufficient (see

below) but not deficient (data not shown) mice using flow cytometry as described above.

186

 

Figure 31. PCR and electrophoresis—based assessment of IL-6 gene disruption in the
liver. Two primers, one to identify the wild type allele and the other one for the mutated
allele, were used. The wild type allele was amplified as a 174 base pair (bp) fragment
(from IL-6 sufficient mice Nos. 1 to 6) while the mutated primer was amplified as a 380
bp fragment (from IL-6 deficient mice Nos. 7 to 12).

IH - 10. Statistical Analysis

Data were reported as mean +/- SE. Differences among groups were analysed by a
one or two-way ANOVA followed by Student-Newman-keuls post hoc test. When
normality or variance equality failed, a Kruskal-Wallis ranked test was conducted. All
analyses were performed using a commercial statistical analysis software (SigmaStat;
Jandel Scientific, San Rafael, CA). Significance was assigned to p values smaller or equal

to 0.05.

187

I V. RESULTS

IV - 1. IL-6 Protein Concentration in the BALF and Plasma of Wild Type Mice

Similar to our previous results, 03 had no effect on the BALF or plasma protein
concentration of IL-6 at any time in wild type mice (Figure 32A-D). However at 9 h, IL-6
protein concentration was elevated in the BALF and plasma of wild type APAP-treated
mice but was greatest when APAP was coexposed with OB, although significance was
not reached in the BALF (Figure 32A-D). No differences in IL-6 concentration were
present between O3 alone, APAP alone or APAP/O3 groups at 32 h (Figure 32A-D). IL-
6 deficient mice had no detectable IL-6 protein concentration in the BALF or plasma at

any time or with any treatment (data not shown).

188

Figure 32. IL-6 protein concentration in the BALF (A and B) or plasma (C and D) of
APAP and O3 exposed IL-6 sufficient mice. Animals were given 0 (saline) or 300 mg/kg
APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm O3 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized and BALF and plasma collected and IL-6
concentration evaluated as described in Material and Methods. Data are expressed as
mean :1: SE, (n = 6). a, significantly different from saline/air group, b, significantly
different from saline/O3 group, c, significantly different from APAP/air group; (p.<_0.05).
ND, not detected.

189

Figure 32

A. BALF IL-6 at 9 h
B. BALF IL-6 at 32 h

  
 
 

 

 

40 b 20
:1 Saline/Air 1:1 Saline/Air
A 30 Saline/03 A 15 m Saline/03
g m APAP/Air — mAPAPI Air
3, - APAP/0.5 03 E - APAP/0. 5 03
c»
5 20 3 10
‘9 «a
=1 10 - -"-l 5
0 293.03”: 0 N! ! M
C. Plasma IL-6 at 9 h D. Plasma IL-6 at 32 h
1200 b 1200
1:1 Saline/Air c :1 Saline/Air
A1000 is: msa'K'tf/m 1000 g films/[£3
— IlaAP Air 2 ir
E 800 - APAP/0. 5 03 E 800 - APAP/0.5 03
a: co
3 600 e 600
_'1 400 3 400
200 200

am

190

IV - 2. Liver Histopathology and Morphometric Analyses

APAP overdose causes centrilobular hepatocellular necrosis in mice. This classic
APAP lesion as well as O3 exacerbation of APAP damage has been described in chapter
2 of this dissertation. O3 exposure did not change ALT activity (a marker of
hepatocellular injury) or cause hepatocellular damage in IL-6 sufficient or deficient mice
compared to control animals (Figure 33A, B). APAP alone caused hepatocellular injury
9 or 32 h after administration in sufficient and deficient mice (Figure 33A-D). Some
inter-individual variability was observed in ALT evaluation, however, morphometric
analyses revealed that no differences were observed between APAP alone and
APAP/O3-treated groups of the sufficient and deficient mice at 9 b (Figure 33A, C). At
32 h, a progression of injury was detected in APAP-treated groups compared to the 9 h
time (Figure 33B, D). At this time, APAP and 03 coexposure resulted in greater necrosis
and degeneration compared to APAP alone in either genotype (Figure 33A-D). IL-6
deletion delayed the onset of APAP-induced hepatocellular toxicity compared to wild
type mice. Indeed, APAP alone and APAP/O3-coexposed groups had significantly less
necrosis and degeneration in IL-6 deficient mice compared to their respective sufficient
counterparts at 9 h (Figure 33A, C). At 32 h, no differences in ALT or hepatocellular
damage were detected between APAP alone or APAP/O3 of the sufficient and deficient

mice (Figure 33B, D).

191

Figure 33. Liver damage induced by APAP and 03 exposure in IL-6 sufficient or
deficient mice. Alanine aminaotransferase (ALT) activity (A and B) in plasma and
morphometric evaluation of hepatocellular damage (C and D). Animals were given 0
(saline) or 300 mg/kg APAP ip and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or
32 h after APAP administration, animals were euthanized, blood and liver tissue were
collected and ALT and liver tissue evaluated as described in Material and Methods. Data
are expressed as mean :1: SE, (n = 6). a, significantly different from respective saline/air
group; b, significantly different from respective saline/O3 group; c, significantly different
from APAP/air KO group; (1, significantly different from APAP/O3 KO group; e,
significantly different from APAP/air WT group; f significantly different from APAP/air

KO group; (pS0.05). ND, not detected.

192

Figure 33

A. Plasma ALT at 9 h

1 0000

8000 ‘
6000 ‘
-' 4000 ‘

2000 -

T (UIL)

A

C. Liver injury at 9 h

dNU-hUIO
GOO

% of Hepatic Parenchyma

OGOO

L

L

 

 

 

 

1:: Air
- O3

Saline

 

a
Air
2 03 b a
‘VVT 'Rii 'VVT' Kr)
Saline APAP

 

193

B. Plasma ALT at 32 h

1 0000

8000'
3
gisoooi
'3 4000 .
.<

2000-

0

 

 

 

D. Liver injury at 32 h

% of Hepatic Parenchyma
O

O

O

O

éNU-FUIOD
O O

O

 

 

 

 

 

- b
1:: AIr
- 03 a b a
TV? 'REI 'VVT 165'
Saline APAP
:1 Air e f
- 03 |
ND ND NDND
'VVT 'Rii 'VVT 165'
Saline APAP

IV - 3. Liver Inflammation

O3 alone did not cause neutrophil accumulation in the liver of IL-6 sufficient or
deficient mice at any time after APAP (Figure 34A, B). At 9 h, liver neutrophil
accumulation was increased in damaged areas of APAP/air and APAP/O3 groups in IL-6
sufficient mice (Figure 34A). At the same time, lL-6 deficient mice given APAP or
APAP and 03 had a nonsignificant increase in liver neutrophils consistent with the
smaller hepatocellular injury measured at this time in these groups compared to the
respective sufficient groups (Figure 34A). At 32 h, APAP alone and APAP/O3-
coexposed mice had similar amounts of liver neutrophils in IL-6 sufficient or deficient

mice (Figure 34B).

194

A. Liver neutrophils at 9 h B. Liver neutrophils at 32 h

 

 

 

 

 

 

 

 

 

400
n- 400 D Air a D Air
u. - 03 I.L - 03
o 300 i 0300
1- a ‘F
E b 3
£200 .5200 1
e e
3 100 1 3100
2 z
0 AWE. _ _ 0 fl _ _
WT KO WT KO W KO WT KO
Saline APAP Saline APAP

Figure 34. Liver neutrophil infiltration in APAP and 03 exposed IL-6 sufficient and
deficient mice. Morphometric evaluation of neutrophil infiltration in hepatic parenchyma
9 or 32 h after APAP. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later
exposed to 0 (air) or 0.5 ppm O3 for 6 h. Animals were euthanized and livers collected
and evaluated as described in Material and Methods. Data are expressed as mean :1: SE, (n
= 6). a, significantly different from saline/air WT group; b, significantly different from
saline/O3 WT group; c, significantly different from saline/air KO group; (p50.05). ND,
not detected; FP, fields of parenchyma.

195

IV- 4. Liver Regeneration

O3 alone did not change the number of cycling hepatocytes in IL-6 sufficient or
deficient mice 32 h after saline administration compared to control animals (Figure 35).
In IL-6 sufficient mice, APAP alone caused a significant increase in the number of
cycling hepatocytes 32 h after its administration (Figure 35). In these animals, 03
exposure following the APAP treatment resulted in a reduction in the number of
proliferating hepatocytes down to control levels (Figure 35). In IL-6 deficient mice,
APAP alone did not cause an elevation of cycling hepatocytes and APAP alone or
APAP/O3-treated IL-6 deficient mice had a number of proliferating hepatocytes similar

to control mice (Figure 35).

196

Liver BrdU at 32 h

I[l

% BrdU Labeled Cells

W E WT— K_O
Saline APAP

Figure 35. Hepatocellular proliferation in APAP and O3 exposed IL-6 sufficient and
deficient mice. Morphometric evaluation of cycling hepatocytes in S phase 32 h after
APAP. Animals were given 0 (saline) or 300 mg/kg APAP ip and 2 h later exposed to 0
(air) or 0.5 ppm 03 for 6 h. Animals were euthanized and livers collected and evaluated
as described in Materials and Methods. Data are expressed as mean 1 SE, (n = 6). a,
significantly different from saline/air WT group; (pS0.05).

197

IV - 5. Lung Histopathology and Morphometric Analyses

Histopathological examination indicated that 03 exposure did not cause airway
epithelial damage or inflammation in IL-6 sufficient or deficient mice compared to
controls. No differences in the nature of lesions (epithelial degeneration and necrosis and
exfoliation of necrotic cells) in the airway epithelia have been observed between IL-6
sufficient and deficient mice given APAP alone or APAP and 03. We also found that in
either genotype, APAP alone or APAP/O3 effects on airway epithelial cells were more
pronounced in the axial airway compared to terminal bronchioles similar to previous
results in wild type animals in chapter 3. These observations are confirmed after
morphometric evaluation of airway epithelial injury or inflammation as reported below.

Morphometric evaluations revealed that in IL-6 sufficient mice, 03 alone had no
effect on the airway epithelium at any time (Figure 36A-D). APAP alone or APAP and
O3 coexposure resulted in a time-dependent increase in epithelial cell loss in the axial
airway and terminal bronchioles between 9 and 32 h (Figure 36A-D). In these sufficient
mice, APAP and O3-coexposed mice had greater damage in either location compared to
APAP alone at the 9 h time (Figure 36A-D). In IL-6 deficient mice, 03 alone had no
effect on airway epithelial cell densities (Figure 36A-D). In these deficient mice, APAP
alone or APAP and O3 coexposure on the other hand caused epithelial cell loss in the
axial airway and terminal bronchioles at 9 or 32 b (Figure 36A-D). In IL-6 deficient
mice, APAP/O3-coexposed mice had more airway epithelial injury than APAP alone at 9
h but not at 32 h (Figure 36A-D). At the early time, IL-6 deficient mice had greater

epithelial cell loss in the APAP/air or APAP/O3 groups when compared to sufficient

198

respective counterparts. At 32 h no differences in airway epithelial losses were detected

between sufficient and deficient mice (Figure 36A-D).

199

Figure 36. Epithelial numeric cell density in the axial airway (A, B) and terminal
bronchioles (C, D) of APAP and O3 exposed IL-6 sufficient and deficient mice. Animals
were injected ip with 0 (saline) or 300 mg/kg APAP and 2 h later exposed to 0 (air) or 0.5
ppm 03 for 6 h. 9 or 32 h after APAP administration, animals were euthanized and left
lung lobes collected, routinely stained and evaluated as described in Material and
Methods. Data are expressed as mean :1: SE, (n = 6). a, significantly different from
respective saline/air group; b, significantly different from respective saline/O3 group; c,
significantly different from respective APAP/air group; d significantly different from
APAP/air WT group; e, significantly different from APAP/O3 WT group; (p_<.0.05). BL,
basal lamina.

200

Figure 36

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A. Axial airway epithelial B. Axial airway epithelial
density at 9 h density at 32 h
E" 200 :1 Air El 200
E - 03 E
g 150 ' E 150 .
m in
= a a =
g 100 I; d b g 100
= C =
g 50 e g 50 .
lilo-l- O __ _ _ _ IB- 0 — _ _ _
m M WT KO WT KO
53""9 APAP Saline —APAP
C. Terminal bronchioles D. Terminal bronchioles
epithelial density at 9 h epithelial density at 32 h
200 Air 200
('5' E 03 E': a 6.3
E 150 a b E150 -
E c a b g a b a
8 10° ‘ 3 8100 - b
35 E
g 50 g 50 .
'3. '3,
In 0 _ _ _ _ "J 0 _ _ _ _
WT KO WT K WT KO WT KO
Saline APAP Saline APAP

201

IV — 6. Lung Inflammation

Neutrophil accumulation was not detected in airways of IL-6 sufficient or
deficient mice exposed to 03 at any time after APAP (Figure 37A-D). IL-6 sufficient
mice given APAP alone or APAP and O3 had no significant neutrophil accumulation in
their airways 9 h after injection (Figure 37A, C). At 32 h, APAP and O3 coexposed
sufficient mice had greater axial airway neutrophil infiltration compared to control mice
(Figure 373, D). In deficient mice, the time course of inflammation was reversed and no
neutrophil accumulation was present in the airways of the APAP/O3 group 32 h after
APAP administration (Figure 37B, D) while significant neutrophil accumulation was
detected in the axial airway and terminal bronchioles at 9 h (Figure 37A, C). At 9 or 32 h
after APAP, APAP alone or APAP/O3-coexposed sufficient or deficient groups had an
increased number of neutrophils in the alveolar septa. No differences in neutrophil
accumulation between treatment (APAP and APAP/O3) or genotype (IL-6 deficient and
sufficient mice) were observed in alveolar septa. Representative pictures of APAP-
induced neutrophils accumulation in alveolar septa from control or APAP and O3-
coexposed mice are presented in figure 38.

In the BALF, no significant total inflammatory cells, macrophages or neutrophils
changes were detected with any treatment regimen or genotype 9 h after APAP (Figure
39A, C, B). At 32 h, APAP/air and APAP/O3 treatment regimen caused significant and
comparable increase of total inflammatory cells and macrophages in IL-6 deficient and
sufficient mice (Figure 39B, D). In the BALF of IL-6 sufficient mice, a statistical

increase of neutrOphils was detected in APAP alone and APAP/O3-coexposed group 32 h

202

after APAP (Figure 39F). No differences were however noticed between these 2 last
groups. IL-6 deficient mice given APAP alone or APAP and O3 had no neutrophil

accumulation in the BALF at any time (Figure 39F).

203

Figure 37. Neutrophil infiltration in the axial airway (A, B) and terminal bronchioles (C,
D) of APAP and O3 exposed IL-6 sufficient and deficient mice. Animals were injected ip
with 0 (saline) or 300 mg/kg APAP and 2 h later exposed to 0 (air) 0.5 ppm 03 for 6 h.
Mice were euthanized 9 or 32 h after APAP. Data are expressed as mean t SE, (n = 6). a,
significantly different from saline/air KO; b, significantly different from saline/air WT;

(p_<_0.05). ND, not detected; BL, basal lamina.

204

Figure 37

A. Axial airway neutrophils

at9h

265'

Q

Neutrophils/mm BL
.h

 

a
2
0 NDND NDND
WT‘ W W KO
Saline APAP
C. Terminal bronchioles
neutrophils at 9 h
10 -
t:Air a
5' 3 -03
E
I” 6
fi 4
2
3 2
Z
0 _ _ _
Wl' KO WI' KO
Saline APAP

B. Axial airway neutrophils

 

at 32 h
8 :1 Air
_, - 03
m
E 6
E
E 4 b
E
D.
g 2
8
Z 0 NDNDND
W W W E
Saline APAP
D. Terminal bronchioles
neutrophils at 32 h
a .
1:1 Alr
n'n‘ - 03
E 6
E
E
E 4
O.
E
'5 2
d)
2
0 NDND ND ND
WT R6 wr K0
Saline APAP

205

A. WTISaline/Air B. WT/APAP/0.5 ijm 03
"7"“: Telf-"1‘ Kill.) v- t- " §:::’V.--“ 110‘,“ “A": fil‘ . .‘ “IT‘J‘ ,‘ ELL‘ ‘2
‘I '”"y ‘ z+ " ' T“ .'-‘ x v‘. "‘V': 3': 1:5, .-.~ . ‘r‘. . TI ‘ - ‘x'rl, gx

 

.‘ *-
1 - J x a...
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.C" . -I‘. ‘ ' \ 1. ‘ -' ‘ i ,,-J ‘ . _ ‘\ w. .l‘
~ - .. ,. . ~, :i.-—.,- ' - ‘ I i '1-‘-",-.
\a . ‘-.

J :4": ""U‘. I!) -':~. 7 Fr". ‘1‘ .1“; ‘r‘. rp'nli'.""'.‘vhl_é" ,1 .‘
C. lL-G KOISaIine/Air D. IL-6 KO/APAP/0.5 ppm 03

Figure 38. Neutrophil accumulation in alveolar septa of lL—6 sufficient and deficient
mice 9 h after APAP. Light photomicrographs of lung sections from wild type (WT) mice
treated with saline/air (A) or APAP/O3 (B) and from 1L-6 knock-out (KO) mice given
saline/air (C) or APAP/O3 (D). Black arrows indicate neutrophils in alveolar septa.

206

A. BALF total cells at 9 h B. BALF total cells at 32 h

 

 

 

 

 

 

 

 

 

100 1:. At, _ 250 . a
g -03 g 935' a b
«e; 80 - €200 . b
O F
5 60 1 25.150 -
é’ a
8 40 . g 100 1
g 20~ E 501 [I I I
l- 13 o
0 _ _ __ ‘ _ _ __ _
W KO WT KO WT KO Wl' KO
Saline APAP Saline APAP

Figure 39. Inflammatory cell accumulation in the BALF of IL-6 sufficient and deficient
mice. Total inflammatory cells (A, B), macrophages (C, D) and neutrophils (E, F) per ml
of BALF. Animals were injected ip with 0 (saline) or 300 mg/kg APAP and 2 hours later
exposed to 0 (air), 0.25 or 0.5 ppm O3 for 6 h. 9 or 32 h after APAP administration,
animals were euthanized and BALF harvested and analyzed as described in Material and
Methods. Data are expressed as mean 1: SE, (n = 6). a, significantly different from
respective saline/air group; b, significantly different from respective saline/O3 group;
(p_<_0.05). ND, not detected.

207

 

Figure 39 (cont’d)

C. BALF macrophages at 9 h

Macrophages (X103)Iml

O

N
0|

.54
GUI

Neutrophils (X103)Iml
0|

whence
OO

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WT' W W"? W
Saline APAP
E. BALF neutrophils at 9 h
1:: Air
- 03
ND N _ ND! ND
WT' K_o' W "K5
Saline APAP

D. BALF macrophages at 32 h

 

 

 

 

 

E 250 1:1Air a
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.. 25 -
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IV - 7. Lung Epithelial Regeneration

BrdU is incorporated in nuclei of cycling epithelial cells in S phase and used as an
indicator of the proliferating epithelial cell pool. O3 alone did not cause significant
change in the number of proliferating epithelial cell in the axial airway and terminal
bronchioles in IL-6 sufficient or deficient mice (Figure 40A, B). APAP administration
resulted in an increase of BrdU-labeled cells in the axial airway and terminal bronchioles
of IL-6 sufficient mice 32 h after APAP (Figure 40A, B). In either location, 03 exposure
resulted in reduction of APAP-induced cell proliferation in coexposed sufficient mice
(Figure 40A, B). In IL-6 deficient mice, APAP administration did not result in an
increase of BrdU-labeled cells in pulmonary airways and APAP/air and APAP/O3 groups
had BrdU labeling indices similar to those of SAL/air control mice (Figure 40A, B). IL-6
deficient mice given APAP alone and APAP/O3 had less BrdU-labeled cells in the axial
airway and terminal bronchioles compared to their respective IL-6 sufficient counterparts

(Figure 40A, B).

209

A. Axial airway cell B. Terminal bronchioles

 

 

 

 

proliferation at 32 11 cell proliferation at 32 h
1'.’ Air .2 Air
3 10 i a 03 a 310 a 03 a
'3 8 13 8 ‘
3 6 l E 6 .
3 b 3
4 4 -
3 e
'- 2 b 1.. 2 .
2 c “3 mini
° 0 —:-_-=—__ _ °‘ 0 . _ _ _ _
WT KO WT KO WT KO WI' KO
Saline APAP Saline APAP

Figure 40. Airway epithelium cell proliferation in APAP and O3 exposed IL-6 sufficient
and deficient mice 32 h after APAP. BrdU-labeled epithelial cells were evaluated in the
axial airway (A) and in terminal bronchioles (B). Animals were injected ip with 0 (saline)
or 300 mg/kg APAP and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. Lung sections
were immunohistochemically stained and evaluated as described in Material and
Methods. Data are expressed as mean :1: SE, (n = 6). a, significantly different from
saline/air WT group; b, significantly different from APAP/air WT group; c, significantly
different from APAP/O3 WT group; (p50.05). ND, not detected.

210

 

Clara cells are part of the cellular armamentarium responsible for airway
epithelial regeneration (Bishop, 2004). O3 exposure did not change the number of Clara
cells lining the axial airway and temrinal bronchioles of IL-6 sufficient or deficient mice
(Figure 41A-D). APAP alone caused significant loss of Clara cell in the axial airway and
terminal bronchioles of sufficient or deficient mice 9 and 32 h after administration
(Figure 41A-D). Similar to our finding after evaluation of total epithelial cell loss, the
main difference between genotypes was observed at the early time where APAP alone or
APAP/O3-cotreated deficient mice had less Clara cells in the axial airway compared to
sufficient mice (Figure 41A-D). At 32 h, no differences in the number of Clara cells were
detected in APAP alone or APAP/O3—coexposed sufficient mice compared to their

deficient counterparts (Figure 41A-D).

211

 

Figure 41. Clara cell density in the axial airway (A, C) and terminal bronchioles (B, D)
of IL-6 sufficient and deficient mice. Animals were injected ip with 0 (saline) or 300
mg/kg APAP and 2 h later exposed to 0 (air) or 0.5 ppm 03 for 6 h. 9 or 32 h after APAP
administration, animals were euthanized, their left lung lobe were collected and
processed as detailed in Material and Methods. Data are expressed as mean 1 SE, (n = 6).
a significantly different from respective saline/air group; b significantly different from
respective saline/O3 WT group; c significantly different from APAP/air WT group; (1
significantly different from APAP/O3 WT group; e significantly different from
APAP/air KO; (p80.05). BL, basal lamina.

212

Figure 41

 

 

 

 

A. Axial airway Clara cell B. Axial airway Clara cell
density at 9 h density at 32 h
80
5' 2 Air 5' 8°
E 03
E 60 E 60
a a a z»
3 40 b c b g 40
_ d _
E a
g 20 g 20
iii 0 _ _ __ _ u‘} 0 _ _ _ _
WT KO WT KO WT KO WT K0
Saline APAP Saline APAP
C. Terminal bronchioles D. Terminal bronchioles Clara
Clara cell density at 9 h cell density at 32 h
:1 All' .
.1100 ' 03 .4100 28';
1:0 in
E 80 a
E b a b E 80 a b a b
g 60 E 60
° 3
° 0
E 40 a 40
3 a
g 20 5 20
a l-
” 0 _ _ _ _ iii 0 _ _ _ _
WT KO WT KO WT KO WT KO
Saline APAP Saline APAP

213

V. DISCUSSION

The main hypothesis behind this work was that OB-induced inhibition of IL-6
expression and protein concentration in APAP-treated mice impaired hepatocellular
regeneration which in turn led to greater hepatocellular damage in the APAP and O3-
coexposed group. We hypothesized that in mice deficient in IL-6, APAP and O3
coexposure will not cause impaired hepatocellular regeneration and will therefore exhibit
injury similar to APAP alone-treated mice. We found that regardless of the genotype,
mice given APAP alone or APAP and 03 had similar hepatocellular damage at 9 h. At 32
h, IL-6 sufficient or deficient mice given APAP and 03 had greater hepatocellular injury
compared to APAP alone-treated mice of respective genotype. In addition, at the 9 h
time, IL-6 deficient mice given APAP alone or APAP and 03 had less hepatocellular
injury and neutrophil accumulation than their respective counterpart of the sufficient
genotype. APAP administration caused a significant increase of BrdU-labeled
hepatocytes in IL-6 sufficient mice 32 h after its administration. 03 exposure caused a
reduction of this APAP-induced hepatocellular proliferation in the coexposed mice. In
IL-6 deficient mice, APAP treatment did not cause hepatocellular proliferation at 32 h
and APAP alone and APAP/O3—coexposed IL-6 deficient groups had BrdU labeling
indices similar to control mice. In summary, contrary to our hypothesis, IL-6 deletion
impaired regeneration in both APAP/air and APAP/O3 groups at 32 h. At the same time,
03 exacerbated APAP-induced livertoxicity in the deficient or sufficient coexposed

group mice. In addition, impaired hepatocellular regeneration in APAP alone-treated

214

deficient mice did not increase liver toxicity compared to sufficient mice of similar
treatment group.

Several teams previously reported the effects of APAP in H.-6 deficient mice
(James et al., 2003a; Masubuchi et al., 2003). In one study, 300 mg/kg APAP in saline
were injected ip in fasted male C57BL/6 mice (Masubuchi et al., 2003). APAP treatment
in these animals caused H.-6 mRNA increase in the liver that reached a peak between 4 h
and 8 h after APAP injection. At this dose of APAP, IL-6 deficient mice had more
hepatocellular injury at 6, 12, 18 or 24 h post-APAP compared to IL-6 sufficient mice. In
a second paper by James and collaborators (2003), 300 mg/kg APAP in saline ip using
the same strain and mouse gender, caused no significant differences in liver toxicity
between IL-6 deficient and sufficient mice 4 or 24 h after APAP. In this last study
however, APAP toxicity was slightly smaller or greater at 4 or 24 h, respectively, in
deficient compared to sufficient mice. At 48 h after APAP, deficient mice had more
biochemical toxicity but less hepatocellular regeneration than sufficient mice (James et
al., 2003a). This is more in line with our results where APAP alone or APAP and 03
liver toxicity was greater in sufficient animals at 9 h while no difference was detected at
32 h between sufficient and deficient mice. In the study by James and collaborators
(2003), it is not clear whether the greater liver toxicity at 48 h was related to the impaired
regeneration detected at the same time. In the same study, APAP-treated deficient mice
still had defective hepatocellular regeneration at 72 h post-APAP while by this time
toxicity resolved in either genotype. In our study, as described in the previous paragraph,
APAP-treated deficient and sufficient mice had similar hepatocellular injury at 32 h while

deficient animals had less BrdU labeled hepatocytes.

215

Our results indicated that in IL-6 deficient mice, the onset of APAP
hepatotoxicity is delayed. The reasons for this protection from APAP toxicity at the early
time remain unclear. IL-6 is apparently not essential for constitutive expression of
cytochromes P450 isoforms including those involved in APAP metabolism such as
Cyp1a2, 2a5, 2e1, and 3a11 (Kovalovich et al., 2000; Siewert et al., 2000; Warren et al.,
2001). In addition, no differences in APAP protein adduct formation or nitrotyrosine
immunostaining were detected between APAP-treated IL-6 sufficient and deficient mice
(James et al., 2003a; Masubuchi et al., 2003). Other factors involved in APAP
detoxication or in hepatic protection from xenobiotics have also been shown to be
similarly produced in IL-6 sufficient or deficient mice. Thus, evaluation of hepatic total
glutathione, glutathione reductase, glutathione peroxidase and glutathione-S-transferase
levels showed no differences between saline-treated IL-6 sufficient and deficient mice
(Warren et al., 2001). The course of hepatic glutathione depletion at 4 h (and resynthesis
at 24 h) after APAP treatment was also similar between the 2 genotypes (James et al.,
2003a). In a recent work, IL-6 was reported to have a role in glutathione metabolism
(Bourdi et al., 2007). This team showed that interleukins 10 and 4 double-deficient mice
were more susceptible to APAP liver effects and had lower hepatic concentrations of
glutathione and high systemic level of IL—6. Administration of an anti-IL-6 antibody or
disruption of IL-6 gene in these double-deficient mice protected from increased, APAP
liver damage and restored liver glutathione level. They speculated that IL-6 directly
stimulates induction of nitric oxide synthase—2 (NOS-2) which then resulted in nitric
oxide (NO) and peroxynitrite formation and decreased glutathione through peroxynitrite

scavenging (Bourdi et al., 2007). Therefore, lack of NOS-2 induction and NO generation

216

might at least partially account for the hepatic protection offered by IL—6 deletion early in
the course of APAP toxicity in our study. Other hepatoprotective factors such as
interleukin—10 or cyclooxygenase-2 or proinflammatory cytokines such as TNF-a or
interferon-gamma were not the basis for differences in toxicity observed between
sufficient and deficient mice (Masubuchi et al., 2003).

O3-induced injury in the lung is dependent upon IL-6 and O3-exposed IL-6
deficient mice have less injury in the lung compared to sufficient animals (Johnston et al.,
2005; Yu et al., 2002). We therefore hypothesized that the course of APAP alone and
APAP/O3 airway toxicity will be similar in IL-6 deficient mice. Instead, we found that at
9 h post-APAP, IL-6 sufficient or deficient mice exposed sequentially to APAP and 03
had more airway epithelial injury than APAP alone-treated mice of respective genotype.
At the later time, APAP alone and APAP/O3 groups had similar airway injury regardless
of the animal genotype. Furthermore, IL-6 deficient mice given APAP alone or APAP/03
had greater injury than their wild type respective counterpart at 9 h whereas at 32 h no
genotype-related differences were detected. Airway neutrophil infiltration was
significantly elevated at 9 h in APAP/O3 deficient mice while this was the case at 32 h in
sufficient mice of the same group. Neutrophil exudation in the BALF was also detected
in APAP alone or APAP/03 sufficient groups at 32 h. In deficient mice, no neutrophil
accumulation was observed in the BALF at any time nor with any treatment. APAP alone
induced a significant increase of airway epithelial cell proliferation 32 h after
administration in sufficient mice. In these animals, 03 exposure suppressed APAP-
induced cell proliferation. In deficient mice, no differences in cell proliferation indices

were detected between treated groups (03 alone, APAP alone or APAP/O3) and control

217

(SAL/air) mice at 32 h. In conclusion, the onset of APAP alone or APAP/O3 toxicity and
inflammation in the airway were accelerated in deficient animals as they had more
epithelial injury and inflammation at the early time compared to sufficient animals. At
this time, APAP/O3-coexposed deficient or sufficient mice had more airway injury than
APAP alone-treated animals of respective genotype. This is not in accordance with our
hypothesis that IL-6 deficient mice would be protected from 03 effects resulting in
comparable airway injury between the coexposure group and animals given APAP alone.
Factors responsible for the accelerated toxicity in the airway of APAP alone or
APAP/O3-treated deficient mice is not known at this time. 03 injury as previously
decribed is dependent upon the presence of IL-6 and deficient animals seem to be
protected from O3 airway damage (Johnston et al., 2005; Yu et al., 2002). To our
knowledge, there is no publication on the airway effects of APAP in IL-6 deficient mice.
In a study of factors involved in lung-liver communication during APAP toxicity, Neff
and collaborators (2003) showed that in C57BL/6 fasted mice, 300 mg/kg APAP resulted
in lung and liver injury while this was not the case in non-fasted, fed mice. In these non-
fasted, fed mice, liver was protected from 300 mg/kg APAP toxicity at 8 or 24 h while
bronchiolar epithelial cells exhibited necrosis and neutrophils accumulation were evident
by 24 h post-APAP (Neff et al., 2003). This team reported that eotaxin is a risk factor for
the lung injury in non-fasted, fed mice as this chemokine was increased in the lung of
these animals but not in fasted mice. Furthermore, immunoneutralization of eotaxin
before APAP injection protected non-fasted, fed mice from APAP lung injury and BALF

neutrophil extravasation (Neff et al., 2003). Although eotaxin has not been evaluated in

218

IL-6 deficient animals in our study, this chemokines might have had a role in the
heightened lung toxicity detected early in IL-6 deficient animals.

Several studies suggest that IL-6 in the lung has protective effects and that its
deletion or suppression constitute results in greater damage as observed with APAP or
APAP and O3 coexposure at the early time point in our study. For instance,
intraperitoneal administration of LPS in IL-6 deficient mice resulted in greater pulmonary
oxidative stress as evidenced by increased lung mRNA expression and protein
concentration of inducible nitric oxide and heme oxygenase-1 as well as greater lipid
peroxidation and 8-hydroxy-2’-deoxyguanosine immunostaining relative to wild type
levels (Inoue et al., 2008). Similarly, Kida and collaborators (2005) using in vitro systems
showed that lung epithelial cells from wild type mice were more resistant compared to
cells isolated from IL-6 deficient mice. Wild type—derived lung epithelial cell resistance
was abrogated by treatment with an anti-IL-6 antibody (Kida et al., 2005).

APAP alone or APAP and O3 coexposure in the wild type animals resulted in
significant extravasation and accumulation of neutrophils into the BALF 32 h after
administration. The absence of significant neutrophil accumulation in the BALF of APAP
alone or APAP/O3-coexposed deficient mice at 9 or 32 h was not expected. Airway
neutrophil accumulation was detected in wild type or deficient mice and correlated with
the extent and timing of the damage. The effects of IL-6 on neutrophil emigration is
unclear as endotoxin exposure resulted in exacerbation of neutrophil lung accumulation
in IL-6 deficient mice (Inoue et al., 2004; Qiu et al., 2004; Xing et al., 1998) whereas the
reverse was observed with 03 or bleomycin treatment (Johnston et al., 2005; Saito et al.,

2008; Yu et al., 2002). Additionally, administration of IL-6 in rabbits resulted in a

219

 

biphasic neutrophilia clue first to systemic release of the marginated pool and
subsequently to accelerated release of neutrophils from the bone marrow (Suwa et al.,
2000). The same team showed that IL—6 administration resulted in preferential
sequestration of neutrOphils into alveolar capillaries (Suwa et al., 2001). This effect of IL-
6 mainly targeted immature neutrophils and was ascribed to the lesser deformability of
these cells due to their high content in F-actin (Suwa et al., 2001). Although these results
seem to be in contradiction with our data, Suwa and collaborators did not report the
effects of IL-6 on extravasation of neutrophils into alveolar spaces or BALF. Species-
related differences could also be an important factor as this team used rabbits while our
studies focused on mice. The alveolar capillaries are the main site of neutrophils
extravasation into alveolar spaces (Wagner and. Roth, 2000). However, the mechanism of
neutrophil extravasation from the alveolar capillary bed compared to the systemic or
bronchial extravasation presents few differences. Those differences include the site of
extravasation (alveolar capillaries into alveolar space or post-capillary venules in
systemic or bronchial circulation), the size of alveolar capillaries (smaller than the
diameter of neutrophils) and molecules involved in the extravasation process (CD18-
dependence or independence) (Wagner and Roth, 2000). These differences could partially
explain the discordant neutrophilia in airways or rather lack thereof in the BALF of
deficient mice.

APAP administration induced airway epithelial cell proliferation in sufficient
mice at 32 h. In those IL-6 sufficient mice, 03 exposure resulted in reduction of APAP-

induced airway epithelial cell proliferation. Airway epithelial cell proliferation was not

detected in APAP or APAP/OB-coexposed deficient mice. Several studies reported

220

 

defective regeneration in mice lacking IL-6 in various organs. IL-6 is important in
successful liver regeneration after partial hepatectomy or chemical injury (Kovalovich et
al., 2000). Furthermore, IL-6 deletion impaired wound repair in the skin while IL-6
promoted post-traumatic repair in the central nervous system (Gallucci et al., 2000;
Gallucci et al., 2001; Sugawara et al., 2001; Swartz et al., 2001). In the lung, IL-6
deficient mice exhibited lower regenerative capacities in animals exposed to 03 or
cigarette smoke and 03 together (Yu et al., 2002). In addition, IL-6 enhanced pulmonary
epithelial cell survival (Kida et al., 2005; Ward et al., 2000). The mechanism behind IL-6
promotion of airway epithelial regeneration is unclear. In one study, administration of
naphthalene in mice lacking STAT3 (a cytoplasmic factor responsible for numerous
transcriptional responses of IL-6) or GP130 (the membrane co-receptor for IL-6 and
activator of STAT3) resulted in defective bronchiolar epithelial cell shape and number
recovery (Kida et al., 2008). Importantly, IL-6 is one of the ligand for the co-receptor
GP130, and STAT3 is an important downstream effector of IL-6 for liver and probably
lung regeneration (Dierssen et al., 2008; Moran et al., 2008; Wuestefeld et al., 2003).

In this study, we found that IL-6 is involved in hepatocellular regeneration after
APAP injury as no regeneration was detected 32 h after administration in deficient mice.
However, no hepatocellular proliferation was detected in APAP alone or APAP/O3-
coexposed deficient mice suggesting that IL-6 is not the mediator of the impaired
regeneration observed in APAP/O3-coexposed sufficient mice. Furthermore, APAP alone
and APAP/O3 groups in deficient mice both had defective proliferation but the second
group had greater hepatocellular toxicity compared to the former one. Taken together,

these observations suggest that IL-6 is not involved in the delayed proliferation observed

221

 

in sufficient APAP/O3-coexposed mice or 03 exacerbation of APAP toxicity. In the
lung, regardless of the genotype, APAP/O3-coexposed mice had more airway epithelial
injury than APAP alone at 9 h while at 32 h no significant differences were detected. In
addition, APAP alone or APAP/O3—coexposed deficient mice had more airway injury
than their respective wild type counterpart at the early time point. No airway epithelial
proliferation was observed in APAP alone or APAP/O3-coexposed deficient mice while
APAP alone caused significant epithelial cell proliferation at 32 h. This seems to indicate
that IL-6 protected the lung from early APAP toxicity and that IL-6 plays a role in airway
epithelial proliferation after chemical injury. Finally, IL-6 had a role in neutrophils
extravasation from alveolar capillaries as no neutrophils were detected in the BALF of
APAP alone or APAP/O3—treated deficient mice while BALF neutrophilia was observed

in sufficient mice.

222

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226

CHAPT ER 5

SUMMARY AND CONCLUSIONS

APAP at high doses targets the liver and causes centrilobular hepatocellular
degeneration and necrosis in laboratory animals and people (Bessems and Vermeulen,
2001; Clark et al., 1973; Davis et al., 1974; Dixon et al., 1975; Dixon et al., 1971; Placke
et al., 1987; Portmann et al., 1975). APAP also causes airway epithelial toxicity in
mammalian species (Amatya et al., 2002; Baudouin et al., 1995; Khanlou et al., 1999;
Neff et al., 2003). 03 exposure on the other hand causes pulmonary airway epithelial
damage and inflammation in rodents and people (Calderon-Garciduenas et al., 2000;
Jorres et al., 2000; Koren et al., 1989; Pino et al., 1992). More recently, 03 exposure in
rat or mouse has been shown to result in nitric oxide and protein synthesis induction in
the liver and a cachexia-like syndrome with modulation of lipid and carbohydrates
metabolisms (Laskin et al., 1994; Last et al., 2005). In the study by Last and collaborators
(2005), O3 inhalation downregulated several cytochromes P450 isoforms and interferon-
gamma-dependent genes in theliver. To our knowledge, there are no published studies on
the effects of APAP and O3 coexposure in the liver and lung. We therefore undertook
this work to address the hypothesis that combined APAP and 03 exposure will result in
greater toxic effects in both organs compared to individual substances.

In the liver, we found that 03 exposure expanded APAP-induced centrilobular
hepatocellular injury from centrilobular toward midzonal areas and increased acute

inflammatory changes. Morphometric evaluation of hepatocellular degeneration and

227

necrosis and evaluation of plasma alanine aminotransferase activity showed that APAP
and O3 coexposure had significantly more liver injury than APAP alone—treated mice.
Although not significant, neutrophil infiltration was also greater in the coexposed group
compared to APAP alone. 03 alone did not cause hepatocellular injury or inflammation.
APAP alone resulted in increased number of cycling hepatocytes (evaluated by BrdU
immunostaining) compared to control saline/air mice. Surprisingly, APAP and 03-
coexposed mice had smaller levels of hepatocellular proliferation compared to the APAP
alone group. In addition, APAP and O3-coexposed mice had slight increase in gene
expression or protein concentration of IL-6, an important initiator of hepatocellular
proliferation, while APAP alone-treated animals had upregulation of IL-6 at the gene and
protein levels.

In the lung APAP caused airway epithelial injury and acute inflammation in our
studies. Injury had an apparent proximal to distal gradient as axial airway at the level of
the fifth bifurcation had more injury than the terminal bronchioles at the level of the
eleventh bifurcation. 03 exposure usually causes airway injury and inflammation,
particularly in distal parts of the airway tree (terminal bronchioles and junction with
alveolar ducts) (Pino et al., 1992). 03 exposure at the dose of 0.5 ppm utilized in our
studies did not cause airway epithelial or inflammatory changes. APAP and O3
coexposure however resulted in airway epithelial injury and acute inflammation greater
than APAP alone-induced airway changes. In the axial airway or terminal bronchioles,
APAP and OB-coexposed mice had epithelial numeric cell densities than APAP alone-
treated animals. In the axial airway, coexposed mice had greater neutrophil infiltration

than APAP alone and a nonsimmmmgnificant trend was observed in terminal

228

bronchioles. Similar to the hepatic proliferation indices, APAP and O3-coexposed mice
had less airway proliferating epithelial cells compared to the APAP alone group. In
reverse of the liver, IL-6 expression was greatest in the lung and systemic circulation of
APAP and OB-coexposed animals compared to APAP or O3-exposed mice.

Mehendale showed that pretreatment of laboratory rodents with a nontoxic dose
of chlordecone potentiated the hepatotoxicity and lethality of carbon tetrachloride,
chloroform or bromotrichloromethane (Soni and Mehendale, 1998). This team showed
that the exacerbation of hepatotoxicity by chlordecone was related to inhibition of the
initial phase of hepatocellular regeneration that resulted in unopposed progression of cell
injury. Moreover, mice deficient in IL—6 exhibited impaired liver regeneration and greater
hepatocellular injury after partial hepatectomy or chemical administration (Cressman et
al., 1996; Kovalovich et al., 2000). We therefore investigated the role of IL-6 in impaired
regeneration of the APAP and O3 co-treated group and the contribution of this cytokine
in the heightened liver toxicity.

We exposed IL-6 sufficient or deficient mice to APAP and/or 03 and found that
both APAP alone and APAP and O3-coexposed groups had impaired hepatocellular
regeneration in deficient animals. At the same time, coexposure of APAP and 03
resulted in greater hepatocellular toxicity compared to APAP alone in deficient mice.
This suggests that IL-6 is involved in hepatocellular regeneration after APAP treatment
but not in impaired regeneration in the APAP/O3 group or in 03 exacerbation of APAP
toxicity. We compared APAP and O3 toxicity in airways of IL-6 deficient and sufficient
mice. Airway epithelial regeneration was also inhibited in APAP alone or APAP/O3-

treated deficient mice suggesting that IL-6 is involved in pulmonary airway regeneration.

229

IL-6 deficiency had no effect on O3 exacerbation of APAP airway toxicity and greater
airway epithelial damage was observed in APAP and O3-coexposed deficient mice
compared to APAP alone-treated animals. IL-6 deficient mice given APAP or APAP and
03 had more airway injury early in time compared to the sufficient respective groups.
Other results presented in this dissertation are summarized in figure 42.

In addition to the impaired regeneration detected in the liver, additional proteins
such as MCP-l and PAI-l or P21 have been down- or upregulated in the group given
APAP and 03, respectively, and could be responsible for the impaired regeneration
detected in this group. MCP-l has been shown to be important in cell regeneration in
different organs and its absence correlated with delayed or impaired regeneration as
discussed in chapter 2. PAL] absence in a mice model of APAP liver injury resulted in
greater injury and delayed hepatocellular regeneration as also discussed in chapter 2. In
our studies, MCP-l and PAI-l exhibited significantly lower levels of expression in the
APAP and O3-coexposed animals and might have been important in the impaired
regeneration observed in this last group. P21, a cyclin-dependent kinase inhibitor, acts as
an important sensor of DNA damage and halts the cell cycle to allow epithelial cell DNA
repair as described in chapter 2. P21 had significantly greater expression in the APAP
and O3-coexposed mice compared to either substance alone-exposed group. As also
discussed in chapter 2, APAP or 03 cause oxidative DNA damage and their combined
exposure might have resulted in greater oxidative DNA damage responsible for the
greater expression of P21 and impaired hepatocellular regeneration in this group.

Clara cells are progenitor cells in the airways and are able to regenerate

themselves but also ciliated cells after injury (Stripp and Reynolds, 2008; Stripp et al.,

230

2000). Mice given APAP and 03 had greater loss of Clara cell and the impaired airway
epithelial regeneration observed in these animals might have been the result of a
depletion of progenitor or a temporal change in the function of remnant Clara cells
toward a more protective phenotype to the expense of their progenitor role. The impaired
airway epithelial regeneration could also be related to a greater DNA oxidative damage
as P21 is also greatest in the coexposure group compared to animals given APAP or 03
alone.

Evidence of increased markers of oxidative stress is detected in the liver of APAP
and O3-coexposed or 03 alone-exposed mice and could be related to the effects on liver
cells of 03 secondary mediators (generated upon the interaction of 03 with lung
epithelial lining fluid or lung epithelial cell membrane). These secondary mediators
comprise but are not limited to hydrogen peroxide, polyunsaturated fatty acids such as
lipid hydroperoxides, endoperoxides and ozonides or end products such as aldehydes
(malonaldehyde, etc).

Similarly, greater induction of oxidative stress markers was observed in the lung
and might have been related to the direct toxic effects of APAP and 03 on airway
epithelial cells as these substances have been reported to each cause oxidant damage
(chapter 3). In the lung, APAP and O3 coexposure produced greater damage to the Clara
cell population and reduced intracellular CCSP staining. CCSP has been shown to protect
the airway epithelium due to its antioxidant and anti-inflammatory properties as
discussed in chapter3 and the lesser staining detected in the combined exposure is

probably another indicator of greater oxidative damage.

231

The role of neutrophils in the progression of epithelial cell death including in
APAP-induced hepatocellular injury has previously been reported (Ho et al., 1996; Roth
et al., 1997). The enhanced neutrophilic inflammation in the APAP and O3-coexposed
group in the liver and lung might have contributed to the oxidative stress described in the
two previous paragraphs. Neutrophils also secrete an array of proteases known to induce
epithelial cell damage which constitute an additional mechanism of toxicity

The hepatocytes at the periphery of necrotic areas in the APAP alone-treated mice
are in a hypoxic state. Hypoxia could have been the functional result, in the poorly
oxygenated centrilobular area, of the greater pulmonary airway injury seen in the APAP
and O3-coexposed mice. Hypoxia could also be related to the known systemic effects of
03 secondary mediators on red blood cell and their capacity to deliver adequate levels of
oxygen to cells (EPA, 2008).

Future studies should be designed to study the role of MCP-l and PAI-l or P21 in
the impaired liver and/or lung epithelial regeneration due to APAP and O3 coexposure.
The role of Clara cells in airway epithelial regeneration should also be further
investigated in this model.

Lastly, oxidative stress seemed a central mechanism in the O3 potentiation of
APAP toxicity as any of the other mechanisms can be linked to oxidative cell damage.
Thus, oxidative damage for instance is one of the main mechanisms of neutrophil-
induced cell injury as discussed in chapter 3. Impaired regeneration could potentially be a
consequence of increased oxidative DNA damage and cell cycle arrest as suggested by
increased P21 in both the lung and liver of APAP/O3-treated animals. APAP-induced

HIF-la accumulation has also been detected in vitro under high oxygen atmosphere and

232

other scientists suggested that this effect was rather a marker of oxidative stress (James et
al., 2006). Therefore, oxidative stress is more likely to be the main player in the
interaction of APAP and O3 and should be further investigated. The definition of
oxidative stress includes 2 components. The first level of this definition comprises
changes in concentrations of antioxidants. Associated to this is an increased generation of
reactive oxygen species. Our results showed some evidence of changes in antioxidant
concentrations during APAP and O3 coexposure. This could be further investigated by
measuring other antioxidant molecules concentrations (superoxide dismutase, catalase,
peroxiredoxins etc). In addition, investigation of reactive oxygen species in the lung, liver
and blood (electron spin resonance spectroscopy, chemiluminescence etc) would further
support an implication of oxidative stress in the greater toxicity seen in APAP and O3-
coexposed animals. The contribution of the enhanced acute inflammation to this

oxidative stress should also be investigated in the APAP and O3-coexposed group.

233

Figure 42. Summary of Results. APAP and O3 coexposure resulted in greater epithelial
injury in the liver and pulmonary airway epithelia in mice. In the APAP and 03-
coexposed mice, impaired epithelial regeneration was detected in both organs. The role of
IL-6 was investigated using IL-6 deficient mice and this molecule was found not to be
involved in this impaired epithelial regeneration or enhanced toxicity seen in the APAP
and O3 coexposure group. Other candidates for the impaired regeneration in the liver
could be MCP—l and PAI-l both shown to be important for hepatocellular regeneration
and significantly downregulated in the APAP and O3-coexposed animals. In the lung,
Clara cells are progenitor cells in the airway epithelium and the greatest loss of these
cells have been detected in the coexposure group. P21, a cyclin-dependent kinase
inhibitor known to stop the cell cycle to allow repair during DNA damage could also be
responsible for the impaired regeneration in both organs. Greater induction of oxidative
stress markers (CCSP, MT -1, GCLC, GSSG, TBARS) was detected in the APAP/O3
group or with O3 alone and could account for the enhanced toxicity seen in APAP and
O3-coexposed animals. The combined exposure resulted in greater neutrophilic
inflammation that could have played a role in this enhanced toxicity. Hepatocytes
surrounding affected areas in APAP-treated animals are in hypoxic (based on glycogen
depletion and intracellular HIF-la accumulation) conditions and died when animals
where coexposed to APAP and 03 also suggesting a role for hypoxia in this model.

234

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