Exploring mesenchymal stem cell derived exosome and tocotrienol (t3) as therapeutic agents in drug induced liver injury (DILI)

EXPLORING MESENCHYMAL STEM CELL-DERIVED EXOSOMES
AND TOCOTRIENOL (T3) AS THERAPEUTIC AGENTS IN
DRUG-INDUCED LIVER INJURY (DILI)





TAN CHEAU YIH
(B. Eng. (Hons.), UTM)







A THESIS SUBMITTED
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
DEPARTMENT OF PHARMACY
NATIONAL UNIVERSITY OF SINGAPORE
2014
i

Declaration



I hereby declare that this thesis is my original work and it has been written by
me in its entirety. I have duly acknowledged all the sources of information
which have been used in the thesis.

This thesis has also not been submitted for any degree in any university
previously.




Tan Cheau Yih
1 August 2014
ii

Acknowledgement
I have never thought of completing another 4 years of graduate
programme after 2 years of master degree. This dissertation marks another
important journey in my life and the completion of it would not be possible
without the support of several people. I would like to express my sincere
gratitude to all of them.
Firstly, I would like to thank my PhD supervisor, Dr. Ho Han Kiat for
his advice and sharing which encouraged me to move on to this journey of
research. Thank you for giving me the opportunity to join the wonderful lab,
the freedom to shape my research works and thoughts, the motivations and
supports throughout my candidature. I truly appreciate all the help and had

enjoyed my 4 years journey working with you. I am also grateful to my co-
supervisor, A/Prof. Dan Yock Young for his precious advice and help given
throughout my PhD pursuit. The enthusiasm, passion and energy he has for
research was contagious and motivational to me. Every meeting with him is a
recharging time to me, especially during hiccup periods in mice work.
Thank you to my thesis committees, Prof. Paul Ho Chi Lui and A/Prof.
Theresa Tan May Chin who have guided me through all these years. Thank
you for the continuous supports, valuable suggestions and recommendations
given. I would also like to extend my deepest appreciation to our collaborators,
Dr. Lim Sai Kiang from Institute of Medical Biology, for the contribution of
MSC-derived exosomes and Dr. Fong Chee Wai from Davos Life Science
Singapore, for the contributions of tocotrienol analogs. This project would not
be possible without their generous supports and professional advices. I am also
iii

grateful to Ruenn Chai for his helps and inputs on the MSC-derived exosomes
and also Judy Saw for her assistance in tocotrienols uptake assay.
The Laboratory of Liver Cancer and Drug-Induced Liver Diseases
Research Group members, Lee Cheng, Yi Yun, Yun Shan, Chun Yan, Angie,
Duan Yan, Sheela and Winnie, thank you for the help, support and great
companies. I am also grateful to the A/Prof. Dans lab members, expecially to
Jaymie and Brian for the help and encouragement given to me to overcome
mice phobia. Mandy, Charmaine, Pan Jing, Luqi, Li Jian, Sudheer, Hua Pey,
Yi Ling, Xiu Ping, Hui Ting and Qiu Yi, thank you for the friendship and joy
that we have shared together. My biggest gratitude goes to Sing Teang, thank
you for being there all the time with me, through ups and downs, thank you for
the all the supports and companion. It is a great pleasure to have all of you
around!
My appreciation also goes to all the supporting staffs in Pharmacy
department for their kind assistance: Johannes, Sek Eng, Sukaman, Kelly,

Timothy, Liza, Napsiah, Ying Ying, Jenny and Mrs Teo. Special thanks to
NUS department of Pharmacy for enrolling me as a postgraduate student and
NUS (President Graduate Fellowship) for the financial support.
Last but not least, I would like to dedicate this dissertation to my
beloved family members and husband for their love and encouragement which
gave me the strength to surmount all challenges.



iv

Table of Contents
Declaration i
Acknowledgement ii
Summary ix
List of Publication xii
List of Tables xiii
List of Figures xiv
List of Supplementary Tables xviii
List of Abbreviations xix

Chapter 1 Introduction 1
1.1 Liver injury and its pattern 2
1.2 Clinical outcomes of drug-induced liver injury 3
1.3 Mechanism of liver injury 5
1.3.1 General drug-induced liver injury mechanism 6
1.3.2 APAP-induced liver injury mechanism 7
1.3.2.1 NAPQI formation 7
1.3.2.2 NAPQI and protein binding 9
1.3.2.3 Mitochondrial superoxide and peroxynitrite formation

10
1.3.2.4 Amplification of mitochondrial oxidant stress 12
1.3.2.5 Apoptosis and necrosis 13
1.3.3 Conclusion 16
1.4 Cellular responses to injury 17
1.4.1 Anti-oxidative responses 18
1.4.2 Anti-apoptosis 21
1.4.3 Liver inflammation 22
1.4.4 Liver regeneration 24
1.4.5 Conclusion 27
1.5 Current management in DILI 28
1.5.1 Protection 28
1.5.2 Treatment and limitations 29
1.6 Proposed strategies in managing DILI 30
v

1.6.1 Potential protective agent: alpha-tocotrienol (-T3) 31
1.6.2 Potential regenerative agent: mesenchymal stem cells (MSC)
derived exosomes 33
1.7 Aims and objectives 36

Chapter 2 Materials and Methods 39
2 .1 Materials 40
2.1.1 Preparation and quantification of MSC-derived exosomes 41
2.1.2 Preparation and quantification of Vitamin E derived -TP and
T3 42
2.2 In vivo studies 43
2.2.1 Animal and diets 43
2.2.2 In vivo CCl
4

induced liver injury model optimization 43
2.2.3 In vivo exosomes route of administration optimization 44
2.2.4 CCl
4
induced acute liver injury induction with exosomes
treatment 44
2.2.5 Measurement of serum ALT and aspartate aminotransferase
(AST) release 45
2.2.6 Histologic examination 45
2.2.7 Immunohistochemistry (IHC) of PCNA 46
2.3 In vitro studies 47
2.3.1 Cell lines and culture conditions 47
2.3.2 In vitro cytotoxicity test of exosomes and Vitamin E analogs
(-TP and T3 isomers) 48
2.3.3 In vitro cellular uptake of Vitamin E analogs (-TP and T3
isomers) 49
2.3.4 In vitro treatment of APAP-induced liver injury model 49
2.3.5 In vitro treatment of hydrogen peroxide (H
2
O
2
)-induced liver
injury model 50
2.3.6 Cell viability assay 51
2.3.7 Isolation of total mRNA from TAMH cells 51
2.3.8 Reverse transcription and qRT-PCR 51
2.3.9 Western blots 53
2.3.10 Determination of GSH content 54
vi

2.3.11 Determination of intracellular reactive oxygen species (ROS) .
54
2.3.12 Determination of intracellular lipid peroxidation (LPO) 55
2.3.13 Determination of membrane potential transition (MPT) 55
2.3.14 Caspase-3 activity assay 56
2.3.15 Combination therapy 56
2.3.16 Statistical analysis 56

Chapter 3 Results on exosomes 57
3.1 Introduction 58
3.2 Influence of exosomes against CCl
4
-induced liver injury in vivo
model 58
3.2.1 Development of CCl
4
-induced hepatic injury in a mouse model
59
3.2.2 Screening of exosomes toxicity and the optimum route of
exosomes administration in a mouse model 61
3.2.3 Effect of exosomes against CCl
4
on biochemical indices of
injury 63
3.2.4 Effect of exosomes against CCl
4
on histopathological patterns
of liver injury 65
3.2.5 Effect of exosomes against CCl
4

on protein expression in liver
regeneration 67
3.2.6 Effect of exosomes on PCNA immunohistochemical staining
(IHC) 69
3.2.7 Summary 71
3.3 Influence of exosomes against APAP- and H
2
O
2
-induced liver injury
in vitro model 72
3.3.1 Exosomes characterisation 72
3.3.2 Effect of exosomes on cell viability in APAP and H
2
O
2
-
induced toxicity 75
3.3.3 Effect of exosomes on gene regulation during priming phase of
liver regeneration 78
3.3.4 Effect of exosomes on the induction of transcription factors
during the G1 phase of cell cycle 81
3.3.5 Effect of exosomes on cell proliferation markers during G1 and
S phase of cell cycle 83
vii

3.3.6 Effect of exosomes on caspase 3 activity and apoptotic gene
Bcl-x
L
85

3.3.7 Effect of exosomes on anti-oxidant gene activity 87
3.3.8 Summary 89
3.4 Discussion 91
3.5 Conclusion 101

Chapter 4 Results on Tocotrienol (T3) 102
4.1 Introduction 103
4.2 Characterization of tocotrienol analogs in TAMH cells 104
4.2.1 Cytotoxicity of T3 analogs in TAMH cells 105
4.2.2 Cellular uptake of different concentration of T3 analogs in
TAMH cells 107
4.2.3 Summary of Vitamin E analogs characterization 110
4.3 Influence of T3 analogs against APAP- and H
2
O
2
-induced in liver
injury in vitro……… 112
4.3.1 Effect of T3 analogs on APAP and H
2
O
2
-induced on cell death
in TAMH cells 112
4.3.2 Effect of lower dosage of -T3 and -T3 against APAP and
H
2
O
2
-induced injury on cell viability in TAMH cells 118

4.3.3 Effect of -TP and -T3 on GSH activity 122
4.3.4 Effect of -TP and -T3 on intracellular ROS 124
4.3.5 Effect of -TP and -T3 on lipid peroxidation (LPO) 126
4.3.6 Effect of -TP and -T3 on antioxidant genes activity 128
4.3.7 Effect of -TP and -T3 on mitochondrial membrane
permeability transition (MPT) 131
4.3.8 Effect of -TP and -T3 on Bcl-x
L
anti-apoptotic gene 133
4.3.9 Effect of -TP and -T3 on caspase 3 activity 135
4.3.10 Effect of -TP and -T3 on gene regulation in ROS induced
inflammation 137
4.3.11 Effect of -TP and -T3 on protein expressions in liver
regeneration 139
4.3.12 Summary 141
4.4 Discussion 143
4.5 Conclusion 155
viii


Chapter 5 Results on combination therapy of exosomes and -T3 157
5.1 Introduction 158
5.2 Effect of exosomes and -T3 against APAP- and H
2
O
2
-induced liver
injury in cell viability 159
5.3 Discussion on the combination therapy of exosomes and -T3 161

Chapter 6 Conclusion and future prospectives 162
6.1 Recapitualtion of overall hypothesis and study aims 163
6.2 Conclusion of MSC-derived exosomes 164
6.3 Conclusion on T3 167
6.4 Conclusion of exosomes and -T3 combination therapy 170
6.5 Overall conclusion 171
6.6 Recommendations for future work 173

References 176
Appendices 194


ix

Summary
Drug-induced acute liver injury (DILI) is a major clinical problem
arising from both diseases and therapeutic misadventures. This issue not only
translates into significant morbidities and mortalities worldwide, but also
causes the repercussions of drug removal from market and socio-economic
burden. Management of DILI is often limited to cessation of drug use and
supportive therapy, as there are no therapeutically proven natural
hepatoprotective agents. Current treatment using N-acetylcysteine (NAC) has
narrow therapeutic window and only effective when administered at a very
early stage of the injury. To address this unmet need, we are interested in
exploring two potential natural hepatoprotective or hepatoregenerative agents,
exosomes and alpha-tocotrienol (-T3) in the acute liver injury model.
Hitherto mesenchymal stem cell (MSC) and the conditioned medium (MSC-
CM) was shown to be effective in treating various organ failure, including
liver. Later, MSC-CM derived exosomes was identified to play a vital
functional role in tissue repair. Nevertheless, the hepatoprotective or

hepatoregenerative effect of exosomes has never been demonstrated. On the
other hand, Vitamin E has been well-known for its antioxidant property with
-tocopherol (-TP) being the most active form. However, recent research
showed that tocotrienols (or T3, another subtype of Vitamin E) analogs exert
better functions in health and disease distinct from -TP, especially -T3 in
overcoming neuronal injury and ischemic perfusion injury. However, the
hepatoprotective effect of specific analogs of T3 has yet to be identified.
Therefore, our overarching aim was to determine if MSC-CM derived
x

exosomes and isoforms of T3 are hepatoregenerative and/or hepatoprotective
in overcoming DILI.
The effect of exosomes (Chapter 3) was investigated in vivo followed
by in vitro whereas the effect of T3 analogs (Chapter 4) was investigated only
in vitro. Exosomes were introduced concurrently with CCl
4
into a mouse
model through different route of administration. Biochemical analysis was
performed based on the blood and liver tissues. Subsequently the exosomes/T3
analogs were evaluated in APAP and H
2
O
2
-toxicants in vitro models. Cell
viability was measured and biomarkers indicative of regenerative and
oxidative biochemical responses were determined to probe into the mechanism
of any hepatoprotective or hepatoregenerative activity observed.
In contrast to PBS-treated mice, CCl
4
injury in mice was attenuated by

concurrent-treatment exosomes, and characterized by an increase in
hepatocyte proliferation as demonstrated with PCNA elevation. Significantly
higher cell viability was demonstrated in the exosomes-treated group as
compared to the non exosomes-treated group in both APAP and H
2
O
2
injury
models. The higher survival rate was associated with upregulation of the
priming phase genes during liver regeneration which subsequently led to
higher expression of proliferation proteins and prevention of apoptosis in the
exosomes-treated group. In contrast to that, -T3 but not other T3 analogs was
found to be effective in preventing both toxicants induced injuries. -T3
preserved cell viability by acting against the build-up of ROS and its
downstream pathway which inhibited the injury and initiation of apoptosis.
Finally, the combination therapy of exosomes and -T3 demonstrated better
restoration of viability compared to respective single treatment, acting in
xi

concert with each other complementing the protection against DILI (Chapter
5).
In summary, these results suggest that MSC-derived exosomes can
elicit hepatoprotective and hepatoregenerative effects against toxicants-
induced injury mainly through activation of proliferative and regenerative
responses while -T3 prevented the hepatocytes injuries caused by oxidative
stress during DILI through its potent antioxidant properties. These two agents
participate distinctively in different aspects of hepatoprotection and
hepatoregeneration, alluding to a potential enhancement of effect when
applied collaboratively.

xii

List of Publication

Publication derived from this thesis:

1. C. Y. Tan, R. C. Lai, W. Wong, Y. Y. Dan, S. K. Lim and H. K. Ho,
Mesenchymal stem cell-derived exosomes promote hepatic
regeneration in drug-induced liver injury models, Stem Cell Research
and Therapy, 5(3):76 (2014).

2. C. Y. Tan, T. Y. Saw, C. W. Fong and H. K. Ho, Comparative
hepatoprotective effects of tocotrienol analogs against drug-induced
liver injury, Redox Biology, 4:308 (2015).


Poster presentations:

1. C. Y. Tan, R. C. Lai, W. Wong, Y. Y. Dan, S. K. Lim and H. K. Ho.
Mesenchymal stem cell-derived exosomes promote hepatic
regeneration in drug-induced liver injury models. ISSX 18
th
North
American Regional Meeting, Dallas, Texas, USA. October 14-18,
2012

2. C. Y. Tan, R. C. Lai, S. Q. Lin, Y. Y. Dan, S. K. Lim, H. K. Ho.
Exploring mesencyhmal stem cell-derived exosome as a

hepatoprotective agent in drug-induced liver injury (DILI). 7
th
ANSC
Scientific Symposium National University of Singapore, Singapore. 6
April 2011

3. C. Y. Tan, R. C. Lai, S. Q. Lin, Y. Y. Dan, S. K. Lim, H. K. Ho.
Exploring mesencyhmal stem cell-derived exosome as a
hepatoprotective agent in drug-induced liver injury (DILI). 6
th

PharmSci@Asia 2011 Symposium, Nanjing University, Nanjing,
China. May 25-26, 2011

4. C. Y. Tan, R. C. Lai, S. Q. Lin, Y. Y. Dan, S. K. Lim, H. K. Ho.
Exploring mesencyhmal stem cell-derived exosome as a
hepatoprotective agent in drug-induced liver injury (DILI). The 21st
Conference of the Asian Pacific Association for the Study of the Liver
(APASL), Bangkok, Thailand. February 17-20, 2011


xiii

List of Tables

Table 1 Sequences of primers used in real time PCR reaction 52

Table 2 Summary of exosomes findings in vivo. 71

Table 3 Summary of exosomes findings in vitro. 89

Table 4 Summary on the effects of -TP and-T3 against APAP and H
2
O
2
-
induced injury in TAMH hepatocytes 141


xiv

List of Figures

Figure 1 Schematic representation depicting the role of metabolism in
acetaminophen toxicity. 8

Figure 2 Mechanism of APAP-induced liver cell injury 11

Figure 3 Death-receptor-mediated and mitochondrial pathways of cell
apoptosis. 15

Figure 4 Defense network against oxidative stress. 20

Figure 5 Acute phase response and induction of acute-phase protein
expression by TNF- cytokines and IL6 cytokines respectively. 23

Figure 6  . 26

Figure 7 Strategies in managing DILI 30

Figure 8 Chemical structure of T3 and TP 32

Figure 9 Summary of proposed aim and management approaches to overcome
DILI 37

Figure 10 ALT over time profile for CCl
4
and dose-dependent CCl
4
induced
liver injury model in mice. 60

Figure 11 Exosomes cytotoxicity in mice. 62

Figure 12 Effect of exosomes on biochemical parameters after CCl
4
treatment
in vivo. 64

Figure 13 Effect of exosomes on hepatocyte injury after CCl
4
treatment in
vivo 66

xv

Figure 14 Effect of exosomes on hepatocyte proliferation after CCl
4
-induced
injury in mice 68

Figure 15 Effect of exosomes on liver tissue proliferation after CCl
4
-induced
injury in mice. 70

Figure 16 Exosomes cytotoxicity tests. 74

Figure 17 Effect of exosomes in cell viability after APAP- and H
2
O
2
-induced
injury 77

Figure 18 qRT-PCR analysis of iNOS, TNF-, COX-2, IL-6, IL-10 and MIP-2
expressions. 80

Figure 19 Effect of exosomes in G1 phase of cell cycle after APAP- or H
2
O
2
-
induced injury in TAMH hepatocytes 82

Figure 20 Effect of exosomes in cell proliferation after APAP- or H
2
O
2
-

induced injury in TAMH hepatocytes. 84

Figure 21 Effect of exosomes on anti-apoptosis in APAP- or H
2
O
2
-induced
injury in hepatocytes 86

Figure 22 qRT-PCR analysis of HO-1, Gpx-4, GSR and MnSOD expressions.
88

Figure 23 Cell cycle with the genes and proteins involved in each stage 92

Figure 24 Proposed summary of exosomes protection mechanisms against
DILI through anti-apoptotic and regeneration pathways. 98

Figure 25 Vitamin E cytotoxicity test in TAMH hepatocytes. 106

Figure 26 Cellular uptake of -TP, -T3, -T3 and -T3 in TAMH
hepatocytes 109

xvi

Figure 27 Summary on the cytotoxicity and cellular uptake of -TP, -T3, -
T3 and -T3 in TAMH hepatocytes. 110

Figure 28 Concurrent effects of -TP, -T3, -T3 and -T3 in cell viability
after APAP- and H
2

O
2
-induced injury in TAMH hepatocytes. 114

Figure 29 Pre-treatment effects of -TP, -T3, -T3 and -T3 in cell viability
after APAP- and H
2
O
2
-induced injury in TAMH hepatocytes. 117

Figure 30 Lower dosage of -TP, -T3 cytotoxicity test in TAMH
hepatocytes 118

Figure 31 Effects of lower dosage of -TP, -T3 in cell viability after APAP-
and H
2
O
2
-induced injury in TAMH hepatocytes. 120

Figure 32 Effects of -TP, -T3 in cell viability after APAP- and H
2
O
2
-
induced injury in TAMH hepatocytes 121

Figure 33 Effects of -TP, -T3 in GSH after APAP- and H
2

O
2
-induced
injury in TAMH hepatocytes. 123

Figure 34 Effects of -TP, -T3 in intracellular ROS after APAP- and H
2
O
2
-
induced injury in TAMH hepatocytes 125

Figure 35 Effects of -TP, -T3 in LPO formation after APAP- and H
2
O
2
-
induced injury in TAMH hepatocytes 127

Figure 36 Effects of -TP, -T3 in antioxidant gene activity after APAP- and
H
2
O
2
-induced injury in TAMH hepatocytes. l. 130

Figure 37 Effects of -TP, -T3 in mitochondrial MPT after APAP- and
H
2
O

2
-induced injury in TAMH hepatocytes 132

Figure 38 Effects of -TP, -T3 in Bcl-x
L
anti-apoptotic gene after APAP-
and H
2
O
2
-induced injury in TAMH hepatocytes. 134

xvii

Figure 39 Effects of -TP, -T3 on caspase 3 activity in APAP- or H
2
O
2
-
induced injury in TAMH hepatocytes 136

Figure 40 Effects of -TP, -T3 on qRT-PCR analysis of iNOS, TNF-, and
IL-6 inflammatory expression 138

Figure 41 Effects of -TP, -T3 on liver regeneration markers after APAP- or
H
2
O
2
-induced injury in TAMH hepatocytes. 140

Figure 42 Proposed protective mechanism of Vitamin E analog in DILI. 143

Figure 43 Proposed -T3 protection pathways in APAP- and H
2
O
2
-induced
liver injury in TAMH cells. 156

Figure 44 Combination therapy of exosomes and -T3 in APAP- and H
2
O
2
-
induced liver injury in TAMH cells. 160

xviii

List of Supplementary Tables

Supplementary table 1 Acute phase plasma proteins in human and rat. 195

Supplementary table 2 Proteomic profile of 3 independently prepared
exosomes as determined by LC MS/MS and antibody arrays 196

xix

List of Abbreviations

AIF
Apoptosis-inducing factor
ALP
Alkaline phosphatase
ALT
Alanine aminotransferase
AP-1
Activating protein-1
APAP
Acetaminophen (paracetamol)
APR
Acute phase response
AST
Aspartate aminotransferase
ATP
adenosine triphosphate
TTP
-tocopherol transfer protein
BSA
Bovine serum albumin
BTI
Bioprocessing Technology Institute
CAT
Catalase
CCl
4

Carbon tetrachloride
CM-H
2

DCFDA
Chloromethyl derivative of 2',7'-dichlorodihydrofluorescein
diacetate
CO
Carbon monoxide
CO
2

Carbon dioxide
COX-2
Cyclooxygenase-2
DAD
Diode array detector
DILI
Drug induced-liver injury
DMEM

DMEM/F12

DMSO
Dimethylsulfoxide
EGF
Epidermal growth factor
FBS
Fetal bovine serum
FGF
Fibroblast growth factor
FLD
Fluorescence detector
Gpx4

Glutathione peroxidase 4
GSH
Glutathione
GSR
Glutathione reductase
GST
Glutathione-s-transferase
H&E
Haematoxylin & Eosin
H
2
O
2

Hydrogen peroxide
HGF
Hepatocyte growth factor
HO-1
Heme oxygenase-1
HPLC
High performance liquid chromatography
i.p
Intraperitoneally
i.s
Intraspleenic
I/R
Ischemia/reperfusion
IACUC
Institutional Animal Care & Use Committee
IHC

Immunohistochemistry
xx

IL
Interleukin
iNOS
Inducible nitric oxide synthase
ITS
Insulin, transferrin, selenium
JNK
c-Jun-N-terminal kinase
LFT
Liver function test
LPO
Lipid peroxidation
MAPK
Mitogen activated protein kinase
MCB
Monochlorobimane
MDA
Malondialdehyde
MHC
Major histocompatibility complex
MI/R
Myocardial infarction/reperfusion
MIP-2
Macrophage inflammatory protein 2-alpha
MnSOD
Manganese superoxide dismutase
MPT

Membrane permeability transition
mrpw
Multiple reads per well
MSC
Mesenchymal stem cells
MSC-CM
MSC-condition medium
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NAC
N-acetylcysteine
NAPQI
N-acetyl-p-benzoquinoneimine
NF-kB
Nuclear factor-kappa B
Nrf-2
Nuclear factor erythroid 2-related factor
NUS
National University of Singapore
PBS
Phosphate buffered saline
PGs
Prostaglandins
PH
Partial hepatectomy
PI3K
Kinases phosphoinositol 3 kinase
PMC
2,2,5,7,8-pentamethyl-6-chromal
qRT-PCR

Quantitative real-time polymerase chain reaction
RNA
Ribonucleic acid
ROS
Reactive oxygen species
ROS/RNS
Reactive oxygen/nitrogen species
SEM
Standard error of means
SM
Silymarin
SOD
Superoxide dismutases
Stat3
Signal transducer and activator of transcription 3
T3
Tocotrienols
TB
Total bilirubin
TBARS
Thiobarbituric acid reactive substances
TFF
Tangential flow filtration
TGF-
Transforming growth factor-alpha
TNF-
Tumor necrosis factor-alpha
TNFR1
TNF receptor 1
TP

Tocopherols
xxi

UDCA
Ursodesoxycholic Acid
ULN
Upper limit of normal
VEGF
Vascular endothelial growth factor

Mitochondrial membrane potential
1


Chapter 1


Introduction

2

1.1 LIVER INJURY AND ITS PATTERN

Liver injury is one of the leading causes of death worldwide, and is the
only major cause of death still increasing year-on-year [1], accounting for
more than 330,000 deaths in United States in 2011 [2]. Histopathologically,
liver injury can be distinguished into hepatocellular, cholestatic, and mixed
injury by measuring the alanine aminotransferase (ALT), alkaline phosphatase
(ALP) and total bilirubin (TB) level in the liver function test (LFT). Among
the three types of liver injury, hepatocellular-predominant injury leads to more

ominous progression in acute liver failure. Clinically, hepatocellular injury is
characterized by disproportionate elevation of ALT compared to TB and ALP
level, with the degree of elevation being more than three times the upper limit
of normal (ULN) and with a ratio of ALT/ALP (referred to as R value) more
than five [3]. Hepatocellular injury reflects damage at the hepatic parenchyma
which leads to apoptosis and cytolytic necrosis [4]. Cholestatic injury on the
other hand involves increase in ALP more than two times ULN with R values
less than two, while mixed liver injury is characterized by equivalents of
elevations in ALT and ALP which is more than two times ULN, and R values
in between two and five [3]. The later injury is often mixed with prominent
features of hepatocellular and cholestatic damage.



3

1.2 CLINICAL OUTCOMES OF DRUG-INDUCED LIVER INJURY

Liver is one of the few organs in the body that has the ability to
regenerate from the replication of mature functioning liver cells, without
recruitment of liver stem cells [5, 6]. However when liver injuries progress
into a state of functional impairment, it can lead impede self-renewal and also
cause severe consequences that include acute liver failure or death [3]. Today,
acute liver injury is a major clinical problem arising from many causes
including diseases and therapeutic misadventures, with approximately 2000
cases each year and 80% mortality. In United States, virally-induced disease
predominated in early 90s but has substantially declined in the past few years,
with most acute injury cases now arising from drug induced-liver injury (DILI)
[7].
In the United States, DILI accounts for more than 50% of acute liver

failure and is the prominent cause of liver transplantation [8]. More than 1,000
therapeutic agents and herbal remedies are believed to be hepatotoxic. Among
which, the drug most often implicated in such cases is acetaminophen (APAP),
also known as paracetamol, which represents 75% of DILI cases and 42% of
U.S acute liver failure cases [9]. This injury is often followed by rapidly
progressive multi-organ failure, exhibiting greater severity of illness than seen
in other causes of liver failure [10]. Nevertheless, other drugs which cause
DILI may also lead to patient morbidity and mortality. Majority of the adverse
DILI events are unpredictable, arising from either immune-mediated
hypersensitivity reactions or of idiosyncratic origins [11]. Idiosyncratic
reactions normally occur in 5-90 days after the causative medication was last