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This is to certify that the
dissertation entitled

MECHANISMS OF SULINDAC/LPS-INDUCED LIVER
INJURY IN RATS: AN ANIMAL MODEL OF DRUG-INDUCED
IDIOSYNCRATIC HEPATOTOXICITY

presented by
Wei Zou

has been accepted towards fulﬁllment
of the requirements for the

ph_ [)_ degree in Microbiology and Molecular Genetics

 

 

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Date

MSU is an Afﬁrmative Action/Equal Opportunity Employer

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K IPrq/Achres/CIRC/Daleoue indd

 

MECHANISMS OF SULINDAC/LPS-INDUCED LIVER INJURY IN RATS: AN
ANIMAL MODEL OF DRUG-INDUCED IDIOSYNCRATIC HEPATOTOXICITY

By

Wei Zou

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

Microbiology and Molecular Genetics

2010

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ABSTRACT

SULINDAC/LPS-INDUCED LIVER INJURY IN RATS: AN ANIMAL MODEL OF
IDIOSYNCRATIC DRUG-INDUCED LIVER INJURY

By

Wei Zou

Idiosyncratic adverse drug response is a type of adverse reaction that
occurs in a minority of patients during drug therapy. Liver is one of the major
organ targets. All of the nonsteroidal anti-inﬂammatory drugs (NSAIDs) have
been associated with hepatic idiosyncratic adverse drug reactions (IADRs) in
patients, and the risk from sulindac (SLD) is reported to be 5-10 fold greater than
for NSAle as a class. However, the mechanism of SLD-induoed hepatotoxicity
has not been clariﬁed because of the lack of experimental animal models.
Previous studies suggest that inﬂammatory stress is a susceptibility factor for
lADRs. The work in this dissertation supports this hypothesis. Cotreatment of rats
with lipopolysaccharide (LPS), which induces modest inﬂammation, and SLD
resulted in liver necrosis, whereas neither LPS nor SLD was hepatotoxic alone.
After we developed a SLD- inﬂammation interaction model of idiosyncratic liver
injury by treating rats with SLD and LPS, the mechanisms of SLD/LPS- induced
liver injury were investigated. SLD/LPS cotreatrnent causes an increase in the
production of tumor necrosis factor-a (T NF), activation of the hemostatic system
and of neutrophils (PMNs) as well as oxidative stress in the liver. Neutralization
of TNF, anticoagulant administration, PMN depletion or antioxidant treatment

attenuated liver injury in this model. Results of neutralization or inhibition studies

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in vivo and in vitro suggest roles for TNF, the hemostatic system, PMNs and
oxidative stress in the pathogenesis of liver injury-induced by SLD/LPS.
Moreover, these mediators are not independent players. They contribute to liver
injury by interacting with each other and with the SL0 toxic metabolite, SLD
sulﬁde. The studies in this dissertation provide an understanding of mechanisms

of liver injury resulting from SLD- inﬂammation interaction.

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ACKNOWLEDGMENTS

In almost ﬁve years at Michigan State University pursuing my Ph.D. degree,
a lot of people have supported and assisted me along the way. Without these
kindnesses, my thesis project might not have been done now, and this
dissertation would not come out. There are no words sufﬁcient to express my
gratitude to them

First of all, I would like to thank my mentor, Dr. Robert Roth. At the ﬁrst
month arriving MSU as well as United State, I attended a seminar in which I was
attracted by the brilliant hypothesis that Bob proposed. I talked to Bob right after
his presentation and expressed my will to join the lab. The more I worked with
Bob, the better I knew it is a decision I would never regret. Bob has given me
tremendous training and help. He leads me to think independently and helps me
without reservation whenever there is an overwhelming obstacle for me in the
project. I am also inﬂuenced by his passion and perseverance in scientiﬁc
research, which sets a role model for me.

Dr. Patricia Ganey is also my mentor in the lab and deserves my gratitude.
Along with Bob, Patti plays a signiﬁcant role in training me throughout my
graduate life. She not only gives me ideas and suggestions in the project, but
also helps me polish my scientiﬁc English in my manuscripts, which I will beneﬁt
from in my whole career.

It is my fortune to work with so many talented and warm-hearted people in
our lab. I would like to thank Shawn Deng. He is the person I closely worked with

during the rotation in the lab. He was always ready to answer all kinds of

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questions I came up with and never weary of showing me lab techniques. Erica
Sparkenbaugh also deserves my gratitude. She helped me a lot with the in vitro
experiments, including hepatocyte isolation. I also thank Kevin Beggs who
worked side by side with me for months on this project. In addition to them, I
would like to thank all the people in the lab who I worked with. They are Dr. Jane
Maddox, Dr. Francis Tukov, Dr. Sachin Devi, Dr. Jesus Olivero—Verbel, Dr. Rohit
Sinhal, Pat Shaw, Christine Dugan, Jingtao Lu, Aaron Fullerton, Kyle Poulsen,
Nicole Crisp, Allen MacDonald, Theresa Eagle and Sandy Newport.

Beside my colleagues in the lab, Dr. Daniel Jones in the Department of
Biochemistry and Molecular Biology also contributed to this project. He spent a
lot of time with me to ﬁgure out the protocol for LC/MS/MS. I would also like to
thank Kazuhisa Miyakawa and Mike Scott from the Department of Pathobiology
and Diagnostic Investigation, who helped me with immunohistochemistry. I also
own a thank to Dr. Husam Younis from Pﬁzer Global Research and
Development, Drug Safety R&D. It was a great experience for me to collaborate
with people from industry.

The last but not the least, I would like to say thanks to my parents. Although
my parents are thousands of miles away from me, I am always their priority. They
never stop caring about me and encouraging me whenever I am down and

discouraged. Without them, I would not be a graduate student and go this far.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. x
LIST OF FIGURES ................................................................................ xi
LIST OF ABBREVIATIONS .................................................................... xvi
CHAPTER 1
GENERAL INTRODUCTION ................................................................................. 1
1.1 Idiosyncratic adverse drug reactions (IADRs) .............................................. 2
1.1.1 Overview of drug-induced idiosyncratic hepatotoxicity ......................... 2
1.1.2 Conventional explanations for IADRs ................................................... 4
1.1.3 Inﬂammatory stress hypothesis as potential eXplanation for IADR ....... 9
1.2 Idiosyncratic adverse drug reactions (IADRs) ............................................ 14
1.2.1 Overview of the inﬂammatory response ............................................ 14
1.2.2 Tumor necrosis factor-a (T NF) ............................................................ 18
1.2.3 The hemostatic system and hypoxia .................................................. 22
1.2.4 Neutrophils (PMNs) ............................................................................ 23
1.2.5 Reactive oxygen species .................................................................... 25
1.3 Models of drug-inﬂammation interaction ................................................... 28
1.3.1 Ranitidine/LPS-induced hepatotoxicity in rats ..................................... 28
1.3.2 Diclofenac/LPS-induoed hepatotoxicity in rats .................................... 30
1.3.3 Trovaﬂoxacin-inﬂammation interaction model of idiosyncratic liver injury
..................................................................................................................... 32
1.4 Sulindac-induced idiosyncratic liver injury ................................................. 35
1.5 Hypothesis and speciﬁc aims .................................................................... 39
CHAPTER 2
HEPATOTOXIC INTERACTION OF SULINDAC WITH
LIPOPOLYSACCHARIDE: ROLE OF THE HEMOSTATIC SYSTEM ................ 41
2.1 Abstract ..................................................................................................... 42
2.2 Introduction ............................................................................................... 43
2.3 Materials and methods .............................................................................. 44
2.3.1 Materials ............................................................................................. 44
2.3.2 Animals ............................................................................................... 44
2.3.3 Experimental protocol ......................................................................... 44
2.3.4 Evaluation of liver injury ...................................................................... 45
2.3.5 Evaluation of serum TNFo concentrations .......................................... 47
2.3.6 Evaluation of hemostasis and ﬁbrin deposition ................................... 47
2.3.7 Evaluation of liver hypoxia .................................................................. 48
2.3.8 Hepatocyte (HPC) isolation and hepatocytotoxicity assessment in vitro
............................................................................................................. 48
2.3.9 Statistical analysis .............................................................................. 49
2.4 Results ...................................................................................................... 50
2.4.1 Dose-response and timecourse of liver injury ..................................... 50

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2.4.3 Effect of SLD/LPS cotreatrnent on serum TNF concentration ............ 57
2.4.4 Activation of the hemostatic system ................................................... 57
2.4.5 Effect of heparin on liver injury induced by SLD/LPS cotreatrnent ...... 62
2.4.6 Effect of low oxygen on hepatocytotoxicity induced by SLD sulﬁde in
vitro ..................................................................................................... 69
2.5 Discussion ................................................................................................. 74
CHAPTER 3

SULINDAC METABOLISM AND SYNERGY WITH TNF IN A DRUG-
INFLAMMATION INTERACTION MODEL OF IDIOSYNCRATIC LIVER INJURY

............................................................................................................................ 81
3.1 Abstract ..................................................................................................... 82
3.2 Introduction ............................................................................................... 83
3.3 Materials and methods .............................................................................. 84

3.3.1 Materials ............................................................................................. 84
3.3.2 Animals ............................................................................................... 84
3.3.3 Experimental protocol ......................................................................... 84
3.3.4 Evaluation of liver injury ...................................................................... 85
3.3.5 Determination of TNF concentration in serum .................................... 86
3.3.6 LC/MS/MS analysis ............................................................................ 86
3.3.7 Evaluation of cytotoxicity of SLD and its metabolites in vitro .............. 87
3.3.8 Cytotoxicity from TNF and SLD metabolites ....................................... 88
3.3.9 Statistical analysis .............................................................................. 89
3.4 Results ...................................................................................................... 90
3.4.1 Timecourse of TNF concentration in plasma ...................................... 90
3.4.2 Effect of TNF inhibition on liver injury ................................................. 90
3.4.3 Effect of LPS on SLD metabolism in rat ............................................. 94
3.4.4 Effect of etaneroept on SLD metabolism in rats ................................ 101

3.4.5 Cytotoxicity of SLD and its metabolites in HepG2 cells and rat primary
hepatocytes ...................................................................................... 101

3.4.6 Effect of TNF on cytotoxicity of SLD and its metabolites in HepGZ and
rat primary hepatocytes .................................................................... 101
3.5 Discussion ............................................................................................... 109

CHAPTER 4

THE CRITICAL ROLE OF TNF AND PAl-1 MEDIATED NEUTROPHIL

ACTIVATION IN A SULINDAC/ LIPOPOLYSACCHARIDE- MODEL OF

IDIOSYNCRATIC LIVER INJURY IN RATS .................................................... 115
4.1 Abstract ................................................................................................... 116
4.2 Introduction ............................................................................................. 1 17
4.3 Materials and methods ............................................................................ 118

4.3.1 Materials ........................................................................................... 1 18
4.3.2 Animals ............................................................................................. 118
4.3.3 Animal model and sample collection ................................................ 118

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........................................................................................................... 1 19
4.3.5 Evaluation of hepatotoxicity .............................................................. 119
4.3.6 Determination of ClNC-1, MIP-1q and PAI-1 concentrations in plasma
........................................................................................................... 120
4.3.7 Evaluation of liver PMN accumulation and activation ....................... 120
4.3.8 Assessment of ﬁbrin deposition in liver ............................................. 121
4.3.9 Statistical analysis ............................................................................ 121
4.4 Results .................................................................................................... 123
4.4.1 Evaluation of PMN accumulation and activation in livers .................. 123
4.4.2 Time course of ClNC-1 and MIP-1q concentrations in plasma ......... 123
4.4.3 Effect of PMN depletion and PMN protease inhibition on SLD/LPS-
induced liver injury ............................................................................ 126
4.4.4 Effect of TNF on PMN accumulation and activation ......................... 131
4.4.5 Role of PAH in liver injury and accumulation and activation of PMNs
........................................................................................................... 131
4.4.6 Effect of TNF on plasma PAl-1 concentration .................................. 131
4.4.7 Effect of TNF and PAl-1 on ﬁbrin deposition ..................................... 136
4.5 Discussion ............................................................................................... 140
CHAPTER 5

OXIDATIVE STRESS IS A POTENTIAL PLAYER IN THE PATHOGENESIS OF
LIVER INJURY INDUCED BY SULINDAC AND LIPOPOLYSACCHARIDE ....‘I47

5.1 Abstract ................................................................................................... 148
5.2 Introduction ............................................................................................. 149
5.3 Materials and methods ............................................................................ 150
5.3.1 Materials ........................................................................................... 150
5.3.2 Animals ............................................................................................. 150
5.3.3 Design of experiments in vivo ........................................................... 150
5.3.4 Gene expression analysis ................................................................ 151
5.3.5 Evaluation of liver injury and protein carbonyls in mitochondria ....... 152
5.3.6 Evaluation of mitochondrial membrane potential .............................. 153
5.3.7 Evaluation of reactive oxygen species in HepG2 cells ..................... 153
5.3.8 Evaluation of cytotoxicity and capase 3 activity ................................ 154
5.3.9 Statistical analyses ........................................................................... 154
5.4 Results .................................................................................................... 155
5.4.1 Gene expression changes regulated by treatment with SLD, LPS or
SLD/LPS ........................................................................................... 155
5.4.2 Gene expression changes speciﬁcally regulated by SLD/LPS point to
oxidative stress ................................................................................. 155
5.4.3 Oxidative stress in SLD/LPS-cotreated rats ..................................... 158
5.4.4 Effect of SLD sulﬁde on mitochondrial membrane potential ............. 158
5.4.5 Effect of SLD sulﬁde on the production of reactive oxygen species in
HepG2 cells ...................................................................................... 164
5.4.6 Cytotoxicity of hydrogen peroxide and TNF to HepG2 cells ............. 164

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5.4.7 Effect of antioxidant treatment and caspase inhibition on cytotoxicity

........................................................................................................... 164
5.4.8 Effect of antioxidant treatment on caspase 3/7 activity induced by SLD
sulﬁde and TNF cotreatrnent ............................................................. 169
5.5 Discussion ............................................................................................... 172
CHAPTER 6

SUMMARY AND CONCLUSIONS ................................................................... 177

6.1 Summary and conclusions ...................................................................... 178
6.2 Commonality and differences among liver injury models of drug-

inﬂammation interaction .......................................................................... 183

6.3 Potential future studies ........................................................................... 187

APPENDICES ................................................................................................... 189

BIBLIOGRAPHY .............................................................................................. 201

ix

LIST OF TABLES

TABLE 1.1 LIST OF IDIOSYNCRATIC DRUGS TO CAUSE LIVER INJURY IN
DRUG/LPS MODELS IN RODENTS .................................................................. 13

TABLE 2.1 MIDZONAL HEPATIC NECROSIS IN LIVERS OF RATS TREATED
WITH SLD/LPS .................................................................................................. 58

TABLE 2.2 SERUM TNFA CONCENTRATION IN SLD/LPS-TREATED RATS .59
TABLE 3.1 EFFECT OF ETANERCEPT ON SLD METABOLISM ................... 102

TABLE 5.1 PATHWAYS ASSOCIATED WITH GENES SPECIFICALLY
CHANGED BY SLD/LPS COTREATMENT ..................................................... 159

TABLE 6.1 CHARACTERISTICS AND UNDERLYING MECHANISMS OF
IDIOSYNCRATIC LIVER INJURY MODELS OF DRUG-INFLAMMATION
INTERACTION ................................................................................................. 184

LIST OF FIGURES
IMAGES IN THIS DISSERTATION ARE PRESENTED IN COLOR
FIGURE 1.1 INFLAMMATION MIGHT PRECIPITATE DRUG— INDUCED IADRS

............................................................................................................................ 11
FIGURE 1.2 SUMMARY OF THE INFLAMMATORY RESPONSE .................... 15
FIGURE 1.3 TNF SIGNAL TRANSDUCTION PATHWAY ................................. 20
FIGURE 1.4 METABOLISM OF SLD ................................................................. 36
FIGURE 2.1 EXPERIMENTAL PROTOCOL FOR ANIMAL TREATMENT ........ 46
FIGURE 2.2 SULINDAC DOSE-RESPONSE IN THE ABSENCE AND

PRESENCE OF LPS .......................................................................................... 51

FIGURE 2.3 DEVELOPMENT OF HEPATOCELLULAR INJURY INDUCED BY
SLD/LPS COTREATMENT ................................................................................ 52

FIGURE 2.4 MARKERS OF HEPATIC CHOLESTASIS INDUCED BY SLD/LPS
COTREATMENT ................................................................................................ 54

FIGURE 2.5 LIVER HISTOPATHOLOGY IN SLD/LPS-COTREATED RATS ....56

FIGURE 2.6 ACTIVATION OF THE HEMOSTATIC SYSTEM ........................... 60
FIGURE 2.7 FIBRIN DEPOSITION IN LIVER .................................................... 63
FIGURE 2.8 HYPOXIA STAINING IN LIVER ..................................................... 64
FIGURE 2.9 EVALUATION OF FIBRIN DEPOSITION AND HYPOXIA IN LIVEF;5
FIGURE 2.10 EFFECT OF HEPARIN ON LIVER FIBRIN AND HYPOXIA ........ 67
FIGURE 2.11 EFFECT OF HEPARIN ON SLD/LPS-INDUCED LIVER INJURY 70
FIGURE 2.12 EFFECT OF HEPARIN ON HEPATIC LESIONS INDUCED BY
SLD/LPS ............................................................................................................ 72
FIGURE 2.13 EFFECT OF HYPOXIA ON SLD SULFIDE- INDUCED
CYTOTOXICITY ................................................................................................. 73

xi

FIGURE 3.1 TIMECOURSE OF TNF CONCENTRATION IN RAT SERUM ...... 91

FIGURE 3.2 EFFECT OF TNF INHIBITION ON LIVER INJURY INDUCED BY

SLD/LPS ............................................................................................................ 92
FIGURE 3.3 EFFECT OF LPS ON PLASMA CONCENTRATIONS OF SLD, SLD
SULFONE AND SLD SULFIDE .......................................................................... 95
FIGURE 3.4 EFFECT OF LPS ON LIVER CONCENTRATIONS OF SLD, SLD
SULFONE AND SLD SULFIDE .......................................................................... 97
FIGURE 3.5 CONCENTRATIONS OF SLD, SLD SULFONE AND SLD SULFIDE
IN GI TRACT AND FECES ................................................................................ 99
FIGURE 3.6 EVALUATION OF CYTOTOXICITY INDUCED BY SLD, SLD
SULFONE OR SLD SULFIDE .......................................................................... 103
FIGURE 3.7 CYTOTOXICITY INDUCED BY TNF AND SLD OR ITS
METABOLITES ................................................................................................ 1 05
FIGURE 3.8 EFFECT OF TNF ON SLD SULFIDE-INDUCED INJURY TO RAT
PRIMARY HEPATOCYTES ............................................................................. 108
FIGURE 4.1 EVALUATON OF PMN ACCUMULATION AND ACTIVATION IN
RAT LIVERS .................................................................................................... 124
FIGURE 4.2 CONCENTRATIONS OF PMN CHEMOKINES IN RAT PLASMA
.......................................................................................................................... 127
FIGURE 4.3 EFFECT OF PMN DEPLETION ON SLD/LPS-INDUCED LIVER
INJURY ............................................................................................................ 129
FIGURE 4.4 EFFECT OF PMN PROTEASE INHIBITION ON SLD/LPS-
INDUCED LIVER INJURY ............................................................................... 130

FIGURE 4.5 EFFECT OF TNF INHIBITION ON PMN ACCUMULATION AND
ACTIVATION .................................................................................................... 1 32

FIGURE 4.6 EFFECT OF PAI-1 INHIBITION ON LIVER INJURY AND PMN ..134

FIGURE 4.7 EFFECT OF TNF INHIBITION ON PLASMA PAl-1
CONCENTRATION .......................................................................................... 137

FIGURE 4.8 EFFECT OF TNF AND PAI-1 ON LIVER FIBRIN DEPOSITION .138
FIGURE 4.9 MECHANISMS OF SLD/LPS-INDUCED LIVER INJURY ............ 146

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FIGURE 5.1 VENN DIAGRAM OF PROBE SETS REGULATED BY SLDNEH,

VEH/LPS OR SLD/LPS .................................................................................... 156
FIGURE 5.2 EVALUATION OF PROTEIN CARBONYL CONCENTRATION IN
LIVER MITOCHONDRIA ................................................................................... 161
FIGURE 5.3 EFFECT OF ANTIOXIDANTS ON LIVER INJURY ...................... 162
FIGURE 5.4 EFFECT OF SLD AND ITS METABOLITES ON MITOCHONDRIAL
MEMBRANE POTENTIAL ............................................................................... 165
FIGURE 5.5 EFFECT OF SLD SULFIDE AND TNF ON PRODUCTION OF
REACTIVE OXYGEN SPECIES IN HEPG2 CELLS ........................................ 166
FIGURE 5.6 CYTOTOXICITY INDUCED BY HYDROGEN PEROXIDE AND TNF
.......................................................................................................................... 167
FIGURE 5.7 EFFECT OF ANTIOXIDANT ON CYTOTOXICITY INDUCED BY
SLD SULFIDE AND TNF ................................................................................. 168
FIGURE 5.8 EFFECT OF ANTIOXIDANT ON CASPASE 3/7 ACTIVITY ........ 170

FIGURE 5.9 EFFECT OF PAN-CASPASE INHIBITOR ON CYTOTOXICITY
INDUCED BY SLD SULFIDE AND TNF .......................................................... 171

FIGURE 6.1 PROPOSED PATHWAY IN THE PATHOGENESIS OF SLD/LPS-
INDUCED LIVER INJURY ............................................................................... 182

xiii

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LIST OF ABBREVIATIONS
ALP - alkaline phosphatase
ALT- alanine aminotransferase
ANOVA— analysis of variance
AST— aspartate aminotransferase
CINC-1- cytokine-induced neutrophil chemoattractant-1
DMEM- Dulbecco's modiﬁed Eagle’s medium
DMSO- dimethyl sulfoxide
FBS- fetal bovine serum
FMO- ﬂavin containing monooxygenase
GGT— y-glutamyltransferase
H&E- hematoxylin and eosin
HOCI- hypochlorous acid
lADRs- idiosyncratic adverse drug reactions
lDlLl- idiosyncratic drug-induced liver injury
LDH- lactate dehydrogenase
LPS- lipopolysaccharide
MPO- myeloperoxidase
MRM- multiple reaction monitoring
MSR- methionine sulfoxide reductase
NSAID- nonsteroidal anti-inﬂammatory drug
PAI-1- plasminogen activator inhibitor-1

PMN- polymorphonuclear neutrophil

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ROS- reactive oxygen species
SLD— sulindac

TAT — thrombin-antithrombin
TNF- tumor necrosis factor-a

UPLC- ultra performance liquid chromatography

XV

CHAPTER 1

General introduction

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1.1 Idiosyncratic adverse drug reactions (IADRs)
1.1.1 Overview of drug-induced idiosyncratic hepatotoxicity

Adverse drug reactions (ADRs) remain a major issue for affected patients as
well as a huge challenge to health providers. The safety of new compounds are
sometimes not well understood until a drug has been on the market for many
years. As a result, serious ADRs commonly emerge after approval of a drug by
the Food and Drug Administration (FDA). More than 10% of newly approved
drugs from 1975 to 2000 in the United States either had to be withdrawn from the
market or received a warning due to adverse reactions (Lasser et al., 2002). The
liver, which plays an important role in the metabolism of drugs, is a frequent
target of IADRs.

There are two types of adverse drug reactions: dose-related reactions (Type
A reactions) and idiosyncratic reactions (Type B reactions). Type A reactions
occur during drug therapy and they are dose-dependent, and most likely occur in
overdosed individuals. A typical example is acetaminophen-induced adverse
reactions that are due to acetaminophen overdose (Amar and Schiff,
2007;Larson et al., 2005). lADRs differ from type A adverse drug reactions in that
they are unpredictable and not apparently dose-dependent. Typically, lADRs
occur only in a minority of patients who are treated with a speciﬁc drug. lADRs do
not relate to the known pharmacologic effects of the drug (Kaplowitz, 2005;
Uetrecht, 2006; Uetrecht, 2007).

Drug-induced idiosyncratic hepatotoxicity, which might result in permanent

disability or death, has great importance to human health. In addition, these

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reactions are a major issue for the pharmaceutical industry, because they lead to
a large number of withdrawals and restrictions to the use of efﬁcient drugs on the
markets. A typical example of a drug which induced idiosyncratic hepatotoxicity
is troglitazone, which contributed 10% of overall idiosyncratic drug reactions
between 1998 and 2001 and was withdrawn from the market by the US. FDA in
2000 (Ostapowicz et al., 2002). The main reason for the withdrawal was that
troglitazone was associated with the development of acute liver failure (Chojkier,
2005). Troglitazone is a peroxisomal proliferator-activated receptor (PPAR)-y
agonist that is used to treat type 2 diabetes. Although troglitazone was beneﬁcial
at improving insulin resistance, among 1.92 million patients who took
troglitazone, ninety-four cases of liver failure were reported (89 acute, 5 chronic)
(Graham et al., 2003).

The risk of idiosyncratic adverse drug reactions is difﬁcult to predict, and
most idiosyncratic reactions are not discovered until a drug is on the market. That
is because a clear mechanistic understanding of IADRs is still absent, and lADRs
are generally not reproducible in traditional animal models. For example, oral
administration of troglitazone to monkeys at large doses (60- to 120—fold larger
than the therapeutic dose) for 52 weeks did not increase serum liver enzymes
and had little gastrointestinal, hematologic or hepatic effects (Rothwell et al.,
2002). The lack of effective preclinical animal models causes mechanisms of
IADRs to be poorly understood, so that appropriate action cannot be applied to

prevent or treat lADRs.

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1.1.2 Conventional explanations for IADRs

Although the mechanisms are still not fully understood, extensive studies on
IADRs have been performed. Several hypotheses have been raised to explain
mechanisms of IADRs, including the metabolic polymorphism hypothesis, the
hapten hypothesis, the danger hypothesis, the mitochondrial abnormality
hypothesis, the failure to adapt hypothesis and the multiple determinant
hypothesis. The detailed hypotheses, their supporting evidence and limitations

are described below.

Metabolic polymorphism hypothesis

The metabolic polymorphism hypothesis suggests that drug metabolites are
responsible for the toxicity of IADRs. Drugs are metabolized into electrophiles or
free radicals, which can covalently bind to proteins and/or unsaturated fatty
acids, or induce lipid peroxidation (Kaplowitz et al., 1986). As a result, cell
functions can be impaired, and cytotoxicity can be causediby inducing a cell
death signaling pathway, causing impaired calcium homeostasis or decreased
energy generation. Cytochrome P450 is a superfamily of enzymes that transform
drugs into their reactive metabolites. Overexpression of P450 in a fraction of
patients can lead to excessive reactive metabolite formation and accumulation in
the liver that can result in hepatotoxicity. Therefore, polymorphism of P450 that is
responsible for drug-induced hepatotoxicity is present in a speciﬁc fraction of

patients.

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Take troglitazone (TGZ) as an example. TGZ is metabolized into reactive
metabolites by CYP3A4, a member of the cytochrome P450 superfamily (He at
al., 2004). CYP3A4 mediates oxidative cleavage of the thiazolidinedione ring,
generating a highly electrophilic metabolite, TGZ quinine, which covalently binds
to cellular macromolecules (Kassahun et al., 2001). This is supported by the
evidence that administrating TGZ to HepGZ cells transfected with CYP3A4 led to
increased cytotoxicity (Vignati et al., 2005). However, another study suggests
that TGZ quinone is less toxic to hepatocytes and HepG2 cells than TGZ itself
(Tettey et al., 2001). Moreover, the role of CYP3A4 in the TGZ-induced toxicity
has not been tested in vivo.

Although many epidemiological studies have been performed attempting to
link susceptibility to drug-induced toxicity with genes involved in drug metabolism
(Kumashiro et al., 2003; Daly et al., 2007), there is no direct evidence proving
that genetic/metabolic polymorphism contributes to idioscyncratic drug toxicity in

vivo.

Hapten hypothesis

The hapten hypothesis is another prevalent theory of IADRs. It suggests that
prodrugs or more likely their reactive metabolites form drug-protein adducts
through covalent binding (Macher and Chase, 1969; Uetrecht, 2006). The drug-
protein adducts are recognized as non-self antigens by the immune system and
are taken up by antigen presenting cells. The processed adduct peptides are

presented to helper T cells. Thus, an active immune response can be elicited

 

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which causes the production of speciﬁc antibodies to adducts. The production of
antibodies may target self proteins and lead to the destruction of host tissues.

This theory is supported by case reports in patients with liver injury.
Antibodies have been detected after exposure to drugs causing idiosyncratic
reactions, including halothane, diclofenac and trogalitazone (Sallie et al., 1991;
Maniratanachote et al., 2005; Nguyen et al., 2008). However, evidence against
this theory is also accumulating. Autoantibodies are present in only a portion of
patients having drug-induced idiosyncratic hepatotoxicity. Moreover, IADRs are
induced in some patients after the ﬁrst exposure to drug (Clay et al., 2006),
whereas a second exposure is required in this theory of adaptive immune
response.

Several animal models of drug-induced autoimmunity have been developed,
including penicillamine-induced autoimmune syndrome and nevirapine—induced
skin rash in rats (T ournade et al., 1990; Shenton et al., 2003). Although
antibodies against penicillin-modiﬁed proteins are present in rats treated with
penicillin, liver injury is not induced in the animal model (Shenton et al., 2004). In
the nevirapine—induced skin rash model, liver injury is not induced either.
Therefore, the results in animal models and human patients suggest that the
autoantibodies are not necessarily pathogenic, and additional evidence

supporting this theory is needed.

Danger hypothesis

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The immune response induced by a foreign antigen is weak in the absence
of adjuvant, which is a possible explanation of why the immune response
induced by a drug itself is insufﬁcient to cause liver injury (Uetrecht, 2006). The
danger hypothesis was proposed as an alternative (Matzinger, 1994; Uetrecht,
1999). It suggests that necrosis or cell stress imposed by reactive drug
metabolites provides a “danger signal” that activates macrophages, antigen
presenting cells or other cells. The danger signals from stressed cells lead to
upregulation of costimulatory molecules or production of cytokines which cause
an enhanced antibody or T-cell-mediated speciﬁc immune response. However, it
is not clear what these danger signals are. Activation of the innate immune
system, eg. inﬂammation, was proposed to be a potential danger signal

(Kaplowitz, 2005).

Mitochondrial abnormality hypothesis

This theory proposes that the mitochondria are targeted by idiosyncratic
drugs, and mitochondrial abnormality is the underlying mechanism of IADRs.
Mitochondria are a critical player in mediating cell death and also a common
target of xenobiotics (Wallace and Starkov, 2000). Numerous drugs associated
with IADRs, such as troglitazone, tolcapone, nimesulide and valproic acid are
reported to cause mitochondrial dysfunction in vitro (Bjorge and Baillie, 1991;
Mingatto et al., 2000; Bedoucha et al., 2001; Haasio et al., 2002). There is also

clinical evidence associating IADRs with mitochondrial dysfunction. In one case

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report, mitochondrial swelling, loss of cristae and reduced matrix density was
observed in hepatocytes of a patient with tolcapone-induced liver injury.

An inherited mitochondrial dysfunction or mutation in mitochondrial DNA
might make people more susceptible to drug-induced toxicity. In one case of
valproate-induced liver injury, an inherited dysfunction of mitochondrial electron
transport chain complexes was observed in the patient (Krahenbuhl et al., 2000).
For many unrelated drugs, age is one of the risk factors for IADRs. Interestingly,
mitochondrial DNA mutations also increase with age, which could explain why
aged people are more sensitive to IADRs (Kujoth et al., 2005).

Models have been developed using an animal model of silent genetic
mitochondrial abnormality. Superoxide dismutase (SOD) eliminates superoxide in
mitochondria, and heterozygous SOD2 mice have a decreased ability to manage
oxidative stress in the liver. Either nimesulide or troglitazone induced
hepatotoxicity in heterozygous SOD2 mice, whereas these drugs had no

hepatotoxic effect on normal mice (Ong et al., 2006; Ong et al., 2007).

Failure to adapt hypothesis

It has been observed that the majority of patients with ALT elevations due to
drugs associated with idiosyncratic liver injury will eventually recover from liver
injury despite continued exposure to the drug (Watkins, 2005). Therefore,
another hypothesis has been raised that a small fraction of patients fail to adapt

to the initial injury, which then leads to the progression of severe liver injury.

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Few animal models have been developed to support this hypothesis, and the
mechanisms underlying injury are not known. One possibility is that a single

inherited defect in adaptation precipitates drug-induced liver injury.

Multiple determinant hypothesis

This theory proposed that idiosyncratic drug toxicity is a result of “the
occurrence of multiple critical and discrete events, with the probability for the
occurrence of idiosyncratic drug toxicity being a product of the probabilities of
each event” (Li, 2002). This theory is not exclusive with some of the other
hypotheses already discussed. The critical events could include chemical
properties of the drug, exposure, environmental factors and genetic factors.

Inﬂammation could be a critical event to take into account.

1.1.3 Inﬂammatory stress hypothesis as potential explanation for IADRs

In addition to the hypotheses mentioned above, a hypothesis has been
proposed that inﬂammation may render an individual susceptible to IADRs (Roth
et al., 2003; Ganey et al., 2004). Inﬂammatory episodes are commonplace in
humans. Inﬂammation can be induced by infections, inﬂammatory diseases or
exposure to endotoxin, which is a potent inducer of inﬂammation.
Lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, is an
inﬂammagen that can cause damage to several organs, including the liver
(Hewett and Roth, 1993). Various conditions, including alcohol consumption,

gastrointestinal distress, changes in diet, antibiotic treatment and surgery can

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increase LPS concentrations in human plasma (Roth et al., 1997). These
conditions are not usually severe enough to cause overt illness, but they may
potentiate the toxicity of xenobiotics through activating inﬂammatory cells and
cytokines. Previous studies proved that an inﬂammatory stress precipitates
hepatotoxicity of numerous xenobiotics, e.g. allyl alcohol and aﬂatoxin (Sneed et
al., 1997; Yee et al., 2000; Barton et al., 2001).

The characteristics of inﬂammation described above led to the inﬂammatory
stress hypothesis for IADRs. As illustrated in Fig. 1.1, the decrease in threshold
for toxicity results from the inﬂammatory episodes that occur in the lifetime of
normal humans. When an individual is undergoing drug therapy, the drug
concentration reaches its therapeutic concentration without causing toxicity under
normal conditions. However, if an inﬂammatory stress decreases the threshold of
toxicity below the drug concentration, an IADR may occur. This hypothesis can
provide a plausible explanation for the characteristics of IADRs. The occurrence
and magnitude of a modest inﬂammatory episode can be unnoticeable in
humans. Therefore, IADRs due to the interaction of an inﬂammatory episode with
idiosyncratic drugs would be expected to have an inconsistent temporal
relationship to exposure and not appear to be dose dependent.

Numerous drug-induced idiosyncratic liver injury models have been
developed in rodents that support the inﬂammatory stress hypothesis.
Speciﬁcally, drugs including diclofenac (DCLF), sulindac (SLD), halothane (HAL),
chlorpromazine (CPZ), trovaﬂoxacin (TVX) and ranitidine (RAN), when

administered at nonhepatotoxic doses, induced signiﬁcant liver injury in rodents

10

Fig. 1.1 I
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Drug Conc. In Plasma

 

 

Fig. 1.1 Inﬂammation might precipitate drug- induced IADRs (Roth et al.,
2003). The subcurve indicates the drug concentration in plasma increases and
remains at a therapeutic concentration during drug therapy. However, the
threshold for drug toxicity (upper dotted line) might be decreased as a result of
inﬂammatory stress. IADRs occur if the threshold for toxicity drops below drug

concentration.

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11

pretreated with LPS (Table 1.1). Levoﬂoxacin and famotidine are in the same
pharmacological class with trovaﬂoxacin and ranitidine, respectively, but they
have far lower tendency to cause IADRs in humans and also failed to induce liver
injury with LPS in rodents. Inﬂammatory stress induced by the viral RNA mimetic,
poly (I:C) also precipitated hepatotoxicity caused by halothane in mice (Cheng et
al., 2009). Therefore, these results suggest that inﬂammation may precipitate the
toxicity of drugs that cause IADRs. Mechanisms underlying drug/LPS interaction
models of idiosyncratic liver injury have been investigated. In the following
sections of this chapter, the innate immune response and mechanisms of several

drug/LPS interaction models are introduced.

12

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models in rodents (Deng et al., 2009).

 

 

LPS + Drug
Drug Human IADRs? Hepatotoxicity
in Rodents?
Diclofenac Yes Yes
Sulindac Yes Yes
Halothane Yes Yes
Chlorpromazine Yes Yes
Trovaﬂoxacin Yes Yes
Levoﬂoxacin No No
Ranitidine Yes Yes
Famotidine No No

 

13

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1.2 Inﬂammation
1 .2.1 Overview of the inﬂammatory response

People are exposed to infectious microorganisms in everyday life.
Inﬂammation is a ﬁrst-line defense mechanism by the innate immune system
against infections. Not only infections, but also tissue trauma and non-infectious
disease can contribute to inﬂammation (Trunkey, 1988). Tissue injury leads to
activation of the innate immune response after trauma, which presents as a
systemic inﬂammatory response syndrome (Lenz et al., 2007). Inﬂammation is
also associated with anemia (Buck et al., 2009), Parkinson’s disease (Barcia et
al., 2003), diabetes (Granic et al., 2009), obesity (Elmarakby and Imig, 2009;
Olefsky, 2009), heart disease and other vasculopathies (Linde et al., 2006) and
cancers (Sevinir et al., 2003; De Marzo et al., 2007).

Inﬂammation is a complex response coordinated by various inﬂammatory
cells (Fig. 1.2). Neutrophils, macrophages, endothelial cells, epithelial cells and
platelets are activated by inﬂammatory stimuli (Ganey et al., 2004).
Transcriptional activation occurs, and several genes are upregulated in
inﬂammatory cells. Proteases and reactive oxygen species are released by
neutrophils and macrophages and can kill parenchymal cells directly.
Proinﬂammatory cytokines, such as tumor necrosis factor-a (T NF) are also
released by macrophages. These cytokines can either activate cell death
signaling pathway or lead to other inﬂammatory events. The coagulation system
as well as the complement system can also be activated as a result of

inﬂammation.

14

Fig. 1.2 Summary of the inﬂammatory response (Ganey et al., 2004).
Inﬂammatory cells are activated by stimulus through receptor binding e.g. TLRs.
As a result, numerous mediators of inﬂammation are released and cause
homeostatic imbalance in the target tissue. These mediators might lead to tissue
injury in aggravated conditions or be inconsequential, beneﬁcial or increase

tissue sensitivity when the response is modest.

 
 

 

   

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Platelet Activating Factor Reactive Oxygen Species

Proteases Nitric Oxide, Etc., Etc.

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Severe inﬂammation can lead to dramatic changes in physiology, such as
redness, swelling, pain and heat at the site of inﬂammation. Moreover, severe
tissue damage or organ failure can result from a marked inﬂammatory response.
More commonly, modest inﬂammatory episodes occur in humans, which may be
unnoticeable. Although modest inﬂammation resolves and may have no
detrimental effect alone, it might potentiate the toxicity of xenobiotics (Sneed et
al., 1997; Yee et al., 2000; Barton et al., 2001).

The Toll-like receptor (TLR) signaling pathway is a well studied pathway
involved in the initiation of the inﬂammatory response (O'Neill, 2008). TLRs are
expressed by inﬂammatory cells and recognize speciﬁc structures conserved
among microorganisms (Takeda et al., 2003). LPS binds to TLR4, which results
in signal transduction leading to a cascade of inﬂammatory events (Linde et al.,
2006). Macrophages are the primary immune cells responding to LPS. TLR4 is
also expressed on the membranes of hepatocytes, endothelium and mast cells
(Migita et al., 2004). TLR4 signals through two intracellular Toll/lL-1 receptor
(T IR) domain-containing adaptors, myeloid differentiation factor 88 (MyD88) and
TlR-containing adapter molecule (T RIF). In the MyD88 dependent pathway,
interleukin-1 receptor-associated kinase (IRAK) 1 and IRAK 4 are recruited to
MyD88. Once phosphorylated, IRAK dissociates from MyD88 and activates TNF
receptor associated Factor (I' RAF) 6. TRAF6 binds to the complex of TGF-B—
activated kinase 1 (TAK1) and TAK-1 binding protein (TAB). In turn, TAK1 is
phosphorylated and leads to the activation of MAPK pathway or NFkB-regulated

genes. In the TRIF dependent pathway, IKB kinase is activated and leads to the

16

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activation of NFKB. TRIF also leads to the activation of interferon regulatory

factor 3 (IRF3) and the expression of interferon-inducible genes.

17

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1.2.1 Tumor necrosis factor-a (TNF)

TNF is a proinﬂammatory cytokine that is produced by many cell types, eg.
macrophages, mast cells, endothelial cells and stellate cells in response to LPS
(Gordon and Galli, 1990; Thirunavukkarasu et al., 2006). Kupffer cells are
resident macrophages attached to the outer surface of endothelial cells in liver
sinusoids (Hewett and Roth, 1993), and they are the major macrophage
population exposed to inﬂammagens in blood. After LPS binding to TLR4, pro-
TNF is produced by Kupffer cells as a result of the activation of NFKB signaling
pathway. Pro-TNF is a 26 kDa membrane-bound precursor form which is not
biologically active (Solomon et al., 1997). TNF converting enzyme (l' ACE), a
membrane bound metalloprotease, recognizes a cleavable signal sequence on
pro—TNF and leads to shedding of the active form of TNF from the cell membrane
(lshisaka et al., 1999; Wullaert et al., 2007).

TNF exerts its biological effects by binding two plasma membrane receptors,
TNF-receptor 1 (TNF-R1) and TNF-R2. In a few cell types, e.g., T cells, the
binding of TNF to TNF-R2 leads to proliferation, NFkB activation and cytokine
production (Rothe et al., 1994; Vandenabeele et al., 1994). However, in most
cells, TNF-R2 has no direct effect on signal transduction and plays an indirect
role in TNF-R1 responses by delivering TNF to the low afﬁnity TNF-R1. The
ligand passing activity of TNF-R2 allows ﬁne-tuning of TNF-R1 mediated signal
transduction.

TNF plays an important role in regulating liver homeostasis (Wullaert et al.,

2007). TNF-R1 activation leads to either hepatocyte proliferation via the

18

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activation of NFkB, the initiation of MAPK cascades or cell death (Fig. 1.3).
Activated TNF-R1 recruits adapter proteins, TNF-R-associated death domain
(TRADD) protein, TNF-R associated factor (TRAF) 2, and receptor interacting
protein (RIP) to the cytoplasmic part of TNF-R1. These proteins form complex I.
TRAF2 possesses ubiquitin ligase activity, which leads to the ubiquitination of
RIP and itself. This serves as an important signal for the binding of TAK1-TAB2-
TAB3 complex. TAK1 leads to the activation of the MAPK pathway as well as lKK
complex, which leads to the expression of NFkB-inducible genes.

On the other hand, activated TNF-R1 can recruit adapter proteins TRADD
and Fas—associated death domain (FADD) as well as pro-caspase 8, which leads
to the formation of the death-inducing signaling complex (DISC). Autoactivation
of procaspase 8 results in the generation of active caspase 8, which directly
cleaves procaspase 3 into caspase 3 and leads to apoptosis (Hehlgans and
Pfeffer, 2005). Caspase 8 converts Bid into truncated Bid (tBid). tBid contributes
to mitochondrial dysfunction via the formation of permeability transition pores
and Bak/Bax pores on the outer mitochondrial membrane, which leads to the
release of cytochrome c and reactive oxygen species to cytosol. Cytochrome c
activates caspase 9 and 3, which lead to apoptosis through activating caspase
activated DNase (CAD). Thereby, cell death is induced as a result of TNF-R1
activation.

Whether TNF leads to cell survival or death is determined by NFkB (Wullaert et
al., 2007). NFkB plays a protective role against cell death by inducing the

expression of several antiapoptotic genes, which include caspase-8 inhibitor c-

19

Fig. 1.3 TI

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Fig. 1.3 TNF signal transduction pathway. TNF can either activate NFkB or

induce cell death.

 

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20

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FLIPL, the Bcl-2 family members Bcl—xL and A1/Bﬂ-1, and X-linked inhibitor of
apoptosis (XIAP) (Zong et al., 1999; Chen et al., 2000; Micheau et al., 2001).
Moreover, prolonged activation of JNK resulting from ROS accumulation or TNF
signaling, causes cell death (Kamata et al., 2005). NFkB can rapidly terminate
JNK activation by upregulating the expression of antioxidant manganese
superoxide dismutase (MnSOD) and ferritin heavy chain (FHC) (Sakon et al.,
2003).

21

1.2.3 1

Va:

1.2.3 The hemostatic system and hypoxia

Vascular hemostasis is regulated by coagulation and ﬁbrinolysis. The
coagulation system is activated in many inﬂammatory diseases (Vrij et al., 2003).
It can be activated through the extrinsic pathway or the intrinsic pathway. In the
extrinsic pathway, tissue factor is exposed to blood and forms a complex with
factor VII, which activates factor X through the activation cascade. Factor X
cleaves prothrombin into thrombin, which converts ﬁbrinogen into ﬁbrin
monomers by cleaving ﬁbrinopeptides. Fibrin can cross-link to form ﬁbrin clots in
blood vessels (Mosesson et al., 2001). Fibrin clots are controlled and dissolved
by the ﬁbrinolytic system, in which plasmin plays an important role by degrading
ﬁbrin clots into D-dimers (Fay et al., 2007). Plasminogen activators (PAs) cleave
plasminogen to active plasmin. PAs can be inhibited by active plasminogen
activator inhibitor (PAl-1), which is synthesized by hepatocytes, endothelial cells
and platelets in response to TNF or LPS (Levi et al., 2003; Westrick and Eitzman,
2007). Therefore, an increase in active PAl-1 can dampen the ﬁbrinolytic system
and enhance ﬁbrin deposition.

Thrombin and PAl-1 are two critical factors in regulating the hemostatic
system, but they also play a role in activating inﬂammatory cells. Thrombin
cleaves and activates G protein-coupled receptors, including protease-activated
receptor-1 (PAR-1), which can signiﬁcantly increase production of pro-
inﬂammatory cytokines (T NF, lL-1 and lL-6) from target cells (Fan et al., 2005).
PAI-1 can potentiate LPS-induced neutrophil activation through a JNK-mediated

pathway in vitro and enhance nuclear translocation of NFkB, which increases

22

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production of the proinﬁammatory cytokines lL-1, TNF and MlP-2 (Kwak et al.,
2006). Previous studies in vivo also suggest that PAl-1 contributes to production
of TNF, IL-10, KC and MCP-1 (Shaw et al., 2009c).

Fibrin deposition controls the magnitude and area of infection, through which
ﬁbrin deposition plays a protective role against inﬂammagen exposure. However,
detrimental effects can also be induced by ﬁbrin deposition in liver sinusoids by
impairing blood ﬂow and thereby causing tissue hypoxia. Hypoxia exerts various
effects on hepatocytes. It activates numerous intracellular signaling pathways
related to transcription of hypoxia-responsive genes, mostly mediated by hypoxia
inducible factor-1a. Reactive oxygen species that accumulate after exposure to
hypoxia lead to the MPT (Qu et al., 2001; Schild and Reiser, 2005). Energy
deﬁcit (loss of ATP) and inhibition of aerobic metabolism are also caused by the
decrease of available oxygen. Hepatic metabolism and function are highly
oxygen-dependent. As a consequence of decreased cellular ATP level,
physiological function is impaired and liver damage can occur (Semenza, 2004).
Furthermore, hypoxia enhances LPS-induced liver damage (Shibayama, 1987)
and potentiates the toxic effects of PMN-derived proteases on hepatocytes in

vitro (Luyendyk et al., 2005).

1.2.4 Neutrophils (PMNs)
PMNs are abundant blood leukocytes and play an important role in the
innate immune response. PMNs are a contributor to tissue injury induced by

ischemia-perfusion and alcohol. (Jaeschke et al., 1990; Hewett et al., 1992;

23

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Jaeschke, 2002). PMNs are involved in several models of drug-induced liver injury
(e.g., acetaminophen) and in drug-LPS interaction models of idiosyncratic liver injury
(Deng et al., 2006; Deng et al., 2007b; Jaeschke and Liu, 2007; Ramaiah and
Jaeschke, 2007; Shaw et al., 2009d).

PMNs are primed and activated by systemic or local exposure to
proinﬂammatory mediators. TNF, lL-1 or CXC chemokines (ClNC-l, MIP-2) may
increase the expression of integrin on the surface of PMNs as well as lCAM-1
and VCAM-1 expression on the surface of endothelial cells (Jaeschke and Smith,
1997; Jaeschke et al., 1998). The binding of an adhesion molecule (lCAM-1 or
VCAM-1) to its counter-receptor (integrin) results in PMNs adhering tightly to
endothelial cells. However, in the RAN/LPS model, neutralization of integrin did
not reduce PMN numbers in the liver (Deng et al., 2007b). Thus, accumulation of
PMNs in vasculature is not necessarily dependent on the binding of adhesion
molecules.

PMN migration from the vasculature and inﬁltration into the parenchyma is a
prerequisite for neutrophil cytotoxicity (Jaeschke and Smith, 1997). During this
process, a signal from parenchymal cells is required. A chemotactic gradient of
CXC chemokines in liver can lead to PMN inﬁltration, which contributes to liver
injury (Okaya and Lentsch, 2003). Necrotic cells can release a mediator called
high mobility group box 1 (HMGB1) which can signal to PMNs and cause their
migration (T sung et al., 2005). Anti-HMGB1 antibody signiﬁcantly reduced the
production of proinﬂammatory cytokines and PMN inﬁltration in an ischemia-
reperfusion model of liver injury (T sung et al., 2005). Interestingly, apoptotic cells

might also trigger PMN migration (Jaeschke, 2006). It was observed that

24

T)

"A

A
:a 3..
’

In. we.

A.
«Jrf'
N

engulfment of apoptotic bodies by Kupffer cells promotes cytokine expression
and PMN activation (Canbay et al., 2003). Alternatively, it has been proposed
that gaps in the sinusoidal endothelial cells may facilitate the direct contact of
PMNs with altered membranes of apoptotic cells (Jaeschke, 2006).

After PMNs cross the endothelial cell barrier, they localize close to
hepatocytes and degranulate, leading to the release of proteases and reactive
oxygen species (ROS) including hydrogen peroxide and hypochlorous acid
(Dahlgren and Karlsson, 1999). Both proteases and ROS contribute to liver injury
in animal models (Jaeschke, 2006). Cathepsin G and elastase are two important
proteases which are released by activated PMNs. They are important mediators
of hepatic parenchymal cell killing (Ho et al., 1996). Inhibition of cathepsin G and
elastase protected against liver injury in a drug-inﬂammation interaction model
(Luyendyk et al., 2005). Besides direct effect on hepatocytes, proteases released
by PMNs can also contribute to ﬁbrin deposition by activating plasminogen
activator inhibitor-1 (PAl-1) (Deng et al., 2007a). The role of ROS in cell death is

discussed in the following section.

1.2.5 Reactive oxygen species

Reactive oxygen species (ROS) are oxygen free radical and nonradical (but
reactive) oxygen species. They include hydrogen peroxide, superoxide, hydroxyl
radicals, etc. ROS are produced by several sources within the cell. Mitochondria,
in which the electron transport chain transfers electrons to oxygen, are a major

source of ROS. During normal cellular respiration, about 2% of electrons escape

25

irt'eas
m-dase
pfasma
gewefa'
cage"
..m.

,u
l-‘IUI I

f??? FA
" «I ﬁt:

r~~ w

VUU'I

and lead to production of superoxide anion (Boveris and Cadenas, 1975). As
described in previous sections, TNF and hypoxia have been shown to cause
increased ROS generation in a mitochondria-dependent manner. Another major
source of ROS is nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase complex. The NADPH oxidase complex assembles on phagosomal,
plasma and granule membranes of activated PMNs and macrophages and
generates ROS (Dahlgren and Karlsson, 1999). NADPH oxidase reduces
oxygen to superoxide at the same time it oxidizes NADPH. Cytochrome b from
NADPH is responsible for the transfer of electrons to oxygen present in
intracellular compartments (Babior, 1999). In addition, many other enzymes,
including xanthine oxidase, cyclooxygenases, lipoxygenases, myeloperoxidases,
hemeoxygenase, monoamine oxidases, aldehyde oxidase, and cytochrome
P450, can cause ROS accumulation. However, the capacity of these enzymes to
generate ROS is less robust than the mitochondrial electron chain complex or
NADPH oxidase (Morgan et al., 2008).

Excessive ROS may tilt the prooxidant-antioxidant balance, causing
oxidative stress in cells. ROS can directly oxidize proteins and lipids as well as
nucleic acids, leading to cellular damage and dysfunction (Morgan et al., 2008).
ROS may induce cell death through a JNK-dependent pathway (Schwabe and
Brenner, 2006). ROS directly inactivate JNK phosphatase, which leads to
prolonged activation of JNK (Kamata et al., 2005). Activated JNK phosphorylates
antiapoptotic Bcl-2 family proteins (Bcl-2, BcI-XL) and inactivates them

(Yamamoto et al., 1999a; Fan et al., 2000).

26

a'ésa'

0'55!“
3U. .

il'sE'S l

ctalier

1r] FA:
U

Oxidative stress plays a role in several models of liver injury. It occurs and
contributes to ischemia/reperfusion injury in mice (Serteser et al., 2002).
Involvement of oxygen free radicals and consequently of oxidative stress has
also been proven in alcoholic liver disease (Koch et al., 2004). R08 are involved
in the pathogenesis of inﬂammatory liver diseases (Jaeschke, 2000). In an
animal model of LPS-induced organ injury, a signiﬁcant decrease in reduced
glutathione and an increase in lipid peroxidation were observed in the lungs and
livers of rats (Suntres and Shek, 1996). The administration of antioxidants after
challenge with LPS resulted in a signiﬁcant alleviation of both lung and liver
injuries. Mice deﬁcient in antioxidant enzyme showed greater susceptibility to
PMN-mediated liver injury (Jaeschke et al., 1999). An NADPH oxidase inhibitor
protected against endotoxin-induced PMN-mediated liver injury, suggesting a
critical role for ROS (Gujral et al., 2004).

Oxidative stress has also been proposed as a potential mechanism of
NSAlD-induced hepatotoxicity (Boelsterli, 2002). Although the role of ROS in
SLD-induced liver injury has not been determined in vivo, SLD and its toxic
metabolite were reported to increase ROS production in several cell lines (Galati

et al., 2002; Adachi et al., 2007).

27

1.3.1 F
Re

*‘ROM
”6 rl
“ V a .

7
ha» 0.-
I :vv \~

1.3 Models of drug-inﬂammation interaction

There are few animal models to study the mechanisms of drug-induced
idiosyncratic hepatotoxicity. The main reason for this is that idiosyncratic
hepatotoxicity-induced by drugs may only occur in a small fraction of animals, as
it often does in humans. However, based on the hypothesis that inﬂammation
precipitates drug toxicity, several idiosyncratic liver injury models based on drug-

inﬂammation interaction have been developed.

1.3.1 RanitidineILPS- induced hepatotoxicity in rats

Ranitidine (RAN) is a histamine-2 (H2)-receptor antagonist used for the
treatment of duodenal ulcers, gastric hypersecretory diseases and
gastroesophageal reﬂux disease. RAN is associated with idiosyncratic
hepatotoxicity with an incidence of less than 1 in 1000 patients taking the drug
(Vial et al., 1991). Famotidine (FAM), although in the same pharmacological
class with RAN, has a decreased propensity to cause idiosyncratic reactions.

An animal model of RAN-induced idiosyncratic liver injury was developed by

pretreating rats with LPS (Luyendyk et al., 2003). In this model, LPS (44.4X106

EU/kg) or its saline vehicle was administered to rats via a tail vein. Two hours
later, RAN (30 mg/kg), FAM (6 mg/kg), or their vehicle (sterile phosphate—
buffered saline) was administered i.v. at a rate of approximately 0.15 mllmin.
Neither RAN nor LPS given alone had a signiﬁcant hepatotoxic effect as
measured by ALT activity compared to control animals. FAM was also not

hepatotoxic to rats in the presence or absence of LPS. In contrast, cotreatrnent of

28

rats wi
ALT. l

quiﬁﬂ'
.LVLV'UI

aorta}

R;

“C

rats with RAN/LPS led to a signiﬁcant increase in markers of liver injury (e.g.,
ALT, AST and GGT) in serum at 6, 12 and 24 hr after RAN treatment.
Histopathology demonstrated the presence of midzonal hepatic necrosis in
animals receiving RAN/LPS cotreatrnent.

RAN but not FAM enhanced the LPS-induced TNF increase before the onset
of hepatocellular injury (Tukov et al., 2007b). It was also observed that a large
concentration of RAN enhanced LPS-induced TNF release in a Kupffer cell-
hepatocyte coculture system. RAN enhanced the activation of p38 induced by
LPS, which led to increased TACE activation (Deng et al., 2008). TACE cleaved
pro-TNF into TNF and led to an increase in TNF concentration in the plasma.
TNF plays a critical role in the pathogenesis of liver injury, which is supported by
the evidence that TNF neutralization protected against liver injury induced by
RAN/LPS. RAN also enhanced the increase in serum interleukin (lL)-1beta, lL-6
and lL-10 induced by LPS.

In plasma of rats treated with RAN/LPS, a decrease in ﬁbrinogen and
increases in thrombin-antithrombin (TAT) dimers and PAl-1 occurred before the
onset of liver injury, suggesting that the hemostatic system was activated by the
cotreatrnent (Luyendyk et al., 2004). Hepatic ﬁbrin deposition was observed in
livers of rats cotreated with RAN/LPS at 3 after RAN (ie, before the onset of liver
injury). The anticoagulant heparin or the ﬁbrinolytic agent streptokinase
signiﬁcantly reduced liver injury induced by RAN/LPS. Hypoxia probably resulting
from sinusoidal ﬁbrin deposition was observed in livers of RAN/LPS-treated rats

at 3 hr, and this was signiﬁcantly attenuated by heparin.

29

I

ccﬁﬂr
(gag.

nct a.“

tasma

PMNs are also critical to RAN/LPS-induced liver injury (Luyendyk et al.,
2006). PMN accumulation occurred in livers of rats treated with LPS/RAN.
Depletion of PMNs using anti-PMN serum protected against liver injury,
suggesting that PMNs are involved in the pathogenesis.

TNF, the hemostatic system and PMNs do not act independently of each
other. TNF contributes to RAN/LPS-induced liver injury by enhancing production
of inﬂammatory cytokines, chemokines (MlP-2) and hemostatic factors including
TAT and PAl-1 (T ukov et al., 2007b). However, hepatic PMN accumulation was
not affected by TNF. Heparin had little effect on liver PMN accumulation or
plasma chemokine concentration, indicating that PMN accumulation is not
affected by ﬁbrin deposition in the liver (Luyendyk et al., 2006). However, both
TNF and PAl-1 contribute to PMN activation (Deng et al., 2007b; Deng et al.,
2008). PMN depletion reduced the plasma concentration of active PAl-1 and
ﬁbrin deposition in livers of rats treated with RAN/LPS, which suggests that
PMNs promote ﬁbrin deposition by increasing PAl-1concentration (Deng et al.,
2007b). As a result, PMNs also promote hypoxia in the liver. These studies
suggest that mediators involved in the pathogenesis of liver injury induced by

RAN/LPS are not isolated, but interact with each other.

1.3.2 Diclofenac/LPS-induced hepatotoxicity in rats
Diclofenac (DCLF) is a nonsteroidal anti-inﬂammatory drug (NSAID)
associated with serious idiosyncratic hepatotoxicity in humans (Aithal, 2004). The

incidence of DCLF-induced liver injury is approximately one to ﬁve cases per

30

IIVer min
DCLF. I.

01' 15 58

“A

‘1‘.
93h.
No

Was E

100,000 persons exposed (Garcia Rodriguez et al., 1994). Liver failure has been
recorded after the administration of DCLF (Greaves et al., 2001). Several
hypotheses of DCLF-induced liver injury have been proposed, including
metabolic polymorphism and allergic hypersensitivity (Daly et al., 2007).
However, in vivo evidence that supports these hypotheses is still lacking.

As in the RAN/LPS-induced liver injury model, a model of DCLF-induced

liver injury in rats was developed by treating rats with a nonhepatotoxic dose of

DCLF, LPS or their vehicles (Deng et al., 2006). Generally, LPS (29 X106 EU/kg)

or its saline vehicle was administered to rats via a tail vein. Two hours later, rats
were given DCLF (20 mg/kg, i.p.) or sterile saline. Neither LPS nor DCLF alone
had an effect on ALT activity. However, cotreatment with LPS and DCLF caused
a signiﬁcant increase in serum ALT activity. Hepatocellular apoptosis,
parenchymal edema, and hemorrhage induced by LPS were also signiﬁcantly
increased by DCLF cotreatrnent.

A gene array study was performed to compare the gene expression patterns
among LPS, DCLF and cotreatrnent groups. Genes encoding the neutrophil
chemokines, such as MlP-2 and MlP-1, and the adhesion molecule lCAM-l,
were greatly increased by DCLF/LPS cotreatrnent compared to LPS or DCLF
alone. Both LPS alone and DCLF/LPS treatment led to hepatic PMN
accumulation. DCLF did not enhance the effect of LPS on PMN accumulation,
although the increase in MlP-2 concentration in serum of rats treated with LPS

was enhanced by DCLF. Anti-PMN serum reduced PMN accumulation in liver

31

rats,

lnAEarﬁ
I V ‘

l“ I

; ”Na

and attenuated liver injury induced by DCLF/LPS. This result suggests that
PMNs play a critical role in the pathogenesis.

Interestingly, a larger dose of DCLF (100 mglkg, i.p.) caused liver injury in
rats, which was attenuated by treatment with nonabsorbable antibiotics
(polymyxin B and neomycin) for 4 days before DCLF administration. This
suggests that bacterial translocation from intestine to liver plays a critical role in

DCLF-induced hepatotoxicity though interacting with DCLF.

1.3.3 Trovafloxacin-inflammation interaction model of idiosyncratic liver
injury

Trovaﬂoxacin (TVX) is a broad spectrum antibiotic which functions through
inhibiting bacterial topoisomerase IV. TVX was approved for marketing in 1997.
Two years later, its use was severely limited due to the risk of hepatotoxicity.
TVX is associated with idiosyncratic hepatotoxicity with an incidence of 1 in
18,000 prescriptions (Stahlmann, 2002). Another ﬂuoroquinolone antibiotic,
levoﬂoxacin (LVX), has not been associated with idiosyncratic liver injury and
was used in model development as a control (Shaw et al., 2007).

Both a rat and mouse model of liver injury induced by TVX/LPS cotreatrnent
was developed (Shaw et al., 2009d). In the mouse model (Shaw et al., 2007),
TVX (150 mglkg), LVX (375 mg/kg) or their vehicle (saline) was administered to
mice by oral gavage. LPS or its vehicle was administered intraperitoneally to
mice 3 hr after the drug. TVX, LVX, LPS alone or LVX/LPS cotreatment did not

increase plasma ALT activity, whereas TVX/LPS cotreatrnent signiﬁcantly

32

increased
after LPS
treated W‘

.09chth fl

et al. 23

liver inju'
.pporfec

”7.qu in

“53}. l

ii”...

50’

increased this biomarker of hepatocellular injury at 9 hr and peaked at 15-21 hr
after LPS administration. Hepatocellular necrosis was observed in livers of mice
treated with TVX/LPS, but not in those treated with TVX, LVX or LPS alone. The
necrotic foci observed in the TVX/LPS-treated group were found in midzonal and
centrilobular regions.

TVX prolonged the appearance of TNF induced by LPS in the plasma (Shaw
et al., 2007). TNF neutralization using etanercept attenuated TVX/LPS-induced
liver injury, suggesting that TNF is an important mediator. This was further
supported by evidence that TVX and TNF cotreatrnent caused signiﬁcant liver
injury in mice, whereas neither TVX nor TNF was hepatotoxic (Shaw et al.,
2009a). Interestingly, TVX prolonged the appearance of TNF in the plasma of
mice cotreated with TVX/TNF compared to mice given only TNF. This prolonged
appearance of TNF was caused by both enhanced production and decreased
clearance of this cytokine. Comparison of hepatic gene expression proﬁles from
mice treated with TVX/LPS to those treated with LPS or TVX alone suggested
that the interferon v (lFNv) signaling pathway was selectively activated by
TVX/LPS (Shaw et al., 2009b). TVX enhanced the appearance of interleukin
(lL)-18 that contributes to the production of lFNy. In turn, IFNy can feedback to
increase lL-18. TVX/LPS-induced liver injury was attenuated in either lFNy -/- or
lL-18 -/- mice, which indicate both lFNy and lL-18 are important mediators in the
pathogenesis of TVX/LPS-induced liver injury.

Compared to LPS, TVX, LVX alone or LVX/LPS cotreatrnent, TVX/LPS

caused a signiﬁcant increase in plasma concentration of TAT dimers and PAl-1,

33

WTlCl'l w
Ezner F
WXLPE
tSnW

PM
TNF is l

Za'lclK

which was accompanied by ﬁbrin deposition in the liver (Shaw et al., 2009c).
Either PAl-1 knockout or heparin treatment reduced liver injury caused by
TVX/LPS, indicating that PAH and ﬁbrin deposition contributed to liver injury in
this model.

PMNs also contribute to TVX/LPS-induced liver injury (Shaw et al., 2009d).
TNF is responsible for the production of PMN chemokines including MlP-1, MIP-
2 and KC in this model (Shaw et al., 2009e).

In addition to the TVX/LPS interaction model of idiosyncratic liver injury,
inﬂammation induced by a Gram-positive stimulus, a peptidoglycan-lipoteichoic
acid (PGN-LTA) mixture isolated from Staphylococcus aureus, also precipitates
TVX-induced liver injury in mice (Shaw et al., 2009d). PGN and LTA activate
TLR2 to induce inﬂammation, which indicates that liver injury induced by drug-
inﬂammation interaction is not necessarily dependent on TLR4 pathway.

Besides the three animal models introduced here, there are other models of
IADRs, in which liver injury is induced by the cotreatrnent of chlorpromazine/LPS,
halothane/LPS and halothane/poly l:C. However, the mechanisms underlying

these models are not yet fully understood.

34

1.4 Sulindac-induced idiosyncratic liver injury

Sulindac (SLD) is a prodrug in the therapeutic class of NSAIDs. SLD was
introduced into the market in 1978 by Merck under the brand name Clinoril to
relieve pain, tenderness, swelling, and stiffness caused by osteoarthritis,
rheumatoid arthritis and ankylosing spondylitis. Typically, the dose of SLD for
human patients is 150-200 mg, twice per day as a result of its 8 hr half life. The
bioavailability of SLD is more than 90%. SLD is absorbed rapidly upon oral
administration, and reaches a peak in human plasma in 2-4 hr (Davies and
Watson, 1997). SLD and its metabolites are secreted into bile and undergo
enterohepatic circulation (Bolder et al., 1999). SLD and its metabolites are
excreted in urine and feces.

Unlike prodrugs that are irreversibly bioactivated to active metabolites, SLD
can be reversibly converted to the active metabolite, SLD sulﬁde, and irreversibly
converted to SLD sulfone (Fig. 1.4). According to previous studies, two enzymes
are responsible for SLD metabolism: methionine sulfoxide reductase (MSR) in
both liver and gut ﬂora reduces SLD to SLD sulﬁde, and a ﬂavin-containing
monooxygenase (FMO) converts SLD to SLD sulfone and also catalyzes the
conversion of SLD sulﬁde to SLD (Etienne et al., 2003b).

The SLD active metabolite, SLD sulﬁde, performs its pharmacological
function by inhibiting cyclooxygenases (COX) -1 and -2 (Lin et al., 1985). Both
COX-1 and COX-2 are responsible for the synthesis of prostaglandins. COX-1 is
constitutively expressed in normal cells and plays a beneﬁcial role, e.g.,maintain

the normal function of GI tract, renal tract, platelet function. COX-2 is inducible

35

Fig. 1.4 Metabolism of SLD (Duggan et al., 1980). SLD can be reversibly
converted to the active metabolite, SLD sulﬁde by methionine sulfoxide

reductase (MSR), and irreversibly converted to SLD sulfone by ﬂavin-containing

monooxygenase (FMO).

F 0|chsz F enzcozn F CHM
We"; 32+ cu; ﬂ‘°_. cu;
ca 73;— CN 0 cu
c333/E:][’ IE::]’ ca§§’E::l'

3‘ 5° 0
0
SULFIDE SULINDAC SULFONE

(SULFOXIDE)

36

enzyme expressed by macrophages and contributes to inﬂammation. Therefore,
the inhibition of COX—2 is effective to resolve inﬂammation.

SLD has been used in the United State for over a decade, during which it
has been associated with increased risk of heart attack, ulcer, stroke and liver
injury (Tarazi et al., 1993). The Food and Drug Administration (FDA) Arthritis
Advisory Committee wrote that “the potential for producing liver injury is a class
characteristic of NSAIDs” (Paulus, 1982; Tarazi et al., 1993). SLD was
associated with a 5—10 fold higher incidence of hepatic injury than other NSAIDs,
which induced liver injury at an incidence of about 1 in 100,000 (Walker, 1997).
According to the analysis of cases reported to FDA, SLD-induced liver injury
often occurred within 8 weeks of taking the drug, whereas about 20% of the
reported reactions occurred after 8 weeks of treatment (T arazi et al., 1993).
Females are more susceptible to toxicity of SLD than males, and two thirds of the
patients were over 50 years of age. Histopathology showed that the pattern of
SLD-associated liver injury can be cholestatic, hepatocellular or mixed. In the
cases of hepatocellular injury, the lesions were spotty and panacinar in most
cases (8 out of 9). Portal inﬂammation and eosinophil inﬁltration was observed in
a portion of patients.

Mechanisms of SLD-induced idiosyncratic liver injury are not well
understood. Because clinical characteristics consistent with hypersensitivity were
observed in some patients, it was proposed that hypersensitivity accounts for a
signiﬁcant proportion of SLD-induced liver injury. However, no direct evidence for

this has been found, and the mechanisms of pathogenesis still require further

37

investigation. Another hypothesis for liver injury induced by NSAIDs including
SLD is through mitochondrial injury. A variety of NSAIDs or their metabolites
(e.g., nimesulide, DCLF and SLD) have a toxic effect to the mitochondria of
hepatocytes in vitro. There is little evidence in vivo supporting this hypothesis,
and an idiosyncratic liver injury model for SLD has not been developed on the

basis of this hypothesis.

38

1.5 Hypothesis and speciﬁc aims

The overall hypothesis of this dissertation is that an inﬂammatory episode
induced by LPS precipitates SLD-induced liver injury in rats. According to
previous studies on the mechanism of LPS- or drug/LPS- induced liver injury, we
hypothesize that TNF, the hemostatic system, PMNs and ROS are elevated by
SLD/LPS cotreatrnent and play a critical role in the pathogenesis of liver injury.
The SLD toxic metabolite, SLD sulﬁde also contributes to liver injury by

synergistically interacting with those inﬂammatory mediators.

Aim 1 Hypothesis
Cotreatment with nonhepatotoxic doses of SLD and LPS causes idiosyncrasy-

like Iiver injury in rats. (Chapter 2)

Aim 2 Hypothesis

The hemostatic system is activated by SLD/LPS cotreatrnent in rats and

contributes to liver injury by causing hypoxia. (Chapter 2)

Aim 3 Hypothesis

TNF, which is increased by SLD/LPS cotreatrnent, plays an important role in liver

injury by interacting with SLD sulﬁde. (Chapter 3)

Aim 4 Hypothesis

39

PMNs are activated in livers of rats treated with SLD/LPS and contribute to liver

injury by releasing toxic proteases. (Chapter 4)

Aim 5 Hypothesis

ROS production is increased in livers of rats treated with SLD/LPS. ROS are
involved in the pathogenesis of liver injury by enhancing the cytotoxicity of TNF.
(Chapter 5)

40

CHAPTER 2

Zou W, Devi SS, Sparkenbaugh E, Younis HS, Roth RA and Ganey PE

(2009) Hepatotoxic interaction of sulindac with lipopolysaccharide: role of

the hemostatic system. Toxicol Sci 108:184-193.

41

2.1 Abstract

Sulindac (SLD) is a nonsteroidal anti-inﬂammatory drug (NSAID) that has
been associated with a greater incidence of idiosyncratic hepatotoxicity in human
patients than other NSAIDs. One hypothesis regarding idiosyncratic adverse
drug reaction (IADRs) is that interaction of a drug with a modest inﬂammatory
episode precipitates liver injury. In this study, we tested the hypothesis that
lipopolysaccharide (LPS) interacts with SLD to cause liver injury in rats. SLD (50

mglkg) or its vehicle was administered to rats by gavage 15.5 hr before LPS

(8.3X105 EU/kg) or its saline vehicle (i.v.). Thirty min after LPS treatment, SLD or

vehicle administration was repeated. Rats were killed at various times after
treatment, and serum, plasma and liver samples were taken. Neither SLD nor
LPS alone caused liver injury. Cotreatment with SLD/LPS led to increases in
serum biomarkers of both hepatocellular injury and cholestasis. Histological
evidence of liver damage was found only after SLD/LPS cotreatrnent. As a result
of activation of hemostasis induced by SLD/LPS cotreatment, ﬁbrin and hypoxia
were present in liver tissue before the onset of the hepatotoxicity. Heparin
treatment reduced hepatic ﬁbrin deposition and hypoxia and protected against
liver injury induced by SLD/LPS cotreatrnent. These results indicate that
cotreatrnent with nontoxic doses of LPS and SLD causes liver injury in rats, and
this could serve as a model of human idiosyncratic liver injury. The hemostatic
system is activated by SLD/LPS cotreatrnent and plays an important role in the

development of SLD/LPS-induced liver injury.

42

2.2 Introduction

Previous studies suggested that mild inﬂammation induced by bacterial
lipopolysaccharide (LPS) could potentiate hepatotoxicity in rodents from IADR-
associated drugs such as chlorpromazine (Buchweitz et al., 2002), ranitidine
(Luyendyk et al., 2003) and trovaﬂoxacin (Shaw et al., 2007). In one of these
LPS/drug interaction models, the hemostatic system proved to be important in
liver pathogenesis (Luyendyk et al., 2005). This system comprises coagulation
and ﬁbrinolytic components. In coagulation, thrombin plays a critical role by
cleaving ﬁbrinogen to ﬁbrin that can form occlusive clots in sinusoids. Meanwhile,
plasminogen activator inhibitor-1 (PAl-1) can inhibit ﬁbrin clearance by the
ﬁbrinolytic system by inhibiting the generation of plasmin from plasminogen.

To evaluate the utility of LPS/drug interaction models to study mechanism(s)
of idiosyncratic liver injury, a broad range of drugs needs to be tested. Therefore,
the purpose of this study was to test the hypothesis that modest inﬂammation
induced by LPS interacts with SLD to cause liver injury in rats. When the results
demonstrated a hepatotoxic interaction, the role of the hemostatic system was

explored.

43

2.3 Materials and methods
2.3.1 Materials
Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich

(St. Louis, MO). The activity of lipopolysaccharide (Lot 075K4038) derived from

Eschen'cia coli serotype O55:B5 was 3.3 X 106 endotoxin units (EU)/mg as

determined by a Limulus amebocyte lysate endpoint assay kit purchased from
Cambrex Corp. (Kit 50-650U; East Rutherford, NJ). The reagents for alanine
aminotransferase (ALT), aspartate aminotransferase (AST), y-
glutamyltransferase (GGT) and total bilirubin were purchased from Thermo Corp.
(Waltham, MA). Alkaline phosphatase (ALP) reagent was purchased from
BioAssay Systems (Hayward, CA), and the kit for total bile acid determination

was purchased from Diazyme Laboratories (Poway, CA).

2.3.2 Animals

Male, Sprague-Dawley rats (Crl:CD(SD)lGS BR; Charles River, Portage, Ml)
weighing 250 to 370 g or 150 to 200 g were used for in vivo or in vitro studies,
respectively. Animals were allowed to acclimate for 1 week in a 12-hr light/dark
cycle prior to use in experiments. They were fed standard chow (Rodent
Chow/Tek 8640; Harlan Teklad, Madison, WI) and allowed access to water ad

libitum.

2.3.3 Experimental protocol

44

Two administrations of SLD were used in the studies (Fig. 2.1). In a dose-
response study, rats were given the ﬁrst administration of SLD (10, 20, 50, 100 or

300 mglkg, p.o.) or its vehicle (0.5% methyl cellulose), and food was removed for

24 hr. 15.5 hr after the ﬁrst administration of SLD, LPS (8.25X 105 EU/kg, i.v.) or

its saline vehicle was administered. Half an hour later, a second administration of
SLD (same dose) or its vehicle was given. Rats were anesthetized with
isoﬂurane and killed at various times after the second administration of SLD. For
subsequent studies, 50 mg/kg was chosen as the dose of SLD. For all studies,
blood was drawn from the vena cava of anesthetized rats, and part was
transferred into vacutainer tubes (Becton Dickinson) containing sodium citrate for
preparation of plasma. The rest of the blood was allowed to clot at room
temperature for preparation of serum. The exterior of the liver was rinsed with
saline, and a portion of the left medial lobe was snap frozen in cooled
methylbutane for immunohistochemistry. Three slices of the left lateral lobe about
3-4 mm thick were ﬁxed in 10% buffered formalin for histological analysis. In
experiments designed to evaluate the role of the hemostatic system,
anticoagulant heparin (3000 Units/kg, so) or its saline vehicle was given to rats

0 and 6 hr after the second administration of SLD.

2.3.4 Evaluation of liver injury
Hepatic parenchymal cell injury was assessed by measuring the activities of ALT
and AST in serum. Cholestatic injury markers, including the activities of ALP and

GGT, as well as the concentrations of total bilirubin and bile acids in serum, were

45

Fig. 2.1. Experimental protocol for animal treatment. Rats were given SLD or

its vehicle (0.5% methyl cellulose) at —16 hr, and food was removed. At -0.5 hr

5
rats received LPS (8.25X10 EUIkg, i.v.) or its vehicle (saline), and 30 min later

they were given a second administration (same dose as 1St administration) of

 

 

 

 

 

SLD.
13t SLD 2"Cl SLD
(10 - 300 mglkg, p.o.) (10 - 300 mglkg, p.o.)
or Veh or Veh
LPS Rats euthanized
5 . samples taken
(8.25 x 10 EUIkg, i.v.)
or Veh l
w k
V ‘ l Ur ' ‘ )
Hr -16 -0.5 0 24

46

also assessed (see above).

Forrnalin-ﬁxed liver slices were embedded in parafﬁn and cut into 6 um
sections. Hematoxylin and eosin (H&E) staining was performed, and sections
were examined under 100X magniﬁcation using a light microscope. Eight,
randomly chosen microscope ﬁelds for each slide were evaluated for midzonal
necrosis and assigned a score of 0-5. 0 represents no liver injury, and 1-5
represents lesions ranging from single cell necrosis (1) to necrotic area
encompassing greater than 30% of the ﬁeld (5). The average score was

determined for each rat.

2.3.5 Evaluation of serum TNFa concentrations
The concentration of TNFa in serum taken at 1 hr after the second
administration of SLD was measured using an ELISA kit purchased from BD

Biosciences (San Diego, CA).

2.3.6 Evaluation of hemostasis and ﬁbrin deposition
Thrombin-antithrombin dimer (TAT) concentration in plasma was used as a
marker of thrombin activation and evaluated using an ELISA kit (catalog number
OWMG15) purchased from Dade Behring, Inc. (Deerﬁeld, Illinois). The
concentration of the active form of plasminogen activator inhibitor-1 (active PAI-
1) was determined using a kit from Molecular Innovations, Inc. (Southﬁeld, MI).
The immunohistochemistry and quantiﬁcation for cross-linked ﬁbrin in liver

were performed as described previously (Copple et al., 2002). Fibrin monomer is

47

solubilized in this protocol, and only cross-linked ﬁbrin in liver is stained. To
investigate whether ﬁbrin deposition occurs before the onset of injury, livers were
collected at 4 hr and ﬁxed for immunohistochemistry. The fraction of positive
pixels averaged from 10 randomly chosen microscope ﬁelds was determined for

each animal.

2.3.7 Evaluation of liver hypoxia

Liver hypoxia was evaluated by quantifying pimonidazole (PlM)-protein
adducts. PIM is a hypoxia probe which is rapidly reduced under low p02
conditions to a reactive intermediate that forms PIM-protein adducts. PIM
hydrochloride (Hypoxyprobe-1, 120 mg/kg; Chemicon International, Temecula,
CA) was given to rats 2 hr before sacriﬁce. Four hr after the second
administration of SLD, livers were collected and ﬁxed for immunohistochemistry.
The fraction of positive pixels averaged from 10 randomly chosen microscope

ﬁelds was determined for each rat (Copple et al., 2004).

2.3.8 Hepatocyte (HPC) isolation and hepatocytotoxicity assessment in
vitro

HPCs were isolated from rat liver as previously described (Tukov et al.,
2006). Isolated cells were suspended in Williams’ Medium E (Gibco BRL,
Rockville, MD) with 10% fetal bovine serum, and cell viability was evaluated

using trypan blue exclusion. The cell viability was always above 80%. The HPCs

were suspended and plated randomly at a density of 2.5 x 105 cells/well in 12-

48

well plates (Corning lnc., Corning, NY). After 2.5 to 3 hr incubation which
allowed HPCs to attach to the plate, serum-containing medium was removed,
and serum-free medium was added. HPCs were treated with 60 uM sulindac
sulﬁde or its vehicle (0.06% DMSO) and incubated in the presence of 20% or 5%
O; (with 5% C02 and balance N2). After 8 hr incubation, the medium was
collected, and the unattached cells were isolated by centrifugation. Both the
remaining attached cells and unattached cells were lysed with 1% Triton X-100.
ALT activity in the medium, attached cell lysate and unattached cell lysate was
determined. Hepatocytotoxicity was assessed by calculating the ALT activity in
the medium plus unattached cells as a percentage of the total ALT activity in the

well (medium + unattached cell lysate + attached cell lysate).

2.3.9 Statistical analysis

One way or two way analysis of variance (ANOVA) was used for data
analysis, and Tukey’s test was employed as a post hoc test. For GGT activity
and necrotic lesion score data, an ANOVA on ranks was performed, and Dunn’s
test was used for multiple comparisons. P < 0.05 was set as the criterion for

statistical signiﬁcance.

49

2.4 Results
2.4.1 Dose-response and timecourse of liver injury

SLD alone did not induce liver injury in rats at any of the doses given. SLD
(2 administrations) at doses of 10 or 20 mg/kg did not cause hepatotoxicity in
LPS-treated rats; however, rats had signiﬁcant liver injury after cotreatrnent with
50, 100 or 300 mg/kg SLD plus LPS (Fig. 2.2). Fifty mg/kg was chosen as the
SLD dose for further study.

In a time course study, SLD or LPS given alone did not increase serum ALT
activity at any time examined (Fig. 2.3A). In the SLD/LPS-cotreated group, ALT
activity remained normal for 4 hr but began to increase by 8 hr after the second
administration of SLD. By 12 hr, a signiﬁcant increase in serum ALT activity was
observed. By 24 hr, ALT activity had decreased to near normal. The activity of
serum AST in rats also reached its peak at 12 hr and showed a pattern similar to
ALT activity (Fig. 2.33).

At 12 hr, the activities of ALP and GGT as well as the concentrations of total
bilirubin and bile acids were also elevated signiﬁcantly in the sera of SLD/LPS-
cotreated rats compared to those of rats treated with SLD or LPS alone (Fig.

2.4).

2.4.2 Histopathological ﬁndings
Hepatocellular lesions were not found in livers of rats treated with VehNeh,
SLDNeh or Veh/LPS (Fig 2.5A, 2.5B and 25C). ln livers of SLD/LPS-cotreated

rats, necrotic foci were present in the midzonal regions (Fig. 2.50). These were

50

Fig. 2.2. Sulindac dose-response in the absence and presence of LPS. Rats
were treated with various doses of SLD (10, 20, 50, 100 or 300 mglkg, p.o.) or its

vehicle (0.5% methyl cellulose) at -16 hr, and food was removed. At -0.5 hr rats

received LPS (8.25X105 EUIkg, i.v.) or its vehicle (saline), and 30 min later they

were given a second administration (same dose as 1st administration) of SLD.
Blood samples were taken at 12 hr after the second administration of SLD, and
ALT activity was measured. *signiﬁcantly different from SLDNeh group at the

same dose. P< 0.05, n=3.

2000 -

-O— SLDNeh
* -O— SLD/LPS

1500 ‘

ALT (UIL)
3
8

500 ‘

 

 

 

 

. . . . 4
O 50 100 150 200 250 300

SLD Dose (mglkg)

 

51

Fig. 2.3. Development of hepatocellular injury induced by SLD/LPS
cotreatrnent. Rats were treated with SLD (50 mglkg, p.o.) or its vehicle (0.5%
methyl cellulose) and LPS or its vehicle as described in Fig. 2.2. Blood samples
were taken at various times (0, 1, 2, 4, 8, 12 or 24 hr), and ALT (A) and AST (B)
activities in serum were measured. *signiﬁcantly different from all other groups at
the same time. #signiﬁcantly different from SLD/LPS group at 0 hr. P<0.05, n=5-
10.

52

>

 

—O— VehNeh
. *# —o— SLDNeh
2000 —v— Veh/LPS
A —¢v- SLD/LPS
E
|-— .
_' 1000
< A
500 .
0 -\/‘\ _“—U——\I——————_—\r
0 4 8 12 16 20 24
Hr after 2nd Sulindac Administration
* + VehNeh
# —o— SLDNeh
200° ‘ + Veh/LPS
—A— SLD/LPS
51500 - ‘
a
|— .
(D 1000 ‘
<
500 ‘

 

——

\ 1 ‘ I ~ ,. 7
..h._. \ \ ’
o T V t

0 4 8 12 16 20 24
Hr after 2"d Sulindac Administration

 

 

53

Fig. 2.4. Markers of hepatic cholestasis induced by SLD/LPS cotreatrnent.
Rats were treated as described in Fig. 2.3, and serum samples were collected at
12 hr. Activities of alkaline phosphatase (ALP) and v-glutamyltransferase (GGT)
as well as concentrations of total bilirubin and bile acids in serum at 12 hr were

evaluated. *signiﬁcantly different from Veh/LPS group. #signiﬁcantly different

from SLDNeh group. asigniﬁcantly different from VehNeh group. p<0.05, n=4-

11.

 

 

 

 

 

m "m l - ,l
8 I7- I: ,l||ll m
I
m “m .mMHU w
m .m. m m w m o m m m .m. o
:23 mEo< BE .53. 35v n_._<
D
*.I
..ll- . s

— Veh
Veh

m
m

1g III

 

 

 

mlamwwsm

w m m w o 33:00

:23 =5§=m .33

55

Fig. 2.5. Liver histopathology in SLDILPS-cotreated rats. Rats were treated
with VehNeh (A), SLDNeh (B), Veh/LPS (C) or SLD/LPS (D) as described in
Fig. 2.3. Liver sections were collected at 12 hr and were stained with hematoxylin
and eosin. The pictures were taken under 200 X maginiﬁcation, and a necrotic

area is indicated with an arrow.

' I
. '. ..‘. 1‘;° 1

I
one.

 

 

 

associated with hemorrhage and neutrophil inﬁltration. The numbers and sizes of
necrotic foci in livers from SLD/LPS-treated rats progressed with time and were
consistent with the elevated ALT and AST activities in rat serum (Table 2.1).
Signiﬁcant liver lesions were observed beginning at 8 hr after the second

administration of SLD and persisted through 24 hr.

2.4.3 Effect of SLD/LPS cotreatrnent on serum TNFa concentration
SLD alone had no effect on the concentration of TNFa in serum (Table 2.2).
Treatment with LPS alone caused a signiﬁcant increase in the serum TNFo

concentration within 1.5 hr. SLD signiﬁcantly enhanced the increase in serum

TNFa induced by LPS.

2.4.4 Activation of the hemostatic system

Thrombin functions as a vital activator of the coagulation system, whereas
active PAl-1 is the major endogenous down-regulator of the ﬁbrinolytic system.
The concentrations of these two regulators were evaluated using ELISA at 1, 4
and 8 hr after the second administration of SLD. Both TAT and active PAl-1
concentrations were elevated by LPS alone at 1, 4 and 8 hr, whereas SLD was
without effect by itself (Fig. 2.6). At 4 hr after the second administration of SLD,
SLD signiﬁcantly enhanced the LPS-induced increases in TAT and active PAI-1
in plasma. Compared to LPS alone, SLD/LPS cotreatrnent also tended to prolong

the elevation in thrombin and active PAl-1 in plasma (Fig. 2.6A, 2.6B).

57

Table 2.1. Midzonal hepatic necrosis in livers of rats treated with SLD/LPS

Liver sections from rats killed at 4, 8, 12 and 24 hr after the second

administration of SLD were evaluated and assigned a score of 0-5 as described

under Materials and Methods. Data are expressed as median score and 25th and

75th quartiles. *signiﬁcantly different from VehNeh group at the same time.

 

 

 

P<0.05, n=5-10.
Treatment Time after 2nd SLD (hr)
4 8 12 24
VehNeh 0.50 (0.34-0.53) 0.50 (0.31-0.66) 0.44 (0.25-0.63) 0.63 (0.47-0.66)
SLDNeh 0.38 (0.34-0.88) 0.88 (047-103) 0.75 (0.63-1.00) 0.75 (0.44-0.88)
Veh/LPS 0.75 (047-097) 0.75 (0.56-1.19) 0.63 (0.44-1.09) 1.00 (0.81-1.26)

SLD/LPS

0.88 (0.72-1.40)

1.88 (1 .63-359)"

58

3.37 (2.25-3.88)*

2.88 (2.25-3.38)‘

Table 2.2. Serum TNFa concentration in SLDILPS-treated Rats. Rats were

treated with SLD (50 mglkg, p.o.) or its vehicle (0.5% methyl cellulose) at -16 hr,

and food was removed. At -0.5 hr rats received LPS (8.25X105 EUIkg, i.v.) or its

vehicle (saline), and 30 min later they were given a second administration (50
mglkg) of SLD. Serum samples were collected at 1 hr after the second
administration of SLD. The concentration of TNFo (ng/mL) in serum was
determined using ELISA. *signiﬁcantly different from corresponding group not

treated with LPS. #signiﬁcantly different from Veh/LPS group. P<0.05, n=3-7.

 

 

Treatment Veh SLD
Veh 0.024 :1: 0.005 0.057 i 0.018
LPS 16.9 e 6.5* 50.2 s 10.7“

 

59

Fig. 2.6. Activation of the hemostatic system. Rats were treated with SLD,
LPS or their vehicles as described in Fig.2.3. They were killed 2, 4 or 8 hr after
the second administration of SLD and plasma was collected. Concentrations of
TAT (A) and active PAl-1 (B) in plasma were measured. *signiﬁcantly different
from VehNeh group at the same time. #signiﬁcantly different from Veh/LPS

group at the same time. P<0.05, n=3-5.

60

Active PAl-1 (nglmL)

“3'

TAT (ngImL)
N
8

 

 

 

 

 

 

 

 

 

 

— VehNeh
1:21 SLDNeh
— Veh/LPS
1:1 SLD/LPS

 

 

 

100 . *
o . .l—A-l-l
2 4 8
Hr after 2"" SLD Administration
1000 ‘ — Veh/Veh
El SLD/Veh
goo . - Veh/LPS *# *
:1 SLD/LPS I
600 1 *
*
40° ‘ *
L *
2m 4
0 . . .
2 4 8
Hr after 2'"I SLD Adrrl'nistration

61

Hepatic ﬁbrin deposition was evaluated 4 hr after the second administration
of SLD because that is a time just before the onset of liver injury as reﬂected by
serum ALT and AST activities (Fig. 2.3). SLD alone and LPS alone were
associated with small increases in ﬁbrin that were not statistically signiﬁcant. In
contrast, SLD/LPS cotreatrnent led to a pronounced elevation in ﬁbrin staining
that was panlobular in distribution (Fig. 2.7 and 2.9A).

As mentioned above, ﬁbrin deposition can lead to hypoxia, which was
evaluated by quantifying PIM-protein adducts in livers. PIM-protein adducts were
at control levels in livers of SLD-treated rats. LPS treatment caused a modest
increase, and SLD/LPS cotreatrnent led to a greater elevation of PIM-protein
adducts, predominantly in midzonal regions of liver lobules by 4 hr after the

second administration of SLD (Fig. 2.7 and 2.8B).

2.4.5 Effect of heparin on liver injury induced by SLD/LPS cotreatrnent
Anticoagulant heparin (3000 Units/kg, s.c.), administered concurrently with
the second administration of SLD, caused a marked decrease in ﬁbrin deposition
in liver as expected (Fig. 2.10A). PIM-protein adduct staining was also
signiﬁcantly reduced by heparin at 4 hr after the second SLD administration (Fig.
2.10B).
To evaluate the role of hemostatic system in liver injury, heparin was given to
rats concurrently with and 6 h after the second administration of SLD, and liver
injury was evaluated at 12 hr. Heparin was without effect on ALT activity in

VehNeh-treated rats but signiﬁcantly attenuated the increase in ALT activity in

62

Fig. 2.7 Fibrin deposition in liver. Rats treated with VehNeh (A), SLDNeh (B),
Veh/LPS (C) or SLD/LPS (D) as described in Fig. 2.4 were killed at 4 hr, and

immunohistochemistry for ﬁbrin was performed.

 

 

 

 

 

 

63

Fig. 2.8. Hypoxia staining in liver. Rats were treated with VehNeh (A),
SLDNeh (B), Veh/LPS (C) or SLD/LPS (D) as described in Fig. 2.4 except that
PIM hydrochloride (120 mg/kg) was administered 2 hr after the second
administration of SLD. At 4 hr after the second administration of SLD, livers were

collected and ﬁxed for immunohistochemistry.

 

3A 8

100m

 

of

 

 

 

 

Fig. 2.9. Evaluation of ﬁbrin deposition and hypoxia in liver. Rats were
treated as described in Fig. 2.3. A) Animals were killed at 4 hr, and livers were
processed for immunohistochemical determination of ﬁbrin deposition. B) Rats
received an additional treatment with PIM hydrochloride (120 mglkg) 2 hr after
the second administration of SLD. They were then killed at 4 hrs, and livers were
processed for immunohistochemical determination of hypoxia. In both panels the

fraction of positive pixels averaged from 10 randomly chosen microscope ﬁelds

(100 X) was determined for each animal. *slgniﬁcantly different from Veh/LPS
group. #signiﬁcantly different from SLDNeh group. asigniﬁcantly different from

VehNeh group. P<0.05, n=4-6.

65

 

 

i

 

—Veh
HELPS

 

e. s u. e. m

0 o o 0 0

3.9.3 e>Eoon *0 .5362“:
55E

Veh

 

 

 

 

h S

e P

V L

E 5m
5 m 5 0
1 1 0 0
0 D 0 n”
0 0 0 0
3.92.. 9:32. no .5568":

£2.25 £39.95...“—

SLD

Veh

66

Fig. 2.10. Effect of heparin on liver ﬁbrin and hypoxia. SLD and/or LPS were
given to rats as described in Fig. 2.3. Heparin (3000 units/kg, so.) was given to
rats at the same time as the second administration of SLD. Rats were killed at
4hr, and liver sections were ﬁxed and stained immunohistochemically for ﬁbrin
(A) or PIM-protein adducts (B). The fraction of positive pixels was determined as
described under Materials and Methods. *signiﬁcantly different from

VehNehNeh group. #signiﬁcantly different from SLD/LPSNeh group.

a
signiﬁcantly different from VehNeh/heparin group. P<0.05, n=4-6.

67

-Veh
1:3l-Iepan'n

 

m
o

A

m.
0
3.9.3 6552. he

ESE

m
o
cows—En:

6.
0

Veh

 

 

Q03}

e m .. .. s
m m
Q Q Q Q Q
3.3.3 02:3“. *0 ceases":

36328 5399.25

m

68

the sera of SLDILPS-treated rats (Fig. 2.11A). The SLD/LPS-induced elevation in
total bile acid concentration in serum was also attenuated by heparin (Fig.
2.11B). Necrotic foci were observed in livers of rats treated with SLD/LPSNeh,

but not in livers of rats treated with SLD/LPSIheparin (Fig. 2.12).

2.4.6 Effect of low oxygen on hepatocytotoxicity induced by SLD sulﬁde in
vitro

To assess whether hypoxia can affect the killing of hepatocytes by SLD, the
active metabolite of SLD, SLD sulﬁde (60 uM) was administered to primary rat
hepatocytes in vitro. Immediately after treatment, hepatocytes were incubated in
oxygen replete (20% 02) or hypoxic (5% 02) atmospheres. The 5% 02 is
equivalent to a nominal p02 of 30 mm Hg, which is the smallest reported oxygen
concentration in blood around hepatic central vein in vivo (Jungerrnann and
Kietzmann, 2000). This degree of hypoxia had no effect on the viability of
hepatocytes after 8 hr of incubation. SLD sulﬁde alone increased ALT activity in
the medium, indicating hepatocellular injury. When HPCs treated with SLD
sulﬁde were exposed to 5% 02, cell death caused by SLD sulﬁde was

signiﬁcantly enhanced compared to incubation in 20% 02 (Fig. 2.13).

69

Fig. 2.11. Effect of heparin on SLDILPS-induced liver injury. Rats were
treated with SLD/LPS as described in Fig. 2.3. Heparin (3000 units/kg, so) or its
vehicle (saline) was given to rats at 0 and 6 h. Rats were killed at 12 hr, and ALT
activity (A) and concentration of total bile acids (B) in serum was determined.
*signiﬁcantly different from VehNeh/heparin group. #signiﬂcantly different from

SLDILPSNeh group. P<0.05, n=3-13.

7O

ALT (UIL)

1200 4

1000 -

800 -

600 -

400 ‘

200 -

3

Total Bile Acid (u_|\[l)

O

 

 

3

3588

*
- Veh
[:3 Heparin
#
A [-3 F-Tl
VehNeh SLD/LPS
*

_ Veh
1:3 Heparin

 

i_jl:l

 

 

Veh

71

Fig. 2.12. Effect of heparin on hepatic lesions induced by SLDILPS. Rats
were treated with SLD/LPS as described in Fig. 2.4. Heparin (3000 units/kg, so.)
or its vehicle (saline) was given to rats at 0 and 6 h. Liver sections were collected
at 12 hr and stained with hematoxylin and eosin. Liver sections of rats treated
with SLDILPSNeh (A) and SLD/LPS/heparin (B) were examined under 200X

magniﬁcation. A necrotic area is indicated with an arrow.

 

72

Fig. 2.13. Effect of hypoxia on SLD sulﬁde-induced cytotoxicity. Rat primary
hepatocytes were isolated as described under Materials and Methods. They were
treated with 60uM SLD sulﬁde and kept in 20% or 5% oxygen. After 8 hr, ALT
release was determined. *signiﬁcantly different from Veh group at the same
oxygen level. #signiﬁcantly different from SLD sulﬁde treatment group at 20%

oxygen. P<0.05, n=3.

— 20%02

 

 

Veh SLD Sulﬁde

73

2.5 Discussion

SLD is an NSAID that is used for the treatment of arthritis. All of the NSAle
have been associated with hepatic IADRs in patients (O‘Connor et al., 2003). The
average risk of serious hepatic injury for NSAle is approximately 1 case in
10,000 patient-years of use, and the risk from SLD is reported to be 5-10 fold
greater than for NSAle as a class (Walker, 1997). The mechanism of NSAID-
induced hepatic IADRs is not clearly understood, and several hypotheses have
been proposed. It is commonly accepted that large amounts of active metabolites
form after exposure to large doses of NSAIDs, and these might lead to protein
adduct formation, oxidative stress and mitochondrial injury that could contribute
to tissue damage (Boelsterli, 2002). However, evidence supporting these
hypothses is mainly obtained from studies in vitro, and animal models of SLD-
induced liver injury are lacking.

Previous studies in rodents suggested that there may be a connection
between inﬂammation and hepatic IADRs for at least some drugs (Buchweitz et
al., 2002; Luyendyk et al., 2003; Roth et al., 2003; Shaw et al., 2007). In these
drug-LPS interaction models, one administration of the drug was sufﬁcient to
produce a hepatotoxic interaction with LPS. In preliminary studies with SLD, we
tried several single-administration regimens using various doses and times
between SLD and LPS treatment. Although hepatotoxic signals (ie, increased
serum ALT activity) were observed in some rats, these were inconsistent.
Changing to a two-administration protocol in which the time between SLD

administrations approximated 3 half lives (Hucker et al., 1973) provided relatively

74

consistent and statistically signiﬁcant liver injury; moreover, this protocol
corresponds to the twice per day treatment regimen typically used therapeutically
in human patients. Using this protocol, SLD or LPS alone did not cause any
lesions in the liver or increase the clinical chemical biomarkers of liver injury.
However, in SLD/LPS-cotreated rats serum markers of both hepatocellular injury
and cholestasis were increased signiﬁcantly, and foci of necrotic parenchymal
cells were found in livers. Interestingly, in a study of 91 cases reported to the
US. Food and Drug Administration, SLD caused hepatocellular injury, cholestatic
injury or mixed liver injury in human patients (Tarazi et al., 1993), consistent with
observations presented here for rats treated with SLD/LPS. These results raise
the possibility that a mild episode of inﬂammation might render human patients
susceptible to SLD-induced liver injury.

Judging by the timecourse of serum ALT and AST activities (Fig. 2.3) and
histological changes (Table 2.1), the onset of liver injury was between 4 and 8 hr.
The serum markers of hepatocellular injury rose until 12 hr and declined by 24 hr.
Despite this decline, histologic evidence of liver injury persisted at 24 hr (Table
2.1). This decline in serum transaminases suggests that injury occurred in the
ﬁrst 12 hrs, and ALT and AST released from hepatocytes were cleared from the
blood thereafter. The short initial half life of ALT/AST in rat plasma
(approximately 5 hr) is consistent with this interpretation (Saheki et al., 1990).

LPS has the potential to inﬂuence toxicity in a number of ways. For
example, it is capable of downregulating the expression of several drug

metabolizing enzymes (Morgan, 1989). SLD is bioactivated by hepatocytes to a

75

more toxic sulﬁde metabolite by methionine sulfoxide reductase (Kitamura et al.,
1980; Kitamura and Tatsumi, 1982; Etienne et al., 2003a). It is not known if LPS
downregulates the expression of this enzyme; however, in a preliminary study,
LPS treatment did not increase the liver concentration of SLD sulﬁde
(unpublished observation).

Cytokines such as tumor necrosis factor-alpha (T NFo), interleukin-1 (IL-1)
and interleukin-6 (IL-6) are upregulated after the activation of Toll-like receptor 4
on Kupffer cells by LPS (Luster et al., 1994; Su, 2002). Some of these changes
have been linked to liver injury from drug/LPS interaction (Shaw et al., 2007;
Tukov et al., 2007b). The observation that ﬁbrin deposited in livers of rats
cotreated with SLD and LPS led us to explore the hemostatic system in this
study. Cytokines are potent modulators of the hemostatic system. For example,
TNFd and lL-1 activate coagulation by upregulating tissue factor expression and
by reducing the ﬁbrinolytic activity of endothelial cells through an increase in PAI-
1 (Schleef et al., 1988; Salgado et al., 1994). The plasma concentrations of TAT
and active PAl-1 increased rapidly in LPS-treated rats, conﬁrming that LPS
induces the activation of the coagulation system and provides conditions for
inhibition of the ﬁbrinolytic system (Fig. 2.6). Although SLD had no effect on
these factors when it was given alone, it enhanced the LPS-mediated changes
and tended to prolong the activation of the hemostatic system. This could be due
to enhanced TNFa release by SLD (Table 2.2). In the liver injury induced by
ranitidine/LPS cotreatrnent, ranitidine enhanced the LPS-induced increase in

TNFa concentration through p38-dependent activation of TNFo converting

76

enzyme (T ACE), which cleaves membrane-bound pro-TNFd to form mature
TNFa (Deng et al., 2008). Whether a similar mechanism is at play in SLD/LPS-
cotreated rats is yet to be determined.

SLD/LPS cotreatrnent signiﬁcantly increased cross-linked ﬁbrin in livers at 4
hr— Le, a time before the onset of liver injury. Although LPS activated the
hemostatic system in rats, it alone was not sufﬁcient to induce marked ﬁbrin
deposition in the liver (Fig. 2.9A). The hemostatic system is activated in
endotoxemia-induced liver injury (Hewett and Roth, 1995), and one possible
consequence is hypoxia in the liver resulting from disrupted blood ﬂow in the
sinusoids. PIM-protein adducts were slightly elevated in livers of rats treated with
LPS alone, indicating that mild hypoxia occurred. In contrast, SLD/LPS led to a
pronounced increase in PIM-protein adducts, suggesting marked hypoxia (Fig.
2.93). Unlike the distribution of ﬁbrin which was panlobular, hypoxia occurred
only in the midzonal regions of livers (Fig. 2.3, 2.4). Interestingly, necrotic foci
were also present predominantly in this region (Fig.2.5).

It has been reported that hypoxia causes hepatocelluar injury in isolated,
perfused rat livers (Lemasters et al., 1981), and liver injury was induced in vivo
in rats exposed for a brief period to a low concentration of oxygen (Fassoulaki et
al., 1984). In studies presented here, the anticoagulant heparin reduced hepatic
ﬁbrin deposition and hypoxia induced by SLD/LPS cotreatrnent (Fig. 2.10). This
suggests that hypoxia might be caused by ﬁbrin clots in liver sinusoids.

Moreover, both hepatocellular and bile ductular injury in SLD/LPS-treated rats

77

was signiﬁcantly attenuated by heparin, supporting the hypothesis that hypoxia
induced by ﬁbrin clots plays an important role in the pathogenesis (Fig. 2.11).

Previous studies suggested that hypoxia can potentiate the toxicity of some
xenobiotics towards hepatocytes (Shen et al., 1982). Compared to SLD or its
sulfone metabolite, SLD sulﬁde is more cytotoxic (Leite et al., 2006). Therefore,
we treated rat primary hepatocytes with SLD sulﬁde. Hypoxia enhanced its ability
to kill these cells (Fig. 2.13). SLD sulﬁde, but not SLD or SLD sulfone uncouples
the mitochondria of HepGZ cells (Leite et al., 2006). Thus, hypoxia could
exacerbate SLD sulﬁde-mediated mitochondrial dysfunction by diminishing the
aerobic metabolism of hepatocytes. Moreover, hypoxia inducible factor 1 (HIF-1)
accumulated in cells under hypoxic stress might induce the expression of
proapoptotic proteins and cause the stabilization of p53, which increases
permeability of the mitochondrial membrane and causes cell death (Greijer and
van der Wall, 2004). In the SLD/LPS idiosyncratic liver injury model, hypoxia
might enhance the mitochondrial toxicity of SLD, providing a synergistic effect on
a mitochondrial pathway to cell death. Although further study is required to test
this, SLD/LPS cotreatrnent in rats could be an animal model supporting the
mitochondrial injury hypothesis of NSAID-induced IADRs described above.

The mitochondrial pathway might not be the only contributor to liver injury.
Hypoxia can also interplay with inﬂammatory factors. For example, when
stimulated by LPS, neutrophils accumulate in sinusoids and transmigrate through
blood vessels to liver parenchyma. At the site of inﬂammation, activated

neutrophils release proteases that can kill rat hepatocytes (Ho et al., 1996), and

78

hypoxia enhanced neutrophil protease-mediated hepatocyte killing (Luyendyk et
al,2005)

Results with SLD are similar to those observed with diclofenac (DCLF),
another NSAID that causes hepatic IADRs in people, in the sense that both
drugs interacted with LPS to cause liver injury in rats. However, a large dose of
DCLF (100 mglkg) caused liver injury by itself in rats in the absence of LPS-
cotreatrnent. This was associated with intestinal injury and translocation of
bacteria to the liver and was prevented by sterilization of the GI tract (Deng et al.,
2006). These results suggested that GI irritation and translocation of bacteria or
LPS caused by DCLF contribute to DCLF-induced hepatotoxicity in rats, and a
similar mechanism might underlie DCLF IADRs in humans. In contrast, large
doses of SLD (up to 300 mglkg) did not cause liver injury in rats by themselves
(Fig. 2.2). This suggests that, at least in rats, SLD is less irritating to the intestine
than DCLF, although the relative gastrointestinal toxicity of SLD and DCLF in
humans is still controversial. One study indicated that SLD is less associated with
gastrointestinal hospitalizations than DCLF (Garcia Rodriguez et al., 1992),
whereas others suggested the opposite (Henry et al., 1993; Savage et al., 1993).
The potential implication for human IADRs is that DCLF may be able to provide
its own inﬂammatory stress through GI irritation, whereas SLD toxicity might
require an additional inﬂammatory stress that arises independently of drug
treatment. It seems possible that the latter could arise from the very condition
that the drug is used to treat- e.g., an inﬂammatory ﬂare of rheumatoid arthritis.

Alternatively, an independently occurring inﬂammatory episode might interact

79

with SLD. The ﬁnding that viral hepatitis may predispose patients to NSAID
hepatotoxicity supports this possibility (Tech and Farrell, 2003).

In summary, SLD/LPS cotreatment caused liver injury in rats, which was not
produced by SLD or LPS alone. The coagulation system was activated while the
ﬁbrinolytic system was inhibited in the cotreated rats. As a consequence, ﬁbrin
clots formed in sinusoids and hypoxia occurred selectively in livers of rats treated
with SLD/LPS. The anticoagulant heparin protected rats against liver injury and
also attenuated ﬁbrin deposition and liver hypoxia. Hypoxia enhanced the
cytotoxicity of SLD sulﬁde in vitro. The results support the hypothesis that
NSAle that cause hepatic IADRs in humans interact with an inﬂammatory stress
to cause liver injury in animals. Hypoxia may play an important role in this
SLD/LPS idiosyncratic liver injury model through synergistic interplay with the
toxic SLD sulﬁde metabolite. These observations do not exclude a role for other

factors, such as cytokines, that are a focus of ongoing investigation.

80

CHAPTER 3

Zou W, Beggs KM, Sparkenbaugh EM, Jones AD, Younis HS, Roth RA and
Ganey PE (2009). Sulindac metabolism and synergy with TNF in a drug-

inflammation interaction model of idiosyncratic liVer injury. J Pharmacol

Exp Ther.

81

3.1 Abstract

Sulindac (SLD) is a nonsteroidal anti-inﬂammatory drug (NSAID) that has
been associated with a greater incidence of idiosyncratic hepatotoxicity in human
patients than other NSAIDs. In previous studies, cotreatrnent of rats with SLD
and a modestly inﬂammatory dose of lipopolysaccharide (LPS) led to liver injury,
whereas neither SLD nor LPS alone caused liver damage. In studies presented
here, further investigation of this animal model revealed that the concentration of
tumor necrosis factor-a (TNF) in plasma was signiﬁcantly increased by LPS at 1
hr, and SLD enhanced this response. Etanercept, a soluble TNF receptor,
reduced SLD/LPS-induced liver injury, suggesting a role for TNF. SLD
metabolites in plasma and liver were determined by LC/MSIMS. Cotreatment
with LPS did not increase the concentrations of SLD or its metabolites, excluding
the possibility that LPS contributed to liver injury through enhanced exposure to
SLD or its metabolites. The cytotoxicities of SLD and its sulﬁde and sulfone
metabolites were compared in primary rat hepatocytes and HepG2 cells; SLD
sulﬁde was more toxic in both types of cells than SLD or SLD sulfone. TNF
augmented the cytotoxicity of SLD sulﬁde in primary hepatocytes and HepG2
cells. These results suggest that TNF can enhance SLD sulﬁde-induced

hepatotoxicity, thereby contributing to liver injury in SLD/LPS-cotreated rats.

82

3.2 Introduction

Several hypotheses have been put forward to explain the basis for IADRs;
however, the modes of action are still unclear, in part because of the lack of
animal models. One hypothesis is that inﬂammatory stress precipitates hepatic
IADRs in humans (Roth et al., 2003; Ganey et al., 2004). In concert with this
hypothesis, cotreatrnent of rats with lipopolysaccharide (LPS), which induces
modest inﬂammation, and SLD resulted in liver necrosis, whereas neither LPS
nor SLD was hepatotoxic alone (Zou et al., 2009b).

In this study, we examined factors that could contribute to the pathogenesis
of liver injury in rats cotreated with LPS and SLD. In vivo, SLD can be
metabolized either irreversibly to SLD sulfone or reversibly to SLD sulﬁde, which
is more cytotoxic than SLD itself. Since LPS can regulate drug metabolism
(Renton, 2001), we tested whether LPS coexposure enhances bioactivation of
SLD. Moreover, we determined the effect of SLD on LPS-induced tumor necrosis

factor-a (T NF) production and its role in the development of liver injury.

83

3.3 Materials and methods
3.31 Materials
Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich

(St. Louis, MO). LPS (Lot 075K4038) derived from Escherichia coli serotype

055235 with an activity of 3.3 X 106 endotoxin units (EU)/mg was used in

experiments. Etanercept was purchased from Amgen Pharmaceuticals
(Thousand Oaks, CA). HepGZ/C3A cells for in vitro studies were obtained from

American Type Culture Collection (Manassas, VA).

3.3.2 Animals

Male, Sprague-Dawley rats (Crl:CD(SD)lGS BR; Charles River, Portage, MI)
weighing 250 to 370 g were used for studies in vivo (rats weighing 290 to 3009
were used to evaluate SLD and its metabolites in GI and feces), and rats
weighing 150 to 200 g were used for primary hepatocyte isolation. Animals were
fed standard chow (Rodent Chow/Tek 8640; Harlan Teklad, Madison, WI) and
allowed access to spring water. They were allowed to acclimate for 1 week in a

12-hr light/dark cycle prior to use in experiments.

3.3.3 Experimental protocol
As described in previous studies (Zou et al., 2009b), rats were given two
administrations of SLD (50 mglkg, p.o.) or its vehicle (0.5% methyl cellulose) with

a 16 hr interval, and food was removed after the ﬁrst administration. Half an hour

before the second administration of SLD, LPS (8.25X 105 EU/kg, i.v.) or its

84

vehicle (saline) was administered via a tail vein. Depending on the purpose of
experiments, rats were anesthetized with isoﬂurane and euthanized at various
times (0, 1, 2, 4, 8 and 12 hr) after the second administration of SLD. For the
collection of plasma, a portion of blood drawn from anesthetized rats was
transferred into vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ)
containing sodium citrate (ﬁnal concentration 0.38%). The rest of the blood was
allowed to clot at room temperature for preparation of serum. Collected plasma
and serum were stored at — 80 0C until use. Three slices (3-4 mm thick) of the left
lateral liver lobe were collected and ﬁxed in 10% buffered formalin for histological
analysis. A portion of the right medial lobe of the liver was ﬂash-frozen in liquid
nitrogen for pharrnacokinetic study of SLD and its metabolites. For determining
drug concentration in the gastrointestinal (GI) tract and feces, each rat was
housed in a separate cage after LPS or vehicle injection and euthanized at 2 hr.
The entire GI tract and its contents were collected. Feces were retrieved from the
cages and were homogenized with the GI tract and its contents for each rat. In

the TNF inhibition study, rats were given etanercept (8mgﬂtg) or vehicle (sterile

water) subcutaneously an hour before LPS (8.25X 105 EU/kg, i.v.) or its saline

vehicle. We have demonstrated that etanercept inactivates TNF activity induced

by LPS administration in rats using this treatment protocol (Tukov et al., 2007b).
3.3.4 Evaluation of liver injury

The activity of alanine aminotransferase (ALT), a marker of hepatic

parenchymal cell injury, was assessed in serum using a diagnostic kit from

85

Thermo Corp. (Waltham, MA). Liver slices ﬁxed in 10% buffered formalin were
embedded in parafﬁn, cut into 6 um sections and stained with hematoxylin and

eosin (H&E) for histological evaluation.

3.3.5 Determination of TNF concentration in serum
The concentration of TNF in serum collected at 0, 1, 2, 4 and 8 hr after the
second administration of SLD was measured by ELISA (BD Biosciences; San

Diego, CA).

3.3.6 LCIMSIMS analysis

Plasma samples, liver homogenates, GI and fecal homogenates or HepGZ
cells in culture medium were mixed with acetonitrile containing diclofenac as
internal standard. After vortex and centrifugation, protein was removed, and the
supernatant was diluted and transferred to ultra performance liquid
chromatography (UPLC) sample vials for LC/MS/MS analysis.

LC/MS/MS analysis was performed using a Waters ACQUITY UPLC System
coupled to a Quattro Premier XE tandem quadrupole mass spectrometer. An
extract volume of 2 uL was injected into the UPLC system and eluted with a
gradient mixture (0-99%) of formic acid and acetonitrile. Electrospray ionization in
positive ion mode was performed for analyses of plasma samples, and the
collision and source cone voltages were optimized independently for each
analyte. Multiple reaction monitoring (MRM) of the following m/z transitions was

used for the quantitative analysis of diclofenac (296.2->214.2), SLD (357.2-

86

>333.2), SLD sulfone (373.2->233.2), SLD sulﬁde (341.2->234.2), SLD acyl
glucuronide (533.1->339.1), SLD sulfone acyl glucuronide (549.1->355.1) and
SLD sulﬁde acyl glucuronide (517.1->323.1).

For samples other than plasma, electrospray ionization was performed in
negative ion mode, and metabolite concentrations were determined by the MRM
of transition of diclofenac (294.2->250.0), SLD (311.2->296.2), SLD sulfone
(327.2->264.2), SLD sulﬁde (295.2->280.2), SLD acyl glucuronide (531.1-
>355.1), SLD sulfone acyl glucuronide (547.1->371.1) and SLD sulﬁde acyl
glucuronide (515.1->339.1).

The LCIMS/MS method achieved low limits of quantiﬁcation (LLOQ) of 30
ng/mL or less for all three forms of sulindac (sulfoxide, sulﬁde, and sulfone) using
both positive and negative ion modes. Analytical reproducibility was judged to be

:I: 12% in the middle of the calibrated range of concentrations.

3.3.7 Evaluation of cytotoxicity of SLD and its metabolites in vitro

HepGZ cells were plated at a density of 4 x 104 cells/well in 96-well plates.

After overnight incubation in Dulbecco's modiﬁed Eagle’s medium (DMEM) with
10% fetal bovine serum (FBS), medium was renewed and SLD, SLD sulfone,
SLD sulﬁde (0- 500 (M) or its vehicle (0.5% dimethyl sulfoxide [DMSO]) was
added to the wells. After a 24-hr incubation, lactate dehydrogenase (LDH)
released into the medium and total cellular LDH were evaluated using a kit from

Promega Corporation (Madison, WI). Cytotoxicity was assessed as the

87

percentage of LDH released into the medium relative to the total LDH in the well
(medium plus lysed cells).

For primary rat hepatocytes, isolation was performed as described
previously (Tukov et al., 2006). Brieﬂy, rat liver was ﬁrst perfused in situ through
the portal vein and then digested with Liver Digest Medium (lnvitrogen Corp,
Carlsbad, CA). The digested liver was combed gently, and hepatocytes were
obtained after centrifugation (100 x g, 30 s).

Hepatocytes were suspended in Williams’ Medium E (lnvitrogen Corp,

Carlsbad, CA) with 10% FBS, and the cell viability was always above 80%.

Hepatocytes were plated at a density of 2.5 x 105 cells/well in 12-well plates and

incubated for 3 hr to attach to the plate. Serum-containing medium was replaced
by serum-free medium, and SLD, SLD sulfone, SLD sulﬁde (0- 120 (M) or
vehicle (0.1% DMSO) was added to the culture wells. After 8 hr incubation,
cytotoxicity was assessed by calculating the ALT activity in the medium plus
unattached cells as a percentage of the total ALT activity in the well as described

previously (Zou et al., 2009b).

3.3.8 Cytotoxicity from TNF and SLD metabolites

SLD sulﬁde or its vehicle (0.5% DMSO) was administrated to HepG2 cells
with recombinant human TNF (200 ng/mL) or its vehicle (medium). After 24 hr
incubation, the percentage of LDH released was evaluated. To determine the

remaining concentration of SLD sulﬁde in each well, HepG2 cells were scraped,

88

and acetonitrile was added to precipitate protein. After centrifugation, the
concentration of SLD sulﬁde in supernatant was determined using LC/MS/MS.

To assess further whether TNF can affect the cytotoxicity of SLD
metabolites, isolated primary rat hepatocytes were treated with SLD sulﬁde (60
uM) in the presence or absence of recombinant rat TNF (2 pg/mL), and the

percentage of ALT released was evaluated 8 hr later.

3.3.9 Statistical analysis

Results are expressed as means 1 SEM. One-way or two-way analysis of
variance (ANOVA) was applied for data analysis as appropriate, and Tukey’s test
was employed as a post hoc test. Student’s t-test was performed when only two
groups were compared. For all studies, P < 0.05 was considered as the criterion

for statistical signiﬁcance.

89

3.4 Results
3.4.1 Timecourse of TNF concentration in plasma

Rats were treated with LPS and two administrations of SLD or their vehicles
as described in Methods, and TNF concentration in serum was evaluated at
various times up to 8 hr after the second administration of SLD. SLD had no
effect on serum TNF concentration in rats. LPS alone led to a signiﬁcant increase
in TNF serum concentration at 0 and 1 hr (ie, 0.5 and 1.5 hr after LPS). The
elevation of TNF concentration induced by LPS was signiﬁcantly increased by

SLD at 1 hr after the second administration of the drug (Fig. 3.1).

3.4.2 Effect of TNF inhibition on liver injury

Etanercept is a soluble TNF receptor that neutralizes the biological activity of
TNF. To investigate the role of TNF in liver injury, rats were treated with
etanercept 1 hr before LPS administration. This treatment protocol inhibits the
activity of TNF in rats (Geier et al., 2003). We have reported previously that
neither LPS nor SLD produces liver injury when given alone at the doses used in
these studies (Zou et al., 2009b). Also consistent with our previous report,
SLD/LPS cotreatrnent increased serum ALT activity signiﬁcantly (Fig. 3.2).
Etanercept signiﬁcantly attenuated this increase, whereas etanercept alone had
no effect on serum ALT activity. Histological examination of H&E-stained livers of
rats revealed a pattern consistent with the ALT activity. That is, midzonal necrotic
foci were present in livers of rats treated with SLD/LPS but were found

infrequently in livers of rats treated with etanercept/SLDILPS.

90

Fig. 3
with fl

cellulo
SLD. L

vein. T
2. 4 or
VehNe
the sar

SLD/L

TNFa (pg/mL)

Fig. 3.1. Timecourse of TNF concentration in rat serum. Rats were treated
with two administrations of SLD (50 mglkg, p.o.) or its vehicle (0.5% methyl

cellulose) with a 16 hr interval. Half an hour before the second administration of

SLD, LPS (8.25X 105 EUIkg, i.v.) or its saline vehicle was administered via a tail

vein. TNF was evaluated by ELISA in serum samples obtained from rats at 0, 1,
2, 4 or 8 hr after the second administration of SLD. *signiﬁcantly different from
VehNeh group at the same time. #signiﬁcantly different from Veh/LPS group at
the same time. P<0.05, n=4-5 for all points except 8 hr group (n=3), Veh/LPS and

SLD/LPS at 1 hr (n=8 and 9, respectiveIY). and SLD/LPS at 0 hr (n=8).

7°°°° ‘ *# —o— VehNeh
-”'- SLDNeh
3 6°°°° T + Veh/LPS
E 50000 - - SLD/LPS
8’
‘5' 40000
”L *r
:: 30000i
I-
20000
10000 .
0

 

 

 

Hr after 2nd Sulindac Administration

91

Fig. 3.2. Effect of TNF inhibition on liver injury induced by SLD/LPS. Rats
were treated with etanercept (8 mglkg, so.) or its vehicle 1 hr before LPS. SLD
and LPS or their vehicles were administered to rats as described in Methods.
ALT activity was determined at 12 hr (A). *signiﬁcantly different from respective
VehNeh group. #signiﬁcantly different from Veh/ SLD/LPS group. P<0.05, n=4
for all groups except SLD/LPS/Etan (n=6). Liver sections from rats treated with
VehNehNeh (B), EtanNehNeh (C), Veh/SLDILPS (D), and Etan/SLD/LPS (E)

were examined. A necrotic area is indicated with an arrow.

92

>
at-

 

zooo — Veh
r:1 Etan
3
.— 1000I
.J
<
500 *#
o .

 

93

Peta:

Cecre

Vt

3.4.3 Effect of LPS on SLD metabolism in rats

SLD and its sulfone and sulﬁde metabolites were determined in rat plasma
at various times after the second administration of SLD. Plasma SLD
concentration reached a peak 1 hr after administration and decreased gradually
over 8 hr (Fig. 3.3A). ln LPS-treated rats, plasma SLD concentration was
signiﬁcantly smaller. SLD treatment increased SLD sulfone concentration in
plasma steadily between 2- 8 hr (Fig. 3.3B). This increase was not observed after
SLD/LPS cotreatrnent, so that the plasma concentration of SLD sulfone was
signiﬁcantly less in SLD/LPS-cotreated rats by 8 hr. Plasma SLD sulﬁde
concentration reached a peak within 4 hr in both groups, and LPS administration
decreased the SLD sulﬁde concentration in plasma signiﬁcantly at 1, 2, 4 and 8
hr compared to that of SLD/vehicle-treated rats (Fig. 3.3C)

ln livers of rats treated with SLD alone, the concentrations of SLD and its
metabolites showed trends similar to those in plasma. LPS cotreatrnent
decreased SLD and SLD sulﬁde concentrations, but SLD sulfone concentration
was unaffected (Fig. 3.4). LPS selectively lowered the SLD concentration in liver
at 1 and 2 hr, and decreased SLD sulﬁde concentration in liver at 2 and 4 hr.

To investigate further the effect of LPS on SLD metabolism, rats were
euthanized at 2 hr and SLD metabolite concentrations were determined in the GI
tract and feces collected between -0.5 and 2 hr. The concentrations of SLD and
SLD sulﬁde in the GI tract and feces were signiﬁcantly increased by LPS (Fig.

3.5). However, the SLD sulfone concentration was not affected by LPS.

94

Fig. 3.3. Effect of LPS on plasma concentrations of SLD, SLD sulfone and
SLD sulﬁde. Rats were treated with-SLD and with LPS or its saline vehicle as
described in Fig. 3.1. They were euthanized, and plasma was collected at 0, 1, 2,
4 and 8 hr after the second administration of SLD. The plasma concentrations of
SLD, SLD sulfone or SLD sulﬁde were determined as described in Methods.
*signiﬁcantly different from SLDNeh group at the same time. P<0.05, n=5 for all

groups except SLD/LPS at 4 hr (n=7).

95

SLD (uglmL)

SLD sulfone (uglmL)

SLD sulfide (uglmL)

 

 

 

 

 

200 —o— SLDNeh
-O— SLD/LPS
150
100 *
* *
so 4 *
0 . .
0 2 4 6 8
Hr after 2nd Sulindac Administration
200 1
150
*
100
50 . . . .
0 2 4 6 8
Hr after 2nd Sulindac Administration
200 1
150 .
100 ~ *
* * l, *
50 - I
0

 

0 2 4 6 8
Hr after 2nd Sulindac Administration

96

Fig. 3.4. Effect of LPS on liver concentrations of SLD, SLD sulfone and SLD
sulﬁde. Rats were treated with SLD and with either LPS or its saline vehicle as
described in Fig. 3.1. Liver concentrations of SLD, SLD sulfone and SLD sulﬁde
were determined at 0, 1, 2, 4 and 8 hr after the second administration of SLD.
*signiﬁcantly different from SLDNeh group at the same time. P<0.05, n=5 for all

groups except SLD/LPS at 4 hr (n=8).

97

>

W

SLD(ng/mg)
«- 8 '8‘ 3’ g s f i

E'

SLD sulfone (nglmg)

s 2

SLD sulﬁde (nglmg)

G
3

ii

 

 

 

 

 

 

 

 

uh J

A

 

0 2 4 6 8
I-I'aferndeindacAd‘rinish'aﬁon

 

 

0 2 4 6 8
Hr after 2nd Sulindac Administration

 

 

0 2 4 6 8
Hr after 2nd Sulindac Administration

98

135.00ncr
rd feces Rat:
tiescnbed in
fer—.01. Two I
r: fie whole I

6 “om-geniz

?: SLD sulﬁd

#187“. group

Fig. 3.5. Concentrations of SLD, SLD sulfone and SLD sulﬁde in GI tract
and feces. Rats were treated with SLD and with either LPS or its saline vehicle
as described in Fig. 3.1. Each rat was housed in a different cage after the LPS
injection. Two hours after the second administration of SLD, feces in the cage
and the whole GI tract and its contents were collected for each rat. The mixture
was homogenized with acetonitrile, and the concentrations of SLD, SLD sulfone
and SLD sulﬁde were determined by LC/MS/MS. *signiﬁcantly different from

SLDNeh group. P<0.05, n=4.

99

 

LPS

 

 

 

6 4 2 0

3:: 3w

 

 

 

1 1

m. n. e s.
1 o o o
3:: one...» Dam

0.00 .

LPS

Veh

 

 

 

5
7.
0

3.5 222.5 3m

0.50 1

5
2
0

1.00 i

0
0
0

 

Veh

100

The l

SLD sulfa:

3.4.4 Effe
SLD
at 8 hr at

metabolit

3.4.5 Cy1
hepatocy
Neitl
increase
SLD 5qu
HM. In
cilotcxlc

C8” deat

The concentrations of acyl glucuronide conjugates of SLD, SLD sulfone and

SLD sulﬁde were below the limit of detection in all of the samples measured.

3.4.4 Effect of etanercept on SLD metabolism in rats
SLD and its sutfone and sulﬁde metabolites were determined in rat plasma
at 8 hr after the second administration of SLD. Etanercept had no effect on SLD

metabolite concentration in plasma of rats cotreated with SLD/LPS (Table 3.1).

3.4.5 Cytotoxicity of SLD and its metabolites in HepG2 cells and rat primary
hepatocytes

Neither SLD nor SLD sulfone at concentrations up to 500 pM led to an
increase in released LDH when applied to HepG2 cells (Fig. 3.6A). In contrast,
SLD sulﬁde induced signiﬁcant LDH release at concentrations greater than 125
pM. In rat primary hepatocytes, SLD and SLD sulfone also produced no
cytotoxicity at the concentrations examined (Fig. 3.68), but SLD sulﬁde caused
cell death at concentrations as small as 30 uM. The cytotoxicity of SLD sulﬁde
was concentration-dependent, and 120 pM SLD sulﬁde killed almost all of the

hepatocytes.

3.4.6 Effect of TNF on cytotoxicity of SLD and its metabolites in HepG2 and
rat primary hepatocytes
TNF alone did not affect the release of LDH from HepG2 cells (Fig. 3.7). Neither

SLD nor SLD sulfone was cytotoxic in the presence or absence of TNF.

101

Table 3.1. Effect of etanercept on SLD metabolism. Rats were killed and
plasma was collected at 8 hr after the second SLD administration. Metabolite

concentrations were determined as described under Methods. n= 4-5.

 

Concentration in plasma (uglmL)

 

 

Treatment SLD SLD sulfone SLD sulﬁde
Veh/SLDILPS 59.6 s 12.2 139.3 s 16.7 62.2 1 12.1
Etan/SLD/LPS 52.8 a: 10.8 153.8 1 13.8 39.8 s 13.3

 

102

‘-

 

..-
I

v.-

 

Fig. 3.6. Evaluation of cytotoxiclty induced by SLD, SLD sulfone or SLD
sulﬁde. SLD, SLD sulfone or SLD sulﬁde was administered at various
concentrations to HepGZ cells (A). The percentage of LDH released in the
medium after 24 hr was determined as a marker of cytotoxicity. (B) Rat primary
hepatocytes were treated with SLD, SLD sulfone or SLD sulﬁde for 8 hr, and the
percentage of ALT activity released into medium was determined as described in
Methods. *signiﬁcantly different from vehicle (0 concentration). #signiﬁcantly

different from SLD or SLD sulfone at the same concentration. P<0.05, n=3.

103

>
3

 

+SLD
-O—SLDSquono
+SLDSulﬂdo

3

 

 

 

 

LDH (% released)
8

 

 

 

    
    

 

 

 

  

20 I J\_ *0
0 100 200 300 400 500 600
Concentration (uM)
B
100 ‘ —o— SLD *# *#
—O— SLD sulfone
A —v— SLD sulﬁde
'5 80 '
0
g *#
_q_; 60‘
f.’
s: 40
'3
< 20 .
0

 

 

0 30 60 90 120
Concentration (uM)

104

t3]. Cytotoxicil
itrere treated \l
agree or absence
371180 after 24
988(1) concentra

late same cor

Fig. 3.7. Cytotoxicity induced by TNF and SLD or its metabolites. HepGZ
cells were treated with SLD (A), SLD sulfone (B) or SLD sulﬁde (C) in the
presence or absence of TNF (200ng/mL). The percentage of LDH released was
determined after 24 hr as described in Methods. *signiﬁcantly different from
vehicle (0 concentration). #signiﬁcantly different from value in the absence of

TNF at the same concentration of SLD or metabolite. P<0.05, n=3.

105

8 8 S

A

LDH (% released)
0

LDH (% released)

LDH (% released)

101

 

+ Veh
-O- TNFa

 

 

 

 

 

 

 

._.,// T . . .
0 100 150 200 250
SLD concentration (uM)
+ Veh
-O— TNFa

 

 

 

 

 

 

‘-H//l . . . f
0 100 150 200 250

SLD sulfone concentration (uM)

 

 

 

 

 

 

* #
+ Veh

-O- TNFa

*
*#
#
1.1-9C” . . . .

0 100 150 200 250

SLD sulﬁde concentration (uM)

106

Small
cytotoxic a
the cytotcr

The r
1981). T0
cells. the
hr after 5
(54:02
(5.32010

A p0
sulﬁde in
the cultui
Significa-
were tree

Smaller concentrations (150, 200 (M) of SLD sulﬁde, which were not
cytotoxic alone, induced cell death in the presence of TNF. TNF also enhanced
the cytotoxicity of a larger concentration of SLD sulﬁde (250 (M).

The conversion of SLD to SLD sulﬁde is a reversible reaction (Duggan,
1981). To evaluate whether TNF affects metabolism of SLD sulﬁde in HepG2
cells, the amount of SLD sulﬁde in medium plus HepG2 cells was measured 24
hr after SLD sulﬁde application to the cells. This amount in wells with TNF
(5.4102 pg) was not signiﬁcantly different from wells that received vehicle
(5.310.109).

A potentiating effect of TNF was also observed on the cytotoxicity of SLD
sulﬁde in primary hepatocytes. TNF alone did not affect ALT activity released into
the culture medium compared to vehicle treatment. SLD sulﬁde alone caused
signiﬁcant release of ALT activity into the medium (Fig. 3.8). When hepatocytes
were treated with SLD sulﬁde and TNF together, TNF signiﬁcantly enhanced the

cell injury induced by SLD sulﬁde.

107

fig.1
hepat
prérhaf

08500

In.

Fig. 3.8. Effect of TNF on SLD sulﬁde-induced injury to rat primary
hepatocytes. SLD sulﬁde (60 0M) and/or TNF (2 ug/mL) was administered to rat
primary hepatocytes. The percentage of LDH released was determined as
described in Methods. *significantly different from corresponding vehicle group.

#signiﬁcantly different from SLD sulfide alone group. P<0.05, n=3.

*#

80 - — Veh

ALT (% released)

 

 

Veh SLD Sulﬁde

108

3.5 Discussion

As reported previously (Zou et al., 2009b), SLD/LPS cotreatrnent induced
severe liver injury in rats. Pro-inﬂammatory cytokines, especially TNF, have
proved to play a critical role in other drug/LPS-induced liver injury models (Shaw
et al., 2007; Tukov et al., 2007b). Moreover, studies suggest that reactive drug
metabolites produced in liver are critical for idiosyncratic hepatotoxicity from
some drugs (Kaplowitz, 2005). Therefore, we focused in this study on the roles of
TNF and the toxic metabolite of SLD as well as their interaction in SLD/LPS-
induced liver injury.

The concentration of TNF in serum was elevated in rats after exposure to
LPS, and SLD signiﬁcantly enhanced the LPS-mediated increase in TNF as early
as 1 hr. Besides SLD, other drugs associated with idiosyncratic hepatotoxicity in
humans, such as ranitidine and trovaﬂoxacin, also had a synergistic effect on the
LPS-mediated increase in TNF in rodents (Shaw et al., 2007; Tukov et al.,
2007b). Sulindac and other NSAIDs enhanced TNF release from LPS-pretreated,
macrophage-derived RAW264.7 cells at concentrations achieved clinically in
humans (Cho, 2007). These ﬁndings suggest that enhancement of serum TNF
concentration might be a common characteristic of drugs that induce
idiosyncratic liver injury. The source of TNF and the mechanism by which SLD
enhances TNF appearance are unknown. After LPS exposure, the increase in
plasma TNF concentration is mirrored by elevated liver concentration
(Femandez—Martinez et al., 2004). Therefore, the source of TNF after LPS

exposure is likely liver. However, the source of enhanced TNF in serum after

109

p'etrei
intibtl
role fc
Stﬁcir
fated
likely i
T
Ueatrr
SLD 1
exam
tract I
SLD
ﬂora l
9361“
SLD.

mGtal

SLD-cotreatment is not known. TNF-converting enzyme (T ACE), which is
required for release of biologically active TNF, is a possible contributor, since
some NSAle can enhance the activity of this enzyme (Gomez-Gaviro et al.,
2002). It is also possible that SLD or its metabolites enhances TNF transcription
or translation or interferes with TNF clearance.

The importance of TNF in SLD/LPS hepatotoxicity was explored by
pretreating rats with etanercept, a soluble receptor that neutralizes TNF. TNF
inhibition protected against SLD/LPS-induced liver injury, suggesting a critical
role for TNF in this model. However, elevation in TNF concentration alone is not
sufﬁcient to cause liver damage, since much larger TNF concentrations have
failed to induced liver injury (Deng et al., 2008). Thus, additional factors are
likely involved in liver toxicity in SLD/LPS-cotreated rats.

The requirement for bioactivation of SLD raises the possibility that LPS
treatment leads to liver injury in SLD-treated rats by increasing the conversion of
SLD to a toxic metabolite. To study the effect of LPS on SLD metabolism, we
examined the concentration of SLD and its metabolites in plasma, liver, and GI
tract plus feces. According to previous studies, two enzymes are responsible for
SLD metabolism; methionine sulfoxide reductase (MSR) in both liver and gut
ﬂora reduces SLD to SLD sulﬁde, and a ﬂavin-containing monooxygenase (FMO)
converts SLD to SLD sulfone and also catalyzes the conversion of SLD sulﬁde to
SLD. SLD was maximally absorbed in 1 hr, and SLD as well as its sulfone
metabolite accumulated in liver, a result consistent with previous ﬁndings

(Duggan et al., 1980). LPS can signiﬁcantly down-regulate the expression of

110

hepatic
oonseq
(Vattan

metabc

hr, res;
SLD fr
omeer
signiﬁc
EXPOSL
feces.
abscrp
The exl

Wiser

hepatic FMO in mice (Zhang et al., 2008). Oxidative stress, a possible
consequence of LPS exposure, can increase the expression of MSR in bacteria
(Vattanaviboon et al., 2005). Therefore, LPS might have an effect on shifting the
metabolism of SLD towards SLD sulﬁde by regulating the expression of these
two enzymes. However, LPS decreased the concentrations of SLD and SLD
sulﬁde in plasma after the second administration of SLD. The liver concentrations
of SLD and SLD sulﬁde were also decreased by LPS at 1 and 2 hr and 2 and 4
hr, respectively. These results suggested that LPS might decrease absorption of
SLD from the GI tract. To address this possibility, we measured metabolite
concentrations in the GI tract and feces at 2 hr, a time at which we found a
signiﬁcant decrease in both SLD and SLD sulﬁde in plasma and liver after LPS
exposure (Fig. 3.5). LPS increased the concentration of SLD in the GI tract and
feces, suggesting that LPS decreased the bioavailability of SLD by reducing its
absorption. This result does not rule out the possibility that LPS has an effect on
the expression of enzymes that metabolize SLD. Moreover, the SLD metabolite
concentrations in the plasma of cotreated rats were not changed at 8hr by
etanercept pretreatment, suggesting that TNF does not play a role in the ability of
LPS to reduce SLD absorption.

The cytotoxicity of SLD and its metabolites were compared in both HepG2
cells and primary rat hepatocytes. SLD and SLD sulfone were not toxic to HepG2
cells even up to 500 uM, yet SLD sulﬁde showed signiﬁcant toxicity. This result is
consistent with previous ﬁndings, although different medium was used and a

different cytotoxicity assay was performed (Leite et al., 2006). It also has been

111

Mddyre
(Khieti
SLD.H0
qhdcxk
sensitive
SLD as
qmncxi
Ah!

mn de;
essentl.
sulﬁde
nhghtl
heDatc
the IN
SUlﬁcle
enhan
SLD tc
S
GEVQK
SYIIGrg

and p

widely reported that SLD sulﬁde can induce apoptosis of other cancer cell lines
(Kim et al., 2005; Bock et al., 2007), which raised interest in treating cancer with
SLD. However, in this study, we found that the active metabolite of SLD was also
cytotoxic to primary hepatocytes and that primary rat hepatocytes were more
sensitive than HepG2 cells (Fig. 3.6). This might have implications for the use of
SLD as an anticancer agent if normal host cells are more sensitive to the
cytotoxic effects of SLD than are cancer cells.

Although the mechanisms of drug-induced idiosyncratic liver injury are still
not clear, it is believed that accumulation of active metabolites in liver is an
essential ﬁrst step for many drugs (Watkins, 2005). Accordingly, excessive SLD
sulﬁde in liver might be critical for SLD- induced idiosyncratic liver injury. This
might be why two administrations of SLD were required in this model to effect
hepatotoxicity. Interestingly, LPS decreased the concentration of SLD sulﬁde in
the livers of rats, suggesting that SLD sulﬁde accumulation alone was not
sufﬁcient to induce liver injury, and that LPS might be activating pathways that
enhance the toxicity of SLD sulﬁde, instead of increasing the concentration of
SLD toxic metabolite.

Since TNF and SLD or its metabolites are both indispensable for the
development of SLD/LPS-induced liver injury, we explored whether TNF acted
synergistically with SLD or its metabolites using an in vitro system. Both HepG2
and primary rat hepatocytes were resistant to TNF toxicity. Even a much greater
concentration of TNF than we used failed to kill HepGZ cells and primary rat

hepatocytes (Adamson and Billings, 1992). SLD or SLD sulfone in combination

112

with T
SLD E
synerg
comblr
HOWE).
liver ll
investi-
activat
particu
inhibit.
NF-kB
Baftim.
‘Iepatc
PlOSur
effect,
Stiﬁde
lead t(
DTOGUC
(Brady
tai'élets

SI
hepatc

nil-Dug!

with TNF was not cytotoxic; in contrast, this cytokine enhanced the toxicity of
SLD sulﬁde to both cell types. There is evidence that SLD and TNF act
synergistically to kill tumor cells in mice, which raised the possibility of using this
combination of agents as a new anticancer therapy (Hiroshi Yasui, 2003).
However, our results suggest that this therapy might also increase the chance of
liver injury. The mechanism of SLD sulﬁde and TNF interaction is under
investigation. TNF can lead either to hepatocyte proliferation through NF-kappaB
activation or to activation of cell death signaling (Wullaert et al., 2007). SLD, and
particularly SLD sulﬁde, are potent inhibitors of the NF-kappaB pathway through
inhibition of lKappa kinase activity (Yamamoto et al., 1999b). It was reported that
NF-kB plays an essential role in preventing TNF- induced cell death (Beg and
Baltimore, 1996). As a result, it is possible that SLD sulﬁde sensitizes
hepatocytes to TNF-induced cell death through inhibition of NF-kappaB
prosurvival signaling. Moreover, SLD sulﬁde and TNF share a common toxic
effect, which may add to enhance cell death. It has been reported that SLD
sulﬁde can induce reactive oxygen species (ROS) in vitro (Sun et al., 2009) and
lead to mitochondrial uncoupling (Leite et al., 2006). TNF can also cause the
production of ROS (Schwabe and Brenner, 2006) and mitochondrial injury
(Bradham et al., 1998). Therefore, ROS and mitochondria are two potential
targets of interaction of SLD and TNF.

SLD sulﬁde and TNF are not the only mediators that contribute to
hepatotoxicity in this model. Previously, we found that liver hypoxia is induced

through the activation of the hemostatic system in SLD/LPS-cotreated rats and

113

that inhibition of coagulation protects from liver damage (Zou et al., 2009b).
Hypoxia might contribute to liver injury through synergistic interplay with SLD
sulﬁde. Furthermore, we cannot exclude the possible roles of other mediators.
For example, proteases released from neutrophils are important in other
drug/LPS models (Luyendyk et al., 2005; Deng et al., 2007a). The
proinﬂammatory cytokine, interferon-gamma, has been shown to exacerbate
TNF-induced cytotoxicity in hepatocytes (Adamson and Billings, 1993). These
mediators might interact with SLD sulﬁde, TNF and/or hypoxia to promote liver
injury.

In summary, SLD and LPS interact to produce liver injury in rats. The LPS-
stimulated increase in the concentration of TNF in rat serum was enhanced by
SLD, and this cytokine plays a critical role in the pathogenesis. SLD sulﬁde was
more toxic than SLD or SLD sulfone in vitro. Although LPS cotreatrnent reduced
the bioavailability of SLD and the production of toxic SLD sulﬁde, the synergy of
this toxic metabolite with TNF was sufﬁcient to cause liver injury in rats. Such
synergistic interactions might be a trigger for idiosyncratic liver injury from SLD in

humans.

114

CHAPTER 4

Wei Zou, Robert A. Roth, Husam S. Younis, Ernst Malle, and Patricia E.
Ganey. The critical role of tumor necrosis factor-a- and plasminogen
activator inhibitor-1- mediated neutrophil activation in a
sulindac/lipopolysaccharide-model of idiosyncratic liver injury in the rat.

(Submitted)

115

4.1Abstr

Prev:
nonsteroi
In this s
investigal
was grea
LPS alor
cotreated
released
liver injur
PMN pro
factorc l
01 liver il
lPAl-i) (
deDOSlIIC
ﬁbrin de;
or inhib,
product},
in livers
C'Olitribui
indeDen.
"‘1qu in

prOmOtlr

4.1 Abstract

Previous studies indicated that lipopolysaccharide (LPS) interacts with the
nonsteroidal anti-inﬂammatory drug sulindac (SLD) to produce liver injury in rats.
In this study, the mechanism of SLD/LPS-induced liver injury was further
investigated. Accumulation of polymorphonuclear neutrophils (PMNs) in the liver
was greater in SLDILPS-cotreated rats compared to those treated with SLD or
LPS alone. In addition, PMN activation occurred speciﬁcally in livers of rats
cotreated with SLD/LPS. We tested the hypothesis that PMNs and proteases
released from them play critical roles in the hepatotoxicity. SLD/LPS-induced
liver injury was attenuated by prior depletion of PMNs or by pretreatment with the
PMN protease inhibitor, eglin C. Previous studies suggested that tumor necrosis
factor-a (T NF) and the hemostatic system play critical roles in the pathogenesis
of liver injury induced by SLD/LPS. TNF and plasminogen activator inhibitor-1
(PAl-1) can contribute to hepatotoxicity by affecting PMN activation and ﬁbrin
deposition. Therefore, we tested the role of TNF and PAl-1 in PMN activation and
ﬁbrin deposition in the SLD/LPS-induced liver injury model. Neutralization of TNF
or inhibition of PAH attenuated PMN activation. TNF had no effect on PAl-1
production or ﬁbrin deposition. In contrast, PAl-1 contributed to ﬁbrin deposition
in livers of rats treated with SLD/LPS. In summary, PMNs, TNF and PAl-1
contribute to the liver injury induced by SLD/LPS cotreatrnent. TNF and PAl-1
independently led to PMN activation, which is critical to the pathogenesis of liver
injury in SLDILPS-treated rats. Moreover, PAl-1 contributed to liver injury by

promoting ﬁbrin deposition.

116

4.2 Introduction

We reported previously that SLD enhanced the LPS-induced elevation of
serum TNF and plasma PAl-1 in rats (Zou et al., 2009b). TNF neutralization
protected against liver injury in this model, suggesting that TNF plays an
important role in the pathogenesis (Zou et al., 2009a). Fibrin deposition in liver
sinusoids resulted from cotreatrnent and contributed to SLD/LPS-induced
hepatotoxicity (Zou et al., 2009b). TNF and PAI-1 participate in other liver injury
models by causing PMN activation and ﬁbrin deposition (Deng et al., 2008).
Therefore, we investigated the role of TNF and PAl-1 in mediating PMN
accumulation and activation as well as ﬁbrin deposition in livers of SLD/LPS-

treated rats.

117

4.3 Materials and methods
4.3.1 Materials

LPS (Lot 075K4038) derived from Escherichia coli serotype 0552B5 with an

activity of 3.3 X 106 endotoxin units (EU)/mg as well as SLD and its metabolites

were purchased from Sigma-Aldrich (St. Louis, MO). Eglin C was provided by
Novartis Phami (Basel, Switzerland). PA|039 was purchased from Axon

Medchem BV (Groningen, Netherlands).

4.3.2 Animals

Male, Sprague-Dawley rats (Crl:CD(SD)lGS BR; Charles River, Portage, MI)
weighing 250 to 370 g were used. Animals were fed standard chow (Rodent
Chow/Tek 8640; Harlan Teklad, Madison, WI) and allowed access to water ad
libitum. They were allowed to acclimate for 1 week in a 12 hr light/dark cycle prior
to use in experiments. All procedures were approved by the MSU Committee on
Animal Use and Care and complied with “Guide for the Care and Use of

Laboratory Animals” published by the National Academy of Sciences.

4.3.3 Animal model and sample collection
The SLD/LPS-induced liver injury model was described previously (Zou et
al., 2009b). Food was removed, and rats were given the ﬁrst administration of

SLD (50 mglkg, p.o.) or its vehicle (0.5% methyl cellulose) 16 hr before the

second administration of the same dose. LPS (8.25X 105 EU/kg, i.v.) or its

118

vehicle (saline) was administered half an hour before the second administration
of SLD. Rats were anesthetized at various times after the second administration
of SLD. Serum and plasma was prepared from blood withdrawn from the vena
cava. Liver tissue from the left lateral lobe was collected and ﬁxed in 10%
buffered formalin for PMN staining. A portion of the left medial lobe of the liver
was ﬂash-frozen in isopentane for determination of hypochlorous acid (HOCl)-

protein adduct staining as well as for ﬁbrin deposition analysis.

4.3.4 Anti-PMN serum, eglin C, PAI039 and etanercept treatment protocols
In PMN depletion experiments, rabbit anti-PMN serum or normal rabbit
serum control was diluted 1:1 in sterile saline and given to rats (0.5 ml per rat,
i.v.) half an hour before the ﬁrst administration of SLD. The efﬁcacy of the anti-
PMN serum in depleting PMNs has been demonstrated in previous studies
(Deng et al., 2007b). A PMN protease inhibitor, eglin C (8 mglkg, i.v.; kindly
provided by Novartis Pharrn AG, Basel, Switzerland) or its saline vehicle, was
administered to rats 4, 6 and 8 h after the second administration of SLD. A PAl-1
inhibitor, PAI039 [{1-benzyl-5-[4-(triﬂuoromethoxy)phenyl]-1H-indoI-3-
yl}(oxo)acetic acid] (6 mglkg, p.o.) or its vehicle (0.5% methyl cellulose) was
administered to rats 1 hr after the second administration of SLD. Etanercept (8
mglkg) or vehicle (sterile water) was given to rats subcutaneously one hour

before LPS or its saline vehicle.

4.3.5 Evaluation of hepatotoxicity

119

The activity of alanine aminotransferase (ALT) in serum was used as a
marker to assess injury to hepatic parenchymal cells. The assay was performed

using a diagnostic kit from Thermo Fisher Scientiﬁc (Waltham, MA).

4.3.6 Determination of ClNC-1, MlP-1a and PAl-1 concentrations in plasma
The concentrations of cytokine-induced neutrophil chemoattractant-1 (ClNC-
1) and macrophage inﬂammatory protein-1o (MlP-1d) in plasma were estimated
by multiplex ELISA. Speciﬁc antibody-coupled beads were purchased from
Millipore Corp. (Billerica, MA). Functionally active PAl-1 was measured by ELISA
using a commercially available test kit from Molecular Innovations, Inc

(Southﬁeld, MI).

4.3.7 Evaluation of liver PMN accumulation and activation

Parafﬁn-embedded liver tissue was cut into 6 um-thick sections on which
PMN immunohistochemistry was performed as described previously (Yee et al.,
2003). Brieﬂy, parafﬁn was removed with xylene, and liver sections were
incubated with polyclonal rabbit anti-PMN lgG as ﬁrst antibody, and then
incubated with biotinylated goat anti-rabbit IgG, avidin-conjugated alkaline
phosphatase, and Vector Red substrate to stain PMNs. The numbers of PMNs
enumerated in 10 randomly selected, 400 X high power ﬁelds were averaged to
assess PMN accumulation in the liver.

The potent oxidant HOCI, generated from hydrogen peroxide by

myeloperoxidase (MPO) in the presence of physiological choride concentrations,

120

EQCHSV
used as
lherhss
ﬁxed ir
phosph
dockec
Probes
whh a

IV-IVI QC
3, 20l
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antiboc
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averag

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F?
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reacts with proteins to form chloramines. These HOCl-protein adducts can be
used as ﬁngerprints (Malle et al., 2006) to directly assess activation of PMNs in
liver tissues (Hasegawa et al., 2005; Deng et al., 2007b). Frozen liver sections
ﬁxed in 4% formalin for 10 min at room temperature were washed with
phosphate-buffered saline (PBS) 3 times for 5 min each. The sections were
blocked for 1 hr at room temperature with 3% [vlv] goat serum (Molecular
Probes, Carlsbad, CA) in PBS, and then incubated for 2 hr at room temperature
with a monoclonal antibody (clone 2D1OG9, subtype lgG2bk; diluted 1:1 in 3%
[vlv] goat serum) speciﬁc for HOCI-modiﬁed epitopes generated in vivo (Malle et
al., 2006) and in vitro (Malle et al., 1995). After another 3 washes with PBS,
slides were incubated with Alexa Fluor 488-labeled goat anti-mouse secondary
antibody (diluted 1:500 in 3% [vlv] goat serum, Molecular Probes, Carlsbad,
California). Ten pictures were taken of 200 X power, randomly selected ﬁelds
using a ﬂuorescence microscope, and the fraction of positive pixels was

averaged for each slide (Deng et al., 2008).

4.3.8 Assessment of ﬁbrin deposition in liver
The immunohistochemistry for cross-linked ﬁbrin in liver was performed as

described previously (Zou et al., 2009b).

4.3.9 Statistical analyses
Results are presented as means 1 SEM. Student’s t-test was performed on

ﬁbrin deposition data. For the rest of the studies, one way or two way analysis of

variance (Ah
Newman-Ker

for statistical

variance (ANOVA) was applied for data analysis, as appropriate, and Student-
Newman-Keuls test was used as a post hoc test to compare means. The criterion

for statistical signiﬁcance was P < 0.05.

4.4 Result
4.4.1Eval
Liver i
administra
(Zou et a
collected 1
SLD/LPS.
(Fig. MA:
with LPS.
LPS and E
HOCI-
Chlorlde s}
MPO of to
“I (not sl
99510085 v
with SLD l
peroxide-t

01 rats treE

“'2 Time

Plasma

PIaSrr

and the C0

4.4 Results
4.4.1 Evaluation of PMN accumulation and activation in livers

Liver injury induced by SLD/LPS occurs between 4 and 8 hr after the second
administration of SLD, and ALT activity in rats signiﬁcantly increases by 12 hr
(Zou et al., 2009b). Accordingly, PMN accumulation was assessed in livers
collected at 4 hr, a time before the onset of hepatocellular injury induced by
SLD/LPS. SLD given alone had no signiﬁcant effect on hepatic PMN number
(Fig. 4.1A). An increase in PMN number was observed in livers of rats treated
with LPS. PMN numbers were signiﬁcantly greater in livers of rats cotreated with
LPS and SLD compared to those treated with LPS alone.

HOCl-protein adducts are generated by the MPO-hydrogen peroxide-
chloride system of activated PMNs (Malle et al., 2006), cells containing up to 5%
MP0 of total cell protein content. Adducts were not elevated in liver sections at 4
hr (not shown). However, at 8 hr pronounced formation of HOCl-modiﬁed
epitopes was found in livers of rats treated with SLD/LPS, but not in rats treated
with SLD or LPS alone (Fig. 4.13). This result indicates that the MPO-hydrogen
peroxide-halide system of PMNs was activated between 4 and 8 hr in the livers

of rats treated with SLD/LPS.

4.4.2 Time course of changes in CINC-1 and MlP-1d concentrations in
plasma
Plasma was collected from rats euthanized at various times (1, 4 and 12 hr),

and the concentrations of ClNC-1 and MlP-1a were measured. LPS increased

123

Fig. 4.1. Evaluaton of PMN accumulation and activation in rat livers. Rats

were treated with two administrations of SLD (50 mglkg, p.o.) or its vehicle (Veh,

0.5% methyl cellulose) with a 16 hr interval. LPS (8.25 X 105 EU/kg, i.v.) or its

saline vehicle was administered half an hour before the second administration of
SLD. (A) PMN staining was performed on livers collected 4 hr after the second
administration of SLD. PMN number in 400 X high power ﬁelds (HPF) was
counted to evaluate PMN accumulation. (B) HOCl-protein adduct staining was
performed on slides of frozen liver collected at 8 hr. Ten random ﬁelds were
photographed for every section, and the fraction of positive pixels was
determined. *signiﬁcantly different from respective group without LPS.

#signiﬁcantly different from Veh/LPS group. P<0.05, n=4-5.

124

 

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CINC-1 and MlP-1d concentrations at 1 and 4 hr (Fig. 4.2). The concentrations of
both chemokines had returned to baseline by 12 hr. SLD treatment had no effect

on ClNC-1 or MIP-1u concentrations in vehicle- or LPS-cotreated rats.

4.4.3 Effect of PMN depletion and PMN protease inhibition on SLDILPS-
induced liver injury

To assess the role of PMNs in SLD/LPS-induced liver injury, rabbit anti-PMN
serum or normal serum was given to rats. In a previous study, anti-PMN serum
selectively reduced PMNs without affecting other leukocyte numbers in blood
(Deng et al., 2007b). Blood PMN number in the anti-PMN serum/SLDILPS group
(499 t 23) was signiﬁcantly smaller than that in the normal serum/SLDILPS
group (2726 :1: 144) at 12 hr. Cotreatment with normal serum/SLDILPS led to
increased serum ALT activity (Fig. 4.3). Pretreatment with anti-PMN serum
abolished the SLD/LPS-induced increase in ALT activity.

Eglin C is a potent and selective inhibitor of elastase and cathepsin G
released by activated PMNs (Schnebli et al., 1985; Braun et al., 1987). Eglin C
had no effect on ALT activity in serum of rats treated with vehicle but attenuated

the elevation in serum ALT activity of rats treated with SLD/LPS (Fig. 4.4).

126

it 42. Cor
=3. ’Mth S.
:‘3 ‘2 hr aft
Fiﬁdtfaticn

*7 15} ma

Fig. 4.2. Concentrations of PMN chemokines in rat plasma. Rats were
treated with SLD and. LPS or their vehicles (Veh) as described in Fig. 4.1. At 1, 4
and 12 hr after the second administration of SLD, plasma was collected and
concentrations of (A) cytokine-induced neutrophil chemoattractant-1 (ClNC-1)
and (B) macrophage inﬂammatory protein-1a (MlP-1or) were evaluated by
multiplex ELISA. *signiﬁcantly different from VehNeh group at the same time.
P<0.05, n=5.

127

CINC-1 (pglmL)

MlP-1a (ngImL)

2500 .

2000 .

1500 .

1000 .

500 -

120 ~
100 .
80 .
60 .
40 .

zol

 

 

 

 

1
hrs after 2nd dose of SLD

 

 

— VehNeh
I: SLDNeh
* — Veh/LPS
1:1 SLD/LPS

 

4 12

 

1

4 12

hrs after 2nd dose of SLD

128

Fig. 4.3. Effect of PMN depletion on SLDILPS-induced liver injury. Rats were
pretreated with either normal serum (NS) or rabbit anti-rat PMN serum (AS) half
an hour before the first administration of SLD. Rats were euthanized 12 hr after
the 2"d dose of SLD, and serum ALT activity was determined. Vehicle (Veh).
*signiﬁcantly different from VehNeh/NS group, #significantly different from

SLD/LPS/NS group. P<0.05, n=3-6.

1000 * — NS
[:3 AS
800 l
600 .
400 -

200- #
o -———-—- [j

VehNeh SLD/LPS

ALT (UIL)

 

 

129

Fig.4.4. Effect of PMN protease inhibition on SLDILPS-induced liver injury.
Eglin C or its vehicle (Veh) was administered to rats 4, 6, and 8 hr after the 2nd
administration of SLD. Rats were euthanized at 12 hr, and ALT activity in serum

was determined. *signiﬁcantly different from VehNehNeh. #signiﬁcantly different
from VehNeh/Eglin C group. asigniﬁcantly different from SLDILPSNeh group.

P<0.05, n=3-6.

2500 - - Veh
l:l Eglin C
*

2000 -

1500
# a

1000 -

ALT (UIL)

500 ‘

 

VehNeh SLD/LPS

130

4.4.4 Effect of TNF on PMN accumulation and activation

As noted above, cotreatrnent with SLD/LPS caused an increase in the
number of PMNs in liver (Fig. 4.1A). Etanercept, which neutralizes TNF and
inhibits its biological effects, did not affect PMN numbers in livers of rats treated
with SLD/LPS (Fig. 4.5A). In contrast, etanercept prevented the elevation in
SLD/LPS-induced formation of HOCl-protein adducts (Fig. 4.5B). These results
suggest that TNF contributes to the release of cytotoxic factors from PMNs but

not to PMN accumulation in the liver.

4.4.5 Role of PAH in liver injury and accumulation and activation of PMNs
A previous study indicated that PAl-1 was selectively increased in the
plasma of SLDILPS- cotreated rats (Zou et al., 2009b); however, its role in liver
injury has not been investigated. The PAl-1 inhibitor, PAI039, greatly attenuated
liver injury, as followed by ALT measurements (Fig. 4.6a) induced by SLD/LPS
cotreatrnent. PAI039 also reduced PMN activation (Fig. 4.60) but not PMN

accumulation at 8 hr (Fig. 4.6b).

4.4.6 Effect of TNF on plasma PAI-1 concentration

In previous studies, TNF was signiﬁcantly increased as early as 1 hr by
SLD/LPS cotreatrnent, and PAl-1 was increased by 8 hr (Zou et al., 2009b). To
evaluate whether TNF regulates the production of PAH, plasma concentration of

PAH was evaluated in rats cotreated with etanercept. Etanercept given at a

131

Fig. 4.5. Effect of TNF inhibition on PMN accumulation and activation. Rats
administered SLD/LPS were pretreated with etanercept or its vehicle (Veh) 1 hr
before LPS. (A) PMN staining was performed on livers collected at 8 hr. The
accumulation of PMNs in livers was evaluated by averaging PMN numbers in 10,
randomly chosen, 400 X ﬁelds. (B) Quantiﬁcation of HOCl-protein adducts in the
livers of rats at 8 hr. *signiﬂcantly different from VehNehNeh group.

#signiﬁcantly different from Veh/SLDILPS group. P<0.05, n=3-6.

132

— VehNehNeh

1:1 VehlSLD/LPS
— EtanISLD/LPS

 
 

 

 

1 . . _ . . m a ..u 5 m 5 .....
M m m w m o m m m m m. m m.
1 o o o 0 o o o
"E: Xeov \ n st Egan e>Emoa .0 5:69”:
36:25 539.... -50:

133

i94.6. Effect
1111311011. Rat
tithiPAIOE
“ stered tc
:‘aiized at
mediation I

5370:3038 t

Fig. 4.6. Effect of PAH inhibition on liver injury and PMN accumulation and
activation. Rats were treated with SLD/LPS as described in Fig. 4.1. PAl-1
inhibitor, PAI039 (6 mglkg, p.o.), or its vehicle (Veh, 0.5% methyl cellulose) was
administered to rats at 1 hr after the second administration of SLD. Rats were
euthanized at 12 hr to measure ALT activity (A) or at 8 hr to assess PMN
accumulation (B) and activation (C). *signiﬁcantly different from VehNehNeh.

#signiﬁcantly different from SLDILPSNeh group. P<0.05, n=4-16.

134

— VehNehNeh
CZ! SLDILPSNeh
* _ SLD/LPS/PAl039

 

 

 

 

800 i

o
6

0 o
4 2

“E: 531:“ 2:...—

 

 

*
o 5 o 5 o 5 o
3 2 2 1 1 W o
h 0 o. h o . n
o o o o o

0. 0
.282.— e>Euoa he 5:62....
2268 522.. .80:

135

dose thal F

increase in

4.4.7 Effec

Fibrin
result in h
were redi
According
causingf

caused b

dose that protected against liver injury (Zou et al., 2009a) had no effect on the

increase in plasma PAl-1 concentration caused by SLD/LPS (Fig. 4.7).

4.4.7 Effect of TNF and PAl-1 on ﬁbrin deposition

Fibrin clots form in the sinusoids of livers of SLD/LPS-cotreated rats and
result in hepatic hypoxia (Zou et al., 2009b). Both ﬁbrin deposition and hypoxia
were reduced by anticoagulant treatment, which protected against liver injury.
Accordingly, we evaluated whether TNF or PAI-1 exerts its toxic effect by
causing ﬁbrin deposition in the liver. Etanercept had no effect on ﬁbrin deposition

caused by SLD/LPS cotreatrnent, whereas PAI039 reduced it (Fig. 4.8).

136

Fig. 4.7. Effect of TNF inhibition on plasma PAl-1 concentration. Rats were
treated with etanercept (Etan), SLD and LPS or their vehicles (Veh) as described
in the legend to Fig. 4.5. Plasma active PAI-1 concentration was determined at 8

hr. * signiﬁcantly different from VehNehNeh. P<0.05, n=4.

— VehNehNeh
C31 VehlSLD/LPS
_ EtanlSLD/LPS

600 - *
400 «

200 i,

N

Plasma Active PAl-1 (ngImL)

 

 

 

 

 

 

137

Fig. 4.8. Effect of TNF or PAI-1 inhibition on ﬁbrin deposition in liver. Rats
were treated with SLD/LPS and etanercept (Etan, A) or PAl-1 inhibitor (B)
respectively. Fibrin deposition was evaluated at 8 hr. * signiﬁcantly different from

SLD/LPSNehicle (Veh). P<0.05, n=4-7.

138

A

 

 

1.

mmmmmmm

. 0 a 0 o. 0 o.
oni 0238a Co .5on“:

55¢

SLDILPS

 

 

mmmmm

o 0 0 0 0
3.9.5 0223.. he c232“:

55E

Veh

SLDILPS

139

4.5 Discussion

PMNs are a double-edged sword in the innate immune response to microbial
infection and tissue trauma (Butterﬁeld et al., 2006). Stimulated by inﬂammatory
signals, PMNs attach to endothelial cells via adhesion molecules and
transmigrate to the site of infection/trauma, where they become activated to
release cytotoxic factors. PMNs can be beneﬁcial by removing invading
organisms and stimulating tissue repair. However, excessive PMN activation
causes tissue injury in many animal models (Jaeschke et al., 1990; Hewett et al.,
1992). PMNs are involved in several models of drug-induced liver injury and in
drug-LPS interaction models of idiosyncratic liver injury (Deng et al., 2006; Deng
et al., 2007b; Ramaiah and Jaeschke, 2007; Shaw et al., 2009d).

As has been reported, LPS administration causes PMNs to accumulate in
liver. Although SLD mildly inhibited the adhesion of PMNs to nylon-wool columns
in vitro (Venezio et al., 1985), it increased the LPS-induced PMN accumulation in
liver before the onset of liver injury (Fig. 4.1A). Two PMN chemokines, MlP-1a
and ClNC-1, are potent inducers of PMN recruitment and extravasation. A
neutralizing antibody to either MlP-1a or ClNC-1 attenuated neutrophil
sequestration in LPS-treated rodents (Standiford et al., 1995; Zhang et al., 1995).
The concentrations of both chemokines were signiﬁcantly increased in plasma by
LPS, whereas SLD had no effect (Fig. 4.2). Thus, both chemokines might
contribute to PMN accumulation in livers of LPS-treated rats; however, other
factors must be involved after SLD/LPS cotreatment. The reason why SLD

enhanced PMN accumulation is unknown, but some possibilities arise from

140

previous results. It is known that SLD/LPS cotreatrnent caused ﬁbrin deposition
in the liver (Zou et al., 2009b). It is possible that entrapment of PMNs in the
meshwork of sinusoidal ﬁbrin occurred. In addition, as a result of ﬁbrin clots in
sinusoids hypoxia occured in livers of cotreated rats (Zou et al., 2009b). Hypoxia
can enhance the adherence of PMNs to human endothelial cells in vitro (Milhoan
et al., 1992), and such an effect might further explain the SLD-induced increase
in PMN accumulation.

Generally, PMNs that sequester in the liver are not injurious unless
extravasation of them into the parenchyma and activation occur (Chosay et al.,
1997). Although PMNs accumulated in livers in rats treated only with a small
dose of LPS (Fig. 4.1), staining for HOCI-modiﬁed proteins, speciﬁc markers for
neutrophil-induced oxidant stress, suggested no activation of PMNs. SLD/LPS
cotreatrnent, however, increased HOCI-protein adducts, indicative that the MPO-
hydrogen peroxide-chloride system of PMNs becomes activated between 4 and 8
hr, when the onset of liver injury occurred. Our observations parallel recent
ﬁndings in patients with steatohepatitis in which liver chemokine expression was
higher in patients with MPO-mediated oxidation products and correlated with
hepatic neutrophil sequestration (Rensen et al., 2009). The role of PMNs in
SLDILPS-induced liver damage was further tested using anti-PMN serum, which
markedly reduced PMNs in the circulation. The protection by anti-PMN serum
shows that PMNs are critical to the development of SLDILPS-induced liver injury
(Fig. 4.3).

141

Activated PMNs release various lysosomal hydrolases including serine
proteases, among which elastase and cathepsin G have been identiﬁed as
primary mediators in hepatocyte killing by PMNs in vitro (Ho et al., 1996). Eglin C
is an inhibitor of elastase and cathepsin G. It attenuated liver injury (Fig. 4.4),
suggesting that PMN proteases also play a role in the pathogenesis. Compared
to the complete protection by PMN depletion, eglin C incompletely reduced
SLDILPS-induced liver injury; thus, the proteases released from PMNs might not
be the only PMN-derived mediators contributing to the pathogenesis. In
numerous inﬂammatory liver injury models, antioxidants attenuated PMN-
mediated liver injury in vivo (Liu et al., 1995; Jaeschke and Smith, 1997). Our
results to date cannot rule out a role for reactive oxygen species other than
HOCI, but this is a topic of current investigation.

TNF neutralization signiﬁcantly attenuated liver injury induced by SLD/LPS in
vivo (Zou et al., 2009a). In addition, TNF directly interacted with SLD sulﬁde to
kill hepatocytes in vitro. TNF can also activate endothelial cells to promote PMN
migration (Smart and Casale, 1994). In results presented here, the number of
PMNs sequestered in the liver was not affected by TNF neutralization, but TNF
neutralization did reduce PMN activation in the liver (Fig. 4.5). These results
suggest that TNF contributes to PMN activation, but not to hepatic accumulation
of these cells.

Like PMN depletion, anticoagulation using heparin abolished the
hepatotoxicity induced by SLD/LPS cotreatrnent of rats (Zou et al., 2009b), which

suggests that there is an interaction between PMNs and the hemostatic system

in the pathogenesis. Hemostatic factors including thrombin and PAl-1 were
increased in plasma by SLD/LPS cotreatrnent (Zou et al., 2009b). Interestingly,
hemostatic factors can bind to PMNs and inﬂuence their accumulation and
activation (Gillis et al., 1997). For example, thrombin can rapidly trigger lysozyme
release from human PMNs and promote PMN activation in perfused rat liver after
LPS exposure (Baranes et al., 1986; Copple et al., 2003).

PAl-1 is an inhibitor of plasminogen activator and a key negative regulator of
ﬁbrinolysis. PAI039, a PAl-1 inhibitor, signiﬁcantly attenuated SLDILPS-induced
liver injury, suggesting that PAl-1 is a mediator of pathogenesis (Fig. 4.6A).
PAI039 decreased ﬁbrin deposition in livers of SLDILPS-treated rats (Fig. 4.83),
which suggests that PAl-1 contributes to ﬁbrin deposition in the SLD/LPS model.

In addition to inhibiting ﬁbrinolysis, PAI-1 can regulate PMN migration and
potentiate LPS-induced PMN activation through a c-Jun N-tenninal kinase-
mediated pathway (Kwak et al., 2006; Roelofs et al., 2009). Consistent with these
ﬁndings, PAl-1 inhibition reduced HOCl-protein adduct staining in livers of
SLDILPS-cotreated rats (Fig. 4.6C), suggesting that PAI-1 is involved in PMN
activation. Therefore, PAI-1 contributed to both PMN activation and ﬁbrin
deposition. It can also play a proinﬂammatory role by stimulating the production
of cytokines and chemokines. For example, in a murine model of
trovaﬂoxacin/LPS-induced liver injury, PAl-1 knockout markedly decreased the
plasma concentrations of interleukin-1B, interleukin-10, keratinocyte
chemoattractant and monocyte chemoattractant protein-1, respectively (Shaw et

al., 2009c). Whether PAl-1 similarly regulates the production of chemokines in

143

SLD/LPS— treated rats and the role of these cytokines in PMN activation are
topics for further investigation.

PMNs can exacerbate ﬁbrin deposition by releasing proteases (Deng et al.,
2007b). For example, proteases from PMNs can release PAl-1 from endothelial
cells and platelets and thereby inhibit ﬁbrinolysis (Pintucci et al., 1992). Eglin C
treatment signiﬁcantly decreased active PAI-1 concentration and ﬁbrin deposition
in a model of ranitidine/LPS-induced liver injury (Deng et al., 2007b). SLD/LPS
cotreatrnent led to ﬁbrin deposition at 4 hr, before the activation of PMNs at 8 hr.
Therefore, PMNs do not contribute to the initial formation of ﬁbrin, but proteases
released by activated PMNs might prolong ﬁbrin deposition.

The concentrations of PAH and TNF in blood were both signiﬁcantly greater
in SLDILPS-treated rats than in rats treated with either LPS or SLD alone. The
peak of PAl-1 (4 hr) in plasma followed the peak of TNF production (i.e.,1 hr; Zou
et al., 2009b). Although it has been reported that both TNF and LPS lead to PAI-
1 release from endothelial cells in vitro (Riedo et al., 1990), inhibition of TNF did
not decrease PAl-1 concentration in SLDILPS-cotreated rats (Fig. 4.7). This
indicates that PAl-1 production does not depend on TNF in this model.
Consistent with this result, TNF did not affect ﬁbrin deposition in liver (Fig. 4.8).
Thus, the activation of hemostatic system is likely a direct effect of LPS but not
mediated through TNF in this model. In contrast, TNF does mediate hemostatic
system activation in ranitidine/LPS- or trovaﬂoxacin/LPS-induced liver injury

(T ukov et al., 2007a; Shaw et al., 2009e). Therefore, these results suggest that

144

TNF does not contribute to liver injury through the same mechanism in all
drug/LPS interaction models.

From results of this and previous studies, we can summarize mechanisms of
liver injury induced by SLDILPS cotreatrnent (Fig. 4.9). Various mediators
including TNF, hypoxia caused by hemostatic system activation and PMNs play
critical roles in the pathogenesis of SLDILPS-induced liver injury. SLD enhances
TNF elevation induced by LPS. SLD/LPS cotreatrnent also leads to the
production of hemostatic factors including thrombin and PAl—1, both of which
contribute to ﬁbrin clot formation in liver sinusoids (Zou et al., 2009b). As a result,
the liver becomes hypoxic. Although the concentration of the toxic metabolite,
SLD suliﬁde, is decreased by LPS in livers and plasma of rats, it synergistically
kills hepatocytes in the presence of TNF and hypoxia (Zou et al., 2009a). PMNs
are another critical player in SLDILPS-induced liver injury (Fig. 4.3). PMN
accumulation in the liver was primarily induced by LPS and this effect was
enhanced by SLD (Fig. 4.1A). Activation of PMNs was observed in livers of rats
treated with SLD/LPS (Fig. 4.18). Both TNF and PAl-1 contribute to PMN
activation independently (Fig. 4.5 to 4.7). When activated, PMNs release
proteases which induce liver injury by interacting with hypoxia (Luyendyk et al.,
2005). In summary, the studies presented here further our understanding of the
roles of various mediators and their interaction in this SLDILPS-induced

idiosyncratic liver injury model.

145

Fig. 4.9. Mechanisms of SLDILPS-induced liver injury. See text for details

 

   
  
  

N
PMN _.___.. PMN _... proteases
accumulation activation

 

Thrombin—9 Tfibrin ——-b hypoxia

PAI-1

146

 

CHAPTER 5

Wei Zou, Robert A. Roth, Husam S. Younis, Lyle D. Burgoon, and Patricia E.

Ganey. Oxidative stress is an important player in the pathogenesis of liver

injury induced by sulindac and lipopolysaccharide cotreatrnent

147

5.1 Abstract

Among all the nonsteroidal anti-inﬂammatory drugs, sulindac (SLD) is
associated with the greatest incidence of idiosyncratic hepatotoxicity in humans.
Previously, an animal model of SLD-induced idiosyncratic hepatotoxicity was
developed by cotreating rats with a nonhepatotoxic dose of LPS. Tumor necrosis
factor-alpha (T NF) was found to be critically important to the pathogenesis. In
this study, we further explored the mechanism of liver injury induced by SLD/LPS
cotreatment by analyzing gene expression in livers of rats before the onset of
liver injury. The results suggested that oxidative stress might be a potential
mediator. Moreover, protein carbonyls, products of oxidative stress, were
elevated in liver mitochondria of SLDILPS-cotreated rats. Antioxidant treatment
witheither ebselen or dimethyl sulfoxide attenuated SLDILPS-induced liver
injury. The role of oxidative stress was further investigated in vitro. SLD sulﬁde,
the toxic metabolite of SLD, enhanced TNF-induced cytotoxicity and caspase 3/7
activity in HepG2 cells. SLD sulﬁde increased dichloroﬂuorescein ﬂuorescence in
HepG2 cells, suggesting generation of reactive oxygen species (ROS). Hydrogen
peroxide and TNF cotreatrnent caused greater cytotoxicity than either treatment
alone. Either antioxidant tempol or a pancaspase inhibitor Z-VAD-FMK
decreased HepG2 cell death as well as caspase 3/7 activity induced by SLD
sulﬁde/1' NF coexposure. These results indicate that SLDILPS treatment causes
oxidative stress in livers of rats and that reactive oxygen species are important in

the cytotoxic interaction of SLD and TNF by activating caspase 3/7.

148

5.2 Introduction

Reactive oxygen species (ROS) include oxygen free radicals and other
nonradical but highly reactive molecules (e.g., hydrogen peroxide). Excessive
generation of ROS tilts the balance between prooxidant and antioxidant
inﬂuences in the cell and results in oxidative stress. ROS can directly oxidize
proteins, DNA or membrane lipids in target cells, and such effects can result in
cell death. One ROS-mediated pathway of cell death is through caspase-
dependent intracellular apoptotic signaling initiated by oxidative stress (Jones et
al., 2000). The oxidative stress in liver can be induced under various conditions
that include consumption of ethanol or other drugs that cause inﬂammatory
stress (Galati et al., 2002; Choi and Ou, 2006; Cederbaum et al., 2009).

In this study, gene expression was analyzed in livers from rats treated with
sulindac and/or LPS. The results of Ingenuity pathway analysis of SLD/LPS-
speciﬁc gene expression proﬁles pointed to the occurrence of oxidative stress.
We tested the hypothesis that oxidative stress plays a role in the pathogenesis of
liver injury induced by SLD/LPS in vivo. In hepatocytes, we evaluated the ability

of SLD and its metabolites to prompt ROS generation and explored its role in

cytotoxicity.

149

5.3 Materials and methods
5.3.1 Materials
Unless othenrvise noted, all chemicals were purchased from Sigma-Aldrich

(St. Louis, MO). The LPS (Lot 075K4038) used in animal experiments was

derived from Escherichia coli serotype O55:B5 and had an activity of 3.3 X 106

endotoxin units (EU)/mg. HepG2/C3A cells were obtained from American Type

Culture Collection (Manassas, VA).

5.3.2 Animals

Male, Sprague-Dawley rats (Crl:CD(SD)lGS BR; Charles River, Portage, MI)
weighing 250 to 370 g were used in this study. They were allowed to acclimate
for 1 week in a 12-hr light/dark cycle prior to use in experiments. Animals were
fed standard chow (Rodent Chowfl’ek 8640; Harlan Teklad, Madison, WI) and
allowed access to spring water ad libitum. Experimental procedures complied
with “Guide for the Care and Use of Laboratory Animals” (National Academy of

Sciences).

5.3.3 Design of experiments in vivo
As described in a previous study (Zou et al., 2009b), rats were given the ﬁrst
administration of SLD (50 mglkg, p.o.) or its vehicle (0.5% methyl cellulose), and

food was removed at this time. Sixteen hours later, SLD at the same dose or its

vehicle was administered to rats. LPS (8.25 X 105 EU/kg, i.v.) or its vehicle

150

 

(saline) was administered via a tail vein half an hour before the second
administration of SLD. As reported previously, this protocol results in liver injury
in the SLDILPS-cotreated rats (Zou et al., 2009b). At various times (4, 8 or 12 hr)
after the second administration of SLD, rats were anesthetized with isoﬂurane,
and blood was drawn from the vena cava. Serum was prepared from clotted
blood. A portion of the right medial lobe of the liver was ﬂash-frozen in liquid
nitrogen for RNA extraction. Another portion was collected and cooled in ice-cold
isolation buffer for mitochondrial preparation. In experiments designed to
evaluate the effect of antioxidants on liver injury, ebselen (50 mglkg, p.o.) or its
vehicle (0.5% methyl cellulose) was given to rats 1.5 hr before the second
administration of SLD; dimethyl sulfoxide (0.3 mL/g, i.p.) was given to rats at the

same time as the administration of SLD.

5.3.4 Gene expression analysis

Total RNA was extracted from frozen liver tissue collected at 4 hr using a kit
purchased from Qiagen Inc. (Valencia, CA) as described previously (Younis et
al., 2006). Microarray analysis was performed using the standard protocol
provided by Affymetrix, Inc. (Santa Clara, CA). Total RNA (10 pg) was reverse
transcribed into cDNA in the presence of oligo dT primer using a Superscript II
Double-Strand cDNA synthesis kit (lnvitrogen, Carlsbad, CA). cDNA was puriﬁed,
and biotin-labeled cDNA was synthesized using the Enzo RNA Transcript
Labeling Kit (Affymetrix, Santa Clara, CA). After the labeled cDNA was puriﬁed

and its quality was evaluated, cDNA was hybridized to a Rat Genome 230 2.0

151

Array (Affymetrix), which comprised more than 31,000 probe sets. The array was
stained with streptavidin-phycoerythrin (Invitrogen, Carlsbad, CA) and scanned to
generate signal intensity ﬁles.

Data normalization was performed using SAS version 9.1 on scanned image
ﬁles (Eckel et al., 2005). Empirical Bayes analysis was used to calculate
posteriori probabilities (P1(t) value) (Eckel et al., 2004). Ratios of gene
expression were calculated by comparing SLDNeh, Veh/LPS or SLD/LPS

groups to the VehNeh group.

5.3.5 Evaluation of liver injury and protein carbonyls in mitochondria

Liver injury was assessed by measuring the activity of alanine
aminotransferase (ALT) in serum using a diagnostic kit from Thermo Corp
(Waltham, MA).

Mitochondria were isolated from livers of rats treated with SLD/LPS or
vehicles at 8 hr using a mitochondrial isolation kit (Sigma, St. Louis, MO). Brieﬂy,
fresh livers (50 mg) were collected and homogenized in HEPES buffer containing
200 mM mannitol, 70 mM sucrose, and 1 mM EGTA. Liver homogenates were
centrifuged at 600xg for 5 minutes. The supernatant fraction was transferred into
a new tube and centrifuged at 11,000xg for 10 minutes. The pellets were washed
and centrifuged at 11,000xg for 10 minutes again. The mitochondrial pellets were
resuspended in HEPES for the evaluation of membrane potential or protein

carbonyl concentration. Protein carbonyl concentration in liver mitochondria was

152

 

measured using a commercially available kit purchased from Cayman Chemical

(Ann Arbor, MI).

5.3.6 Evaluation of mitochondrial membrane potential

The effect of SLD and its metabolites on mitochondrial membrane potential
was evaluated using JC-1 mitochondrial membrane potential assay kit from
Cayman Chemical (Ann Arbor, MI). Mitochondria isolated from normal rats (2 ug
protein) were incubated with various concentrations of SLD or its metabolites at a
ﬁnal volume of 200 uL. After 30 min, the JC-1 dye solution (0.2 uL) was added.
Fluorescence was read for JC-1 agglomerates and monomers respectively. The
ratio of JC-1 agglomerates (excitation/emission=560l595 nm) to monomers
(excitation/emission= 485/535 nm) in mitochondria was calculated, and the data
were expressed as a percentage of vehicle control (0.5% dimethyl sulfoxide).
The decrease in ratio of JC-1 agglomerates to monomers was associated with a

decrease in membrane potential (Reers et al., 1995).

5.3.7 Evaluation of reactive oxygen species in HepGZ cells
ROS in HepG2 cells were assessed using 5-(and-6)-chloromethyl-2’,7”-

dichlorodihydroﬂuorescein diacetate, acetyl ester (CM-H2DCFDA) purchased

from lnvitrogen, Inc. (Carlsbad, CA). HepG2 cells (4 x 105 cells/mL) in

suspension were incubated with 10uM CM-HZDCFDA in Dulbecco’s Modiﬁed
Eagle’s medium (DMEM) for 45 min. The cells were washed twice with DMEM

with 10% fetal bovine serum. Cells were plated in 96-well plates and treated with

153

250 UN
absenc

DCF ﬂL

5.3.8 E

Hi

DMEM
reneWI
08008!
(200-
vehicle
percel
et al.,

HepG

5.3.9

variar
Keuls
Criteﬁ
e”‘lpil

250 uM SLD sulﬁde or its dimethyl sulfoxide (DMSO) vehicle in the presence or
absence of recombinant human TNF (200 ng/ml) dissolved in DMEM vehicle.

DCF ﬂuorescence intensity was read at 0, 0.5, 1 and 3 hr after treatment.

5.3.8 Evaluation of cytotoxicity and capase 3 activity

HepGZ cells were plated in 96-well plates at a density of 4 x 105 cells/mL in

DMEM with 10% fetal bovine serum. After overnight incubation, medium was
renewed, and HepG2 cells were treated with tempol (0.2- 1 M) or with the
pancaspase inhibitor, Z-VAD-FMK (Z-VAD,10uM). Half an hour later, SLD sulﬁde
(200— 250 uM), hydrogen peroxide (0.2- 2 mM), TNF (200 ng/mL) or their
vehicles were added depending on the purpose of the experiment. The
percentage of LDH released was evaluated at 24 hr as described previously (Zou
et al., 2009a). Caspase 3” activity was evaluated at 6 hr after treatment in

HepGZ cells using a Caspase-Glo 3/7 assay kit (Promega, Madison, WI)

5.3.9 Statistical analyses

Results are expressed as means i S.E.M. One-way or two-way analysis of
variance was applied for data analysis as appropriate, and Student-Newman-
Keuls test was used as a post hoc test to compare means. For all studies, the
criterion for statistical signiﬁcance was P < 0.05. For gene array analysis,
empirical Bayes analysis was used and posterior probability (P1(t)- value) > 0.9

was set as the criterion for signiﬁcance.

154

5.4 Results
5.4.1 Gene expression changes regulated by treatment with SLD, LPS or
SLDILPS

In previous studies, neither LPS nor SLD was hepatotoxic when given alone;
however, cotreatrnent with SLD/LPS caused severe liver injury in rats (Zou et al.,
2009b). SLDILPS-induced liver injury occurred between 4-8 hr after the second
administration of SLD. Genes regulated by SLD/LPS at the onset of liver injury
i.e., 4 hr, might be involved in the pathogenesis. Thus, livers were collected 4 hr
after treatment with SLD and/or LPS or their vehicles, and gene expression was
analyzed. To compare the number of gene expression changes (relative to
VehNeh) caused by treatment with SLDNeh, Veh/LPS or SLD/LPS, a Venn
diagram was generated (Fig. 5.1). A large number of genes (1476) were changed
by LPS administration. In contrast, SLD only caused a small number of genes
expression changes (79). SLD/LPS cotreatrnent led to expression changes in
2040 genes. Not surprisingly, most of the gene expression changes caused by
LPS (1309/1476) were represented in the genes regulated by SLD/LPS
cotreatrnent. However, there were 721 genes regulated by SLD/LPS that were

not affected by either SLD or LPS treatment alone (presented in appendix).

5.4.2 Gene expression changes speciﬁcally regulated by SLDILPS point to
oxidative stress
SLD/LPS was the only treatment that resulted in liver injury (Zou et al.,

2009b). Accordingly, some of the 721 genes selectively regulated by SLD/LPS

155

F151. Venn d
101%. Rats w

tiehlcle (0.5% r
:e second admin

csadministered
fer the second

stression was 8‘
I-‘ite sets Chang
silthehNeh as
iailment, and :
linent groups

1113160 083

Fig. 5.1. Venn diagram of probe sets regulated by SLDNeh, Veh/LPS or
SLDILPS. Rats were treated with two administrations of SLD (50 mglkg, p.o.) or

its vehicle (0.5% methyl cellulose) with a 16 hr interval (n=5). Half an hour before

the second administration of SLD, LPS (8.25X 105 EUIkg, i.v.) or its saline vehicle

was administered via a tail vein. Livers were collected from rats euthanized at 4 hr
after the second administration of SLD. RNA was isolated from liver, and gene
expression was evaluated as described in Materials and Methods. The numbers of
probe sets changed by SLDNeh, Veh/LPS or SLD/LPS cotreatrnent was derived
using VehNeh as baseline. S indicates SLDNeh treatment, L indicates Veh/LPS
treatment, and SL indicates SLD/LPS cotreatrnent. The intersection (A) of
treatment groups represents the gene expression changed after both or all three of

the indicated treatments.

156

are most
were furll
the genes
by SLD/L
ddennhh
metabolis

pathway

5.4.3 Oxi

P101l
treated I
SQHmCar
ht Sugg
cotreatm

Pret
53). The

etheret

5.4.4 En
MnOChm
Sulﬁde 0

“TIM dic

are most likely to be involved in the pathogenesis of liver injury. These genes
were further subjected to Ingenuity Pathway Analysis, which annotated 576 of
the genes, including 83 that were upregulated and 493 that were downregulated
by SLDILPS. A list of toxicity pathways selectively affected by the 576 genes was
determined (Table 5.1). Genes associated with pathway involved in fatty acid
metabolism, LPS/IL—1-mediated inhibition of RXR function, NFkB signaling

pathway and oxidative stress were highly impacted by SLD/LPS cotreatrnent.

5.4.3 Oxidative stress in SLDILPS-cotreated rats

Protein carbonyl concentration was not affected in liver mitochondria of rats
treated with SLD or LPS alone (Fig.5.2). In contrast, SLD/LPS cotreatrnent
signiﬁcantly increased protein carbonyls in mitochondria of livers collected at 8
hr, suggesting that hepatic oxidative stress was associated with SLD/LPS
cotreatrnent.

Pretreatment of rats with either ebselen or DMSO reduced liver injury (Fig.
5.3). That is, ALT activity at 12 hr was signiﬁcantly increased by SLD/LPS, and

either ebselen or DMSO markedly decreased serum ALT activity.

5.4.4 Effect of SLD sulﬁde on mitochondrial membrane potential
Mitochondria isolated from livers of untreated rats were incubated with SLD, SLD
sulﬁde or SLD sulfone. Compared to vehicle treatment, SLD or SLD sulfone up to

1mM did not change the ratio of JC-1 aggregates and monomers, a marker of

158

lable 5.1. F
SLDILPS cotl
SLDILPS. The
Zhways was
Cisewed nurr
Either eXpe.

“59101 ger

Table 5.1. Pathways associated with genes speciﬁcally changed by
SLDILPS cotreatrnent. Expression of 721 genes was changed speciﬁcally by
SLD/LPS. These genes were imported into Ingenuity Pathway Analysis. A list of
pathways was derived and ranked by p value, which indicates the deviation of
observed number of genes for each pathway found in the imported list from the
number expected to occur by chance. Ratio indicates the percentage of the

number of genes affected in a particular pathway.

159

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Toxicity Lists -Log(P) Ratio (%) Genes
ALDH1A1,PECI,CYP4B1,A

Fatty Acid 3 27 11 0 UH,ADH1C,CYP2D6,

Metabolism ' ' CYP2J2, ACAD9,ADHFE1,
HSD17B4,ACSL1,GCDH
PRKACB,GSTM2,ALDH1A

PXR/RXR Activation 2.95 12.7 1,AKT1,PRKACA,IL6,HNF4
A,RXRA
ALDH1 L1 ,SLCO1A2,MAOB

LPS/lL-1 Mediated ,GSTM2,ALDH1A1,MGST2,

Inhibition of RXR 2.88 8.5 E6629219,XPO1,SULT1E1

Function ,LBP,RXRA,ACSL1,SULT1
B1 ,MGST3,ALDH6A1
GSTM2,MGST2,CYP4B1 ,A

Xenobiotic 2 55 9 1 DH1C,

Metabolism ' ' ALDH1 L1 ,CYP206,CYP2J2
,ADHFE1, MGST3
lL1A,GSTM2,ALDH1A1,MG

Aryl Hydrocarbon 2 43 8 6 ST2,MAPK1,CDK4,ALDH1L

Receptor Signaling ' ' 1,NFlB,lL6,RXRA,ALDH6A
1,MGST3
PRKACB,IL1A,NFKBIA,PR

. . KACA,SLCO1A2,LBP,IL6,H

Hepatic Cholestasts 2.18 8.6 NF 4 A,RXRA, CYPBB 1 ,IRAK
2

Mechanism of Gene

Regulation by PRKACB,IL1A,NFKBIA,MA

Peroxisome 1 .66 8.60 PK1 ,PDGFA,ME1,RXRA,H

Proliferators via SD17B4

PPARo

- - PRKACB,TLR2,|L1A,AKT1,
ngB s'gna'mg 1.53 7.6 GHR,NFKBIA,PRKACA,MA
athway P3K8

Cytochrome P450

Panel - Substrate is

a Fatty Acid 1.43 20.0 CYP4B1,CYP2J2

(Human)

Oxidative Stress 1.28 8.93 ‘ng3'VCAM1'GSTM2'ME‘
PRKACB GHR NFKBIA MA

PPARd/RXR ' ' '

Activation 1.26 6.37 PK1,PRKAA2,PRKACA,PL

160

CG1,|L6,ADIPOR2,RXRA

Fig.

Rats
were
isola
diffel

P<0.

Fig. 5.2. Evaluation of protein carbonyl concentration in liver mitochondria.
Rats were treated with SLD, LPS or their vehicles as described in Fig. 5.1. They
were euthanized at 8 hr, and the livers were collected. Liver mitochondria were
isolated, and protein carbonyl concentration was determined. *Signiﬁcantly
different from SLDNeh group. #Signiﬁcantly different from Veh/LPS group.

P<0.05, n=4-8.

S"
o

*#

 

N
o-

 

 

 

.N
o

..s
O

P
01

 

Protein Carbonyl
(nmollmg mitochondrial protein)
0|

P
o

 

 

 

Veh SLD

161

fig. 5.3. Eﬁ
:ehylcellulc
Serum ALT

group. #Sigr

Fig. 5.3. Effect of antioxidants on liver injury. (A) Ebselen, its vehcle (0.5%
methylcellulose) or (8) DMSO was administered to rats treated with SLD/LPS.
Serum ALT activity was evaluated at 12 hr. *Signiﬁcantly different from VehNeh

group. #Signiﬁcantly different from SLD/LPS group. P<0.05, n=3-9.

ALT (UIL)

1400

1200 ‘
1000 ‘

600 -

ALT (UIL)

400 -
200 -

1 200

1000

800-

600‘

 

 

 

 

— VehNehNeh
* E Veh/SLDILPS
— EbselenlSLD/LPS

 

 

  
   

  
   

- VehNeh
1:1 SLDILPS
— DMSO/SLDILPS

 

163

me

del

5.4

He

SL

ad

5y
Th

RC

Ne

9‘1

DE

C)

di

membrane potential (Fig. 5.4). SLD sulﬁde caused a concentration-related

decrease in mitochondrial membrane potential.

5.4.5 Effect of SLD sulﬁde on the production of reactive oxygen species in
HepGZ cells

SLD sulﬁde increased DCF ﬂuorescence in HepGZ cells at 0.5, 1 and 3 hr after
addition (Fig.5.5), indicating that SLD sulﬁde induced ROS production. TNF plays
a critical role in the pathogenesis of SLDILPS-induced liver injury in part by
synergistically killing hepatocytes with SLD sulﬁde (Zou et al., 2009a). However,
TNF alone had no effect on DCF ﬂuorescence, and the effect of SLD sulﬁde on

ROS generation was not inﬂuenced by TNF.

5.4.6 Cytotoxicity of hydrogen peroxide and TNF to HepGZ cells

Neither TNF (200ng/mL) nor hydrogen peroxide (up to 1 mM) was toxic to
HepGZ cells (Fig. 5.6). At 2 mM, hydrogen peroxide caused very modest
cytotoxicity. When HepG2 cells were treated with TNF (200ng/mL) and hydrogen
peroxide (1-2 mM) together, signiﬁcant cytotoxicity occurred, as marked by a
pronounced increase in LDH release. This indicated that TNF enhanced the

cytotoxicity of hydrogen peroxide.

5.4.7 Effect of antioxidant treatment and caspase inhibition on cytotoxicity

SLD sulﬁde (250 uM) killed HepG2 cells (Fig. 5.7). Tempol, which is a superoxide

dismutase mimetic, reduced the cell death caused by SLD sulﬁde. As shown

164

Fig. 5.4. Effect of SLD and its metabolites on mitochondrial membrane
potential. Mitochondria were isolated from the livers of normal rats and
incubated with SLD or its metabolites, SLD sulfone or SLD sulﬁde, for 30 min.
Mitochondiral membrane potential was evaluated. *Signiﬁcantly different from
any other group at the same concentration. #Signiﬁcantly different from vehicle

group. P<0.05, n=3.

 

140 1 -O- SLD
-O- SLD sulfone

120 ‘ + SLD sulﬁde

 

 

 

—L
O
O

  
 

co
0

60-

 

JC-1 aggregates/m onomers
(% control)

 

4O . . . .
0.00 0.25 0.50 0.75 1.00

Concentration (mM)

165

Fig.
spec
in it
spec
eval

the :

Fig. 5.5. Effect of SLD sulﬁde and TNF on production of reactive oxygen
species in Hesz cells. SLD sulﬁde (250 uM) was administered to HepGZ cells
in the presence of TNF (200nglmL) or its medium vehicle. Reactive oxygen
species generation at various times (0, 0.5, 1 and 3 hr) after treatment was
evaluated using DCF ﬂuorescence. *Signiﬁcantly different from VehNeh group at

the same time. P<0.05, n=4.

 

 

 

 

 

 

+ VehNeh
—O— Sulﬁde/veh
+ Veh/T NF
80 w —A— Sulﬁde/1' NF

2:

._ * *

g 75 *

E 70 - * *

g 65 «

5

o 60 ‘

m

2 55 -

3

E 50 -

3 45 .

D

40 . . . .
0 0.5 1 3
Hr after treatment

166

Fig. 5.6. Cytotoxicity induced by hydrogen peroxide and TNF. Hydrogen
peroxide was administered to HepG2 cells in the presence of TNF (200nglmL) or
its medium vehicle. After 24 hr incubation, the percentage of LDH released from
cells was evaluated as a marker of cell injury. *Signiﬁcantly different from 0
concentration hydrogen peroxide group. #Signiﬁcantly different from

corresponding hydrogen peroxide/vehicle group. P<0.05, n=4.

 

 

 

 

 

80- +Veh *#
.5
8
«I 60‘
2
at
h 40
,,\° *# *
E 20-
..l
0

 

‘1

0.0 0.5 1.0 1.5 2.0 2.5
Hydrogen peroxide (mM)

167

Fig. 5
TNF.
admin

percel

diffe re

group

Fig. 5.7. Effect of antioxidant on cytotoxicity induced by SLD sulﬁde and
TNF. Tempol was administered to HepG2 cells half an hour before the
administration of SLD sulfide (250 uM), TNF (200nglmL) or their vehicles. The
percentage of LDH activity released was determined after 24 hr. *Signiﬁcantly
different from SLD sulfide group. #Significantly different from SLD sulﬁde/TNF

group. P<0.05, n=5.

or Q
G O

LDH (% released)
.h
O

 

 

20 1
0
SLD sulﬁde - - - + + + + + +
TNF - - - - - - + + +
Tempol - 0.2 1 - 0.2 1 - 0.2 1

168

previously (Zou et al., 2009a), a nontoxic concentration of TNF (see Fig. 5.6)
signiﬁcantly enhanced the cytotoxicity of SLD sulﬁde. Tempol decreases the

cytotoxicity due to the interaction of SLD sulﬁde and TNF.

5.4.8 Effect of antioxidant treatment on caspase 3” activity induced by SLD
sulﬁde and TNF cotreatrnent

Either SLD sulﬁde or TNF alone at the concentrations used did not increase
caspase 3/7 activity in HepGZ cells (Fig. 5.8). In contrast, coadministration of
SLD sulﬁde and TNF activated caspase 3/7 activity at 6 hr. Pretreatment of
HepGZ cells with antioxidant tempol or a pancaspase inhibitor Z-VAD abolished
the increase in caspase 3/7 activity caused by SLD sulﬁde and TNF cotreatrnent.
Z-VAD failed to protect HepG2 cells from the cytotoxic effect of SLD sulﬁde when
it was given alone but reduced the cytotoxic effect of SLD sulﬁde/T NF

coadministration (Fig. 5.9).

169

Fig. 5.8. Effect of antioxidant on caspase 3” activity. Tempol (200 uM), Z-
VAD (10 uM) or their medium vehicle was administered to HepGZ cells. Half an
hour later, SLD sulﬁde (250 uM) and/or TNF (200 ug/mL) was administered.
Acitivity of caspase 3/7 in HepGZ cells was determined after 6 hr incubation.

*Signiﬁcantly different from any other group. P<0.05, n=4.

 

 

7.
I— 6‘
>A
6315‘
(BC
:2“
€00
09-5 3‘
85
au- 2‘
mv
:3 1-
0‘
SLDsquide - + - + + +
TNF - - + + + +
Tempol - - - - + -
Z-VAD - - - - - 4.

170

Fig. 5.9. Effect of pan-caspase inhibitor on cytotoxicity induced by SLD sulﬁde
and TNF. Z-VAD (10 uM), SLD sulﬁde, TNF or their vehicles were administered to
HepG2 cells as described in Fig.5.8. The percentage of LDH activity released from
HepG2 cells was determined after 24 hr. *Signiﬁcantly different from
corresponding SLD sulﬁde-free group. #Signiﬁcantly different from corresponding

SLD sulﬁde/T NF group in the absence of Z-VAD. P<0.05, n=3.

 

 
   

 

 

 

 

 

-e— VehNeh *

.3 80 , -O— TNFNeh

a: + VehIZ-VAD #

m + TNFIZ-VAD

<0 .

2 60

Q)

" 4o -

°\° *#

V

:l: 20 i

Q E

_I

0 - / L. . .

0 150 200 250

SLD sulﬁde concentration (uM)

171

5.5 Discussion

SLD/LPS cotreatrnent induced liver injury in rats, whereas the doses of
either SLD or LPS employed were not hepatotoxic when given alone. To
investigate the interaction between SLD and LPS, gene array analysis was
performed on livers collected at 4 hr, a time before the onset of liver injury. Gene
expression changes caused by SLD, LPS and SLD/LPS were compared. The
results suggested that SLD alone had only a modest effect on gene expression
(Fig. 5.1). As expected, LPS treatment caused the changes in the expression of
numerous genes. Interestingly, SLD interacted with LPS to cause a large
number of genes to be expressed speciﬁcally after SLD/LPS cotreatrnent.

The pathway most impacted by SLDILPS is fatty acid metabolism (Table
5.1). All the 12 genes in that category are downregulated, and half of them
(ACSL1, AUH, ACAD9, ADHFE1, HSD17B4, GCDH) locate in the mitochondria,
which might indicate an impaired function of mitochondria. Several LPS related
pathways (LPS/lL-1-mediated inhibition of RXR function, NFkB signaling
pathway) are highly impacted by SLD/LPS cotreatrnent. These results suggest
SLD has an effect on the signaling pathway driven by LPS. Ingenuity pathway
analysis indicated that genes related to oxidative stress were inﬂuenced by
cotreatrnent. Two important genes involved in detoxiﬁcation of reactive oxygen
species (glutathione peroxidase 3 and glutathione S-transferase mu 2) were
signiﬁcantly downregulated by SLD/LPS. Glutathione peroxidase partially
determines the susceptibility of cells to oxidative stress (Yang et al., 2006), and

downregulation of glutathione S-transferase mu 2 has been associated with

172

mane

SUQQ
beﬁ>

For
repe
asa
m v
509

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eat
an

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of

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rat

inj

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Ti

increased levels of superoxide anion (Zhou et al., 2008). Accordingly, this result
suggests that the cellular defense system against oxidative stress was impaired
before the onset of liver injury.

Oxidative stress has been associated with numerous models of liver injury.
For example, ROS play a role in liver injury induced by alcohol and ischemia-
reperfusion (Wiseman, 2006; Cederbaum et al., 2009). Hepatotoxic drugs such
as acetaminophen can induce oxidative stress in mouse liver and in hepatocytes
in vitro (Adamson and Harman, 1993; Lores Arnaiz et al., 1995). It has been
suggested that ROS play a role in the idiosyncratic liver injury caused by
NSAle (Boelsterli, 2002). SLD induces oxidative stress in cultured cell lines
(Seo et al., 2007; Park et al., 2008). However, SLD has not been reported to
cause oxidative stress in an animal model. In the model of SLD-LPS interaction,
an increase in protein carbonyl concentration in isolated liver mitochondria was
observed at 4 hr (Fig. 5.2), which suggests oxidative stress was induced in livers
of rats cotreated with SLD/LPS before the onset of liver injury. Ebselen is a
glutathione peroxidase mimic, and DMSO is a scavenger of ROS. Although
DMSO can suppress conversion of the prodrug sulindac to its bioactive sulﬁde
metabolite (Swanson et al., 1983), both of these agents decreased ALT activity in
rats treated with SLD/LPS, suggesting that oxidative stress contributes to liver
injury in this model.

Injured mitochondria can be a major source of ROS arising from leakage of
electrons from the electron transport chain (Zorov et al., 2006; Orrenius, 2007).

There are numerous reports that NSAle lead to mitochondrial dysfunction by

173

acting as mitochondrial uncouplers or causing mitochondrial membrane
permeability transition pore opening (Moreno-Sanchez et al., 1999; Al-Nasser,
2000; Boelsterli, 2002). In isolated rat mitochondria, SLD sulﬁde decreased
mitochondrial membrane potential, whereas SLD or SLD sulfone showed no
effect (Fig. 5.4). Consistent with this result, a previous study using the JC-1
assay also suggested that SLD sulﬁde may lead to dissipation of mitochondrial
membrane potential in HepG2 cells (Leite et al., 2006). Decreased ATP
synthesis can be a direct consequence of decreased mitochondrial membrane
potential, which might explain why SLD sulﬁde is more hepatotoxic compared to
SLD or SLD sulfone (Zou et al., 2009a).

SLD sulﬁde also induced ROS generation in HepGZ cells (Fig. 5.5).
However, exposure to SLD by itself failed to increase liver protein carbonyl
concentration in rats; coexposure to LPS was necessary for this effect. Hypoxia,
which occurs in the livers of rats cotreated with SLD/LPS as a result of
hemostasis (Zou et al., 2009b), is a potential contributor of oxidative stress in
vivo (Arteel et al., 1999). Moreover, PMNs accumulate and become activated in
the livers of rats cotreated with SLD/LPS and contribute to liver injury at least in
part by releasing cytotoxic proteases. During PMN activation that results in
protease release, , NADPH oxidase assembles on the PMN plasma membrane
and ROS are generated (Dahlgren and Karlsson, 1999). Thus, activated PMNs
might contribute to ROS generation and consequent oxidative stress in this

model.

174

A previous study revealed that TNF potentiated the cytotoxicity of SLD
sulﬁde in HepG2 cells and primary hepatocytes in vitro (Zou et al., 2009a).
However, TNF had no effect on ROS generation induced by SLD sulﬁde in
HepGZ cells (Fig. 5.5), indicating that TNF does not contribute to cell death by
enhancing oxidative stress. Interestingly, TNF enhanced the cytotoxicity of
hydrogen peroxide in HepGZ cells (Fig. 5.6). This is consistent with previous
observations that hydrogen peroxide and TNF synergistically kill primary mouse
and rat hepatocytes in vitro (lmanishi et al., 1997; Han et al., 2006). These
results might explain the synergistic killing by SLD sulﬁde and TNF; that is,
although TNF does not enhance SLD sulﬁde-induced oxidative stress, it does
render cells sensitive to ROS-mediated cell killing. This is supported by the
observation that antioxidant tempol decreased the cytotoxicity of SLD sulﬁde and
signiﬁcantly reduced the cytotoxic interaction between TNF and SLD (Fig. 5.7). In
vivo, it seems unlikely that SLD sulﬁde concentrations become great enough to
cause ROS-mediated liver injury; rather, TNF production caused by LPS
coadministration renders the liver more sensitive to otherwise noninjurious ROS
generation. The gene expression results discussed above suggest that
compromised antioxidant protective mechanisms could play a role in this
heightened sensitivity.

JNK is a common target of TNF and ROS and regulates apoptosis (Kanda
and Miura, 2004; Schwabe and Brenner, 2006). TNF-induced JNK activation
leads to caspase activation, which in turn leads to liver injury (Wang et al., 2006).

Activation of caspase 3f7 was only observed in HepG2 cells cotreated with SLD

175

sulﬁde and TNF (Fig. 5.8), whereas TNF or toxic concentration of SLD sulﬁde
alone had no effect. This indicates that the cytotoxicity of SLD sulﬁde alone in
vitro depended on ROS but not on caspase activation. However, both ROS and
caspase activation were critical in the synergistic killing induced by TNF and SLD
sulﬁde (Figs. 5.7 and 5.9). The observation that tempol reduced the activation of
caspase 3l7 (Fig. 5.8) suggests ROS contribute to caspase activation induced by
SLD sulﬁde/T NF interaction.

In summary, this study further revealed the mechanisms of SLD/LPS-
induced liver injury in rats. According to the comparison of gene expression in the
liver of rats treated with SLD and/or LPS or their vehicles, genes associated with
oxidative stress were selectively regulated by SLD/LPS. SLD/LPS cotreatment
led to an increase in protein carbonyl in the mitochondria of rat livers, and
antioxidants protected against liver injury, which suggests oxidative stress is
involved in SLDILPS-induced liver injury. SLD sulﬁde exerts its cytotoxicity
through decreasing mitochondrial membrane potential and increasing the
production of ROS in vitro. The synergistical interaction of SLD sulﬁde and TNF
to kill HepG2 cells is dependent on the oxidative stress induced by SLD sulﬁde.
Under oxidative stress TNF leads to the activation of caspase 3/7, which

contributes to the cytotoxicity of SLD sulﬁde/T NF interaction.

176

CHAPTER 6

Summary and conclusions

177

6.1 Summary and conclusions

The goal of this project was to test the hypothesis that LPS precipitates SLD-
induced liver injury in rats. A model of SLDILPS-induced liver injury was
developed, and the mechanisms were further investigated. These studies provide
additional evidence that supports the hypothesis that inﬂammation is a
susceptibility factor for idiosyncratic drug hepatotoxicity. This project also
enhances our understanding of the mechanisms of drug-LPS interaction.

First, to develop a liver injury model of SLD and LPS interaction, rats were
treated with two administrations of SLD with a 16 hr interval. Two administrations
of SLD were chosen because treatment with one administration of SLD with LPS
was not sufﬁcient to induce liver injury in rats. Half an hour before the second
administration of SLD, a nonhepatotoxic dose of LPS was administered to rats
via a tail vein. The ALT activity in rat serum increased at 8 hr and was signiﬁcant
at 12 hr (Fig. 2.3). Midzonal necrosis was also observed only in the livers of rats
cotreated with SLD/LPS (Fig. 2.5). These results suggest that SLD interacts with
the inﬂammatory stress induced by LPS, which leads to liver injury.
Subsequently, several mediators potentially involved in the pathogenesis were
investigated.

Hemostatic factors, including PAH and thrombin, were elevated by LPS in
rat plasma (Fig. 2.6) and were enhanced by SLD cotreatment. As a result,
signiﬁcant ﬁbrin deposition was observed in liver sinusoids of rats cotreated with
SLDILPS compared to those treated with SLD or LPS alone (Fig. 2.9).

Presumably resulting from the impaired blood ﬂow, hypoxia occurred in the liver

178

of rats treated with SLD/LPS. Anticoagulant heparin, which attenuated ﬁbrin
deposition and hypoxia in the liver, protected against liver injury induced by
SLD/LPS (Fig. 2.10 and 2.11). These results suggest that the hemostatic system
and hypoxia resulting from ﬁbrin deposition are critical to SLDILPS-induced liver
injury.

TNF is another mediator involved in the pathogenesis of liver injury induced
by inﬂammation or inﬂammation-xenobiotic interaction. LPS led to an increase in
TNF concentration in serum, which was enhanced by SLD cotreatrnent (Fig. 3.1).
TNF neutralization using etanercept decreased serum ALT activity in rats
cotreated with SLD/LPS, which shows that TNF plays an important role in the
pathogenesis (Fig. 3.2).

The concentrations of SLD, SLD sulfone and SLD sulﬁde in plasma and
livers of rats were decreased by LPS cotreatrnent (Fig. 3.3 and 3.4). In contrast,
their concentrations in rat GI tract and feces were increased by LPS (Fig. 3.5).
SLD is bioactivated to SLD sulﬁde, and this metabolite was much more cytotoxic
to HepG2 cells and primary rat hepatocytes than SLD in vitro (Fig. 3.6).
However, in rats the amount of SLD sulﬁde produced was insufﬁcient to cause
liver injury by itself. Presumably, it is this bioactivated metabolite that acts
synergistically with LPS to precipitate liver injury in vivo. In vitro, SLD sulﬁde
synergistically interacted with TNF to kill cells, whereas SLD or SLD sulfone had
no interaction with TNF (Fig. 3.7 and 3.8).

PMNs are a critical contributor to liver injury in other drug/LPS interaction

models. LPS led to PMN accumulation in the liver, and SLD enhanced the

179

accumulation of PMNs induced by LPS (Fig. 4.1). However, HOCI-protein
adducts were increased only in the livers of rats treated with SLD/LPS,
suggesting that SLD/LPS cotreatrnent causes PMN activation in the liver. Either
anti-PMN serum or the PMN protease inhibitor, eglin C, attenuated liver injury,
which suggests that proteases released from activated PMNs participate in the
pathogenesis (Fig. 4.3 and 4.4). Previous studies also showed that proteases
synergistically interacted with hypoxia to kill hepatocytes (Luyendyk et al., 2005).
This same interaction might contribute to SLDILPS-induced liver injury. Several
mediators are involved in PMN activation which is a prerequisite for protease
release. Inhibition of TNF or PAI-1, both of which are important mediators in liver
injury induced by SLDILPS, decreased HOCl-protein adducts but had no effect
on PMN numbers in the liver (Fig. 4.5 and 4.6). Thus, PMN activation but not
accumulation is dependent on TNF and PAl-1. Neutralization of TNF had no
effect on PAI-1 production, suggesting an independent relation between TNF and
PAl-1 (Fig. 4.7).

SLD/LPS cotreatrnent produced a speciﬁc gene expression proﬁle in the
liver compared to treatments with SLD alone or LPS alone. Analysis of the genes
speciﬁcally changed by SLD/LPS at 4 hr suggested that a pathway associated
with oxidative stress is inﬂuenced before the onset of liver injury (Table 5.1).
Antioxidants protected against liver injury induced by SLD/LPS in rats and
reduced cytotoxicity caused by SLD sulﬁde and TNF interaction in vitro. These
results suggest oxidative stress is also involved in the pathogenesis of SLD/LPS-

induced liver injury. Moreover, ROS not only kills hepatocytes itself but also

180

interacts with other mediators. A study in vitro showed that TNF enhanced the
cytotoxicity of hydrogen peroxide in HepG2 cells (Fig. 5.6).

Mechanisms of SLDILPS-induced liver injury are summarized in Fig. 6.1.
TNF, the hemostatic system, hypoxia, PMNs as well as reactive oxygen species
play critical roles in the pathogenesis of SLDILPS-induced liver injury. SLD
enhances the activation of hemostatic factors, including thrombin and PAl-1,
induced by LPS. An activated hemostatic system leads to ﬁbrin clot formation in
liver sinusoids. As a result, the liver becomes hypoxic which renders hepatocytes
susceptible to injury. SLD also enhances the elevation in TNF induced by LPS.
LPS decreased the bioavailability of SLD in rats, but the liver is nevertheless
apparently able to produce enough SLD sulﬁde to precipitate a toxic interaction
with LPS. SLD sulﬁde synergistically kills hepatocytes along with TNF and
hypoxia in LPS-cotreated rats. PMNs are another critical player in SLDILPS-
induced liver injury. PMN accumulation in the liver was primarily induced by LPS,
and this effect was enhanced by SLD. Activation of PMNs was only observed in
livers of rats cotreated with SLDILPS. TNF and PAI-1 contribute to PMN
activation independently. Activated PMNs release proteases which induce liver

injury by itself or by interacting with hypoxia.

181

Fig. 6.1. Proposed pathway in the pathogenesis of SLDILPS-induced liver
injury. See text for details.

 

 
 
       
  
 
 

W
PMN _. PMN _. Proteases
accumulation activation

 

 

 

 

Thrombin——-> Fibrin ———-e Hypoxia

PAI-1

6.2 Comm

  
  
   

inflammatiO
So far.
hypothesis
injury induc
humans. Th
(TVX), suli
characteristi'
to various e:
PreVIOL
inﬂammage
been evalue
0f gram-n1
lipoteichoic
routes of d
liver injury
the Ollly m
to indUCe ;
a‘dminlstra
RAN,
investigate
acilVation

 

6.2 Commonality and differences among liver injury models of drug-
inﬂammation interaction

So far, the evidence in animal models supporting the inﬂammatory stress
hypothesis has been accumulating. In animals, inﬂammation precipitates liver
injury induced by several drugs associated with idiosyncratic liver injury in'
humans. These drugs include ranitidine (RAN), diclofenac (DCLF), trovaﬂoxacin
(TVX), sulindac (SLD), halothane (HAL) and chlorpromazine (CPZ). The
characteristics and mechanisms involved in the pathogenesis have been studied
to various extents, and the results are summarized in table 6.1.

Previous studies suggest that inﬂammation induced by different kinds of
inﬂammagens precipitates drug-induced liver injury. The inﬂammagens that have
been evaluated so far in drug interaction models include the cell wall components
of gram-negative bacteria (LPS), Gram-positive stimuli (a peptidoglycan-
lipoteichoic acid (PGN-LTA) mixture) and the viral RNA mimetic (polyl:C). The
routes of drug administration are different among the models, which suggest that
liver injury is not dependent on a speciﬁc route of drug administration. SLD is
the only model in which the drug was administered twice; one dose of SLD failed
to induce a signiﬁcant liver injury in rats. Times between drug and inﬂammagen
administration needed to elicit a toxic response vary among different models.

RAN, TVX and SLD models are three that have been relatively well
investigated using LPS as an inﬂammatory stimulus. Enhanced TNF production,
activation of the hemostatic system and PMN accumulation in liver have been

observed in these models. Neutralization or inhibition studies showed that TNF,

183

Table 6.1. Characteristics and underlying mechanisms of idiosyncratic liver
injury models of drug-inﬂammation interaction. Y means the phenomenon or
mechanism is observed in the model. N means the listed item is not true in the
model. A blank entry means it has not been tested or reported. *indicates the

inﬂammagen used in the mechanism studies. (See text for references)

184

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RAN DCLF TVX SLD CPZ HAL
LPS“ or Polyl:C*
Inﬂammagen LPS LPS P GN+LT A LPS LPS or LPS
Administration route of the i v p o i p p o i p i p
drug . . . . . . . . . . . .
””"be' °f 9mg 1 1 1 2 1 1
administrations
Time (hr) drug was given -15.5
- 2 —6
relative to LPS 2 2 3 and 0.5
Hr after the drug
12 ft
administration when liver 6 6 12 a er 24 15
. . 2nd SLD
Injury was observed
Enhanced TNF production Y Y Y Y
TNF Involved In Y Y Y Y
pathogenesrs
Hemostatic system activated Y Y Y
Fibrin deposition and
. . Y Y Y
hypoxra involved
PMN accumulation Y Y Y Y Y
PMN activation Y Y
PMNs involved In the Y Y Y Y
pathogenesrs
IFN / lL-18 dependent Y
ROS involved Y
Hemostasis affected by TNF Y Y N
PMN accumulation
N
increased by TNF N N
PMN accumulation affected
. N
by hemostasis
PAl-1 increased by TNF Y Y N
PMNs activated by PAH and Y Y
TNF
Kupffer cells and NK cells Y

involved

 

 

 

 

 

 

 

185

 

hypoxia and PMNs are critical mediators in the pathogenesis of liver injury
induced by the interaction with LPS for all three of these drugs. Their roles in
DCLF, HAL and CPZ models need to be tested. These results suggest that TNF,
hypoxia and PMNs are important players shared by many liver injury models of
drug-inﬂammation interaction. These mediators are not independent but interact
with each other. However, their interactions differ among models.

Some mediators have been found to be critical in the pathogenesis in a
single model but have not been tested in other models. For example, lFNy and
lL-18 are both involved in TVX/LPS-induced liver injury. Oxidative stress proved
to play a role in SLDILPS-induced liver injury. The roles of these mediators in
other models need to be investigated.

In summary, evidence is accumulating to support the hypothesis that
inﬂammation precipitates or enhances drug-induced liver injury. TNF, the
hemostatic system and PMNs are involved in the pathogenesis of all of the liver
injury models of drug-inﬂammation interaction in which their roles have been
tested. The roles of oxidative stress or proinﬂammatory cytokines (lFNy and IL-

18) and other potential mediators remain to be determined in other models.

186

6.3 Potential future studies

This project generally clariﬁed several important mediators involved in the
liver injury induced by SLD/LPS coexposure. However, the mechanisms of
SLD/LPS interaction remain incompletely understood. Potential directions for
future studies of this idiosyncratic liver injury model are discussed below.

Although it was observed that SLD enhances the appearance of TNF and
hemostatic factors, the mechanisms have not been clariﬁed. SLD has been
reported to be toxic to the GI tract and to cause ulcers. A possible explanation for
the effect of SLD on TNF, thrombin and PAI-1 is that SLD causes bacterial
translocation to the liver resulting from gastrointestinal toxicity of SLD. In concert
with this hypothesis, diclofenac at large dose exerts hepatotoxiticy to rats by
causing bacterial translocation from the GI tract. Another possible mechanism is
that SLD directly acts on the TLR4 signaling pathway. For example, ranitidine
speciﬁcally enhanced the activation of p38, which led to enhanced TACE
activation and TNF production. Therefore, the activity of signaling proteins in
TLR4 pathway is worth evaluation. Activation of TACE is a potential target of
SLD.

The cellular sources of several mediators have not been identiﬁed. Kupffer
cells are a major source of TNF in many inﬂammatory models. However, it is not
known if they are the only source of TNF in the SLD/LPS model; hepatocytes and
endothelial cells can also contribute to TNF production (Zhaowei et al., 2007).
Kupffer cell depletion is a feasible approach to investigate the role of these cells

in this model. The mechanisms by which the ROS are produced have not been

187

identiﬁed either. PMNs, Kupffer cells, hypoxia and TNF might all contribute to
generation of ROS in the SLD/LPS model. Their roles can be investigated
through their inhibition or depletion.

The relation and interaction among these critical mediators are not fully
understood. According to results in the ranitidine model, PMN proteases
exacerbate ﬁbrin deposition by upregulating PAl-1. ROS might contribute to the
activation of PMNs (Jaeschke, 2006). In the TVX/LPS model, PAl-1 contributes
to the production of proinﬂammatory cytokines. These interactions could be
investigated in the SLD/LPS model.

Despite of the discovery of various mediators including TNF, hypoxia, PMNs
and ROS, there could be some participants yet to be discovered. IFNy, which
was upregulated in plasma by SLD/LPS and plays a critical role in the TVX/LPS

model, is a potential candidate.

188

APPENDICES
Genes selectively regulated by SLDILPS cotretrnent. The number of gene
expression changes (relative to VehNeh) caused by treatment with SLDNeh,
Veh/LPS or SLDILPS were depicted using Venn diagram (Fig. 5.1). According to
the diagram, there were 721 probe sets regulated by SLD/LPS that were not
affected by either SLD or LPS treatment alone. The genes in this group were

listed as below.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ID :3: Symbol ID :23: Symbol
1 367589_at -2.674 A002 1 376009_at -2. 121 CTAGE5
1367614 J1 3.064 ANXA1 1376094_at -1.901 HINT3
1367636_at -1.696 IGF2R 1376105_at 4.540 COL14A1
1367672_at -1.542 HSD17B4 1376321_at 1.883 FAM38A
1367673_at -3.673 SELEN 8P1 1376337_at -1 .667 SMARCA2
1367695_at -1 .708 QDPR 1376569_at 1 .774 KLF2
1367721_at 1.563 3004 1376692_at 4.514 HIPK2
1367729_at -1 .814 OAT 1376706_at -2.293 TMEM47
1367750_at -2.102 PRPSAP1 1376715_at -1.671 CBARA1
1367771_at -2.808 TSC2203 1376727_at -1 .663 YIPF4
1367807_at -2.164 PLODl 1376758_at -2.106 ING1
136781 §_at -1.682 SLCSA6 1376771_at -1.797 PPM1 L
136781Lat -1.884 HDGF 1376796_at -1.664 RAB14
1367818_at -1.716 0003 1376862_at -2.386 UBE4B
1367857_at -4.297 FADS1 1376930_at -1.614 MRPL51
1 367869! at -2.537 OXR1 1377049_at -1.555 PNPLA7
1367874_at 3.210 RHOQ 1377060Aat -1.862 MCCC2

 

 

189

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1367889_at -1.820 CAMK1 1377166_at -1 .522 ALSZ
1367896!at -2.700 CA3 1377209_at -1 .619 KLH L25
1367933_at -1 .658 AMD1 1377307_at 2.444 FAM89A
1367940_at 2.018 CXCR7 137760Lat -2.121 SNX24
1367998_at 2.628 SLPI 1377654_at -1 .605 FAM3A
1368021_at -1.676 ADH1 C 1377657_at -1.663 IBTK
1368057_at -2.056 ABCD3 1377745_at -1.751 LRRC40
1368067_gt -1.776 ZNF148 1377758_at -2.274 HSD17B13
136807 Lat -1 .775 MOSC2 1377810_at -1 .593 RALGPS2
1368085_at -1 .589 GCHFR 1377995_gt -1 .860 ITFGS
1368091_at -3.031 OPLAH 1378027_at -2.013 PVRL3
1368096_at -1 .693 RAB7L1 1378146_at -1 .741 T801 024
1368115_at -2.670 CLDN3 1 3781 8; at -1.556 NXT2
1368117_at -1.685 GPHN 1378394_at -1.819 MPPE1
1368122_at -2.453 RNF103 1378842_at -2.071 GABARAPL1
1 3681 31; at -1.598 MPDZ 1379044_at -1.894 LARP2
1368144_at 2.208 RG82 1379101!at -1.549 DHX36
1368183_at -1.787 PLCG1 1379315_at -1.664 RASSF7
1368215_at -2.300 TPP1 1379353_at -1.737 AASDHPPT
1368265!at -1 .802 CYP2T4 1 3793754 at 1 .601 PDGFA
1368272_at -3.178 GOT1 1379441_at -2.343 RNF160
1368277_at -2.073 PPP3CA 1379456_at ~2.233 MCART2

1 368378! at -1.699 ALDH1 L1 1379499_at 1 .965 LTB
1368387_at -1.990 BDH1 1379525_at -1.780 CRLS1
1368427_at -1.577 AKAP1 1 1379578_at -1.807 ZBTB20
1368435_at -4.049 CYP8B1 1379606__at -3.277 RAB30
1368437_at 2.305 CA4 1379645_at -1.859 PBRM1

 

190

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1368446_at -2.661 SPINK1 1379784_at -1.796 PEX7
1368453Aat -2.931 FADSZ 1379794_at 2.159 GZMB
1368474_at 1.989 VCAM1 1379803_at 1 .675 LMO4
1368482_at 1.829 BCL2A1 1379850_at -2 . 093 PSMC6
1368509_at -1.541 3882 1379901_at -1.582 TBC1 D17
1368514_at -1.993 MAOB 1379909_at -1.968 GKAP1
1368519_at 4.112 SERPINE1 1379935_at 2.280 CCL7
1368545_at 1.614 CFLAR 1380063_at 3.086 CH25H
1368592_at 1 .688 1L1 A 1380229_at 1 .790 MAFF
1368657_at 3.211 MMP3 1381012_at -1.547 SERPINF1
1368702_at 1 .585 PAWR 1 381 193_at -1 .936 LPGAT1
136871 1_at -2.039 FOXA2 1381768_at -1 .523 MTHFS
1368733_at -1.784 SULT1 E1 1381973_at -1.569 SLC25A30
1368814_at -1 .592 ALDH6A1 1 382024_at 1 .906 DNAJ 36
13688603t 3.143 PHLDA1 1382101_at -1.722 HSZST1
1368862_at -1 .755 AKT1 1382150_at -1 .581 SLC25A46
1368869_at 3.134 AKAP12 1382200_at -2.381 CENPV
1368914_at 2.918 RUNX1 1382216_§t -1.574 GEMIN6
1368924_at -3.846 GHR 1 382274; at 1.703 RARRES1
1368960_at -1 .855 LGALSB 1382285_at -1 .656 NAGA
1369063_at -2.462 AN P32A 1382 325_at -1 .750 GCAT
1369069_at -2.219 AKAP1 1382332_at -1 .736 STAG2
1369070_at -1.549 PEX12 1382371_at -1 .538 DRAMZ
1369078_at -1.734 MAPK1 1382402_at -1 .961 ULK1
1369150_at -2.259 PDK4 1382496_at -1 .624 HN F4A
1369169_at -1.577 SLC23A1 1382602_at -1.696 UBR3
1369191_at 2.780 1L6 1382843_at -1.907 SGPL1

 

191

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1369268_at 3.449 ATF3 1 382 935_at -2 . 046 KIAA0141
1369278_at -1.660 GNA12 1383004_at -1.550 AHCYL1
1369393_at 2.703 MAP3 K8 1383037_at -1 .693 POLDI P2
1369453_at -1.737 EPN1 1383050_at -2.286 CENPV
1369492_at -1.816 AADAC 1383118_at -1.901 TMEM209
1369654_at -2.490 PRKAA2 1383155_at -1.610 FAM1 17B
1369785_at -1.900 PPAT 1383159_at -1 .936 TOM1 L2
1369837_at -1.685 GULO 1383282_at -1 .689 THAP1 1
1369922_at -1.733 PLBDZ 1383358_at -2.712 AKAP1
1369926_at -1 .507 GPX3 1383359_at -1 .628 LNX2
1369931_at 1 .577 PKM2 1383395_at -1 .878 AGMAT
136993643t 1.687 CALM1 1383462_at -1 .678 RNF160

1 369942_at -1 .539 ACTN4 1 383463_at -1 .565 ZFP91
1369950_at -2.125 CDK4 1 383474_at 2.182 IRAK2
1369956_at -1.764 IFNGR1 1383732_at -3.490 BC021614
1369961Lat -1.593 FXYD1 1383863_at 1.529 LM02
1369982_at -1 .524 AP2A2 1383933_at -1 .607 KIAA0564
1369989_at -1.978 PNPO 1383960_at -1.712 PEX16
136999Lat -1 .577 DVL1 1384029_at -1 .646 XPA
1370029_at -1.643 CTBP1 1384131_at -2. 195 ATL2
1370036_at -2.348 SUOX 1384205_at -2. 590 NGLY1
1370047_at -1.679 EN PP1 1384254_at 2.056 OTUD1
1370067_at -2.173 ME1 1384293_at -1.962 CZOORF191
13701 12_at -1.865 PTEN 1384383_at -1 .700 AGPAT6
1370121_at -1.711 ADD1 1384628_at -1.722 IYD
137017Lat 2.146 PPP1R15A 1384903_at -2.443 GPT2
1370177_at 3. 345 PVR 1385160_at -1 .621 STAB2

 

192

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1370190Lat 1.529 H3F3C 1385266!at -1.590 NLK
1370200_at -1.557 GLUD1 138556Lat 1.767 AKAP2
1370236_at -1.525 PPT1 1385690_at -1.948 MUT
1370249_at 2.047 TSPO 1385845_at -1.753 D730039F16R|K
1370285_at -1.690 CALCOCO1 1385889_at -1 .927 C20RF64
1370319_at -2.026 PPIF 1386280_at -1.574 METTL7B
1370322_at -1 .896 STK16 1 386764_at 1 .946 AKAP2
1370329_at -1 .957 CYPZDG 1386895_at -2. 147 MAGED1
1370334_at -1.877 PLEKHB1 138697Lat -3.225 CA3
1370359_at -1 .809 AMY2A 1387006_at -2.31 5 EG629219
1370360_at -1 .738 C3ORF34 1387018_at -1 .566 SORBSZ
1370375_at -2.340 GLSZ 138702Lat -1 .921 ALDH 1A1
1 370399_at -2.607 CYP4B1 1 387023_at -3. 142 GSTM2
1370501_at -1.843 UBEZG1 1387093_at -1.683 SLCO1 A2
1370516_at 1 .679 SLC15A3 1387094_at -2.694 SLCO1 A2
1370548_at -1.807 SLC16A10 1387186_at -1.732 RABQA
137080843t -2.317 CY85R3 1387188_at -1 .866 SLC17A1
1370814_at -1.501 DH RS4 1387190_at -1.542 DGKA
1370818_at -1.892 DECR2 1387209_at -2.259 SEC168
1370848_at 1.688 SLC2A1 1387214_at -1.997 E822
1370875_at 2.092 EZR 1387219_at 1 .927 ADM
1370881_at -1.504 TST 1387244_at -2.280 CGRRF1
1370891_at 1.770 CD48 1387296_at -2. 141 CYP2J2
1370905_at -1.728 DOCK9 1387314_at -1.763 SULT1B1
1370939!at -1.571 ACSL1 138737Lat -2.454 KHK
1371034_at -2.912 ONECUT1 138756Lat -1.863 SLCO1A1
137107Lat 1.819 ZBP1 1387652_at -1.638 IDE

 

193

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1371317_at 4.606 L081 1367725_at -2041 GULO
1371322_at 1.956 LAMC1 1387782_at 4.676 DYNLL2
1371362_at 4.700 . KDELR3 1387786_at -1.610 MTPN
1371385_at 4.651 PSMG1 1387790_at -2.156 PAICS
1371388_at 4.576 PDHB 1387821_at 4.570 RAB3IP
1371400_at 2.932 THRSP 1387852_at 2.101 THRSP
1371404_at 4.935 EIF4B 1387857_at 4.707 srx7
1371405_at 4.671 CZORF64 1367661_at -1.693 AES
1371432_at 4.544 VAT1 1387864_at -1.936 KIDIN8220
1371443_at 4.626 C1ORF174 1387865_at 4.630 our
1371445_at 1.935 LRRC59 1387868_at 1.863 LBP
1371447_at 2.064 PLACB 1387900_at 4.926 CDIPT
1371460_at 4.646 C12ORF62 1387901_at -1.746 PTPRS
1371461_at 4.602 FAM548 1387920_at 4.505 MANZC1
1371464_at 4.542 ZFAND6 1387921_at 4.524 2031414
1371471_at 4.626 GLTSCR2 1387948_at 4.521 ICK
1371463_at 4.669 NNT 1387959_at -1.989 ASPG
1371493_at -2.337 AP2A2 1388136_at 4.522 TIMM9
1371525_at 4.666 SLC12A7 1388150_at 4.707 XPO1
1371527!at 1.766 EMP1 1388155_at 1.510 KRT18
1371531_at 4.564 LOC678880 1388167_at 4.991 NFIB
1371553_at 4.955 MRPL36 1388300_at -2.351 MGST3
1371560_at 4.656 IRF3 1388324_at -2.044 NIT1
1371571; at -2.016 MRPS36 1388338_Lat -2.086 PPP2R4
1371578_at 4.993 PRKACA 1388364 _at 4.576 NDUFS3
1371563_at 3.100 RBM3 1388382_at -1.605 C1ORF43
1371611_at 4.713 EXT2 1388404_at 4.673 GM12751

 

194

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1371620_at 4.726 PRELID1 1388410_at 4.733 ucpz
1371634_at -2.053 TMEM126A 1388430_at 4.915 PTOV1
1371637_at 4.660 HP1BP3 1388441_at 4.920 LOC689574
1371668_at -3.060 RXRA 136647131 4.574 TCP11L2
1371697_at 4.641 PNPLA2 1388474_at 4.569 UBE2I
137171631 4.507 SMARCCZ 136646631 4.596 DPP8
137171731 4.751 MFN1 1388497_at 4.599 ACOT13
137174731 -2.748 PPDPF 136650331 -2.238 EID1
137179931 4.909 GAA 136650731 1.727 EIF6
1371835_at -2.283 PRKACB 136651631 4.902 FBXW5
137165331 4.631 MRPL42 1366533_a1 -2.024 crosm
137192031 4.604 POLDIP2 1388540_at 4.744 MAZ
1371948_at 4.972 PIGP 1388549_at 4.691 NCOA4
1371956_at -1.968 LOC683077 136656731 2072 THUMPD1
1371963_at 4.760 PCCA 1388617_at 4.766 BPHL
1371964_at -2291 GRSF1 1366636_a1 4.554 RNF167
1371982_at -2.173 DPY30 1388658_at 4.970 SURF2
1371983_at 4.763 JOSD1 136666631 1.796 ENC1
137199331 4.516 CPNE3 1388672_at -2.232 ZCCHC24
137207131 4.533 c0320 1388680_at 4.563 C1GALT1C1
137207331 4 .625 GATADZA 1388685_at 4.905 DGCR2
1372074_ at 4.752 NUDT3 1388725_at 4.622 LEPROT
1372085_at 4.959 ATL2 136673231 4.549 SLC35F5
137209931 4.663 FAM21A 136675531 4.966 SEC23A
137210231 4.667 C200RF191 136676331 -1.596 HMGB1
137212431 2491 EIF4B 1388809_at 4.757 SMPDL3A
137214931 4.664 AUH 136661531 4.933 SAPS1

 

195

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1372158_at -1.531 MACROD1 1388817_at -1 .578 FAM63A
1372214_at -1.645 MRPS33 1388823_at -2.642 RABSB
1372217_at -1.727 TMEM199 1388831_at -1.735 SLCQA3R2
1372281_at -1.768 LYPLAL1 1388833Jt -1.548 POLE3
1372284_at -2.080 TRAPPC3 1388877_at -1 .718 MRPSS
1372286_at -1.558 TSPAN6 1388908_at -1.755 PECI
1372295_at -1.635 NARF 1388913_§t -1.978 PPAP2C
1372306_at -1.987 ETHE1 1388965_at -2.086 PPP2R5E
1372310_at -1 .667 |SOC1 1388976_at -1 .507 BOLA3
137237331 -1.894 CMBL 1388995_at -2.779 RNF14
1372389_at 2.456 IER2 1389072_at -1.532 MTMR4
1372394_at -2.382 HECTD1 1389128!at -2 .226 WDFY3
1372395_at -1 .632 MARCH6 1 389139Aat -2.060 TTC1 5
1372408_at -1 .731 GGA2 1389146_at -1 .547 FAM1 07B
1372409_at 1 .609 MADZL1 BP 1389167Aat -1 .643 MAPKAP1
1372421_at -1.772 AGA 1389176Aat -1.737 |NPP5F
1372426_at -2.115 ADAMTSL4 1389196_at -1.944 2310039H08R|K
1372459_at 1 .633 VASP 1389199_at -1 .647 CBORF58
1372463_at -2.216 FCH02 1389215_at -1.609 SEPHS1
1372469_at ~2.001 LOC68631 0 1 389253_at -2.605 VNN1
1372475_at -1.749 PINK1 1389329_at -1.533 LGALSB
1372507_at -1 .606 TCTA 1389338_at ~2.228 TMEM126B
137252Lat -2.098 FLCN 1389339_at -1.634 ARSA
1372562_at -1.682 C80RF82 1389351_at 1.854 LRRFIP1
1372571_at -1.793 MARCH2 1389358_at -1.931 LPGAT1
1372597_at -1.671 MRPL14 1389361L§t -1.829 ADI1
1372599_at -1.536 MGST2 1389386_at -1.585 CSORF23

 

196

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1372612_at -2.414 DYNLL2 1389407_at -1 .626 DH RS1
1372624_at -1.590 AN06 1389538_at 2.149 NFKBIA
1372630_at -2.108 RADZ3A 1 389540_at -1 .747 LOC686590
1372650_at -1.765 DNMBP 1389548_at -1.613 ADHFE1
1372663_at -1 .616 PTDSSZ 1389567_at -2 . 071 SCAP

1 37272Q_ at -1.530 BTBD1 1389676_at -1.612 CCDC101

1 372723_ at -1 .588 IP09 1389738_at -1 .682 UNG
1372729_at 1 .727 PROCR 1389844_at -2.556 FKBP4
1372814_at -2.170 SFT2DZ 1389906_at -1 .775 FDFT1
1372819_at -1 .815 COG4 1389918_at -1 .726 LOC290704
1372828_at -1.733 MSRBZ 1389998_at -1 .543 NR2F2
1372835_at 1 .692 RHOJ 1390102_at -1 .910 DI RC2
1372845_at -1 .509 RPP21 1390189_at -1 .506 ZN F277
1372854_at -1 .653 TTC17 1390312_at 2.041 SAM 09L
1372860_at -1.576 LHPP 1390374_at -1 .650 FGFRL1
1372871_at -1 .877 C2ORF24 1390445_at -1 .669 LOC6881 33
1372888_at -1 .929 UBE4A 1390478_at -1 .942 ORC4
1372907_at -1 .912 ATP6VOE2 1 390526_at -1 .546 KLH L9
1372946_at 1 .654 CXORF4OA 1390591_at -1 .582 SLC1 7A3
1372947_at -1 .997 PLS3 1390699_at -1 .625 KIAA2026
1372996_at -2.171 L00684270 1390717_at -2.121 CRLS1
1373036_at -1.877 RGD1561455 1390819_at -1.696 TEF

1 373080; at -1.548 PAPOLA 1 390943_ at -2.030 C1 ORF63
1373145_at -1.640 VPS41 1390989_at -2.23O MOSPD2
1373157_at -1.817 USP47 139107Lat -1.707 RFC1
1373162_at -1.797 LOC681708 1391270_at -1.732 CNNM3
1373182_at -1.718 CLDN12 1391282_at -1.605 C60RF192

 

197

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1373201_at -2.221 DBT 1391433_at -2.137 ACOT2
1373228_at -1.842 HGSNAT 1391483_at -1.992 CREB3L3
1373239_at -1 .879 SNX33 1391 507_at -2.042 ZN F467

1 373287_at -1 .829 ATOH8 1 391 527_at -1 .501 STAT6
1373305_at 1 .664 SNX4 1391702_at -1 .607 ZN F446

1 373389_at -1 .664 ACADQ 1 391 807_at -1 .756 TFCP2
1373409_at -1 .676 UBE3C 1392280_at 2.246 TLR2
1373426_at -1 .537 MAPK1 1392502_at -1 .940 AHCTF 1
1373450_at -1.847 USP38 1392534_at 1 .680 PMEPA1
1373469_at -2.332 RGD1565496 1392543_at -1 .903 RBBP4
1373492_at -1 .752 SDHAF2 1 392547_at 1 .538 C150RF48
1373502_at -1 .739 DYM 1392888_at 1 .624 GPC4
1373512_at -1.870 ILVBL 1392912Lat -1.800 CACYBP
1373523_at 2.919 FCGR3A 1392916_at -1 .827 MAP7
1373547_at -1.985 C7ORF25 1392929_at -1.642 CZOORF194
1373570_at -1.921 NPEPL1 1392955_at -1.793 SEL1 L
1373578_at -1.510 TRI M2 1 392975_ at -1 .765 LOC686393
1373625_at -1 .696 SHMT1 1 392979_at -1 .882 CACYBP
1373664_at -1.525 PIGC 1392984_at -1.544 CPN E3
1373686_at -1.864 SERPINA6 1393005_at -1 .500 SFT2 DZ
1373826_at -1.631 YPEL5 1393110_at -1.968 MPV17L
1373829_at 1.556 FGF R2 1393140!at 1.556 ZC3H12A
1373874_ at -1.871 SGPP1 1393171_at ~1.707 TMEM47
1373906_at -1.788 FAM173B 1393218_at -1.923 ATGZB
1373921_at -3.628 ECHDC3 1393351_at -1.635 RDH1O
1373923_at -1.736 RDH10 1393414_at -1.632 LOC679161
1373954_at -2.247 SU D83 1393615_at -1 .566 DEPDC6

 

198

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1373984!at 1.500 SLC39A14 1393826_at -2.020 APON
1374039_at -1.558 CAR14 1393862_at -1.585 1700019617RIK
1374045_at -1.574 COGS 1394737_at -1.928 C90RF64
1374154_at -1.530 SFRSZIP 1395338_at -1.677 LRPPRC
1374303Lat -1.505 ALKBH2 1395565_at -1.691 COPS4
1374331_at 1.806 RQCD1 139561Lat -1.593 COPS4
1374396_at -1.733 ATP6V1C1 1396112_at -1.676 MTMR1O
1374467_at -1.621 TRAP1 1397268_at -1 .548 SLC17A4
1374487_at -1 .508 FAM96A 1397363_at -1 .514 PVR L3
1374554_at -1.718 C1 ORF128 1397419_at -1.633 MPP6
1374571_at -1.725 PIGX 1397519_at -1.562 ADI POR2
1374612_at -1 .966 PAPD5 1397526_at -1 .995 GCDH
1374669_at -1 .527 BRWD2 139752Lat -1 .512 DDX17
1375034_at -1.654 PLAZG15 1398249_at -1.764 SLCZ5A20
1375170_at 2.108 GM5068 1398282_at -3.201 KYNU
1375173_at -1.523 PLBD2 1398286_at -1.814 CSAD
1375298_at -1.664 PPPZRSC 1398295_at -1.525 SLC29A1
1375357_at -1.57O TOR1A 1398341_at -1.625 CISD3
1375429_at -1.864 ZH16 1398350_at 2.334 BASP1
1375431_at -1.752 C20RF69 1398472_at -1.651 C1 OORFZ
1375524_at -1.949 AR|D1A 1398514_at -2.151 HGD
1375536_at -1.919 NUMB 1398591_at 2.643 CCRL2
1375634_at -2.387 CCDC53 1398642_at -2.039 MTRR
1375638_§t -1.547 SDPR 1398808_at -1.999 IMPA1
1375869_at -2.210 ULK1 1398891_at -2.331 MRPL15
1375934_at -1.967 RNF128 1398902Aat -1.737 KIAA0664
1375951_at 1.799 THBD 1398976_at -1.956 C2OORF191

 

199

 

 

 

1375977_at

-1.805

CETN2

, 139696131

-2.146

TRIAP1

 

200

 

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