apoptosis and liver diseaseepubs.surrey.ac.uk/855229/1/27558529.pdf · apoptosis and liver disease...
Post on 14-Oct-2020
2 Views
Preview:
TRANSCRIPT
APOPTOSIS AND LIVER DISEASE
ITS ROLE IN PARACETAMOL HEPATOTOXICITY
By
Dr Hasan EL-Hassan (MBBS, MSc)
School of Biomedical and Molecular Sciences (SBMS)
University of Surrey
United Kingdom
A thesis submitted in accordance with the requirements of the University of
Surrey for the Degree of Doctor of Philosophy
January 2004
ProQuest Number: 27558529
All rights reserved
INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.
In the unlikely event that the author did not send a com p le te manuscript and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.
uestProQuest 27558529
Published by ProQuest LLO (2019). Copyright of the Dissertation is held by the Author.
All rights reserved.This work is protected against unauthorized copying under Title 17, United States C ode
Microform Edition © ProQuest LLO.
ProQuest LLO.789 East Eisenhower Parkway
P.Q. Box 1346 Ann Arbor, Ml 48106- 1346
ACKNOWLEDGEMENTS
Many thanks to my supervisor Dr George E N Kass for giving me the opportunity to
undertake this study and work with him as a research fellow. I sincerely thank Dr Kass
for his invaluable input and for his friendship. Without Dr Kass’s forbearance,
guidance, and help this PhD would never have been finished. Many thanks also to my
co-supervisors Dr Richard H Hinton and Dr Shirley C Price for their invaluable advice
and assistance.
I also like to extend sincere thanks to my colleague Dr Khurshid Anwar with whom I
worked throughout this project. I am very grateful to Dr Anwar for allowing me to share
some o f his results, including Figure 9, Figure 14, Figure 15, and Figure 16.
I also thank my PhD colleague Dr Patricia Macanus-Pirard for her friendship and
invaluable assistance.
I am grateful to all the Consultant Physicians I worked with at Royal Gwent Hospital
(RGH), University Hospital o f Wales (UHW), Llandough Hospital, and at Nevill Hall
Hospital (NHH), for their encouragement, motivation, and unlimited support.
I am indebted to my wife for her patience, support, and understanding throughout this
project.
Finally many thanks to the Sir Jules Thom Charitable Tmst for supporting this project.
SUMMARY
Paracetamol (Acetaminophen, V-acetyl-p-aminophenol, AAP) is a popular domestic
analgesic and antipyretic for adults and children. It has a high margin o f safety when
used in therapeutic doses. However, acute paracetamol overdose results in hepatic (and
renal) damage. It is believed that such liver damage occurs in two phases. The first
phase is due to formation o f the highly reactive oxidation product, V-acetyl-p-
benzoquinone-imine (NAPQI). After consuming the hepatic reservoir o f glutathione,
NAPQI oxidises thiol (SH-) groups o f key enzymes, which causes cell death, liver
failure, and occasionally death. The pathophysiology o f the second phase o f liver
damage is poorly characterised and probably results from the continuing damage
inflicted by persistently high levels o f serum paracetamol.
Although it is generally accepted that centrilobular hepatocyte necrosis is characteristic
o f paracetamol hapatotoxicity, several sporadic reports have previously presented
evidence for the occurrence o f some morphological and biochemical (such as DNA
fragmentation) changes that are suggestive o f apoptosis. Therefore, in this in vivo study,
a more in-depth investigation o f the role o f apoptosis in paracetamol-induced hepatic
injury was carried out using different modulators o f the induction o f parenchymal cell
apoptosis by paracetamol.
Six hours after paracetamol administration to BALB/c mice, a significant loss o f hepatic
mitochondrial cytochrome c was observed that was similar in extent to the loss observed
after in vivo activation o f the apoptosis inducing death receptor CD95. Paracetamol-
induced loss o f mitochondrial cytochrome c coincided with the appearance in the cytosol
o f a fragment corresponding to truncated Bid (t-Bid). At the same time, t-Bid became
detectable in the mitochondrial fraction, and concomitantly, Bax was found translocated
to mitochondria. However, paracetamol failed to activate the execution caspases-3 and -
7 as evidenced by a lack o f procaspase processing and the absence o f an increase in
caspase-3-like activity.
On the other hand, pretreating BALB/c mice with the broad-spectrum caspase inhibitor
benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-fmk) or the caspase-
3 family specific inhibitor Z-Asp-Glu-Val-Asp-chloromethylketone (Z-DEVD-cmk)
nearly completely abrogated paracetamol-induced damage as reflected by the degree o f
histopathological lesions (centrilobular necrosis, haemorrhagic damage and congestion),
the rise in serum transaminases, and DNA damage (as assessed by the TUNEL method
and DNA agarose gel electrophoresis). At the same time, the analogue
benzyloxycarbonyl-Phe-Ala-fluoromethylketone (Z-FA-fmk) that does not inhibit
caspases did not prevent the development o f paracetamol-induced liver injury or the
appearance o f apoptotic parenchymal cells. This demonstrates that caspase inhibitors
protected the liver by pharmacologically targeting caspases.
I found that Z-VAD-fmk was protective when administered up to two hours after
paracetamol dosing. This protective effect against paracetamol-related liver damage by
Z-VAD-fmk also correlated with the inhibition o f the processing o f Bid to tBid.
However, Z-VAD-fmk failed to prevent both the redistribution o f Bax to the
mitochondria and the loss o f cytochrome c.
IV
In contrast, pretreatment o f mice with gadolinium chloride to block Kupffer cell function
did not prevent liver injury but completely blocked paracetamol-induced parenchymal
cell DNA fragmentation (as assessed by the TUNEL method).
Our findings also suggest that the development o f liver injury in paracetamol toxicity is
not necessarily a direct consequence to parenchymal cell apoptosis but instead, damage
to the sinusoidal endothelial cells may play a major role through haemorrhagic
congestion and shut down o f hepatic microvascular perfiision.
In conclusion, apoptosis is an important causal event in the initiation o f the hepatic
injury inflicted by paracetamol and is primarily triggered by Kupffer cell-derived
factors. As suggested by the lack o f activation o f the main execution caspases, apoptosis
is not properly executed and degenerates into necrosis. However, the causal role o f
apoptosis in paracetamol-induced hepatic damage meant that an opportunity for
pharmacological intervention to protect the liver from drug-induced injury was
uncovered.
V
PUBLICATION
The following original publication is based on the work presented in this thesis:
El-Hassan, H., Anwar, K., Macanas-Pirard, P., Crabtree, M., Chow, S. C., Johnson, V.
L., Lee, P. C., Hinton, R. H., Price, S. C. and Kass, G. E. (2003). Involvement o f
mitochondria in acetaminophen-induced apoptosis and hepatic injury: roles o f
cytochrome c, Bax, Bid, and caspases. Toxicology and Applied Pharmacology
191, 118-129.
VI
LIST OF ABBREVIATIONS
AAP Acetaminophen: A-acetyl-p-aminophenoI
Ac-DEVD-afc N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin
Ac-LEHD-afc Ac-Leu-Glu-His-Asp-7-amido-4-trifluoromethylcoumarin
AFC Amino-4-trifluoromethylcoumarin
AIF Apoptosis-inducing factor
AK Adenylate kinase
ALD Alcoholic liver disease
ALF Acute liver failure
ALT Alanine transaminase
ANT Adenine nucleotide translocator
Anti-Fas Human anti-Fas receptor antibody
AP-1 Activator protein-1
Apaf-1 Apoptotic protease activating factor-1
AST Aspartate transaminase
ATP Adenosine triphosphate
Bad Bcl-2 family member; proapoptotic protein
Bak Bcl-2 homologous antagonist/killer
Bax B-cell lymphoma X protein
Bcl-2 B-cell lymphoma-2
B cI-Xl B-cell lymphoma X protein, long
Bid BH3-interacting domain death agonist
CAD Caspase-activated deoxyribonuclease
Caspases Cysteine aspartate proteases
CDC Chenodeoxycholate
CLD Cholestatic liver disease
Con A Concavalin A
COX Cytochrome c oxidase
CTL Cytotoxic T-lymphocytes
VII
CYP-2E1 Cytochrome P450 2E1
CYP-450 Cytochrome P450
C ytc Cytochrome c
DcR Decoy receptors
DD Death domains
DED Death effector domain
DISC Death-inducing signalling complex
DR Death receptor
dUTP Deoxyuridine-triphosphate
FADD Fas-associated protein with death domain
FasL Fas ligand
FasR Fas receptor
FLICE FADD-like interleukin 1-p-converting enzyme (FADD-like ICE)
FLIP FLICE inhibitory protein
G-6-PD Glucose-6-phosphate dehydrogenase
GaIN D-galactosamine
GCDC Glycochenodeoxycholate
GdCla Gadolinium chloride
GSH Reduced glutathione or L-y-glutamyl-L-cysteineylglycine
GSSG Oxidised glutathione
GVHD Graft versus host disease
H&E Haematoxylin and Eosin stain
H2O2 Hydrogen peroxide
HAV Hepatitis A virus
HBV Hepatitis B virus
HBx Hepatitis B virus X protein
HCC Hepatocellular carcinoma
HCV Hepatitis C virus
HEB Hypotonic extraction buffer
lAPs Inhibitors o f apoptosis
ICAD Inhibitor o f caspase-activated deoxyribonuclease
VIII
ICE
IkB
JNK
LAP
LDH
Ipr
LPS
M P T (orP T )
M PT P(or PTP)
NAC
NAD
NADH
NADP
NADPH
NAPQI
NF-kB
NIK
NK
NO
PAPS
PARP
PBC
PBS
PCD
PI
PKC
PSC
RBCs
Rh-TRAIL
RIP
ROS
SDH
Interleukin 1-p-converting enzyme
Inhibitor o f NF-kB
c-JUN N-terminal kinase
Latency associated peptide
Lactate dehydrogenase
Lymphoproliferation
Lipopolysaccharide
Mitochondrial permeability transition
Mitochondrial permeability transition pore
N-acetylcysteine (Parvolex)
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide reduced
Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate reduced
A-acetyl-/7-benzoquinone-imine
Nuclear factor-kappa B
NF-k B-inducing kinase
Natural killer cell
Nitric oxide
3 ’ -phosphoadenosyl-5 ’ -phosphosulphate
Poly(ADP-ribose)polymerase
Primary biliary cirrhosis
Phosphate buffered saline
Programmed cell death
Propidium iodide
Protein kinase C
Primary sclerosing cholangitis
Red blood cells
Recombinant human TRAIL
Receptor interacting protein
Reactive oxygen species
Succinate Dehydrogenase
IX
SDS-PAGE SDS-polyacrylamide gel electrophoresis
SEC Sinusoidal endothelial cell
Smac/DIABLO Second mitochondria-derived activator o f caspases
Smad Zinc finger transcription factor Schnurri mothers against decapentaplegic
TdT Terminal deoxynucleotidyl transferase
TEM Transmission electron microscopy
TGF Transforming growth factor
TGF-P Transforming growth factor beta
TGF-P R-I Transforming growth factor beta receptor I
TG F-PR-II Transforming growth factor beta receptor II
TNF Tumour necrosis factor
TN F-a Tumour necrosis factor alpha
TNF-Rl Tumour necrosis factor receptor 1
TNF-R2 Tumour necrosis factor receptor 2
TRADD TNF-Rl-associated death domain
TRAF-2 TNF receptor associated factor-2
TRAIL TNF-related apoptosis-inducing ligand
TUNEL TdT-catalysed dUTP-fluorescein nick end labeling
UDCA Ursodeoxycholic acid
UDP Uridine diphosphate
UDPGA UDP-glucuronic acid
UV Ultraviolet (radiations)
VDAC Voltage-dependent anion channel
Z-DEVD-cmk Benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone
Z-FA-fmk Benzyloxycarbonyl-Phe-Ala-fluoromethylketone
Z-VAD-fmk Benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone
TABLE OF CONTENTS
ACKNOWLEDGEMENTS II
SUMMARY m
PUBLICATION VI
LIST OF ABBREVIATIONS VII
TABLE OF CONTENTS XI
LIST OF TABLES XIX
LIST OF FIGURES XX
CHAPTER 1: INTRODUCTION 1
1.1 Apoptosis 2
1.1.1 Historical review 2
1.1.2 Apoptosis versuss Necrosis 4
1.1.3 Pathways of apoptosis 7
1.1.4 The role of cell surface receptors in apoptosis 8
)0
1.1.4.1 Fas(CD95/Apo-l) 10
1.1.4.2 Tumour Necrosis Factor 13
1.1.4.3 TRAIL 19
1.1.4.4 TGF-P 23
1.1.5 The role of perforin/granzyme B system in apoptosis 25
1.1.6 The role of mitochondria in apoptosis 25
1.1.6.1 General 25
1.1.6.2 Mitochondria and apoptosis 26
1.1.7 Apoptotic cell death depends on the action of caspases 30
1.1.8 Bcl-2 family proteins and apoptosis 35
1.1.9 Other inhibitors and regulators of apoptosis 40
1.1.10 Physiological role of apoptosis 42
1.1.11 Apoptosis and pathology 43
1.2 Apoptosis and the liver 46
1.2.1 Structure of the liver 46
1.2.2 Apoptosis and liver disease 48
_
1.2.2.1 Cholestatic liver diseases (CLD) 48
1.2.2.1.1 General 48
1.2.2.1.2 Primary biliary cirrhosis and Primary
sclerosing cholangitis 50
1.2.2.1.3 Obstructive jaundice 51
1.2.2.2 Apoptosis and viral hepatitis 51
1.2.2.3 Apoptosis and tumours of the liver 53
1.2.2.4 Apoptosis and Wilson’s disease 54
1.2.2.5 Apoptosis and drug-induced liver disease 55
1.2.2.5.1 Alcoholic liver disease (ALD) 55
1.2.2.5.2 Paracetamol and hepatotoxicity 58
CHAPTER 2: MATERIALS AND METHODS 65
2.1 Materials 66
2.2 Solutions and Buffers 71
2.2.1 General 71
2.2.2 Caspase activity assay buffers 71
m
2.2.3 Agarose Gel Electrophoresis 72
2.2.4 Succinate dehydrogenase (SDH) assay 73
2.2.5 SDS-PAGE and Western Blotting 73
2.3 Methods 77
2.3.1 Animals and in vivo experiments 77
2.3.2 Serum Biochemistry 78
2.3.3 Histology 78
2.3.4 Determination of DNA fragmentation 79
2.3.4.1 DNA isolation and purification 79
2.3.4.2 Agarose gel electrophoresis 80
2.3.5 Succinate dehydrogenase (SDH) assay 81
2.3.5.1 Isolation of mitochondria for SDH assay 81
2.3.5.2 Assaying for SDH 82
2.3.6 Adenylate kinase (AK) assay 83
2.3.7 Cytochrome c oxidase (COX) assay 84
XIV
2.3.8 Determination of caspase activation 85
2.3.8.1 Isolation of thymocytes (positive controls) 85
2.3.8.2 Preparation of total liver homogenates 86
2.3.8.3 Measurement of caspase activation 86
2.3.9 SDS-PAGE and Western Blotting 86
2.3.9.1 Isolation of mitochondria 86
2.3.9.2 Polyacrylamide Gel Electrophoresis 87
2.3.9.3 Western Transfer of Proteins 87
2.3.9.4 Immunoblotting 88
2.3.10 Bio-Rad protein assay 89
2.3.11 Statistical analysis of the data 90
CHAPTERS: PARACETAMOL HEPATOTOXICITY 91
3.1 Histopathology and Biochemistry in AAP overdosing 92
3.2 Role of phagocytes in paracetamol overdosage 98
3.3 Role of caspase inhibitors in paracetamol hepatotoxicity 101
_ _
3.4 Role of sinusoidal endothelial cells in paracetamol toxicity 103
3.5 Role of reactive FMK-moiety in AAP toxicity 107
CHAPTER 4: CASPASES AND PARACETAMOL 109
4.1 Role of caspases in paracetamol hepatotoxicity 110
4.2 Effector caspases and paracetamol toxicity 117
CHAPTERS: DNA FRAGMENTATION AND
PARACETAMOL 121
5.1 Paracetamol overdosage causes loss of genomic DNA
through fragmenatation 122
5.2 Time-course for protection from paracetamol-induced
hepatotoxicity by Z-VAD-fmk 126
CHAPTER 6: MITOCHONDRIA AND
PARACETAMOL 130
6.1 Mitochondrial cytochrome c and paracetamol hepatotoxicity 131
6.2 Release of cytochrome c from mitochondria correlates with
the release of adenylate kinase 136
XVI
6.3 Release of cytochrome c from mitochondria correlates with
the loss of succinate dehydrogenase activity 139
6.4 Role of pro-apoptotic members of the Bcl-2 family in
paracetamol-induced hepatic injury 142
CHAPTER 7: DISCUSSION 147
7.1 Purpose of study 148
7.2 Background 148
7.3 Mode of cell death after paracetamol overdose in BALB/c
mice: Necrosis or Apoptosis? 151
7.4 TUNEL assay and DNA fragmentation as markers of
apoptosis in paracetamol hepatotoxicity 153
7.5 Initiation of paracetamol-induced apoptosis involves
mitochondria: Role of Bcl-2 family members 156
7.6 Lack of execution caspase activity in paracetamol toxicity 162
7.7 Sinusoidal endothelial and Kupffer cells in paracetamol
hepatotoxicity: Role of hepatic congestion and cytokines 167
7.8 Conclusion 170
XVII
CHAPTER 8: REFERENCES 172
CHAPTER 9: APPENDIX 211
XVIII
LIST OF TABLES
Table 1: Differentiation between apoptosis and necrosis 5
Table 2: Regulation o f apoptosis by the Bcl-2 family proteins 36
Table 3: Effects o f Gadolinium chloride treatment on paracetamol-
induced alterations in serum liver enzyme levels 99
XIX
LIST OF FIGURES
Figure 1: The two main pathways o f apoptosis 9
Figure 2: Apoptosis signalling by CD95 12
Figure 3: Pro-apoptotic signalling by TNF-Rl 15
Figure 4: Anti-apoptotic signalling by TNF-Rl 17
Figure 5: Apoptotic signalling by TRAIL-Rl (DR4) and TRAIL-R2 (DR5)
and its modulation by decoy receptors 22
Figure 6: General mechanisms o f caspase activation 34
Figure 7: The Bcl-2 family members 39
Figure 8A: Architecture o f the liver 47
Figure 8: Proposed metabolic activation o f paracetamol to the toxic, reactive
intermediate NAPQI 64
Figure 9: Histopathological documentation o f paracetamol-induced acute liver
injury and failure o f GdClg-pretreatment to prevent such injury 93
Figure 10: Z-VAD-fmk and Z-DEVD-cmk prevent the rise in serum alanine
transaminase (ALT) after paracetamol intoxication 94
Figure 11: Z-VAD-fink and Z-DEVD-cmk prevent the rise in serum aspartate
transaminase (AST) after paracetamol intoxication 95
Figure 12: Z-VAD-fink and Z-DEVD-cmk prevent the rise in serum lactate
dehydrogenase (LDH) after paracetamol intoxication 96
XX
Figure 13: Z-VAD-fink does not abolish hepatic injury after intoxication by
higher doses o f paracetamol 97
Figure 14: Detection o f DNA fragmentation in the liver following paracetamol
intoxication using the TUNEL assay: Gadolinium chloride (GdClg)
and Z-VAD-fink prevent DNA fragmentation 100
Figure 15: Z-VAD fink prevents liver damage in response to paracetamol 105
Figure 16: Paracetamol induces sinusoidal endothelial cell damage in
the liver 106
Figure 17: Effect o f FMK moiety o f caspase inhibitors on AAP toxicity in
BALB/c mice 108
Figure 18: Paracetamol-induced apoptosis does not involve the increase in
caspase 3-like activity 112
Figure 19: Time-Course o f o f Ac-DEVD-afc cleavage activity during
paracetamol-induced liver injury 113
Figure 20: Time-Course o f inhibition o f Ac-LEHD-afc cleavage during
paracetamol-induced liver injury 114
Figure 21: The failure o f liver homogenates from AAP-treated animals
to decrease DEVDase activity o f thymocyte apoptotic-extract
indicates the absence o f an endogenous inhibitory activity in
those homogenates 115
Figure 22: Lack o f DEVDase activity in serum from paracetamol-treated
BALB/c mice 116
Figure 23: Processing o f procaspases-3 and -7 in paracetamol-induced liver
injury in BALB/c mice 120
XXI
Figure 24: Early experiment showing a smear o f DNA fragmentation 123
Figure 25: Time-Course o f paracetamol-induced effects on the electrophoretic
behaviour o f genomic DNA from livers o f BALB/c mice 125
Figure 26: Z-VAD-fmk prevents DNA fragmentation at different intervals
after paracetamol administration 128
Figure 27: Z-VAD-fmk prevents the rise in serum ALT after paracetamol
intoxication 129
Figure 28: Presence o f mitochondria is confined to the mitochondrial
fraction. No mitochondrial contamination o f the cytosol 133
Figure 29: Paracetamol induces cytochrome c release from liver mitochondria 135
Figure 30: Cytochrome c release from liver mitochondria after in vivo
administration o f acetaminophen or anti-CD95 activation:
Role o f the pan-caspases inhibitor Z-VAD-fink 137
Figure 31: Release o f adenylate kinase from mitochondria by paracetamol
and after activation o f CD95 138
Figure 32: Mitochondrial succinate dehydrogenase (SDH) activity after
AAP and anti-CD95 treatments 141
Figure 33: Paracetamol induces Bid processing and translocation to the
mitochondria in vivo 145
Figure 34: Paracetamol induces Bax translocation to mitochondria in vivo 146
Figure 35: Schematic representation o f in vivo paracetamol-mediated apoptosis
and caspase activation 171
XXII
Chapter 1
Introduction
1.1 Apoptosis
1.1.1 Historical review
In Greek, apo means “from”, ptosis means “fall”; apoptosis used to describe the
“dropping o f f ’ or “falling o f f ’ o f petals o f flowers, or leaves from trees (Kerr et ah,
1972; Majno and Joris, 1995). The word apoptosis was first adopted by Kerr et al in
1972 to depict the phenomenon o f controlled cell death as an antithesis to mitosis in the
regulation o f cell populations (Kerr et al., 1972). The same authors had previously
named this mode o f cell death as “shrinkage necrosis” reflecting that cells undergoing
apoptosis become condensed and compacted (Kerr, 1971).
Although apoptosis is a fashionable topic now, it was largely neglected for almost a
century after its discovery. Both Flemming and Nissen first reported the observation o f
apoptosis in 1885, under the name “chromatolysis” (Majno and Joris, 1995; Wang and
Wang, 1999).
In a 1914 paper “A new joint o f view regarding the elimination o f cells” (translated from
German), Graper proposed chromatolysis to be a mechanism counterbalancing mitosis
and responsible for the elimination o f unwanted cells, and he was the first to note the
debris o f dead cells was taken up by neighbouring cells (Graper, 1914; Majno and Joris,
1995).
In 1950s, Glucksmann extensively described a “form o f physiological cell death” among
vertebrate embryos, and his descriptions were generally consistent with what we now
know to occur during apoptosis (Glucksmann, 1951). Glucksmann realised that what he
had observed was a new type o f cell death distinct from necrosis (Majno and Joris, 1995).
However he did not designate a new term to specify it. This was left until 1964 when
Lockshin and Williams used the term “programmed cell death” (PCD) to refer to the
death o f given cell types in predictable sites at predictable times during ontogeny
(Lockshin and Williams, 1964).
In 1971 the term “shrinkage necrosis” was used to differentiate a newly identified type o f
cell death from classical necrosis (Kerr, 1971), and in 1972 the term “apoptosis” was
used to describe cell death with characteristic morphology (Kerr et al., 1972).
Kerr et al defined apoptosis as a form o f cell death with a pivotal role in embryogenesis,
metamorphosis, and maturation o f immune systems, as well as natural tissue turnover
whereby apoptosis maintains the balance between cell loss and cell gain and is therefore
regarded as nature’s way o f eliminating unwanted, senescent, and damaged cells from
multicellular organisms (Kerr et al., 1972).
From 1976 through 1981, three independent groups noted that chromatin breaks into
pieces the size o f multiple nucleosomes. This was termed “DNA laddering” which is a
distinctive double strand cleavage o f nuclear DNA at the linker regions between
nucleosomes (Skalka et ah, 1976; Yamada et al., 1981; Zhivotovsky et al., 1981).
However, Wyllie, in 1980 was the first to link DNA laddering and activation o f
endogenous endonuclease to apoptosis (Wyllie, 1980).
1.1.2 Apoptosis versus Necrosis
It is important to recognise that these terms (apoptosis and necrosis) relate to a
morphological description o f cell death only, and it remains possible that the two
processes might actually be part o f a continuum, with morphological necrosis being the
ultimate fate for cells that undergo apoptotic changes initially (Dong et aL, 1997).
As outlined in Table 1 and in contrast to necrosis, apoptosis is an active process
associated with cell shrinkage, loss o f contact with neighbouring cells, release o f
cytochrome c from mitochondria, caspase activation, plasma membrane blebbing,
phosphatidylserine externalisation on the plasma membrane, chromatin condensation at
the nuclear membrane, and nuclear and cytoplasmic fragmentation with formation o f the
so called “apoptotic bodies” that can be taken up and degraded by neighbouring cells
(Allen et aL, 1997; Thompson, 1995; Wilson, 1998).
During apoptosis, plasma membrane is kept intact thus preventing cytosolic contents
from leaking to the exterior. Therefore apoptosis is not associated with an inflammatory
response (Wyllie, 1997b).
Necrosis, on the other hand, is a passive form o f cell death that tends to affect contiguous
cells, is associated with inflammation, and is often due to an overwhelming cellular
insult. As shown in Table 1, necrosis is characterised by swelling o f the cytoplasm and
organelles, breakdown o f plasma membrane, release o f lysosomal enzymes, and cell lysis
with spillage o f pro-inflammatory intracellular constituents into the extracellular milieu
which results in an inflammatory response in the surrounding tissue (Fadeel et aL, 1999;
Trump and Berezesky, 1995).
Table 1, Differentiation between apoptosis and necrosis (adopted, with modification,
from Leist et al (Leist et al., 1998)).
Changes Apoptosis Necrosis
Distribution Scattered single cells Contiguous group o f cells
Nucleus Convolution o f nuclear outline and breakdown
Initially condensation (pyknosis), later mostly disintegration (karyorrhexis) and dissolution (karyolysis)
Nuclearchromatin
Condensation and margination, with formation o f uniformly dense masses, often yielding characteristic crescent-shaped figures
Clumping, not sharply defined
DNAbreakdown
Characteristic high molecular weight (50-300 kbp) DNA fi*agmentation. Often oligonucleosomal cleavage into « X 180 bp DNA fi-agments
Random DNA degradation
Cytoplasm Condensation o f cytosol. Dense packaging o f relatively intact organelles. Occasional dilatation o f endoplasmic reticulum
Oedematous swelling o f organelles and rupture o f intracellular membranes
Plasmamembrane
Blebbing with maintenance o f membrane integrity
Blebbing with eventual rupture o f plasma membrane and spillage o f intracellular contents
Cell Formation o f apoptotic bodies Swelling and later disintegration
Phagocytosis Rapid removal by professional phagocytes and by neighbouring cells
Clearance by infiltrating phagocytes after disintegration o f cells
Exudativeinflammation
Absent Present, with occasional scar formation
Studies suggest that apoptosis and necrosis generally occur simultaneously and that both
intracellular energy levels and the severity o f the insult are important determinants o f the
mode o f cell death (Ankarcrona et aL, 1995; Bonfoco et aL, 1995; Leist et aL, 1997b;
Nicotera c? a/., 1998).
While both types o f cell death are associated with DNA damage, the pattern o f DNA
degradation is different between the two. In apoptosis there selective DNA cleavage at
intemucleosomal sites, whereas in necrosis degradation is random (Dong et aL, 1997;
Leist a/., 1998).
Apoptosis-associated nuclear condensation and DNA fragmentation is usually
accompanied by the activation o f endonucleases that first cleave the DNA into HMW
(50-300 kb) pieces and then further into smaller intemucleosomal fragments o f 180-200-
base pairs in length, the latter gives the classic DNA-laddering constituting an important
biochemical criterion found in most apoptotic cells (Duke et aL, 1983; Wyllie, 1980;
Wyllie et aL, 1984).
A specific endonuclease called CAD (caspase-activated DNase) cleaves the chromosomal
DNA in a caspase-dependent manner. CAD is normally synthesized with the help o f
ICAD (inhibitor o f CAD), which works as a specific chaperone for CAD and is found
complexed with it in proliferating cells. When cells are induced to undergo apoptosis,
effector caspases (particularly caspase-3) dissociate and degrade the ICAD, which, in
turn, liberates CAD resulting in the induction o f nuclease activity, nuclear condensation,
and DNA fragmentation (Enari et aL, 1998; Mitamura et aL, 1998; Nagata, 2000;
Sakahira c /a /., 1998).
However, DNA fragmentation is not pathognomonic to apoptosis as it is also observed in
some cell deaths with ultrastructural features o f necrosis (Collins et aL, 1992). Also
reports o f failure to detect intemucleosomal DNA cleavage in some apoptotic cells raise
further doubts about the validity o f using DNA electrophoresis in isolation to categorise
cell death (Cohen et aL, 1992a). Therefore, intemucleosomal DNA cleavage should not
be the sole criterion for identifying apoptosis (Collins et aL, 1992; Dong et aL, 1997).
1.1.3 Pathways of apoptosis
Since the proposal o f the term “apoptosis” and its implications within the context o f
human cell biology and diseases in the early 1970s (Kerr and Searle, 1972a; Kerr and
Searle, 1972b; Kerr et aL, 1972) and the work on apoptosis carried out by Wyllie
(Wyllie, 1980) and Duke (Duke et aL, 1983) in the 1980s, our ability, over the last two
decades, to understand the remarkable and genetically complex processes involved in
apoptosis is attributed to the use o f powerful molecular biology techniques.
We now have a good, albeit incomplete, understanding o f the wide variety o f genes
involved in the regulation o f apoptosis. Such genes have been subjected to fairly
thorough analysis to determine the complex interactive pathways o f apoptosis. As new
apoptosis genes continue to be identified, and new apoptosis-related functions are
assigned to familiar proteins, our understanding o f this rapidly growing subject will
remain in a state o f flux for some time to come (Afford and Randhawa, 2000).
As outlined in Figure 1, it is generally accepted that apoptosis occurs by one o f two
pathways: (1) Receptor (or death receptor) pathway, and (2) Mitochondrial pathway
(Green, 1998).
1.1.4 The role of cell surface receptors in apoptosis
Apoptosis can be triggered by specific cell surface death receptors engaging specific
ligands. It is well established that a number o f ligand/receptor interactions cause
apoptosis and these include:
1. The TNF receptor superfamily (also called death receptors) which includes Fas
receptors (also called Fas, FasR, or CD95/Apol), TNF receptors TNF-Rl and TNF-R2,
and TRAIL (TNF-related apoptosis-inducing ligand) receptors.
2. Transforming growth factor p receptor (TGF-pR) family which mediate the action o f
TGF-pi (Patel e/fl/., 1998).
The TNF superfamily receptors are type 1 membrane spanning glycoproteins. The N-
terminus o f which faces to the outside o f the cell and contains 1-6 ligand binding
domains. The C-terminus faces towards the interior o f the cell and contains a region o f
about 60-70 residues. The receptors oligomerize upon activation with a trimeric ligand
(or membrane-bound ligand) to form a cluster, which then interacts with cytosolic
proteins to form the DISC (death inducing signalling complex) with its active “death
domain”.
TNF or CD95L or TRAIL
TNFRl orCD95 orDR4/DR5
c-FLIPB id {p22) 0 ^ 0
Bax monomers
Bcl-2
Caspase-8
Procaspase-3
Caspase-3
ApoptoticSubstrates
O a if
Bcl-x
t-B id {o l5 )
A poptosom e
N ucleus
Sm ac/D IA B L O
APOPTOSIS
Figure 1. The two main pathways of apoptosis: Relationship between the death receptor
pathway (left) and the mitochondrial pathway (right) and their interaction with members o f
the Bcl-2 family. Details o f the much-disputed pathways initiated by the TGF-ft family have
been omitted.
1.1.4.1 Fas (CD95/Apo-l)
While expression o f some TNF receptors appears to be restricted to certain cell types, Fas
(CD95/Apol) receptors are fairly well distributed across a wide range o f cell types and
are thought to have a central role in triggering apoptosis (Desbarats et aL, 1998; Loweth
et aL, 1998; Luo et aL, 1997).
Transduction o f the Fas signal has been mainly studied in lymphoid cells (Nagata, 1997).
The expression o f Fas ligand (FasL) is essentially restricted to CD8^ cytotoxic T
lymphocytes (CTL) and natural killer (NK) cells. The normally membrane-bound FasL
acts as a major effector o f their cytotoxic effects (Hanabuchi et aL, 1994; Montel et aL,
1995).
In rat liver, Kupffer cells and sinusoidal endothelial cells (SECs) slightly express FasL-
mRNA in the basal state, and this expression can be enhanced in culture when EPS is
added to the medium (Muschen et aL, 1998). In contrast, FasL is not normally expressed
in hepatocytes, although FasL-mRNA may be expressed in culture after exposure to
dexamethasone and during several conditions causing oxidative stress (Galle et aL, 1995;
R u getaL , 1997; Muller c /a /., 1997; Strand c /a /., 1998).
The intraperitoneal administration o f the monoclonal anti-Fas antibody/Jo2
(lOOpg/animal) rapidly killed wild type mice but not Ipr (lymphoproliferation) mice,
which have a Fas gene defect (Ogasawara et aL, 1993).
Despite the fact that Fas receptor is expressed in many organs, the administration o f the
anti-Fas antibody mainly damages the liver, possibly because hepatocytes do not express
(or poorly express) Bcl-2. In Bcl-2 transgenic mice (mice expressing human Bcl-2 gene
_
product in their hepatocytes), hepatic apoptosis induced by the administration o f anti-Fas
antibody (lOpg/animal) was delayed and reduced and most o f the animals survived,
whereas the nontransgenic mice suffered from hepatic apoptosis and death (Lacronique et
a/., 1996).
As shown in Figure 2, FasL forms the Fas-associated death domains (FADD) which
binds to procaspase-8 and causes its activation and the initiation o f the caspase cascade
(Boldin «/., 1996).
It is however believed that Fas signalling involves two major pathways termed type I and
type II (Scaffidi et aL, 1998). In type I pathway, sufficient amounts o f caspase-8 are
recruited (at the level o f the DISC) to directly activate caspase-3, thereby bypassing the
need for mitochondrial cytochrome c (see below). Type II pathway results in the indirect
activation o f caspase-3. It occurs when caspase-8, present in insufficient levels at the
DISC, cleaves the BH3 interacting domain death agonist (Bid) which induces the release
o f mitochondrial cytochrome c to form the apoptosome complex that activates caspase-3
(Luo et aL, 1998; Thomberry and Lazebnik, 1998; W olf and Green, 1999). This could
explain why Bcl-2 is only capable o f preventing Fas-mediated apoptosis in type II
(Scaffidi era/., 1998).
Tissue specificity is presumed to be the main factor determining which o f these two
pathways to use. Hepatocytes are presumed to use the type II pathway as hepatocytes
from Bid-deficient (Bid-/-) mice were shown to be relatively resistant to Fas-induced
apoptosis, whereas thymocytes from the same (Bid-/-) mice were shown to be sensitive to
Fas-mediated apoptosis (Yin et aL, 1999).
11
CD95L
FADD
Œ
Caspase-8
CD95/Fas/Apo1
DED-containingprocaspase-8
(FLICE)
Pro-caspase-3 ►Caspase-S
DISC Complex
ProteinSubstrates
Figure 2. Apoptosis signalling by CD95. The oligomerization, most probably, trimerization, o f
CD95 is required for the transduction o f the apoptotic signal. The death-inducing signalling complex
(DISC) forms within seconds o f receptor engagement. First the adaptor FADD (Fas-associated death
domain protein, also known as Mortl) binds via its own death domain (DD) to the clustered death
domains o f CD95. FADD also carries a so-called death-effector domain (DED), and, again, by
homologous interaction, recruits the DED-containing procaspase-8 (also known as FLICE) into the
DISC. Upon recruitment by FADD, caspase-8 oligomerization drives its proteolytic activation
through self-cleavage based on “induced-proximity” model (Figure 6: panel A). This releases active
caspase-8 from the DISC into the cytoplasm in the form o f a heterotetramer o f two small subunits
(plO) and two large subunits (p20). Active caspase-8 cleaves various proteins in the cell including
procaspase-3, which results in its activation and the completion o f the apoptotic process.
12
1.1.4.2 Tumour Necrosis Factor
Tumour necrosis factors (TNFs) are a family o f ligands that exists as soluble or cell-
bound forms. TNFs play a significant role in the modulation o f survival/apoptosis in
cells o f a variety o f lineages, and they act via a TNF receptor superfamily comprising a
minimum o f 19 members, with a wide range o f biological effects, not solely limited to
the regulation o f apoptosis. Whereas at least four o f them can trigger apoptosis directly,
it seems that some family member which are incapable o f signalling because they lack a
cytoplasmic tail, can function as “decoy receptors”. At present, the exact bio-function of
this receptor family has not been clearly defined yet (Nagata and Golstein, 1995; Orlinick
and Chao, 1998; Smith et aL, 1994).
TNF-a is mainly produced by activated lymphocytes, monocytes, and macrophages,
including Kupffer cells (Tracey and Cerami, 1993). In humans, the TNF-a gene is
located on chromosome 6 in the human leukocyte antigen (HLA) region (Beutler and
Cerami, 1988). The TNF-a promoter region contain both NF-kB and activator protein-1
(AP-1) binding sites (Zwacka et aL, 1998).
TNF-a is initially synthesized as a 26-kDa membrane-bound form o f 233 amino acids,
which is then proteolytically cleaved between Ala and Val to the 17-kDa secreted form
consisting o f 137 amino acids (Tracey and Cerami, 1993). Secreted TNF-a is an
unglycosylated polypeptide that is active in its trimeric form (Jones et aL, 1989; Smith
and Baglioni, 1987). The cell surface membrane-bound form o f TNF-a is also active
(Tracey and Cerami, 1993). In the latter case, TNF-a bearing cells kill target cells
13
through cell-to-cell contact (Decoster et aL, 1995; Kriegler et aL, 1988; Perez et aL,
1990).
LPS can enhance TNF-a production in macrophages and Kupffer cells, thus increasing
DNA binding o f both NF-kB and AP-1 (Tran-Thi et aL, 1995). TNF-a induces cell death
in cultured rat and mouse hepatocytes and in mouse liver in vivo. The effects o f TNF are
mediated by two receptors, the 55 kDa TNF-Rl and the 75 kDa TNF-R2. As outlined in
Figure 3, both TNF-a and TNF-(3 can bind to TNF-Rl and induce apoptosis by an
intracellular cascade identical to the Fas receptor except that it first incorporates TRADD
(TNF-receptor-associated death domain) which then recruits FADD (Heller and Kronke,
1994; Leist et aL, 1995b).
Signalling through TNF-Rl is, however, more complex than CD95. Indeed, in addition
to recruiting adaptor molecules that bind and activate caspases, TNF-Rl recruits proteins
that engage various signal transduction pathways, some o f which abrogate the apoptotic
response. For example, in addition to recruiting FADD, TRADD also binds the serine-
threonine kinase RIP (receptor-interacting protein) thereby coupling stimulation o f TNF-
R l to the activation o f NF-kB and offering protection against TNF-induced apoptosis
(MacFarlane, 2003).
The role o f TNF-R2 in apoptosis is not well established. It has been shown that under
conditions o f transcriptional arrest, TNF-Rl alone was shown to be responsible for the
apoptotic signal transduction upon TNF binding (Leist et aL, 1995b; Leist et aL, 1998).
14
TNF“ 1
TNFR1 m
Œ
C aspase-8
i
FADD
TRADD
Pro-caspase-3 Caspase-3
DED-conta in ing procaspase-8
(FLICE)
DISC C o m p le x
Protein Substra tes
Figure 3. Pro-apoptotie signalling by TNF-Rl. Upon binding, TNF trimerises TNF-Rl and induces
the receptors’ death domains (DD). Subsequently, an adaptor termed TRADD (TNF receptor
associated death domain) binds through its own death domain to the clustered receptor death domains.
TRADD functions as a platform adaptor that recruits several signalling molecules to the activated
receptor. The association o f TRADD with FADD mediates the activation o f apoptosis.
15
The fact that pretreatment with the transcription inhibitor D-galactosamine (GalN)
sensitises hepatocytes to TNF-a (and LPS) and leads to apoptosis, suggests the presence
o f TNF-induced genes which are anti-apoptotic (Lawson et aL, 1998; Leist et aL, 1994).
Such anti-apoptotic effects o f TNF-a were confirmed to be mediated by the
transcriptional activator NF-kB which, upon translocation to the nucleus, promotes
transcription o f survival genes (Beg and Baltimore, 1996; Van Antwerp et al., 1996;
Wang et al., 1996a). As demonstrated in Figure 4, the activation o f NF-kB by TNF-a is
attributed to the capability o f TRADD to bind other proteins such as RIP and TRAF-2
(TNF receptor associated factor-2) which cause phosphorylation and degradation o f IkB
(inhibitor o f NF-kB) that normally maintains NF-kB in an inactive cytosolic complex
(Hsu et aL, 1996; Hsu et aL, 1995; Regnier et aL, 1997).
RIP possesses a functional N-terminal kinase domain and can autophosphorylate itself,
but its kinase activity is not required for activation o f NF-kB. TRAF-2 has likewise been
implicated in the activation o f NF-kB-inducing kinase (NIK). However, studies using
RIP and TRAF2-deficient cells indicate that RIP is most likely responsible for NF-kB
activation, whereas TRAF2 preferentially activates c-JUN N-terminal kinase (JNK) (Yeh
et aL, 1999). Other studies have shown that RIP could be cleaved by caspase-8 to
produce a dominant negative fragment which inhibits TNF-induced NF-kB activation,
and therefore promotes apoptosis (Lin et aL, 1999; Martinon et aL, 2000).
The anti-apoptotic effect o f NF-kB at least partly is accounted for by upregulation o f
lAPs (Inhibitors o f apoptosis) which inhibit executioner caspases (e.g. caspases-3 and
capases-7) (Chu et aL, 1997; Wang et aL, 1998).
16
TNF
TN FR 1
T R A D D
RIP RIP
T R A F 2
NIK
IKKi
M E K K 1 ?
I -kB / N F - kB J N K K
: iN F- kB J N K
c-JUN
Figure 4. Anti-apoptotic signalling by TNF-Rl. Stimulation o f TNF-Rl by ligand-induced
cross-linking initiates the recruitment o f various adaptor molecules (through homophilic death
domain-DD-interactions) and results in activation o f the NF-kB and JNK signalling pathways
through RIP and TRAF-2, respectively.
17
Thus, in addition to its pro-apoptotic effect, TNF-a can also promote growth o f
hepatocytes (Diehl et ah, 1994) and may be required to initiate liver regeneration as
observed in mice after partial hepatectomy (Yamada et aL, 1997). However, the role o f
TNF-a in human liver diseases is not as well established as it is for TGF-(31 (Patel et aL,
1998).
A model o f TNF/TNF-Rl-dependent liver injury is the Concavalin A (Con A)-model.
Con A is a T-lymphocyte mitogenic plant lectin that leads to the in vivo production o f
pro-inflammatory cytokines (TNF-a, IFN-y, IL-1, IL-2, IL-6, IL-10) and polyclonal T-
cell proliferation. Due to its preferential binding to the liver, Con-A leads to a liver-
selective injury with no other organs being affected. Mice, when injected with Con A,
tend to develop an acute, partly apoptotic, hepatic injury that is subsequently overlaid by
massive necrosis (Gantner et aL, 1995; Tiegs et aL, 1992) and, eight hours after
intravenous injection, a picture o f fulminant hepatitis is observed (Louis et aL, 1997).
TNF-a is produced early in the course o f Con A-induced hepatitis, and blockade o f its
action is protective against liver injury (Gantner et aL, 1995; Mizuhara et aL, 1994).
CD" T lymphocytes have been identified as being the effector cells in Con A-induced
hepatitis (Tiegs et aL, 1992).
Intravenously administered Con-A predominantly binds to sinusoidal endothelial cells
(SEC), and not to Kupffer cells. Loss o f SEC barrier function may expose the underlying
hepatocytes for further attacks by activated T-lymphocytes (Knolle et aL, 1996).
Intravenous Con A, when given alone, induced TNF-mediated hepatotoxicity that is
dependent on both TNF-Rl and TNF-R2. Under this model, the broad spectrum caspase
inhibitor Z-VAD-fmk, failed to protect animals from Con A-mediated liver injury. On
18
the other hand and, under transcriptional arrest (with GalN), Con A induces TNF-Rl-
dependent, but not TNF-R2-dependent, activation o f casapase-3-like proteases and
zVAD-fmk was shown to protect animals from Con A-mediated liver injury under these
conditions. Therefore, it may be concluded that Con A initiates liver destruction without
activation o f caspase-3-like proteases, and that TNF-induced caspase-3-like proteases
activity is triggered and required for apoptosis only under conditions o f transcriptional
arrest (Kunstle et aL, 1999).
1.1.4.3 TRAIL
In 1995, TRAIL (TNF-related apoptosis-inducing ligand), also called Apo-2L, a new
member o f the TNF cytokine family, was identified purely on the basis o f sequence
homology to other members o f the TNF family (Wiley et aL, 1995). Five receptors for
this cytotoxic ligand were characterised. As outlined in Figure 5, two o f these receptors,
TRAIL-Rl (DR4) and TRAIL-R2 (killer, DR5, TRICK2), contain classical cytoplasmic
death domains and are able to transduce an apoptotic signal. The signal transduction
pathways for the two pro-apoptotic TRAIL receptors (DR4 and DR5) also seems to use
both FADD and caspase-8 which then activates downstream caspases or cleaves RIP
directly, in keeping with their molecular brothers (Bodmer et aL, 2000; Kischkel et aL,
2000; Sprick et aL, 2000).
Analysis o f the TRAIL DISC revealed that FADD was present together with caspase-8
and that in a cell line lacking either FADD or caspase-8, TRAIL-induced apoptosis was
completely abrogated (Bodmer et aL, 2000; Kischkel et aL, 2000; Sprick et aL, 2000).
19
An obligatory role for FADD in TRAIL signalling was further corroborated by studies in
FADD'^ fibroblasts expressing individual TRAIL receptors, where TRAIL-induced
apoptosis was abolished (Kuang et ah, 2000). However, other studies have shown that
TRADD too may be recruited by both DR4 and DR5 (Chaudhary et ah, 1997; Schneider
et aL, 1997) and this raises the possibility that TRAIL, similar to TNF, may signal for
NF-kB and/or JNK (MacFarlane, 2003). Indeed, it was demonstrated that DR4 or DR5
can activate the NF-kB (Figure 5) or JNK signalling pathways, most likely through RIP
and TRAF-2, respectively, analogous to that shown for TNF-Rl (Figure 4) (Hu et aL,
1999; MacFarlane, 2003). More recently, it has been shown that TRAIL-induced
apoptosis results in a caspase-dependent activation o f JNK, but significantly, JNK
activation does not appear to be required for the induction o f apoptosis (MacFarlane et
aL, 2000).
As with the TNF pathway (Lin et aL, 1999; Martinon et aL, 2000), caspase-8 mediated
cleavage o f RIP could significantly inhibit the capacity o f TRAIL-sensitive cells to
activate NF-kB (Harper et aL, 2001). Instead, in cells that are relatively resistant to
TRAIL-induced apoptosis, the predominant TRAIL signalling event seems to be NF-kB
activation (MacFarlane, 2003).
The other three TRAIL receptors lack fimctional death domains and are not able to
promote cell death. These include two additional cell bound receptors (called
“decoyDcR” receptors), TRAIL-R3 (LIT, D cR l) and TRAIL-R4 (TRUNDD, DcR2),
and, last a soluble receptor called osteoprotegerin (OPG). So far the activities o f OPG
have been shown to be inhibition o f osteoclastogenesis and increased bone density in
vivo. The existence o f multiple receptors for TRAIL suggests an unexpected complexity
20
to TRAIL-mediated biological functions. (Degli Esposti, 1999; Emery et ah, 1998; Pan
et aL, 1997; Pan et aL, 1998; Sheridan et aL, 1997).
Pre-clinical studies in mice and non-human primates have previously shown that
administration o f recombinant human TRAIL (rh-TRAIL) can induce apoptosis in human
tumours but yet does not induce apoptosis in normal cells. This would make TRAIL o f
great potential to use in the treatment o f human cancers (Ashkenazi et aL, 1999; Pitti et
aL, 1996; Walczak et aL, 1999).
However, in contrast with previous reports, recent results have shown that rh-TRAIL
does induce extensive apoptosis in normal human hepatocytes in culture but still confirms
the pre-clinical data which showed that hepatocytes from other species (mouse, rat, and
monkey) are not killed by exposure to rh-TRAIL. This suggests species differences in
sensitivity to TRAIL, and that substantial liver toxicity might result if TRAIL were used
in human cancer therapy. Caspases involved in apoptosis induced by rh-TRAIL were
similar to those involved in apoptosis induced by TNF and Fas systems, and included
both the initiator caspases 8, 9, and 10, as well as the effector caspases 3 and 7 (Jo et aL,
2000).
21
n p v r & U i f I i x m u .
DcR1DcR2
+ TRADD + RIP
ActiveI - k B / N F - k BCaspase-8
DISC C o m p l e xCaspase Cascade ^
N F - K B
Figure 5. Apoptotic signalling by TRAIL-Rl (DR4) and TRAIL-R2 (DR5) and its modulation
by decoy receptors. This “extrinsic” cell death pathway is launched by death-receptor ligands
(TRAIL/Apo2L) that trigger caspase-8 oligomerization and proximity-induced autoproteolytic
activaion via the adaptor molecule FADD/Mortl. Activation is regulated by decoy receptors (DcRl
and DcR2) which preclude the binding o f TRAIL to the functional receptor. DR4 or DR5 can also
activate NF-kB or JNK signalising pathways, most likely through RIP and TRAF-2, respectively.
JNK activation pathway (not shown) appears not to be required for induction o f apoptosis.
22
1.1.4.4 TGF-p
Transforming growth factor p (TGF-P) is a member o f a large superfamily, including the
activins, inhibins, bone morphogenic proteins, and several other growth and
differentiation factors (Grande, 1997).
Like TNF-a, TGF-P has two reeeptors: TGF-p receptor type 1 (TGF-p R-1) and TGF-p
receptor type 2 (TGF-p R-11). These receptors are transmembrane proteins with
serine/threonine kinase activity in their intracellular domains (Heldin et aL, 1997). The
dimeric TGF-p protein first binds to a dimer o f TGF-p R-11, which then associates with a
TGF-p R-1 dimer, and phosphorylates the cytoplasmic domains o f the TGF-p R-I dimer
(Heldin et aL, 1997).
Thus TGF-P, TGF-p R-Il, and TGF-p R-1 form a ligand-bound, phosphorylated
tetrameric receptor complex responsible for signal transduction (Heldin et aL, 1997).
Cytosolic Smad (zinc-finger transcription factor Schnurri mothers against
decapentaplegic) proteins seem to play an essential role in transducing the TGF-p signal
into the nucleus (Heldin et aL, 1997; Massague et aL, 1997).
The most abundant isoform is TGF-pl; this particular form has a wide range o f effects,
including the triggering o f apoptosis in a large variety o f normal and tumour cells
(Grande, 1997; Rizzino, 1988). TGF-pl is also a potent inhibitor o f DNA synthesis in rat
and human hepatocytes as well as in regenerating rat livers in vivo; (Carr et aL, 1986;
RussqW et aL, 1988).
23
TGF-pl is synthesized in several cells and tissues (Grande, 1997), In the liver, TGF-pl
is expressed in Kupffer cells, sinusoidal endothelial cells, and fat-storing perisinusoidal
Stellate (Ito) cells (Bedossa and Paradis, 1995; Date et aL, 1998).
Active mature TGF-pl is a 25 kDa disulphide linked homodimer which gets activated
after dissociation from the N-terminal TGF-pl latency associated peptide (LAP)
(Massague, 1990).
The first evidence that TGF-pl can cause cell death in primary cultures o f normal rat
hepatocytes was reported by Oberhammer et al (Oberhammer et aL, 1991; Oberhammer
et aL, 1993; Oberhammer et aL, 1992b). The ability o f TGF-pl to cause hepatocyte
apoptosis in vivo was demonstrated in transgenic mice overexpressing hepatic TGF-pl
(Sanderson et aL, 1995).
The initial mechanisms that trigger TGF-pl-induced hepatocyte apoptosis are not
completely understood (Neuman, 2001). Although Smad molecules are involved in the
transduction o f the TGF-pl signal (Massague et aL, 1997; Shi and Massague, 2003), their
possible implication in the TGF-P 1-induced apoptosis has not been studied. However,
more is known about later events whereby TGF-pl-induced apoptosis is associated with
the activation o f caspase-1, caspase-2, and caspase-3 (Cain et aL, 1996; Choi et aL, 1998;
Inayat-Hussain etaL , 1997).
24
1.1.5 The role of perforin/granzyme B system in apoptosis
In addition to the Fas/FasL and TRAIL/DR&DR5 systems, activated CTL can kill target
cells through the perforin/granzyme B system (Kagi et al., 1994). Perforin makes holes
in the cell membrane and, possibly, in postendosomal intracellular vesicles, which may,
respectively, allow the entry o f extracellular and endocytosed granzyme B into the
cytosol (Pinkoski et aL, 1998). Although granzyme B is a serine protease, it also cleaves
proteins after aspartate (as do caspases) and thus can activate the caspase cascade
(Darmon et aL, 1995). Thus, the executioners o f apoptosis are similar in Fas/FasL
system and the perforin/granzyme B system (Nagata, 1997).
1.1.6 The role of mitochondria in apoptosis
1.1.6.1 General
Mitochondria are the cell’s powerhouse involved in fatty acid p-oxidation, the
tricarboxylic acid cycle, and oxidative phosphorylation, which provide most o f the cell
energy. Mitochondria are also the main endogenous source o f reactive oxygen species
(ROS) in the normal cell (Fromenty and Pessayre, 1995).
Mitochondria have two membranes: a circular outer membrane limiting the
intermembranous space, and an inner membrane with inner folds (or cristae), which
encircle the mitochondrial matrix and helps to maintain the mitochondrial inner
transmembrane potential (Av|/m). Recent studies suggested that the induction o f
mitochondrial permeability transition (MPT) results in loss o f Ai|/m and the opening o f the
25
mitochondrial permeability transition pore (MPTP) and that this effect is prevented by the
MPT inhibitor cyclosporin A (Loeffler and Kroemer, 2000; Yang and Cortopassi, 1998).
The MPTP is a large channel consisting o f both inner and outer mitochondrial membrane
proteins, such as adenine nucleotide translocator (ANT) and voltage-dependent anion
channel (VDAC), respectively, and is formed at the contact sites o f the two membranes
(Crompton, 1999).
Cytochrome c is one o f the components o f the mitochondrial electron-transport chain. It
is involved in the production o f ATP by shuttling electrons between respiratory chain
complexes III (cytochrome reductase) and IV (cytochrome c oxidase). It is synthesized
from two inactive precursor molecules: r^o-cytochrome c and heme, ^^o-cytochrome c
is encoded by a nuclear gene and synthesized in the cytosol. It is imported into the
mitochondrial intermembrane space in an unfolded configuration and, when covalently
linked to the heme moiety, it undergoes conformational changes to yield holo-
cytochrome c (better known as cytochrome c). Cytochrome c is a 13-kDa protein
normally confined to the mitochondrial intermembrane space. It is only the holo-
cytochrome c, albeit in a reduced or oxidised form, that is capable o f activating pro-
caspase-9 (Hampton et aL, 1998; Kroemer, 1999).
1.1.6.2 Mitochondria and apoptosis
Insofar as apoptosis is concerned, mitochondria were originally believed not to be
involved in apoptotic cell signalling as cells lacking mitochondrial DNA (mtDNA) were
still capable o f undergoing apoptosis (Jacobson et aL, 1993). However, subsequent
26
reports supported a role o f mitochondrial participation because membrane fraction
containing mitochondria was required for nuclear apoptosis (Green and Reed, 1998; Liu
etaL , 1996;Newmeyer^/nr/., 1994).
Mitochondria, which normally tend to spread over the whole cell, manifest “perinuclear
clustering” in apoptotic cells (De Vos et aL, 1998). Moreover, from a morphological
point o f view, mitochondria are reduced in size and the matrix becomes hyperdense in
apoptosis. This is referred to as “mitochondrial pyknosis” (Hackenbrock, 1968; Mancini
et aL, 1997; Martinou et aL, 1999; Zhuang et aL, 1998).
Unlike nuclear DNA, mitochondrial DNA remains intact during the apoptotic process.
This further supports the notion that DNA fragmentation during apoptosis is a specific
nuclear event (Murgia et aL, 1992).
Mitochondria are central targets for oxidative stress. When stressed, mitochondria
release a set o f molecules, namely cytochrome c and Apaf-1 (apoptotic protease
activating facor-1) which are vital to the activation of caspase-9 (Kroemer and Reed,
2000; Li etaL , 1997b).
Cytochrome c release from mitochondria has been confirmed to be an event o f paramount
importance in the execution o f apoptosis both in in vitro and in vivo (Kluck et aL, 1997;
Yang et aL, 1997). Although the mechanism o f how exactly cytochrome c (and other
mitochondrial inter-membranous proteins) manage to cross the mitochondrial outer
membrane is not yet known, it is clear that the Bcl-2 family members are intimately
involved in the regulation o f this process. The fact that cytochrome c release can precede
27
(Zhuang and Cohen, 1998) or even occur in the absence (Gross et al., 1999a) o f any
change in AYm suggest that MPT can not be the sole mechanism o f cytochrome c release
and, at the same time, indicate that MPT? can not be the sole target o f the Bcl-2 family
protein.
As outlined in Figure 1, once in the cytosol, cytochrome c associates with the adaptor
protein Apaf-1, in the presence o f dATP (or ATP), and then with procaspase-9 which
results in the proteolytic activation o f the caspase-9 zymogen. Activated caspase-9 then
converts procaspase-3 to caspase-3 (Li et al., 1997b; Liu et al., 1996; Zou et al., 1997). It
is now thought that the cytochrome c/Apaf-1 /caspase-9 complex, otherwise known as the
“apoptosome”, to actually represent the true form o f active capase-9 (Rodriguez and
Lazebnik, 1999).
The fact that apoptosis could be switched to necrosis when ATP levels are reduced (Leist
et ah, 1997b; Nicotera et al., 1998) might be related to reduced caspase activity and/or
the formation o f the apoptosome complex (Leist et al., 1997b; Li et al., 1997b)
In the presence o f d-ATP and cytochrome c, the monomeric Apaf-1 is transformed into
an oligomeric complex made o f at least eight subunits (Hu et al., 1998; Saleh et al., 1999;
Zou et al., 1999). The importance o f Apaf-1-induced activation o f caspase-9 has been
highlighted by reports that demonstrate that Apaf-1 or caspase-9 deficiency results in
embryonic lethality due to defective neuronal apoptosis (Cecconi et ah, 1998; Hakem et
al., 1998; Kuida et al., 1998; Yoshida et al., 1998). On the other hand, Apaf-1
overexpression was shown to trigger mitochondrial Av|/m, cytochrome c release into the
cytosol, and apoptosis through a mechanism that involves the cleavage and activation o f
28
caspase-9 and caspase-3 but not caspase-8 and Bid (Perkins et al., 1998; Perkins et al.,
2000).
Several pro-apoptotic (like Bax and Bak) and anti-apoptotic (such as Bcl-x^) members o f
Bcl-2 family can directly bind to VDAC and, respectively, accelerate and close its
channel activity and, consequently, regulate cytochrome c release (Shimizu et al., 1999).
Apoptosis induced by death receptors often bypasses the mitochondrial pathway, which
renders such deaths insensitive to the protective effect o f Bcl-2, and in this case,
cytochrome c release is likely to be the result o f caspase activation rather than its cause
(Scaffidi e ta l , 1998).
As shown in Figure 1, many other molecules are released from the mitochondria in the
presence o f pro-apoptotic stimuli. These include several procaspases (mainly
procaspase-2, -3, and -9) (Loeffler and Kroemer, 2000), Smac/DIABLO (Du et al., 2000;
Verhagen et al., 2000), apoptosis-inducing factor (AIF) (Susin et al., 1999a; Susin et ah,
1997a; Susin et ah, 1996), and adenylate kinase isozyme 2 (AK2) (Kohler et ah, 1999;
Single et ah, 1998).
Smac (second mitochondria-derived activator o f caspases) or DIABLO is a novel
mitochondrial protein (with a monomeric Mr o f 21k) released into the cytosol when cells
undergo apoptosis. In the cytosol, it physically binds to inhibitor o f apoptosis proteins
(lAPs) and neutralizes their inhibitory activity (Figure 1). Smac promotes apoptosis
through at least two mechanisms; (1) Inducing the proteolytic activation o f procaspase-3
by promoting caspase-9 activity in the apoptosome complex and (2) Promoting the
29
enzymatic/catalytic activity o f the active caspase-3. Both o f these fonctions depend on
the ability o f Smac to bind lAPs (Chai et al., 2000; Du et al., 2000).
AIF is a flavoprotein (Mr~57k) normally confined to the mitochondrial intermembrane
space. When apoptosis is induced, it translocates to the nucleus causing chromatin
condensation and large-sized DNA fragmentation (to fragments o f ~50kbp) in a caspase-
independent pattern (Figure 1). AIF is also capable o f inducing phosphatidylserine
extemalization, dissipation o f the mitochondrial Ai}/m, and inducing the mitochondria to
release cytochrome c, activate caspase-9 and, consequently, activate caspase-3 (Lorenzo
e ta l., 1999; Susm etal., 1999b; S u s i n a / . , 1997b).
1.1.7 Apoptotic cell death depends on the action of caspases
Caspases (cysteine aspartate proteases) are interleukin 1-(3-converting enzymes (ICE)-
like cysteine proteinases that form a central part o f the apoptotic cascade and are believed
to be the central executioners o f apoptosis. They can be found from humans all the way
down to insects, nematodes, and hydra. (Budihardjo et al., 1999; Cikala et al., 1999).
Caspases cleave their substrates after aspartic acid residues (at Asp-Xxxx bonds). The
four amino acids (Xxxx) distal to the cleavage site determine the individual caspase's
distinct substrate specificity. Activation o f caspases leads to the selective cleavage o f a
certain set o f target proteins, usually at one, or at most a few aspartate residues in the
primary sequence. Caspase substrates range from single polypeptide chain enzymes (like
PolyADP-ribose polymerase) to more complex macromolecules (such as actin, (3-catenin,
30
and the nuclear lamin network). It is the cutting o f these structural macromolecules that
leads to the disassembly o f cell structures that characterise apoptosis (Eamshaw et al.,
1999; Nicholson, 1999; Thomberry and Lazebnik, 1998).
As cleavage o f lamins leads to the collapse o f nuclear lamina and chromatin condensation
(Buendia et al., 1999; Rao et al., 1996), cleavage o f cytoskeletal proteins (like fodrin and
gelsolin) contributes to the morphological changes seen in apoptosis (Kothakota et al.,
1997). It is also believed that cleavage o f PAK2 (a member o f the p21-activated kinase
family) mediates the cell blebbing observed in apoptotic cells (Rudel and Bokoch, 1997).
Caspases are commonly divided into two groups based on the primary structure o f their
NH2-terminal prodomain: long prodomain “initiator” caspases (caspase-2, -8, -9, and -
10), and short prodomain “effector” caspases (caspases-3, -6, and -7) (Nicholson, 1999).
However, caspases may also be divided on the basis o f their substrate specificities (Rano
et al., 1997; Thomberry et al., 1997) into three subgroups: (1) Group I caspases (1, 4, 5,
and 13) which are mainly involved in cytokine processing and inflammation, but do not
have a substantial role in apoptosis. (2) Group II caspases (2, 3, and 7) which are the
major effectors o f cell death. (3) Group III caspases (6, 8, 9, and 10) constitute the
upstream activators o f the group II effector caspases.
This molecular ordering o f group III and group II caspases has been upheld in several
cases. Thus, for instance, caspase-8 mediates the activation o f caspase-3 and -7 in the
CD95 (Fas, A pol) system (Boldin et al., 1996; Muzio et al., 1996). Similarly, caspase-9
mediates the activation o f caspase-3 in the Apaf-1/cytochrome c pathway (Li et al.,
1997b). Both o f these examples have been substantiated in caspase-8 or -9 knockout
mice (Hakem et al., 1998; Juo et al., 1998; Kuida et al., 1998; Varfolomeev et al., 1998).
31
However, there are exceptions to this molecular ordering. One such exception is whether
caspase-6 (a group III caspase) has an effector role (e.g. lamin proteolysis) instead o f its
putative activation role, or in addition to it (Lazebnik et ah, 1995). Another exception
may be caspase-2 which appears to be self-activating (Ahmad et aL, 1997; Duan and
Dixit, 1997; Thomberry e/ <7/., 1997).
As previously described, the activation o f caspases occurs via receptor-ligand binding
and the formation o f the receptor-ligand complex, which according to Boldin (Boldin et
aL, 1996) becomes the recognition molecule for the precursor enzyme procaspase-8. But
the formation o f such ligand-receptor complex is by no means the sole trigger for caspase
activation as a number o f agents were shown to directly activate the caspase cascade,
bypassing the receptor pathway (Buckley et aL, 1999). Other agents can penetrate the
cell directly and modulate the apoptotic process in the absence o f specific cell surface
receptors. Example o f such agents include heat shock/stress factors (Thompson, 1995),
free radicals (Buttke and Sandstrom, 1994), ultraviolet radiation (Yamada and Ohyama,
1988), numerous dmgs and synthetic peptides (Schwall et aL, 1993), toxins (Sachs and
Lotem, 1993), and the potent lymphocyte enzymes (granzymes) (Hayes et aL, 1989).
All caspases are present constitutively and synthesized in enzymatically inert precursor
forms (procaspases/zymogens) o f -30-55 kDa that must be proteolytically cleaved in
order to be activated. Each procaspase consists o f an NH2-terminal prodomain, a large
(p20: -2 0 kDa), and a small (plO: -1 0 kDa) subunit (Whyte and Evan, 1995). The
mature enzyme is a heterotetramer containing two p20/pl0 heterodimers and two active
sites (Eamshaw et aL, 1999).
32
As demonstrated in Figure 6, three general mechanisms o f caspase activation have been
described so far and they include induced proximity, proteolytic cleavage by an upstream
caspase, and the holoenzyme (apoptosome) formation (Hengartner, 2000).
The “induced proximity” model (Figure 6: panel A) is evident when, upon the binding o f
FasL to Fas receptor, several molecules o f the initiator procaspase-8, with the help o f
adaptor molecules (such as FADD/MORTl), are brought into a crowded proximity o f
protein-protein interactions to form the death inducing signalling complex (DISC). This
permits autoprocessing (autocatalytic activation) o f the initiator procaspases-8 (Muzio et
ah, 1998). This mechanism may also mediate the activation o f caspase-2 (Yang et ah,
1998).
The “proteolytic cleavage” (Figure 6: panel B) model is seen when the above-mentioned
active form o f the initiator/upstream caspase-8 goes to proteolytically cleave and activate
the downstream effector procaspases (procaspases-3, -6, and -7). The initiator caspase-8
cleaves (at Asp-X sites) between p20 and plO domains, and usually also between the
prodomain and the p20 domain (Thomberry et aL, 1997). Therefore a “caspase cascade”
which acts as an amplification loop is generated and culminates in the activation o f the
downstream effector caspases. Activated effector caspases lead to most o f the
morphological and biochemical features o f apoptosis (Cryns and Yuan, 1998).
The apoptosome (holoenzyme) formation model (Figure 6: panel C) applies to the
activation o f procaspase-9 in association with cytochrome c and Apaf-1 in the presence
o f d-ATP (Cecconi, 1999). This will further activate other caspases, including
procaspase-3, in a positive amplification loop (Green and Reed, 1998; Kroemer and
Reed, 2000).
33
A
+
pro p20 plO n + 1
B UpstreamCaspases
pro p20 plOA
d-ATP+
am Apaf-1 # Cytochrome c
Active caspase
Figure 6. General mechanisms of caspase activation. Caspases are activated by
proteolytic cleavage at Aspartate-X sites. This takes place through three main
mechanisms: induced proximity (panel A), protelytic cleavage by upstream caspase
(panel B), and holoenzyme formation (panel C)
34
1.1.8 BcI-2 family proteins and apoptosis
Members o f the Bcl-2 (B-cell lymphoma/leukaemia-2) family o f proteins play a major
role in regulating apoptosis. At least 15 Bcl-2 family members have been identified in
mammalian cells and others in viruses. Many members o f this family are anti-apoptotic
but others are pro-apoptotic (Table 1) but how exactly these proteins modulate apoptosis
is still unclear. All members o f Bcl-2 family contain at least one o f four domains denoted
Bcl-2 homology (BH) domains (BH1-BH4). As shown in Figure 7, Bcl-2 family
members are classified into three functional groups based on these BH domains (Adams
and Cory, 1998; Antonsson and Martinou, 2000; Gross et aL, 1999a).
The activity o f these proteins appears to be regulated, at least partly, by formation of
homo- and hetero-complexes. Heterodimerization between pro-apoptotic (like the Bcl-2
associated x protein, better known as Bax) and anti-apoptotic Bcl-2 family members
(such as Bcl-2 and B c1 -X l) is a common feature (Borner et aL, 1994; Knudson and
Korsmeyer, 1997; Yin et aL, 1994). In the pro-apoptotic proteins Bax and Bak, the BH3
domain is o f central importance in mediating such protein-protein interactions and
modulating cell death (Chittenden et aL, 1995; Hunter and Parslow, 1996; Kelekar and
Thompson, 1998; Wang et aL, 1996b; Zha et aL, 1997).
Heterodimerization may titrate one another’s function depending on their relative
concentration (Boise et aL, 1993; Oltvai et aL, 1993; Sedlak et aL, 1995).
Heterodimerization is achieved when the BH3 domain o f one molecule binds into a
hydrophobic pocket formed by the B H l, BH2, and BH3 domains o f another family
members (Sattler et aL, 1997).
35
Table 2. Regulation of apoptosis by the Bcl-2 family proteins.
Bcl-2 family members Effects1. Bcl-2 subfamily:
Bcl-2 Anti-apoptotic (promotes survival)Bcl-XL Anti-apoptotic (promotes survival)Bcl-xs Pro-apoptotic (promotes death)Bcl-w Anti-apoptotic (promotes survival)
2. Bax family:Bax Pro-apoptotic (promotes death)Bak Pro-apoptotic (promotes death)Bok Pro-apoptotic (promotes death)
3. BH3 subfamily:Bid Pro-apoptotic (promotes death)Bad Pro-apoptotic (promotes death)Bik Pro-apoptotic (promotes death)Blk Pro-apoptotic (promotes death)
Bcl-2 family members may directly regulate caspases via adaptor molecules or they may
do so indirectly by modulating mitochondrial ftinction (Chau et aL, 2000; Ng et aL, 1997;
Zhang et aL, 2000b).
Mounting evidence points to Bax and other pro-apoptotic family members as the central
regulators o f the release o f cytochrome c from mitochondrial intermembranous space.
Although overexpression o f Bax in cells or the addition o f purified recombinant Bax
directly to mitochondria triggers the release o f cytochrome c, the exact mechanism
through which Bax triggers the permeability o f the outer mitochondrial membrane is
unclear (Jurgensmeier et aL, 1998; Rosse et aL, 1998).
The latter may be achieved through the interaction between pro-apoptotic Bcl-2 family
proteins (including Bax and Bak) with other mitochondrial membrane proteins (like
36
VDAC and ANT o f the MPTP). Although evidence for this occurring in vivo is still
lacking, whatever the mechanism is, the outcome is to generate a pore in the outer
mitochondrial membrane for cytochrome c release (Eskes et ah, 1998), a process
inhibited by Bcl-xL (Finucane et aL, 1999). Alternatively, Bax could stimulate
cytochrome c release indirectly by altering mitochondrial homeostasis (for example, ion
exchange or oxidative phosphorylation), or even to form a weakly selective ion channels
(Jurgensmeier et aL, 1998; Narita et aL, 1998; Reed, 1997; Reed et aL, 1998; Shimizu et
a/., 1999).
As outlined in Figure 1, the BH3-domain-only proapoptotic protein Bid exists in the
cytosolic fraction o f living cells as an inactive precursor that becomes activated upon
cleavage by caspase-8. Such cleavage occurs at internal Asp-sites and generates the
active 15-kDa COOH-fragment o f Bid, termed “truncated” Bid (t-Bid), which still
contains the BH3 domain (Wang et aL, 1996b). After cleavage, t-Bid translocates onto
the mitochondria where it also induces the release o f cytochrome c and leading to the
activation o f caspase-9 followed by caspase-3 (Li et aL, 1998; Luo et aL, 1998;
McDonnell et aL, 1999). This process also seems to be regulated by BcL-xL (Gross et
aL, 1999b).
Bid initiates the release o f cytochrome c apparently without evoking gross mitochondrial
swelling or permeability transition. This suggests that the loss o f mitochondrial
membrane potential is not the cause for Bid-mediated cytochrome c release (Luo et aL,
1998).
Bid may induce a conformational change in Bax favouring the translocation o f cytosolic
Bax to the mitochondria (Desagher et aL, 1999). As shown in Figure 1, it has recently
37
been reported that following binding to Bid, Bax oligomerizes and then integrates in the
outer mitochondrial membrane where it forms a pore o f sufficient size to enable the
release o f cytochrome c (Eskes et aL, 2000).
While oligomers o f Bax (-140-160 kDa) formed channels in liposomes and triggered
cytochrome c release from mitochondria, monomeric Bax (-22-24 kDa) was inactive in
both respects. This suggests that it is only oligomeric Bax that possesses such channel-
forming activity whereas monomeric Bax has no such activity (Antonsson et aL, 2000).
Recent studies showing the capability o f Bax to trigger the release o f cytochrome c from
liposomes estimated the quaternary structure o f the cytochrome c-conducting Bax-
channels to be a tetramer (Saito et aL, 2000). Oligomers o f Bax, which possibly form
complexes with yet unidentified mitochondrial proteins, were found inserted in the outer
mitochondrial membrane. Also, it was demonstrated that in the presence o f BcL-2, Bax
oligomer formation and insertion into the mitochondrial membrane was inhibited
(Antonsson et aL, 2001).
38
BH4 BH3 BHl BH2 TM
Group I Bcl-2
Group II Bax
Group III Bid
Figure 7. The Bcl-2 family members. Three subfamilies (groups I-III) are indicated. BH l to
BH4 are conserved sequence motifs. Members o f group I (like Bcl-2 and B c1-xl) possess the four
domains (BH1-BH4) as well as the C-terminal hydrophobic tail (TM) which localises the proteins
to the outer surface o f the mitochondria with the bulk o f the proteins facing the cytosol. Proteins in
this group are anti-apoptotic. Members o f group II and III are pro-apoptotic. Members o f group II
which includes Bax and Bak are structurally similar to group I but lack the N-terminal BH4
domain. Members o f group III (such as Bid and Bik) share the presence o f the (-12-16 amino
acid) BH3 domain.
39
1.1.9 Other inhibitors and regulators of apoptosis
As described in previous section, members o f the Bcl-2 family play a major regulatory
role in apoptosis through modulating cytochrome c release from the mitochondria. This,
however, is by no means the only way to control apoptosis as other proteins have been
shown to regulate the apoptotic process by targeting the caspases (Nicholson, 2000).
Caspase modulation is indeed highly sophisticated, and little is known about the
regulation o f the interaction between procaspases and their cofactors. Consider, for
instance, the discovery o f cellular FADD-like ICE inhibitory proteins (c-FLIP) (Irmler et
ah, 1997; Shu et aL, 1997). These proteins are similar in sequence to procaspase-8,
except that they lack essential catalytic residues. As shown in Figure 1, these proteins
probably compete with procaspase-8 for binding to its cofactor, F ADD, thus preventing
caspase activation (Koseki et aL, 1998).
Inhibitors and regulators o f apoptosis may also target the complex proteolytic caspases.
Identification o f caspase inhibitors has come out o f work on viruses that attenuate
apoptosis to circumvent the normal host immune response to infection. Three distinct
classes o f viral inhibitors have been described: the cowpox virus CrmA (Ray et aL,
1992a), the baculovirus protein p35 (Bump et aL, 1995; Xue and Horvitz, 1995), and a
family o f endogenous cellular inhibitors o f apoptosis (LAPs) (Uren et aL, 1998).
Currently, five members (c-IAP-1, c-IAP-2, XIAP, survivin, and NAIP) o f the lAPs
family o f proteins have been identified in humans and at least three o f these have been
40
shown to directly inhibit specific caspases. Members o f the lAPs family o f proteins are
the only endogenous inhibitors o f caspases known in mammals (Deveraux et aL, 1999).
The precise caspase targets o f the lAPs remain elusive. Potent selective inhibition o f
caspase-3 and -7 was observed in vitro with XIAP (Deveraux et aL, 1997) suggesting that
lAPs inhibit apoptosis through inhibition o f effector caspases (Figure 1). The story is not
so simple, however, because lAPs also prevent the activation o f these enzymes upon
overexpression, suggesting that effector caspase proenzymes or other proteins in the
activation complex are the real targets in cells (Deveraux et aL, 1998; Seshagiri and
Miller, 1997).
Although the lAPs do not inhibit caspase-1, -6, -8, or -10 (Deveraux et aL, 1997), they
are however capable o f binding to inactive procaspase-9 and interfering with its
processing (Deveraux et aL, 1998; Takahashi et aL, 1998).
As shown in Figure 1, both death receptor and mitochondrial pathways converge at the
level o f caspase-3 activation, and activity o f the latter is antagonised by lAP proteins
(Deveraux et aL, 1997) which are in turn antagonised by the Smac/DIABLO protein
released from the mitochondria (Du et aL, 2000; Verhagen et aL, 2000). This adds an
extra dimension to the already complex role o f mitochondria in apoptosis and suggests
that mitochondria may even be as important in regulating the apoptotic process as they
are in activating it.
41
1.1.10 Physiological role of apoptosis
In biology, it might seem illogical to think o f cell death as a component o f normal growth
and morphogenesis, although it is recognised that loss o f a tadpole’s tail, which is
mediated by genetically programmed cell death, is part o f the metamorphosis o f a frog
(Steller, 1995; Tata, 1966). The self-destructive process o f apoptosis is considered by
biologists to play four main physiological roles (Wang and Wang, 1999):
(1) Sculpting the body: Embryologists view the massive loss o f cells through PCD or
apoptosis, or both as the chisel used to sculpt the body during embryogenesis,
metamorphosis, and adult life across species (Wang and Wang, 1999). Examples
include the elimination o f surplus cells to allow for the formation o f the digits o f the
limbs (Garcia Martinez et aL, 1993), the hollowing o f solid structures to create
lumina (Jacobson et aL, 1997), the disappearance o f the human tail (Fallon and
Simandl, 1978), and the elimination o f endometrial cells at the end o f each menstrual
cycle (Otsuki et aL, 1994).
(2) Developmental plasticity and selection o f the fittest cells as seen in brain
development (Oppenheim, 1991) and immune system development (Healy and
Goodnow, 1998).
(3) Homeostasis: Examples include the ovarian follicle development from the foetal
period through reproductive ages (Kaipia and Hsueh, 1997), the renewal o f
endometrium during each menstrual cycle (Tabibzadeh, 1996), and the
haemotopoietic system and its default apoptotic death in the absence o f survival
42
cytokines (McKenna and Cotter, 1997). The latter constitutes the “social control
hypothesis” proposed by Raff (Raff, 1992).
(4) Protection o f organisms from threats created by their own deleterious cells: As in the
“better dead than wrong” maxim pointed out by Cohen et al (Cohen et aL, 1992b), the
purpose o f negative selection is to eliminate autoreactive cells that bear self-reactive
T-cell receptors to avoid autoimmune response (Healy and Goodnow, 1998). In
addition, genomic damage resulting from insults such as ultraviolet (UV) or ionising
radiation can trigger apoptosis (mainly through p53-dependent pathways) in genome-
damaged cells, thereby safeguarding against potential tumorogenesis (Wang and
Wang, 1996). Also, organisms use apoptosis to delete unwanted, injured or virus-
infected cells (Peter et aL, 1997).
1.1.11 Apoptosis and pathology
As homeostasis o f cell numbers depends on the careful balance between proliferation and
apoptosis, dysregulation o f cell death by either deficiency or excess o f apoptosis may
result in a wide range o f pathological conditions including cancer, neurodegenerative
disorders, viral infections (including acquired immunodeficiency syndrome AIDS),
autoimmune diseases (McKenna and Cotter, 1997; Thompson, 1995), and liver diseases
(Neuman, 2001).
There is substantial evidence that failure to activate apoptosis after DNA injury may be
one route to carcinogenesis (Griffiths et aL, 1997; Lowe et aL, 1994; Lyons and Clarke,
43
1997; Wyllie, 1997a), which may provide a credible explanation for the frequency with
which pro-apoptotic regulatory genes such as p53 are deleted in the majority o f human
cancers (Wang and Wang, 1996; Wyllie, 1997b). Also there is accumulating evidence
that some tumour cells can escape immune detection by expressing Fas ligand on their
surface and so activating apoptosis o f Fas-bearing cytotoxic T cells (Strand et aL, 1996a).
Excessive apoptosis is also observed in many chronic neurodegenerative disorders
(Bredesen, 1995) such as Alzheimer’s disease (Kim et aL, 1997), Parkinson’s disease
(Jenner and Olanow, 1996), Huntington’s disease (Yuan and Yankner, 2000); and
amyotrophic lateral sclerosis (Friedlander et aL, 1997; Kostic et aL, 1997).
Viruses, for their own proliferative advantage, may inhibit apoptosis o f host cells
(Roulston et aL, 1999; Young et aL, 1997) through viral anti-apoptotic proteins which
include EIB-19k from adenoviruses (Debbas and White, 1993), BHRFl from Epstein
Barr Virus (Henderson et aL, 1993), CrmA from cowpox virus (Tewari et aL, 1995) and
the FLICE-inhibitory proteins (FLIPs) from kaposis’s sarcoma-associated human herepes
virus-8 (Thome et aL, 1997) and human molluscum contagiosum virus (Berlin et aL,
1997).
On the other hand, CD4-T cell depletion seen in AIDS patients probably relates to the
upregulation o f FasL in these cells and therefore indicates that Fas (CD95/Apol)
signalling pathway plays a vital role in the pathogenesis o f AIDS (Aupeix et aL, 1997;
Thompson, 1995).
As mentioned previously, apoptosis is essential for removal o f autoreactive lymphocytes
(autoimmune cells) both during development and after completion o f an immune
44
response (Thompson, 1995). Failure o f this very delicate process can result in a variety
o f autoimmune diseases (Benoist and Mathis, 1997; Cohen et aL, 1992b; Drappa et aL,
1996).
Evidence is also accumulating that apoptosis is a significant part o f many pathological
processes that have long been considered to be necrosis-dependent, such as stroke
(Uyama et aL, 1992) and myocardial infarction (Olivetti et aL, 1997; Olivetti et aL,
1996).
From a toxicological point o f view, exposure to a wide range o f chemicals, biological
agents, and various environmental contaminants can result in cell damage and apoptosis
(Robertson and Orrenius, 2000).
45
1.2 Apoptosis and the liver
1.2.1 Structure of the liver
The liver is a large organ making up about 3.5% o f the body weight o f an adult rat or 2%
o f the body weight o f an adult human. Approximately 80% o f the blood supply o f the
liver is derived from the portal vein, and the remaining 20% comes from the hepatic
artery. The bulk o f the liver is composed o f a single cell type, the hepatocyte (Hinton and
Grasso, 2000).
Hepatocytes form approximately 90%of the volume o f the liver parenchyma but only
constitute 60% o f the total cell number. Hepatocytes are assembled into sheets, each a
single cell thick, which bifurcate and fuse to give a very complex network. Through this
network run the liver capillaries, termed sinusoids (Figure 8A). Sinusoids are lined by
sinusoidal endothelial cells (SECs). Unlike the endothelia o f normal capillaries, SECs do
not form a continuous barrier but are penetrated by fenestrations about 100 nm in
diameter which act like a sieve preventing blood cells from direct contact with the
hepatocytes. However, these fenestrations will allow a free exchange o f proteins between
the blood within the sinusoids and the “Space o f Disse” which lies between the SECs and
the adjacent hepatocytes (Figure 8A). The extracellular matrix o f the space o f Disse is
maintained by the modified fibroblasts and fat storing Stellate (Ito) cells (Hinton and
Grasso, 2000).
Within the sinusoids lie Kupffer cells which are fixed macrophages which form
attachments both to the walls o f SECs and, by means o f processes pushed through the
fenestrations, to the hepatocytes (Hinton and Grasso, 2000).
46
I K
lOum
Figure 8A. Architecture of the liver. Diagram showing the structural relationship
between Hepatocytes (Hep), Space o f Disse (SOD), Sinusoidal endothelial cells (SEC),
Sinusoids (S), Hepatic arteries (HA), Portal veins (PV), Bile ducts (BD), and Bile
canaliculi (BC). Bile duct lining cells contact hepatocytes directly, the junction zone
being termed the canal o f Hering (H). Adopted (with permission) from Hinton and
Grasso (Hinton and Grasso, 2000).
47
1.2.2 Apoptosis and liver disease
Even before the term apoptosis was described by Kerr et al in 1972 (Kerr et ah, 1972),
the morphological appearance o f this mode o f cell death in the liver had long been
referred to as acidophilic or councilman bodies (Biava and Mukhlova, 1965; Klion and
Schaffiier, 1966).
Often, liver disease is either associated with enhanced hepatocyte apoptosis (as seen in
cholestatic disease, viral and autoimmune hepatitis, and metabolic disorders) or
disruption o f apoptosis such as in hepatocellular carcinoma (Neuman, 2001).
Use and abuse o f certain drugs, especially alcohol and paracetamol, have also been
associated with increased apoptosis and liver damage (Rust and Gores, 2000).
1.2.2.1 Cholestatic liver diseases (CLD)
1.2.2.1.1 General
Cholestasis, defined as impairment o f bile flow, is observed in a variety o f human liver
diseases, occurring in primary biliary cirrhosis (PBC), Primary sclerosing cholangitis
(PSC), biliary atresia, allograft rejection, graft versus host disease (GVHD), and drug
related liver diseases. CLD, which results from destruction o f bile duct epithelial cells
(chlangiocytes), is better termed “cholangiopathies”. In cholagiopathies, the initial insult
results in cholagiocyte apoptosis, which is likely to be CTL-mediated. Cholangiocyte
48
apoptosis results in retention and accumulation o f hydrophobic, toxic bile salts within the
hepatocytes, which leads to bile salt-mediated apoptosis o f hepatocytes. Thus, apoptosis
o f both cell types (cholangiocytes and hepatocytes) contributes to liver dysfiinction in
cholangiopathies. Toxic bile salts induce hepatocyte apoptosis by direct activation o f the
Fas death receptor pathway (Faubion et aL, 1999; Miyoshi et aL, 1999).
Glycochenodeoxycholate (GCDC), a toxic bile salt, induces hepatocyte apoptosis in a
ligand-independent activation o f Fas by GCDC, which subsequently incorporates
caspase-8 and eventually activating effector proteases, including downstream caspases
and cathepsin B (Faubion et aL, 1999; Patel et aL, 1994b; Roberts et aL, 1997; Spivey et
aL, 1993). GCDC-induced hepatocyte apoptosis also appears to incorporate protein
kinase C (PKC) which mediates the increase in intracellular magnesium concentration
and activation o f Ca^^/Mg^^-dependent endonucleases (Patel et aL, 1994a).
Ursodeoxycholate (UDCA), a hydrophilic bile salt o f confirmed value in the treatment o f
cholangiopathies, has an anti-apoptotic cytoprotective properties (Beuers et aL, 1998;
Guicciardi and Gores, 1998; Rodrigues et aL, 1998).
Both in intact hepatocytes and cholangiocytes, UDCA has been shown to have a direct
cytoprotective effect. Such an effect is mediated by preventing mitochondrial membrane
permeability transition (MPT), inhibiting the production o f ROS, and interrupting the
release o f cytochrome c during pro-apoptotic stimuli induced by GCDC (Botla et aL,
1995; Que et aL, 1999; Rodrigues et aL, 1998; Rodrigues et aL, 1999) and
chenodeoxycholate (CDC) (Neuman et aL, 1999b).
49
1.2.2.1.2 Primary biliary cirrhosis and primary sclerosing cholangitis
Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by
progressive destruction o f intrahepatic small bile ducts. Primary sclerosing cholangitis
(PSC) is a chronic inflammatory process associated with fibrosis o f intrahepatic and
extrahepatic bile ducts (Neuman, 2001).
In PBC, cholangiocytes exhibit both necrosis and apoptosis (Bemuau et aL, 1981).
Infiltrating lymphocytes (CTL) may cause apoptotic death o f cholangiocytes through
Fas/FasL and Perforin/granzyme B-dependent mechanisms (Harada et aL, 1997).
In humans, the administration o f UDCA slows disease progression in PBC and improves
liver function in both PBC and PSC (Poupon et aL, 1987; Rubin et aL, 1994). In addition
to its direct cytoprotective effect (as discussed above) in cholangiopathies, UDCA may as
well act indirectly through reducing the terminal ileal absorption o f toxic/hydrophobic
bile acids (Beuers et aL, 1998).
It has been shown that UDCA, when given to patients with PBC, significantly decreases
DNA fragmentation (Koga et aL, 1997) and modulates the expression o f Fas /FasL
(Neuman et aL, 1999a).
In both PBC and PSC, endotoxin (like LPS) accumulates abnormally in cholangiocytes
and leads to apoptosis. Therefore, UDCA treatment in PBC may provide a beneficial
effect on the intrahepatic metabolism o f endotoxins (Hamada et aL, 1999; Sasatomi et aL,
1998).
50
1.2.2.1.3 Obstructive jaundice
Bile duct ligation has long been used as an animal model for obstructive jaundice.
Although Bcl-2 is not expressed in hepatocytes under physiological conditions, it was
expressed in hepatocytes during chronic cholestasis in bile duct-ligated rat. This may
represent an adaptive mechanism to inhibit apoptosis by toxic bile salts (Kurosawa et ah,
1997). In contrast, cholangiocytes, which are in direct contact with bile, do express Bcl-
2. It was demonstrated that apoptosis o f cholangiocytes in bile duct-ligated rats may be
regulated by the interaction o f Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) (Stahelin et
a l , 1999).
1.2.2.2 Apoptosis and viral hepatitis
A variety o f acute and chronic viral diseases, including hepatitis A, hepatitis B, and
hepatitis C affect the liver. Even though the involvement o f apoptosis in acute viral
hepatitis is not well defined, the identification o f apoptotic (councilman) bodies is a
characteristic feature o f acute viral hepatitis. Fas protein expression was found to be
enhanced in some patients with fulminant hepatitis B virus infection, suggesting the
involvement o f Fas-mediated apoptosis in acute hepatitis (Rivero et aL, 1998).
On the other hand, apoptosis plays an important role in chronic viral hepatitis (hepatitis B
and C) and is believed to be mediated by the host cytotoxic T-lymphocytes (CTL). Both
the Fas-Fas ligand as well as the perforin/granzyme B systems have been implicated in
this CTL-mediated apoptosis o f virus-infected hepatocytes (Kagi et aL, 1994; Lowin et
aL, 1994;Nakamoto e ta l , 1997; Shrestac/a/., 1998).
51
Hepatitis C infection induces apoptosis via the TNF-a signaling pathway (Zhu et ah,
1998). Plasma TNF-a levels are raised in chronic hepatitis B and C-infected patients
(Lim et aL, 1994; Sheron et aL, 1991). Despite the fact that the expression o f TNF-a and
TNF-a receptors is increased in patients with chronic hepatitis B infection (Fang et aL,
1996), only minimal apoptosis was observed when TNF-a was used to treat chronic
hepatitis B virus infection (Sheron et aL, 1990). In rat hepatocytes cell lines, TN F-a-
induced extensive apoptosis only in those hepatocytes expressing high levels o f hepatitis
B virus (Guilhot et aL, 1996). This suggests that viral components, like hepatitis B virus
X (HBx) antigen and hepatitis c virus core protein, may play a role in sensitizing
hepatocytes to the TNF-a and promoting apoptosis (Kim et aL, 1998; Zhu et aL, 1998).
Sometimes, and acting in a selfish manner, viruses attempt to prevent removal o f virally
infected hepatocytes in order to complete their replication, and therefore may develop a
mechanism to block apoptosis (Fujita et aL, 1996).
The expression o f TGF-(31 appears to be upregulated by HBx antigen in chronic hepatitis
B infection (Castilla et aL, 1991; Yoo er aL, 1996) and its serum levels are also increased
in chronic hepatitis C infection (Nelson et aL, 1997). Depending on the physiological
status o f the cells, HBx antigen can be pro- or anti- apoptotic after interacting with the
tumour suppressor and pro-apoptotic gene the P53, and occasionally HBx can directly
induce apoptosis in a p53-independent pattern (Chirillo et aL, 1997; Puisieux et aL, 1995;
Terradillos et aL, 1998; Wang et aL, 1995).
52
1.2.2.3 Apoptosis and tumours of the liver
Currently it is believed that “carcinogenesis” involves sequential changes in the
phenotype from normal to malignant cells and occurs as a multi-step process that
includes initiation, promotion, and metastasis. Many o f these steps can be viewed as a
failure o f apoptosis (Que and Gores, 1997).
The available evidence indeed demonstrates disruption o f apoptosis at several steps in the
development o f hepatocellular carcinoma (HCC). Analysis o f 22 HCC patients revealed
a partial or complete loss o f Fas receptor (Fas) which is normally expressed constitutively
in hepatocytes. It is presumed that loss o f Fas expression is one means through which
malignant cells escape the Fas-mediated killing by cytotoxic T-lymphocytes (Higaki et
ah, 1996).
Another theory (called “Fas counterattack”) through which cells o f HCC can evade
immune destruction has been proposed. According to this theory, HCC cells express Fas
ligand (FasL) and induce apoptosis o f activated Fas" ^ T lymphocytes (Strand et ah,
1996b).
As TGF-pi plays a major role in induction o f apoptosis and in regulating liver size,
disruption o f TGF-pi signal may contribute to the development o f HCC by preventing
apoptosis in target cells (Thorgeirsson et al., 1998). Cells o f HCC are usually resistant to
apoptosis by TGF-pl, but often secrete TGF-pl to induce apoptosis o f neighbouring
hepatocytes in a paracrine mechanism (called tumour directed apoptosis o f adjacent
benign tissue) aimed to achieve non-policed tumour expansion and spread within the
liver. Those results also suggest that TGF-pl plays an important role in the altered
53
metabolism o f collagen in HCC (Murawaki et ah, 1996; Shirai et ah, 1994).
Occasionally, this paracrine mechanism o f producing TGF-(31-mediated apoptosis o f
neighbouring hepatocytes may as well be adopted by activated Stellate (Ito) cells in
alcoholic liver disease (Bissell et ah, 1995; Gressner et ah, 1996).
On the other hand, all human hepatoblastomas (like many other tumour cells) co-express
both Fas and its ligand (FasL) on their surface. It has always remained a mystery why
such cells do not simply kill themselves. This was attributed to the existence o f “Fas-
resistance pathways” which may include the expression o f Bcl-2 and Fas-associated
phosphatase-1 (FAP-1), and the expression o f soluble Fas (sFas). Hepatoblastomas even
express some o f these Fas-mediated apoptosis inhibitors (like FAP-1) in their tumour
cells. These results suggest that it is probably due to the action o f inhibitory molecules o f
the Fas pathway that the tumour cells o f hepatoblastomas do not kill themselves in an
autocrine-driven cycle and that in this manner hepatoblastomas avoid apoptosis and thus
proliferate (Lee et ah, 1999)
1.2.2.4 Apoptosis and Wilson’s disease
Wilson’s disease (WD) is a congenital (autosomal recessive) disorder o f copper
metabolism that is due to a defect in the gene coding for a copper transporting P-type
ATPase (Bull et ah, 1993; Tanzi et ah, 1993). Disturbed export o f copper from
hepatocytes to bile and decreased incorporation o f copper into ceruloplasmin results in
accumulation o f copper in a number o f organs and particularly the liver and brain
(Stremmel et ah, 1991).
54
Copper accumulation results in the generation o f ROS and oxidative stress o f the liver.
Therefore, ROS may be a major contributor to apoptosis seen in WD (Jacobson, 1996;
Mansouri et ah, 1997).
The demonstration o f high Fas protein expression on hepatocytes o f patients with
fulminant hepatic failure due to WD, combined with the appearance o f Fas ligand mRNA
in the cytoplasm o f other adjacent hepatocytes suggest that, in WD, hepatocytes kill each
other in a paracrine (or autocrine) pattern termed “fratricidal killing” (Strand et ah, 1998).
1.2.2.5 Apoptosis and drug-induced liver disease
Several hepatotoxins such as alcohol (Benedetti et ah, 1988; Goldin et ah, 1993;
Kawahara et ah, 1994; Patel and Gores, 1995), carbon tetrachloride (LeSage et ah,
1999a; LeSage et ah, 1999b), paracetamol (Ray et ah, 1993), cocaine (Cascales et ah,
1994), or dimethylnitrosamine (Ray et ah, 1992b) have been shown to induce DNA
fragmentation in vitro and the formation o f apoptotic bodies has been observed after
treatment o f animals with cocaine, dimethylnitrosamine, D-galactosamine, lead, or
thioacetamide (Feldmann, 1997).
1.2.2.5.1 Alcoholic liver disease (ALD)
Although chronic alcohol consumption has been shown to cause hepatocyte apoptosis,
the exact pathophysiology o f apoptosis in ALD is still unclear (Slomiany et ah, 1999).
However, several mechanisms seem to be involved in the alcohol-induced apoptosis o f
55
hepatocytes. Experimental evidence has linked alcohol-induced liver injury to oxidative
stress caused by the CYP2E1-dependent production o f reactive oxygen species (ROS)
and lipid peroxides (Higuchi et al., 1996; Kurose et al., 1997). The associated increase in
the anti-apoptotic Bcl-2 protein concentration in rats with experimental ALD may reflect
an adaptive defensive mechanism against ALD (Yacoub et al., 1995).
Oxidation of alcohol by alcohol dehydrogenase generates acetaldehyde, which is capable
o f inducing apoptosis (Zimmerman et ah, 1995). Oxidation o f alcohol also results in
alteration o f the cell redox potential and the generation o f free radicals (Rust and Gores,
2000). Prolonged glutathione (y-glutamyl-cysteinyl-glycine, better known as GSH)
consumption chronically decreases mitochondrial GSH and sensitizes mitochondria to
MPT and apoptosis (Colell et al., 1998; Pastorino et al., 1999).
Glutathione depletion, however, may have dual effect on apoptosis. While prolonged
depletion o f glutathione results in increased death receptor-mediated hepatocyte
apoptosis (Chiba et al., 1996; Colell et al., 1998; Xu et al., 1998), its acute depletion is
associated with decreased apoptosis (Hentze et al., 2000; Hentze et al., 1999; Lawson et
a/., 1999).
The combination o f chronic depletion o f hepatocyte glutathione reserves (both cytosolic
and mitochondrial) and hepatic iron overload catalyse the formation o f free radicals and
enhance oxidative stress on the liver (Garcia-Ruiz et ah, 1994; Sadrzadeh et al., 1994).
The demonstration o f increased TNF-a activity in patients with ALD (and in ethanol-fed
rats), and the increase in the number o f TNF-a receptors 1 (TNF-Rl) on hepatocytes after
chronic ethanol administration, may suggest the direct involvement o f TNF-a in
56
mediating apoptosis in ALD (Deaciuc et ah, 1995; Leist et ah, 1997a; McClain et ah,
1993; Nanji et ah, 1994; Spengler et al., 1996). Pastorino and Hoek have reported that
TNF-a-induced cytotoxicity is potentiated by ethanol exposure in HepG2, and in rat
primary hepatocytes (Pastorino and Hoek, 2000). The rapid rise in intracellular levels o f
ROS seen after stimulation o f TNT receptors may indicate another, but indirect, role o f
TNF-a in apoptosis in ALD (Larrick and Wright, 1990).
In normal human primary hepatocytes, ethanol signaling for apoptosis is initiated by Fas
ligand (Neuman et al., 1999c). Exposure o f hepatocytes to ethanol generates ROS that
induce the expression o f Fas ligand, and because hepatocytes also express the Fas
receptor, this suggests that hepatocytes might mediate their own death in a fratricidal
pattern (Galle et al., 1995; Hug et al., 1997; Nanji, 1998).
TGF-pl, produced by Kupffer and hepatic Stellate (Ito) cells in ALD, may have a double
impact on the progression o f hepatic injury: promoting fibrogenesis and killing
hepatocytes by apoptosis (Gressner et al., 1996; Oberhammer et al., 1993; Oberhammer
et al., 1992a).
The capability o f UDCA (ursodeoxycholic acid) and TUDCA (tauroursodeoxycholic
acid) to protect Hep G2 cells from ethanol-induced apoptosis has been attributed to their
capability to decrease (or abolish) the glutathione depletion seen in ALD (Neuman et al.,
1998).
57
1.2.2.5.2 Paracetamol and hepatotoxicity
Since its introduction in the mid-1950s, paracetamol (acetaminophen, Y-acetyl-p-
aminophenol, AAP) has been used as an over-the-counter analgesic and antipyretic.
However, when taken in an overdose it is hepatotoxic. Hepatic necrosis resulting from
massive overdosage with paracetamol was first reported in rats by Boyd and Bereczky in
1966 (Boyd and Bereczky, 1966), and a report o f this complication in man soon followed
(Davidson and Eastham, 1966; Prescott et al., 1971).
In the UK alone, paracetamol overdoses cause 200 death per year. Paracetamol-induced
hepatic damage is the most common form o f toxic liver damage experienced in clinical
practice in the UK and USA (Meredith and Vale, 1984).
It is well documented that under light microscopy, AAP-induced hepatotoxicity in mice
is characterised by hepatomegaly and massive centrilobular congestion which precede the
appearance o f necrosis. The initial increase in liver size is attributed to result from
plasma accumulation due to endocytic vacuolation o f hepatocytes and Disse space
enlargement in centrilobular regions. These events occur without any increase in
intrahepatic or portal venous pressure. There are two major consequences o f
acetaminophen-induced hepatotoxic congestion. First, blood and plasma volumes fall
significantly, and it was suggested that hypovolaemic shock contributes to early mortality
after acetaminophen. Second, impaired circulation within the congested liver probably
aggravates the initial injury. Early lesions are almost always evenly distributed around
central veins. However, the pattern o f damage after 24 hours o f toxicity could be
variable. Occasionally, large confluent areas o f congestion and necrosis were observed.
58
which is consistent with the concept that secondary ischaemic damage can develop in
AAP-induced hapatotoxicity (Walker et al., 1985).
In 1975, Dixon, et al, carried out an in-depth examination (by light and electron
microscopy) o f livers from rats killed at various time-intervals (up to 48 hr) after a single
large dose o f AAP (3g/kg). His results revealed glycogen depletion, loss o f ribosomes,
and cytoplasmic matrix swelling commencing 3-6 hr after AAP administration which, in
centrilobular hepatocytes, progressed to frank coagulative necrosis at 12-24 hr. Midzonal
cells showed more prominent aqueous swelling with vésiculation o f the endoplasmic
reticulum, and, in some cells, gross hydropic vacuolation (Dixon et ah, 1975).
The exact mechanism o f paracetamol-induced liver cell death is still controversial but,
until recently, it has been considered to be entirely the consequence o f its metabolism by
the cytochrome p-450 (CYP-450) system within the hepatocytes (Nelson, 1990).
Paracetamol, in therapeutic doses, is safely metabolized (and eliminated) by hepatocytes
to non-toxic conjugates o f sulphate and glucuronic acid. However, when the enzyme
systems involved in these processes become saturated, metabolism via CYP-450 systems
becomes increasingly important (Mitchell et al., 1973a).
As shown in Figure 8, CYP-450 converts AAP to the highly reactive electrophillic
metabolite A-acetyl-/7-benzoquinone-imine (NAPQI) by a pathway utilizing oxygen and
NADPH. NAPQI is o f interest as a proposed ultimate toxic metabolite in AAP overdose
(Hinson et al., 1981; follow et al., 1974).
The potentially toxic effect o f NAPQI is initially largely prevented via the conjugation
with intracellular reduced glutathione (GSH) before its renal excretion (Coles et al..
59
1988; Mitchell et oL, 1973b). However, excess NAPQI depletes the stores o f GSH
(Dahlin et al., 1984; Nelson, 1995) and the free NAPQI binds to cysteine-rich liver
proteins and key enzymes, particularly those controlling calcium homeostasis (Boobis et
ah, 1986). NAPQI also binds to the DNA (Moore et al., 1985) and, in addition, may
induce peroxidation o f plasma membrane lipid in the liver (Muriel et al., 1992;
Ozdemirler c/fl/., 1994).
Despite the fact that there are marked interspecies variations in susceptibility to
paracetamol (AAP), acute liver damage is relatively reproducible across a wide range o f
laboratory animals. Mice and hamsters are known to be more sensitive to AAP than rats,
guinea pigs, or rabbits (Davis et al., 1974; Liu et al., 1991).
As outlined in Figure 8, rapidly biotransformed AAP is predominantly excreted as
glucuronide and sulphate conjugates (Kane et al., 1995; Kim et al., 1995). However
marked interspecies differences were observed in the ratio o f the two conjugation
pathways. Rats were found to excrete 40-65% o f the AAP dose as sulphate conjugate
(Thomas et al., 1974), whereas other species, including mice, only excreted 4-14% o f the
dose as conjugate o f sulphate (Gregus et al., 1988; Whitehouse et al., 1977).
When HepG2 cells are exposed to 5mM acetaminophen plus 40 mM ethanol, there is a
synergistic induction o f CYP2EI and therefore o f apoptosis (Shear et al., 1995). In
chronic alcoholics, taking paracetamol in therapeutic doses can also cause liver damage
because ethanol induces the CYP-450 system and thereby leads to increased production
NAPQI (S eeffe/« /., 1986).
60
Centrilobular hepatic necrosis is a characteristic feature seen in paracetamol overdose
(Dixon et al., 1975). Recently, it has been hypothesized that a significant number o f
hepatocytes also die by apoptosis. Ray et al has even concluded that, in paracetamol-
induced liver cell injury, the majority o f hepatocytes predominantly choose to die via
programmed apoptotic pathway rather than facing the unprogrammed consequences o f
necrosis (Ray et al., 1996).
The irreversible inhibition o f mitochondrial respiration by NAPQI, and the reversible
inhibition by paracetamol results in massive drop in intracellular energy status which
negatively affects the cellular protective capacity (Burcham and Harman, 1991; Esterline
et ah, 1989; Ramsay et ah, 1989). Depletion o f glycogen and ATP, featured in
paracetamol-induced liver damage, are energy-linked processes dependent on
mitochondrial function. Therefore, it would not be surprising in the future to realize that
opening o f the MPT, disruption o f mitochondrial membrane potential, and the release o f
cytochrome c are all integral parts o f paracetamol-induced hepatotoxicity (Ray et ah,
1999).
As paracetamol is a notorious producer o f ROS, the production o f ROS and the induction
o f oxidative stress (or vice versa) have been proposed as a possible mechanism o f
paracetamol-induced hepatotoxicity (Wendel, 1983; Wendel, 1984; Wendel et ah, 1979;
Wendel and Hallbach, 1986; Wendel and Jaeschke, 1982; Wendel et ah, 1982).
In addition to being a mitochondrial poison, paracetamol can also target several other
intracellular organelles, e.g. nucleus and endoplasmic reticulum. The disruption o f
cellular Ca homeostasis after hepatotoxic paracetamol overdose in mice leads to
sustained elevation in intracellular and nuclear Ca^ levels and, consequently, the
61
activation o f Ca^^-dependent endonucleases leading to DNA fragmentation (Boobis et ah,
1990; Ray et ah, 1990; Ray et ah, 1991). The latter effect was abolished when mice were
pretreated with calcium antagonists like verapamil and chlorpromazine (Ray et ah, 1993).
In mouse liver the following Bcl-2 family are constitutively expressed: B C L -x l , B C L -x s ,
Bak, Bad, and Bax. In contrast, Bcl-2 is not expressed in mouse whole liver
homogenates (Patel et ah, 1998). However, western blot analysis done 6 and 18 hours
after the intraperitoneal administration o f paracetamol (500mg/kg) to male ICR mice
showed a shift to a higher molecular weight band o f the B C L -x l protein. Such a band
may represent a phosphorylated form o f B C L -x l (called B C L -x l -p ) . The appearance o f
B C L -x l -p was associated with massive apoptotic cell death. In the same time period, the
level o f expression o f B C L -x l and BCL-xs remained unaltered and BCL-2 remained
unexpressed (Ray and Jena, 2000).
Since B C L -xl is localised to the mitochondria (Gonzalez-Garcia et ah, 1994), it was
hypothesized that paracetamol-induced modification o f B C L -xl protein (possibly by
phosphorylation) points to the mitochondria as being one o f the main targets in
paracetamol-induced liver toxicity. Phosphorylation o f B C L -xl is presumed to result in
the inactivation o f B C L -xl protein, and thus the potentiation o f apoptotic cell death (Ray
and Jena, 2000).
The fact that human macrophages, when cultured with AAP, secrete increased quantities
o f the inflammatory mediator-TNF suggests that macrophages play a potentially
important direct role in paracetamol-induced liver damage (Goldin et ah, 1996).
62
In vivo studies in rats reported that modifying macrophage functional status using
macrophage inhibitors such as gadolinium chloride (GdClg) could alter the degree o f
hepatic injury induced by AAP; and these protective effects o f GdClg were attributed to
neither the alterations in AAP metabolism nor the suppression o f NAPQI formation
(Laskin et al., 1995). However these findings do not appear to be specific for AAP
because a number o f different studies have described similar results o f inhibiting
macrophage activity on hepatic injury induced by toxicants such as galactosamine, LPS,
carbon tetrachloride, allyl alcohol, and 1,2 dichlorobenzene (Arthur et al., 1985; Edwards
et al., 1993; elSisi et al., 1993; Gunawardhana et al., 1993; Przybocki et ah, 1992;
Shiratori et ah, 1988; Shiratori et ah, 1986).
63
NH—COCH UDPGA UDP
UDP-glucuronosyl transferase
OH
NH—C O C H 3
ParacetamolOQHgOg
Paracetamolglucuronide
(N
OH—N—COCHNH —COCH
3'-Phosphoadenosyl-5-phosphate
OH
N -hy droxyparacetamol Paracetamolsulphate
Rearrangem ent
I— COCHNH—COCHNH —COCH
Glutathione-S-Transferase Urinary
ExcretionGSHSCHoCHCOOHS-glutathione
OHOH
N-acetyl-p-benzoqu- inone imine [NAPQI]
Covalent binding to liver proteins
Hepatocellular death
GlutathioneConjugate
Paracetamol- mercapturic acid
Figure 8. Proposed metabolic activation of paracetamol to the toxic, reactive
intermediate NAPQI. Scheme depicting formation and decomposition pathways for
NAPQI.
64
Chapter 2
Materials and Methods
65
2.1 Materials
Reagents used in this study are listed according to the name o f the suppliers
Amersham International pic, Buckinghampshire, UK
Hybond C nitro-cellulose membrane (cat # RPN 203W) and polyoxyethelenesorbitan
(Tween-20).
Bachem, Bubendorf, Switzerland
Benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD-FMK; cat # N-1510),
Benzyloxycarbonyl-Asp-Glu-Val-Asp-chloromethylketone (Z-DEVD-CMK; cat # N-
1580), N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin (Ac-DEVD-AFC;
cat # 1-1725) and Ac-Leu-Glu-His-Asp-7-amido-4-trifluoromethylcoumarin (Ac-LEHD-
AFC; cat #1-1820).
Bio-Rad Laboratories Ltd, UK
Kaleidoscope prestained protein molecular weight standards (cat # 161-0324), Whatmann
3MM filter paper, Bio-Rad protein assay dye reagent concentrate (cat # 500-0002), and
Bio-Rad bovine y-globulin standards for protein assay (cat # 500-0005).
Boehringer Mannheim Ltd, Mannheim, Germany
Bovine serum albumin fraction V (BSA), nonidet p40 (10%^^Q, aprotinin, pepstatin,
pefabloc, leupeptin, HEPES, and Tris base.
66
British Drug House (BDH)/AnalaR Ltd, UK
Glycine, methanol, ethanol, industrial methylated spirit (IMS), sodium chloride, disodium
hydrogen orthophosphate (Na2HP0 4 ), sodium dihydrogen orthophosphate
( N a 2 H 2 P 0 4 .2 H 2 0 ) , potassium dihydrogen orthophosphate (K H 2 P O 4 ), magnesium
chloride (MgCh), potassium chloride (KCl), trichloroacetic acid (TCA), boric acid,
sodium malonate, sodium succinate, and [ethylene glycol-bis(P-aminoethyl ether)
N,N,N ,N ]-tetraacetic acid (EGTA).
Calhiochem-Novabiochem Ltd, Nottingham, UK
Rabbit anti-Caspase-3 polyclonal antibody (cat # 235412).
Camlab Ltd, Cambridge, UK
Biophenol/chloroform mixture (1:1 ratio) (stabilised).
DAKO Ltd, Cambridge, UK
Horseradish Peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin (cat #
P0447), HRP-conjugated rabbit anti goat immunoglobulin (cat # P0449), and HRP-
conjugated goat anti-rabbit immunoglobulin (cat # P0448).
Enzyme Systems Products, California, USA
The negative control Benzyloxycarbonyl-Phe-Ala-fluoromethylketone (Z-FA-FMK, cat #
Fk-029386).
67
Gibco-BRL, Paisley, Scotland, UK
Ultra-pure agarose.
Molecular Probes, Netherlands
Mouse monoclonal anti cytochrome oxidase subunit IV.
Oncor, Gaithersburg, MD, USA
ApopTag Direct kit.
Phone Merieux Limited, UK
Pentobarbitone sodium (60 mg/ml).
Pierce chemical company, Roclrford, USA
Supersignal enhanced chemiluminescent (ECL)-substrate (cat # 34080T).
Pharmacia Biotech, UK
Commassie brilliant blue (R250).
Pharmingen, San Diego, California, USA
Hamster anti-CD95 antibody (Jo2: cat # 1541 CD) and purified mouse anti cytochrome c
monoclonal antibody (lgG2b: cat # 65981A).
68
Premiere Brands, UK
Dried skimmed marvel milk (99% fat-free)
Promega corporation, UK
Lambda DNA/EcoRI Hindlll DNA markers (cat # G 1731) and Blue/Orange 6 x DNA
loading dye (cat #G1881).
R & D Systems, UK
Goat anti-human/mouse Bid polyclonal antibody (cat # AF860).
Roche, UK
Kits used for the estimation o f lactate dehydrogenase (LDH), alanine aminotransferase
(ALT), and aspartate aminotransferase (AST) were from Roche.
Santa Cruz Biotechnology, USA
Bax mouse monoclonal antibody (lgG2b: cat # Sc-7480).
Sigma-Aldrich Chemical Co. Ltd, UK
Acetaminophen (99% pure), gadolinium chloride, lipoplysaccharide, galactosamine, p-
iodonitrotetrazolium violet (INT), ethyl acetate, 40% acrylamide/bis-acrylamide (37:1
ratio), ethidium bromide, proteinase-K, RNase A, ^-glycerophosphate, ponceau S,
pyronin Y, D-mannitol, amino-4-trifluoromethylcoumarin (AFC), 2-{3-mercaptoethanol,
69
bromophenol blue, lauryl sarcosine, minimum essential medium (MEM), glycerol, lauryl
sulfate (SDS), magnesium chloride-hydrous (MgCE.bHiO), dexamethasone, and
ethylenediaminetetraacetic acid (EDTA), sulfosalicylicic acid, and N,N,N,N-
tetramethylethylenediamine (TEMED).
StressGen Biotechnologies Corp, Victoria, BC Canada
Rabbit anti-caspase-9 polyclonal antibody (cat # AAP-149).
TAAB Laboratories, UK
TAAB resin.
University o f Leicester, Leicester, UK
Cytochrome c was a gift from Dr SC Chow. Rabbit anti-caspase-7 polyclonal antibody
was a gift from Professor GM Cohen.
Zymed Company, UK
Mouse lgG2a monoclonal B C - X l primary antibody (cat # 33-6300)
70
2.2 Solutions and Buffers
2.2.1 General
Phosphate buffered saline (PBS):
137 mMNaCl, 2.7 mM KCl, 4.3 mM Na2HP0 4 , 1.4 mM KH2PO4, pH 7.4.
2.2.2 Caspase activity assay buffers
Hypotonic extraction buffer {HEB)\
25mM Hepes, 5mM MgCb, ImM EGTA, ImM pefabloc, Ipg/ml pepstatin, Ipg/ml
leupeptin and Ipg/ml aprotinin, pH adjusted to 7.5 (with NaOH).
Cell lysis buffer (CLB):
40mM (3-glycerophosphate, 50mM NaCl, 2mM MgCl2-6 H2 0 , 5mM EGTA, lOmM
Hepes, pH adjusted to 7.0 (with NaOH). The buffer was stored at 4°C and used within
one month o f preparing.
Caspase assay buffer (CAB):
lOOmM Hepes, 10% (w/v) sucrose, 0.1% (v/v) nonidet P-40, lOmM dithiothreitol, pH
adjusted to pH to 7.25 (with NaOH). The buffer was stored at 4°C and used within one
month o f preparing.
71
2.2.3 Agarose Gel Electrophoresis
Lysis buffer:
Û.1M Tris, lOmM EDTA, 0.5% (w/v) sodium N-lauroylsarcosine, adjust pH to 7.8 using
HCi.
Tris-EDTA, p H 8 {TE-8):
lOmM Tris base, ImM EDTA, pH 8.0.
lOx TBE (per litre):
890mM Tris base (108.0g), 890mM Boric acid (55.0g), 25mM EDTA (9.3g), pH 8.0.
Autoclave and store at +4°C.
Ix TBE (per litre):
Add 900mls o f Milli-Q water to lOOmls o f 10 x TBE.
Proteinase K (25 mg/ml):
125 mg was dissolved in 6 mis o f Milli-Q water and stored at -20°C in 300 pi aliquots.
RNase ^ (50 mg/ml):
50 mg was dissolved in 1 ml o f 10 mM Tris (pH 7.5) containing 15 mMNaCl. This was
then boiled for 15 minutes and stored at -20°C in lOOpl aliquots.
72
Ethidium Bromide (Carcinogenic 0):
lOmg/ml in Milli-Q water.
2.2.4 Succinate dehydrogenase (SDH) assay
MSH-EDTA buffer: 2 1 0 mM mannitol, 70mM sucrose, 3mM Hepes, and ImM EDTA,
pH 7.4 (with KOH). Buffer was stored at 2-8°C and was not used after four weeks o f
preparing.
Substrate: 0.3M sodium succinate, pH 7.4
Inhibitor: 0.3M sodium malonate, pH 7.4
Chromogen: 1.5 mg/ml o f p-iodonitrotetrazolium violet (INT) freshly dissolved in
0.3M o f sodium dihydrogen orthophosphate (Na2H2? 0 4 .2 H2 0 ) buffer, pH 7.4
Stopping Reagent: 6 % (w/v) o f Trichloroacetic Acid (TCA)
2.2.5 SDS-PAGE and Western Blotting
Homogenisation buffer {HB)\
210mM mannitol, 70mM sucrose, 3mM Hepes, and ImM EDTA, ImM pefabloc, Ipg/ml
pepstatin, Ipg/ml leupeptin and Ipg/ml aprotinin, pH adjusted to 7.1 (with KOH). Buffer
was stored at 2-8°C and was not used after four weeks o f preparing.
73
2x Resolving Gel buffer (AX2) (per litre):
750mM Tris (90.6g) were dissolved in 800ml o f Milli-Q water. Then the pH was
adjusted to 8 .8 with concentrated HCI and then made up to a final volume o f IL and
stored at 2-8°C.
2x Stacking Gel buffer (BX2) (per litre):
250mM Tris (30.24g) were dissolved in 800ml o f Milli-Q water. Then the pH was
adjusted to 6 .8 with concentrated HCI and then made up to a final volume o f IL and
stored at 2-8°C.
10% Ammonium persulphate {w/v):
Made up by dissolving lOOmg o f Ammonium persulphate in 1ml o f Milli-Q water. Fresh
solution was always prepared prior to every experiment.
Resolving and Stacking gels:
These were prepared as in the following table:
Resolving gel (15%) (Enough for two gels)
Stacking gel (4%) (Enough for two gels)
Milli-Q Water 2 ml 3.75 ml2x Resolving gel buffer (AX2) 1 0 ml2x Stacking gel buffer (BX2) 5 mlAcryl :bis-acrylamide (40% w/v) 7.6 ml 1 ml20% SDS 2 0 0 pi 1 0 0 pi10% Ammonium persulphate 300 pi 1 0 0 piTemed 6 pi 30 pi
74
Both resolving and stacking gels set at room temperature after 30 and 15 minutes,
respectively.
2x Loading buffer:
Mix 5mls BX2 with 2mls 20% SDS, 4.0g glycerol, 0.5ml p-mercaptoethanol, and 0.1ml
o f 1% bromophenol blue. Then take the final volume to 10ml using Milli-Q water.
Buffer was stored at room temperature and was used within two weeks o f preparing.
Running Buffer (per litre):
Weigh 3.02g Tris base and 14.44g glycine and add 5ml o f 20% SDS. Then take the final
volume to IL using Milli-Q water. The pH should be to 8.12 (do not adjust). Fresh
solution was always prepared prior to every experiment.
Transfer buffer (per litre):
Weigh 3.02g Tris base and 14.44g glycine and add 600ml o f Milli-Q water and 200ml o f
methanol. Then take the final volume to IL using Milli-Q water. Fresh solution was
always prepared (and left to cool for two hours at 4°C) prior to every experiment.
lOx Tris buffered saline-Tween buffer (lOx TBS-T):
Make two litres by adding 48.4g Tris base, 160g NaCl, and take volume up to 1.8L using
Mill-Q water. Adjust pH with HCI to 7.6. Then add 20 ml Tween-20 and take the final
volume to 2 L using Milli-Q water. Keep stored at 4°C.
75
Ix Tris buffered saline-Tween buffer (TBS-T):
Add 900ml o f Milli-Q water to 100ml o f lOx TBS-T.
Membrane blocking solution:
5% (w/v) non-fat powdered milk (Marvel) in TBS-T.
Commassie brilliant blue stain (gel-staining dye):
To prepare 100ml, weigh 0.25g o f commassie brilliant blue. Add 45 ml o f Milli-Q water
and 45ml o f methanol. Finally add 10ml o f acetic acid.
Gel de-staining solution (per litre):
Made by mixing 300ml methanol and 100ml acetic acid and taking the final volume to
IL using Milli-Q water.
lOx Ponceau S stain (stock):
Mix 2g Ponceau S, 30g o f trichloroacetic acid, and 30g o f sulfosalicylicic acid. Using
Milli-Q water, take to a final volume o f 100ml.
Ix Ponceau S stain:
Add 90ml o f Milli-Q water to 10ml o f lOx Ponceau S.
76
2.3 Methods
2.3.1 Animals and in vivo experiments
Male BALB/c mice were purchased from Bantin & Kingman (B&K / UK) at a weight o f
25 g (6 - 8 weeks old). They had ad libitum access to standard powdered chow and water.
The animals were kept in our experimental biology unit (EBU) to acclimatise in a
controlled environment (21-24 °C, 30-40% humidity and an alternating 12-hour cycle o f
light and dark) for at least one week prior to all experiments.
Paracetamol, dissolved in phosphate-buffered saline (PBS), was injected intraperitoneally
at a dose o f 500mg/kg. Anti-CD95 antibody; dissolved in PBS; was also injected
intraperitoneally at a dose o f 40pg/animal. Where indicated, animals received an
intravenous injection (through a caudal vein) o f Z-VAD-fmk (10 mg/kg, dissolved in
2.5% dimethylsulfoxide in PBS) 15 minutes prior to paracetamol or Anti-CD95 antibody
administration. Control animals always received an equivalent volume o f the
corresponding solvent via the corresponding route.
Mice were sacrificed at the indicated time points by an intraperitoneal injection o f a lethal
dose o f sodium pentobarbital ( 6 mg/animal). Where indicated, blood, obtained by
intracardiac puncture, was immediately collected into heparinised tubes (for plasma) or
into plain tubes (for serum) and then centrifuged at 2500g for 10 minutes to get plasma or
serum respectively.
Pieces o f liver tissue to be processed for light and transmission electron microscopy were
instantly fixed in 10% neutral buffered formalin. On the other hand, pieces o f liver tissue
77
to be processed for biochemical analyses, caspase assays, agarose gel electrophoresis, and
western blotting were snap frozen by immersion into a bath o f hexane cooled by a
mixture o f industrial methylated spirit (IMS) and dry ice and then stored in glass tubes at
-80°C.
2.3.2 Serum Biochemistry
Plasma activities o f ALT, AST and LDH were measured using commercial kits (Roche)
using a Cobas Bios auto-analyser according to the manufacturer’s instructions. Quality
controls were run with every assay.
2.3.3 Histology
For light microscopic examination, hepatic tissue specimens were fixed in 10% neutral
buffered formalin, embedded in paraffin and cut at 5-pm thickness sections using a rotary
microtome. The sections were stained with hematoxylin and eosin (H&E) for routine
examination.
The in situ labeling o f apoptotic cells was performed using the ApopTag Direct TdT-
catalysed dUTP-fluorescein nick end labeling (TUNEL) method as specified by the
supplier (Oncor) (Gavrieli et aL, 1992). The TUNEL-stained sections were
counterstained with propidium iodide and mounted with antifade (Oncor) and examined
by fluorescence or confocal laser scanning microscopy (Leica).
78
For transmission electron microscopy (TEM), liver tissue specimens o f approximately
Imm^ were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) overnight,
followed by several 10-min washes o f the tissues with 0.1 M cacodylate buffer (pH 7.4).
Following counterfixing with 2% osmium tetroxide in cacodylate buffer, the specimens
were dehydrated in ethanol and finally embedded in TAAB resin. Ultrathin sections were
cut, stained with uranyl acetate and analysed by TEM.
2.3.4 Determination of DNA fragmentation
2.3.4.1 DNA isolation and purification
Phenol/chloroform extraction o f DNA was carried out (with minor modifications) as
previously described (Ray et al., 1993; Ray et al., 1992).
1 0 % (w/v) liver homogenates o f frozen liver sections were homogenised in ice-cold lysis
buffer using a glass-tefion homogeniser. The 10% homogenates were digested with
proteinase K (0.2 mg/ml) for 6 hours at 50°C on a shaking water bath. Triple
phenol/chloroform extraction was carried out by adding phenol/chloroform to the
homogenate in a 3:l(v/v) ratio, respectively. Mixing was carried out by gently shaking
the tubes for 5 minutes. The solution was then centrifuged at 3,000 rpm for 5 minutes at
room temperature. The upper, clear aqueous layer (where the DNA is found) was
collected. Phenol/chloroform extraction was repeated three times.
79
To the aqueous layer, cold absolute ethanol (kept overnight at -20°C) was added to make
up to a volume o f 18mls. Finally, 2mls o f 2.5M sodium acetate were added to the
mixture which was kept overnight at -20°C.
Next day, the mixture was centrifuged at 3,000rpm/4°C/20min. The supernatant
(ethanol) was discarded, and the DNA pellet resuspended in cold 6 6 % ethanol (kept
overnight at -20°C) to make up to a volume o f 18mls. Again, 2mls o f 2.5M sodium
acetate were added to the mixture, which was kept at -20°C for one hour, and then
centrifuged at 3,000rpm/4°C/20min. The washing in 6 6 % ethanol was repeated three
times.
The DNA sample was resuspended in 100-200 pi o f TE-8 buffer and digested with
RNase A (Ipg/ml) for 30 minutes at 37°C in a non-shaking water bath.
2.3.4.2 Agarose gel electrophoresis:
After preparing 1.6% (w/v) ultrapure agarose gel in IxTBE, the required volume o f
ethidium bromide was added to make a final concentration o f 0.4pg/ml. After the
mixture was heated in the microwave (at full power) for 3-4 minutes, the agarose was
allowed to cool (at room temperature) to around 60°C. It was then poured into the gel
cast and the comb was placed into the gel, which was allowed to set for one hour. After
the cast was placed into a IL-submarine tank which was filled with IxTBE, the comb
was carefully removed, taking care not to split the gel.
80
A volume o f 30pl (15pl o f DNA sample and 15pl o f DNA loading buffer) was loaded
into each well. The gel was run at 10 volts overnight (at room temperature). At the end
o f the run, the gel was removed from the tank and placed in a plastic tray and given a
quick rinse in distilled water. Then it was put on a shaker and washed three times (x3) in
TE- 8 buffer. Each wash was for 15-30 minutes.
Finally the ethidium bromide-stained bands were visualised under an ultra-violet
transilluminator and photographed on a gel documentation system (UVP imagestore).
2.3.5 Succinate dehydrogenase (SDH) assay
2.3.5.1 Isolation of mitochondria for SDH assay
Mitochondriae were isolated from mice pre-frozen liver tissues which were homogenised
in ice-cold MSH-EDTA buffer using a glass-teflon homogeniser to make a 25% (w/v)
homogenate. The homogenate was centrifuged at 500g for 10 minutes at 4°C and the
supernatant at 12,000g for 10 minutes. The 12,000g supernatant was used as the
cytosolic fraction and the 12,000g pellet was used as the mitochondria. Mitochondrial
pellet was washed by resuspending in the MSH-EDTA buffer and gently homogenised by
hand in a glass-teflon homogeniser and centrifuged again at 1 2 ,0 0 0 g for 1 0 minutes at
4°C. Mitochondrial pellets were resuspended in MSH-EDTA and protein concentrations
were determined using Bio-Rad protein assay.
Mitochondrial samples to be used for the SDH assay were diluted in MSH-EDTA buffer
to give a final protein concentration o f 2mg/ml. The final mitochondrial fractions were
_
divided into aliquots and used immediately for the assay, or otherwise kept in -80°C
freezer.
2.3.S.2 Assaying for SDH
Measurement o f SDH activity was based (with slight modification) on the Pennington
assay (Pennington, 1961).
In LP3 tubes, duplicates o f the following mixtures (as shown in the table) were prepared
and pre-incubated in a 37°C-preheated water bath for at least four minutes.
Standard blank Tissue blank Test sample
Mitochondrial samples (pi) # # # 150 150
Substrate (pi) # # # # # # 75
Inhibitor (pi) 75 75 # # #
MSH-EDTA buffer (pi) 150 # # # # # #
Then, taken tube by tube and at specified times, 75pi o f pre-warmed (to 37°C)
chromogen was added to each tube, whirlmixed, and immediately placed back into the
bath. Timing for the incubation period started the moment the tube is replaced back into
the bath.
Each tube was incubated for a precisely timed 20 minutes, then the reaction was stopped
by adding 1 .2 ml stopping reagent (must be kept at room temperature) to make a final
82
volume o f 1.5ml. Immediately after adding the stopping reagent, the tube was lifted out
o f the waterbath, whirlmixed, and then placed in a rack at room temperature. Strict
timing is critical.
The red fromazan formed in the reaction was extracted into 4ml o f ethyl acetate and
gently centrifuged (at 500g for 3-5 minutes at room temperature) to get rid o f
occasionally encountered turbidities. Finally, the absorbance o f each tube was measured
at 490nm using the spectrophotometer Kontron Uvikon 932.
2.3.6 Adenylate kinase (AK) assay
The enzyme assay was performed by a modification o f the procedure o f (Passarella et ah,
1988) and (Barile et al., 1994). 0.5ml o f reaction buffer A (70mM triethanolamine and
1.4mM EDTA in Milli-Q water, pH adjusted to 7.4 with concentrated HCl) was aliquoted
into a quartz glass cuvette. Then, 5pi o f reaction buffer B (2.8mM glucose (Sigma) and
7mM MgCl2 (BDH) in Milli-Q water) was also added, both buffers were prewarmed at
37°C. An aliquot o f the isolated mitochondria, containing 35pg o f protein per sample,
5 pi o f carboxyatractyloside (CAT) (mitochondrial inhibitor. Sigma), 2pl oligomycin
(mitochondrial inhibitor. Sigma), 2pi hexokinase (Roche), Ipl glucose-6 -phosphate
dehydrogenase (G6 PD) (Roche), 5pi NADP^ (Roche), and 5pi ADP (Sigma) were then
added. The cuvette was rapidly mixed and NADP^ reduction was measured at 340nm in
a time-drive o f 2 minutes with 150 absorbance readings per minute using a Kontron 932
spectrophotometer. The rate o f NADP^ reduction is directly related to the AK activity.
The temperature o f reaction was set at 2>TC.
83
2.3.7 Cytochrome c oxidase (COX) assay
COX is an enzyme found attached to the inner mitochondrial membrane. COX activity is
proportional to the amount o f mitochondria present in the samples and thus COX activity
was used as a marker for data normalisation. The enzyme assay was performed by a
modified procedure from (Balaban et al., 1996; Miro et al., 1998; Williams et al., 1998).
Mitochondria, 70pg o f protein/sample were incubated with digitonin (0.106mg
digitonin/mg o f protein as mentioned in (Boyer et ah, 1993)) (Sigma) and lOOpl enzyme
reaction buffer (0.3M sucrose and lOmM KH2PO4) in an eppendorf and kept in ice for 15
minutes. Meanwhile, lOpl o f 30mM cytochrome c (Sigma) solution prepared in enzyme
reaction buffer was aliquoted into an Eppendorf and 30pl o f lOmM dithionite (Sigma)
solution , also prepared in assay buffer, was added. The contents were carefully mixed to
allow reduction o f cytochrome c. Cytochrome c reduction was checked
spectrophotometrically by a quick wavelength scan between 450-600nm. Reduction o f
cytochrome c showed a distinct peak at 550nm.
After the 15 minutes incubation period, the assay was performed by the addition o f 780pl
reaction buffer, prewarmed at 37®C, into a 1.5ml plastic cuvette. Followed the addition
o f lOOpl o f incubated mitochondria and 40pl reduced cytochrome c. The cuvette was
rapidly mixed and the reaction was followed at 550nm in a time-drive o f 2 minutes with
150 absorbance readings per minute using a Kontron 932 spectrophotometer. The
temperature o f reaction was set at 37®C.
84
2.3.8 Determination of caspase activation
2.3.8.1 Isolation of thymocytes (positive controls):
This was carried out (with slight modification) as previously described (Chow et aL,
1995). A three-week old male Wistar rat was sacrificed by an intraperitoneal injection o f
9mg o f sodium pentobarbital. The thymus was dissected out, removing the parathymie
nodes and connective tissue, and was kept in ice-cold minimum essential medium
(MEM). The thymus was chopped and squeezed between the frosted ends o f two glass
slides and filtered using 10ml o f ice-cold MEM through a bolton cloth (to remove the
debris) into a sterile universal tube. Thymocytes were washed once by centrifuging at
l,250rpm for 5 minutes at 4°C. The thymocytes-pellet was resuspended in 10ml o f ice-
cold MEM in a clean universal tube and poured into a 20ml-culture flask and were
incubated at 37°C in an atmosphere o f 95% O2: 5% CO2 in a humidified incubator in the
presence o f IpM dexamethasone for 6 hours. After the incubation period, thymocytes
were washed by centrifuging at l,250rpm for 5 minutes at 4°C and the supernatant was
carefully removed using a tap water-powered suction system. Thymocytes-pellet was
resuspended in 1ml o f ice-cold cell lysis buffer (CLB), poured into an eppedorf tube,
bench-centrifuged at l,250rpm for 5 minutes at 4°C, and then resuspended again in 50pl
o f CLB and kept overnight at -80°C. Next day, x2 cycles o f thawing and re-freezing (in a
mixture o f IMS and dry ice) were carried out. Finally, thawed thymocytes were bench-
centrifuged at 16,000rpm for 30 minutes at 4°C. The supernatant was collected and
divided into eppendorfs o f lOpl aliquots that were immediately stored at -80°C.
85
Protein concentration in the thymocytes solution was determined using the Bio-Rad
protein assay.
2.3.5.2 Preparation of total liver homogenates:
33% (w/v) total liver homogenates (Test samples) were prepared in ice-cold HEB by
homogenising frozen liver sections using a glass-teflon homogeniser.
Protein concentration in the homogenates was determined using the biuret method
(Gomall et ah, 1949).
2.3.5.3 M easurement o f caspase activation:
Caspase activity was assayed at room temperature using a fluorescence microtitre plate
reader and a fluorimeter (WellFluor, Denley) in a reaction mixture containing 40pM o f
the fluorogenic substrate peptides Ac-DEVD-AFC or Ac-LEHD-AFC as previously
reported (Jones et ah, 1998), with the exception that each well contained Img protein.
Amino-4-trifluoromethylcoumarin (AFC) in a ranging concentration (0-20nmoles) was
used for plotting a standard curve each time the assay was performed. Appropriate
blanks were also carried out.
2.3.9 SDS-PAGE and Western Blotting
2.3.9.1 Isolation of mitochondria
Frozen liver sections were homogenised in ice-cold homogenisation buffer (HB) using a
glass-teflon homogeniser to make a 25% (w/v) homogenate. The homogenate was
centrifuged at 500g for 10 minutes at 4°C and the supernatant at 12,000g for 10 minutes.
86
The 12,000g supernatant was used as the cytosolic fraction and the 12,000g pellet was
used as the mitochondria. The mitochondrial pellet was washed by resuspending in HB
and gently homogenised by hand in a glass-teflon homogeniser and centrifuged again at
12,000g for 10 minutes at 4°C. The final mitochondrial pellet was resuspended in the HB
buffer. Both cytosolic and mitochondrial fractions were divided into aliquots and kept
frozen at -80°C. Protein concentrations were determined using Bio-Rad protein assay.
2.3.9.2 Polyacrylamide Gel Electrophoresis
Cytosolic and mitochondrial fractions underwent SDS-15% polyacrylamide gel
electrophoresis. A 4% stacking gel was always used. The gels were cast in a Bio-Rad
mini gel system using the previously described recipes. Cytosolic or mitochondrial
samples were diluted one fold in 2x loading buffer, boiled for 3-5 minutes and loaded in a
final volume o f lOpl onto the wells o f the stacker. The total amount o f protein loaded in
each well ranged from lOpg to 50pg. Electrophoresis was carried out at room
temperature for 80-90 minutes at lOOV until the tracking dye o f the loading buffer was
within a few millimetres o f the lower edge o f the gel.
2.3.9.3 Western Transfer of Proteins
Whatmann 3MM filter papers, Hybond C nitrocellulose membrane, and scotchbrite pads
were soaked in pre-cooled transfer buffer for 10 minutes. Transfer o f proteins was
carried out by layering two pieces o f filter paper, followed by the gel, and then the
nitrocellulose membrane. This was then layered by two more pieces o f filter paper,
sandwiched within the scotchbrite pads and then placed in the Bio-Rad transfer system.
The transfer tank was filled with the pre-cooled transfer buffer. The transfer was usually
87
done overnight at 34V at room temperature. Occasionally, cool transfer (with the cooling
element in place) at lOOV for 2-3 hours was carried out. Following transfer, the protein
profile was checked on the membrane by staining with Ponceau S stain for 2-3 minutes,
followed by de-staining in Milli-Q water. The efficiency o f the transfer process was
determined by staining the gel in Commassie brilliant blue stain for 1-2 hours (on a
shaker), and de-staining for several hours (or overnight) using gel de-staining solution.
/2.3.9.4 Immunoblotting
The membrane was then blocked either overnight (at 4°C) or for one hour (on a shaker)
in 5% (w/v) membrane blocking solution. The membrane was then probed with a
suitable dilution o f primary antibody (or a mixture o f two primary antibodies) made in
5% (w/v) membrane blocking solution. Used dilutions o f primary antibodies against
cytochrome c (Ipg/ml or 1:500 dilution), cytochrome oxidase subunit IV (0.25pg/ml or
1:1,000 dilution), Bax (Ipg/ml or 1:200 dilution). Bid (Ipg/ml or 1:1,000 dilution), BCl-
x l (2pg/ml or 1:500 dilution), caspase-3 (1:2,500 dilution), caspase-7 (1:2000 dilution),
caspase-9 (1:500 dilution), and caspase- 8 (1:1,000 dilution) were all in accordance with
the recommendations o f the corresponding manufacturers.
Incubation in the primary antibody was always carried out at room temperature and for a
one hour duration except in the case o f antibodies against Bid and the caspases (caspase-
3, caspase-9, and caspase-8 ) when incubation was carried out overnight and at cold room
temperature (4-8°C). The membrane was washed 3x 10 minutes in TBS-T on a shaker.
After washing, incubation for one hour with a 1:2,000 dilution (in 5% (w/v) membrane
blocking solution) o f the appropriate HRP-conjugated secondary antibody was carried
88
out. Finally the membrane was washed again for 3x 10 minutes in TBS-T on a shaker
before being visualised with the chemiluminescent detection system as described by the
manufacturer (Pierce chemical company). Premixed detection reagent was added and
incubated for 5 minutes. Excess reagent was drained o ff and the blot was wrapped in
Saran wrap. Exposure to the x-ray film was for a variable period ranging from 5 seconds
to 20 minutes. Band densitometry was carried out using MCID 4 (Gels-Lane Analysis).
2.3.10 Bio-Rad protein assay
Protein concentration was assessed using Bio-Rad protein assay. The test is a dye-
binding assay based on the differential colour change o f a dye in response to various
concentrations o f protein.
As the dye reagent is provided as a five-fold concentrate, one volume o f the dye reagent
concentrate was diluted with four volumes o f Milli-Q water and then filtered through a
Whatmann No. 1 paper. This dilute dye reagent was always discarded after two weeks.
Several dilutions o f protein standard 1 (containing from 0.2 to 1.4 mg/ml o f bovine y
globulin) were prepared and used in generating a standard curve each time the assay is
performed.
Aliquots o f lOOpl o f standards and appropriately diluted samples were placed in clean,
dry test tubes. Corresponding “Blank” test tubes were prepared using aliquots o f lOOpI
o f the sample buffer on its own. To each test tube, 5.Omis o f diluted dye reagent were
added, and the mixture was vortexed (avoid excessive foaming). After a period ranging
89
from 5 to 60 minutes, measurement o f OD595 (versus reagent blank) was carried out and
plotted against concentration o f standards and read unknowns using the standard curve.
2.3.11 Statistical analysis of the data
All data are given as means + SE o f the number o f animals (SEM) indicated in the text.
Comparison o f treatments against controls was made using Dunnett’s t-test. Where
appropriate, Bonferroni’s multiple comparisons test was performed to determine the
significance between different treatments. The significance level chosen for all statistical
analyses was p < 0.05. The statistical analyses were carried out with the SPSS statistical
package.
90
Chapter 3
Paracetamol Hepatotoxicity
91
3.1 Histopathology and Biochemistry in AAP overdosing
Earlier experiments (data not shown) carried out in our laboratory confirmed that the
minimal toxic dose o f AAP in BALB/c mice was 500mg/kg (Anwar, 2001). After six
hours o f overdosing BALB/c mice with a single intraperitoneal injection o f AAP
(500mg/kg), haematoxylin and eosin (H&E) staining revealed histopathological evidence
o f acute liver injury (Figure 9). This was paralleled by a rise in the serum liver enzymes
ALT (Figure 10), AST (Figure 11), and LDH (Figure 12). Similar results were obtained
with higher doses o f AAP (Figure 13).
TUNEL staining o f liver sections from AAP-overdosed mice also confirmed the loss o f
genomic DNA through fragmentation (Figure 14).
Adoption o f the “six-hour” figure throughout most o f our project was based on
histopathological and biochemical results on earlier studies (data not shown) carried out
on the time-course o f AAP-induced hepatic damage (Anwar, 2001). In summary, these
studies revealed that one hour post-AAP (500mg/kg) overdosing, small vacuolations
were observed in centrilobular (zone 3) hepatocytes. By two hours, vacuolations were
more prominent and extended to involve zone 2 as well. By the third hour, scattered
necrotic cells (with some RBCs) were observed in zone 3. By four to five hours, large
numbers o f RBCs were widely spread throughout zones 1-3 with very few necrotic cells
remaining. By the sixth hour, only a very small number o f necrotic cells were visualised
(mainly in zone 3). Serum liver transaminases (and LDH) were rapidly rising after the
fourth hour post-AAP overdosing. Therefore it was felt that in order to identify the early
events leading to the development o f liver toxicity, subsequent studies should mainly
concentrate on the changes found in the liver six hours after AAP overdosing.
92
Figure 9. Histopathological documentation of paracetamol-induced acute liver injury and failure of GdClg-pretreatment to prevent such injury. BALB/c mice were pretreated with intravenous GdCL (lOmg/kg in 20% Tween-80 in normal saline) or vehicle control (20% Tween-80 in normal saline) 24 hours prior to receiving intraperitoneal AAP (500mg/kg in PBS). All animals were sacrificed after six hours. Hepatic tissues were fixed in 1 0 % neutral buffered formalin, embedded in paraffin, cut into 5-pm thick sections, and stained with H&E as outlined in Methods earlier. Section A: “Control”; Section B: “AAP- treated”; and Section C: “AAP-treatment following GdCL”. Magnification lOOX. This work was carried out in collaboration with my colleague Dr K Anwar (Anwar, 2001).
93
3500
3000
2500
P2000
H1500
1000
500
t
0
1oV
Qq ><e > QN N4- +PM
< < << < < S
Figure 10. Z-VAD-fmk and Z-DEVD-cmk prevent the rise in serum alanine transaminase (ALT) after paracetamol intoxication.
“Control” mice received ip-PBS (0.5ml). “AAP” group received ip-AAP (500mg/kg). “AAP+Z-VAD” group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to dosing with AAP. “AAP+Z-DEVD” group received iv Z-DEVD-cmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to dosing with AAP. “Z-VAD” group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) and ip-PBS (0.5 ml) 15 minutes later. All animals were sacrificed after 6 hours. ALT activity in serum was measured using commercial kits (Roche) on a Cobas Bios auto-analyser according to the manufacturer’s instructions. Data given representsMean ± SEM. Each group consisted o f five animals. *: Significantly different from control(p<0.05). f : Significantly different from AAP-treated group (p<0.05).
94
1500
1250
1000
IH%
750
500
250
IO
U
Q<S3 > Q
.2a +
?Py PM PM< < << < N
Figure 11. ZL-VAD-fmk and Z-DEVD-cmk prevent the rise in serum aspartate transaminase (AST) after paracetamol intoxication.
“Control” mice received ip-PBS (0.5ml). “AAP” group received ip-AAP (500mg/kg). “AAP+Z-VAD” group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to dosing with AAP. “AAP+Z-DEVD” group received iv Z-DEVD-cmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to dosing with AAP. “Z-VAD” group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) and ip-PBS (0.5 ml) 15 minutes later. All animals were sacrificed after 6 hours. AST activity in serum was measured using commercial kits (Roche) on a Cobas Bios auto-analyser according to the manufacturer’s instructions. Data given representsMean ± SEM. Each group consisted o f five animals. *: Significantly different from control(p<0.05). f : Significantly different from AAP-treated group (p<0.05).
95
25000
20000
IMQ
15000
10000
5000
IoU
Q<a > 9
.2 N SI03 + +PM
< <3 << < <
Figure 12. Z-VAD-fmk and Z-DEVD-cmk prevent the rise in serum lactate dehydrogenase (LDH) after paracetamol intoxication.
“Control” mice received ip-PBS (0.5ml). “AAP” group received ip-AAP (500mg/kg). “AAP+Z-VAD” group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to dosing with AAP. “AAP+Z-DEVD” group received iv Z-DEVD-cmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to dosing with AAP. “Z-VAD” group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) and ip-PBS (0.5 ml) 15 minutes later. All animals were sacrificed after 6 hours. LDH activity in serum was measured using commercial kits (Roche) on a Cobas Bios auto-analyser according to the manufacturer’s instructions. Data given representsMean ± SEM. Each group consisted o f five animals. *: Significantly different from control(p<0.05). f : Significantly different from AAP-treated group (p<0.05).
96
2 5 0 0
2 0 0 0
1 5 0 0
H
0 0 0
5 0 0
Otao
U
O)a'a
î
Figure 13. Z-VAD-fmk does not abolish hepatic injury (reflected by serum ALT) after intoxication by higher doses of paracetamol.
“Control” mice received ip-PBS (0.5ml). “AAP” group received ip-AAP (600mg/kg, ip). “AAP+Z-VAD” group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to overdosing with AAP (600mg/kg, ip). All animals were sacrificed after 6 hours. ALT activity in serum was measured using commercial kits (Roche) on a Cobas Bios autoanalyser according to the manufacturer’s instructions. Data given represents Mean + SEM.Each group consisted o f five animals. *: Significantly different from control (p<0.05).
97
3.2 Role of phagocytes in paracetamol overdosage
In earlier studies, phagocytes (consisting mainly o f in situ activated Kupffer cells and
chemotactically attracted blood monocytes to the site o f injury and inflammation) have
been hypothesised, through the release o f cytotoxic and proinflammatory mediators, to
play a major role in the hepatotoxicity o f AAP (Ferluga and Allison, 1978; Laskin and
Pilaro, 1986; Laskin a/., 1986).
Pretreatment o f animals with gadolinium chloride (GdCls), a recognised inhibitor o f
hepatic macrophage function, is a well known model in biological sciences (Husztik et
aL, 1980; Laskin e /o /., 1995).
Laskin (Laskin et aL, 1995) reported that pretreatment o f rats with intravenous
gadolinium chloride (GdClg, 7mg/kg) for 24 hours, prior to overdosing with
intraperitoneal AAP (800mg/kg), completely blocked hepatic necrosis and prevented the
rise in serum transaminase levels induced by AAP. Laskin also concluded that the effects
o f GdCls were not due to suppression o f NAPQI formation
In our project, we found that intraperitoneal AAP overdosing (500mg/kg) o f BALB/c
mice was associated with marked rise in all o f the serum liver enzymes (Table 3). On the
other hand pretreatment with GdCb (lOmg/kg, iv) for 24 hours prior to overdosing with
AAP did not prevent AAP-related liver injury as evidenced by the continuing rise in
serum liver enzymes (Table 3) and the persistence o f damaged hepatic tissue (Figure 9).
However, when examining histological sections for DNA damage as assessed by TUNEL
staining, it was found that pretreatment with GdClg has prevented the formation of
apoptotic cells in AAP-overdosed animals (Figure 14). Thus, we concluded that although
98
pretreatment with GdCb was very successful in preventing apoptosis, it could not stop
liver damage from taking place. This suggests that cytokines released from Kupffer cells
are not the major effectors o f AAP-induced hepatic damage in BALB/c mice model.
Table 3. Effects o f Gadolinium chloride treatment on paracetamol-induced alterations in
serum liver enzyme levels.
Treatment n ALT (lU/L) AST (lU/L) LDH (lU/L)
Control 61 ±14 286 ± 126 3300 ±1056
Control + GdCls 69 ±19 119 ±44 1878 ±476
Paracetamol 9 1835 ±621" 546 ± 175 6137 ± 1039'
Paracetamol + GdClg 10 1979 ± 609" 1030 ± 221" 10400 ± 1872'
NOTE: BALB/c mice were pretreated with Gadolinium chloride (GdClg) (10 mg/kg, iv) or
vehicle control (20% Tween 80 in normal saline) for 24 hours prior to dosing with
paracetamol (500 mg/kg, ip). After six hours, the animals were sacrificed and serum
prepared for the measurement o f the liver-derived enzymes ALT, AST and LDH as
described under Methods.
All data are given as Mean ± SEM. Significantly different from control (p<0.05).
99
l U N E L PI TUNEL + PI
Control
AAP
AAP
+
GdCIs
AAP+
ZVAD-fmk
H H 1B
H i BBKgm
Figure 14. Detection of DNA fragmentation in the liver following paracetamol intoxication using the TUNEL assay: Gadolinium chloride (GdCL) and Z-VAD-fmk prevent DNA fragmentation. Mice were pretreated with gadolinium chloride (GdCb; 10 mg/kg, in 20% Tween 80 in normal saline, iv) 24h before or with Z-VAD-fmk (10 mg/kg; in 2.5% DMSO in PBS, iv) 15 min prior to dosing with AAP (500 mg/kg, ip). After six hours, all animals were sacrificed and hepatic tissue specimens were collected and fixed in 10% neutral buffered formalin, embedded in paraffin and cut at 5-pm thickness sections. The sections were processed for TUNEL staining as described under Methods and visualized by confocal laser scanning microscopy. The left panel shows the TUNEL stained cells (green), the middle the propidium iodide (PI) counterstained nuclei (red) and the right panel the merged images. Note that nearly all TUNEL stained cells are parenchymal liver cells with condensed nuclei, suggesting that the TUNEL assay identifies apoptotic cells under the present conditions. The merged images from the section prepared from AAP-treated and AAP- and GdClg-pretreated animal also show the presence o f congested areas with large numbers o f erythrocytes (dark green fluorescence). This work was carried out in collaboration with my colleague Dr K Anwar (Anwar, 2001).
100
3.3 Role of caspase inhibitors in paracetamol hepatotoxicity
Z-VAD-fmk is widely used as a “broad specificity” irreversible inhibitor o f caspases. As
previously reported by Kunstle et al (Kunstle et al., 1997), pretreatment o f mice with Z-
VAD-fmk (lOmg/kg) was protective from CD95- and TNF-alpha-induced liver injuriy
(as determined by plasma ALT) and also from hepatocyte apoptosis (assessed by DNA
fragmentation). Similar results were found when apoptosis was initiated via TNF-alpha
or via CD95 in primary murine hepatocytes or in various human cell lines. These findings
indicate that, in mammals, both the CD95 and the TNF-a receptors share a signaling
pathway that involves caspases (Kunstle et ah, 1997).
In our project, pretreatment o f BALB/c mice with Z-VAD-fmk (lOmg/kg in 2.5% DMSO
in PBS, iv) proved to be significantly protective against AAP-induced liver injury, when
the latter was given at a dose o f 500mg/kg. This also was evidenced biochemically by
the marked decrease in serum liver enzyme levels [ALT (Figure 10), AST (Figure 11),
and LDH (Figure 12)].
Z-VAD-fmk pretreatment also managed to abolish the pathological changes (Figure 15)
and prevented the appearance o f TUNEL positive parenchymal cells (Figure 14) due to
AAP overdosing. However, this hepatoprotective effect o f Z-VAD was lost with doses
o f AAP higher than 500mg/kg (Figure 13)
After the sixth hour o f AAP-overdosing, livers from AAP-treated mice were grossly
different from the control animals, they looked dark, haemorrhagic, and were massively
swollen and congested. On the other hand, livers from mice pretreated with Z-VAD-fmk
before receiving AAP looked very healthy and were generally indistinguishable from
101
those in the untreated group in both gross appearance and under the microscope (Figure
15).
Protection from paracetamol-induced toxicity with Z-VAD-fmk may suggest that
caspase-3 is o f pivotal importance in paracetamol-induced apoptosis as Z-VAD-fmk
specifically inhibits the processing o f caspase 3 (CPP32) to its active form (Slee et aL,
1996).
In addition to the protective effect o f Z-VAD-fmk, we also observed that the caspase-3
like inhibitor Z-DEVD-cmk also alleviated, albeit to a lesser extent, the damage induced
by AAP (Figures 10-12). However, one notable difference between the two was that, on
histopathology, Z-VAD-fmk blocked the accumulation o f neutrophils in the liver whereas
large number o f neutrophils was still observed in the Z-DEVD-cmk-treated mice. This
difference may be attributed to the difference in caspase specificity between the two
peptide inhibitors, with Z-DEVD-cmk expected to show a greater degree o f selectivity
towards the group 2 caspases as compared to the broad spectrum acting Z-VAD-fmk
which also block the pro-inflammatory group 1 caspases (Nicholson and Thomberry,
1997).
102
3.4 Role of sinusoidal endothelial cells in paracetamol toxicity
The fact that both GdCls and Z-VAD-fmk were capable o f preventing DNA damage on
TUNEL assay (Figure 14) may suggest that at least two different mechanisms (namely
the inhibition o f cytokine rlease from macrophages/Kupffer cells and the prevention o f
caspase processing, respectively) are involved in preventing apoptosis.
However, while GdCb pretreatment was unable to protect the liver against AAP-induced
injury (Figure 9), Z-VAD-fmk was protective (Figure 15). This suggests that cytokines
secreted by Kupffer cells play a major role in apoptosis o f hepatocytes in AAP toxicity.
The liver is the major detoxifying organ in humans and because o f their location
(between blood stream and hepatic parenchyma), sinusoidal endothelial cells (SECs) are
probably more vulnerable to toxins than any other type o f cell in the body. When
Walker, et al investigated (using TEM) AAP-induced hepatotoxicity and its associated
hepatic congestion, he realised that AAP (750 mg/kg, orally) causes changes in the
relationship between hepatocytes and SECs. He then observed endocytic vacuolation at
lateral and sinusoidal margins o f centrilobular hepatocytes, loss o f microvilli. Disse space
enlargement, dilatation o f bile canaliculi, and disappearance o f the studlike projections
from hepatocyte lateral surfaces. He concluded that RBCs enter the enlarged Disse space
and endocytic vacuoles via enlarged pores in SECs, thereby collapsing the sinusoids.
However he reported that lining SECs are not lost, but apparently held in position by
preservation o f intercellular junctions, cytoplasmic projections from hepatocytes, and
anchorage by fat-storing cells within the Disse space. He finally observed that
congestion can abate by 24 hours, indicating that RBCs can return to the general
circulation from the Disse space (Walker et al., 1983; Walker et al., 1985).
—
Also it is reported that treatment o f mice with anti-CD95 leads to damage o f SECs cells
and hepatic microvascular perfusion failure. Intravenously administered anti-CD95 (10
microgram/mouse) antibodies to mice leads to sinusoidal perfusion failure and loss o f
integrity o f SECs as early as Ihour post dosing. Histological evidence o f hepatocellular
apoptosis and haemorrhagic necrosis were most pronounced at 6 hours. Blocking o f
caspase activity using Z-VAD-fmk attenuated the sinusoidal perfusion failure, fully
protected the liver from apoptotic damage, and preserved an intact microarchitecture
(Wanner gr a/., 1999).
In our project, results o f histology (Figures 9 & 15) confirmed the presence o f severe
hemorrhagic damage/congestion after six hours o f overdosing BALB/c mice with AAP
(500mg/kg, ip). To investigate whether this may be due to changes in the relationship
between hepatocytes and SECs, we used TEM to look for any abnormalities in the
morphology o f SECs. As shown in figure 16, there was clear evidence o f direct damage
to SECs evidenced by marked blebbing (open arrows) and in numerous places SECs were
found detached from the adjacent hepatocytes (closed arrows) leaving hepatocytes in
direct contact with RBCs (asterisks) both intra-and extra-vascularly.
We therefore hypothesise that protection against AAP overdose may be dependent on an
intact sinusoidal endothelium. This may suggest that the protective property o f Z-VAD-
fmk against AAP-induced liver injury could primarily be due to anti-apoptotic effects on
the SECs (and in turn the prevention o f hemorrhagic damage) rather than through its
effects on the hepatocytes.
104
ï
SK .
-.'iSCi
Figure 15. Z-VAD-fmk prevents liver damage in response to paracetamol. BALB/c mice were dosed with AAP (500mg/kg in PBS, ip) or pretreated with Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS, iv) 15 minutes prior to dosing with AAP (500mg/kg, ip). Animals were sacrificed after six hours. Hepatic tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, cut into 5-pm thick sections, and stained with H&E as described in Methods section. Control (A); AAP treatment (B); AAP treatment following Z-VAD-fmk (C). Magnification lOOX. Each section is representative o f the sections obtained from 8-10 animals per treatment group. This work was carried out in collaboration with my colleague Dr K Anwar (Anwar, 2001).
105
t '
-, -%
L'
Figure 16. Paracetamol induces sinusoidal endothelial cell damage in the liver. Mice were
dosed with AAP (500 mg/kg, ip) and after six hours were sacrificed. Hepatic tissue specimens
were collected and were fixed in 4% glutaraldehyde, processed and examined by TEM as described
under Methods. This micrograph shows that damage had occurred to sinusoidal endothelial cells-
SECs (white asterisk) with extensive blebbing (open arrows) and detachment from adjacent
hepatocytes (closed arrows). Red blood cells (* ) are also observed, some o f which having
accumulated in the space o f Disse. Bar represents 2 pm scale. This work was done in
collaboration with my colleague Dr K Anwar who also showed that SECs from control animals
(receiving ip PBS on its own) were normal (not shown) (Anwar, 2001).
106
3.5 Role of reactive FMK-moiety in AAP toxicity
Caspases are a family o f cysteine proteases which play a crucial role in apoptosis and
inflammation. The involvement o f caspases in these processes can be demonstrated by
their irreversible inhibition with fluoromethyl ketone (fink-) and chloromethyl ketone
(cmk)-derivatives o f peptides resembling the cleavage site o f known caspase substrates.
These inhibitors irreversibly alkylate the cysteine residue in the active site o f caspases.
Z-VAD-fmk also efficiently affinity-labels cathepsin B and cathepsin H, and along with
other caspase inhibitors (like Z-DEVD-fmk and Ac-YVAD-cmk), it efficiently inhibits
cathepsin B activity in vitro and in tissue culture cells at concentrations that are generally
used to demonstrate the involvement o f caspases (Schotte et ah, 1999).
To rule out any non-specific effect o f the reactive FMK-moiety o f the caspase inhibtors
towards cathepsins or the bioactivation pathway for AAP-induced liver injury, we
investigated the effects o f Z-FA-fmk (benzyloxycarbonyl-Phe-Ala-fluoromethylketone:
lOmg/kg as 0.2ml in PBS, iv) on AAP-induced hapatotoxicity. Unlike the caspase
inhibitors, Z-FA-fmk did not prevent hepatic injury (Figure 17). Also, work carried out
by K Anwar (data not shown) clearly demonstrated that the number o f TUNEL positive
cells did not diminish (as compared to AAP-treatment) in the absence o f the dipeptide
(Anwar, 2001).
107
3000
2500
20 00
1 500
1000
500
Io
U
o&oes
î+ î +
SFigure 17. Effect of FMK moiety of caspase inhibitors on AAP toxicity in BALB/c mice.
“Control” mice received 0.2ml o f 0.9% normal saline (clinical, iv) 24 hours prior to receiving
PBS (0.5ml, ip). “AAP” group received AAP (500mg/kg as 0.5ml PBS, ip). “AAP+Z-VAD”
group received iv Z-VAD-fmk (lOmg/kg in 2.5% DMSO in PBS) 15 minutes prior to dosing
with AAP. “AAP+Z-FA-fmk” group received FMK (lOmg/kg, in 0.2ml in PBS, iv) 15
minutes prior to AAP (500mg/kg as 0.5ml in PBS, ip). All animals were sacrificed 6 hours
after receiving AAP. ALT activity in serum was measured using commercial kits (Roche) on
a Cobas Bios auto-analyser according to the manufacturer’s instructions. Data given
represents Mean ± SEM. The numbers in parasthesis refer to the number o f animals in
individual groups. *: Significantly different from control (p<0.05). f : Significantly different
from AAP-treated group (p<0.05).
108
Chapter 4
Caspases and paracetamol
109
4.1 Role of caspases in paracetamol hepatotoxicity
Although the two main apoptosis receptors o f mammalian cells (the 55 kDa TNF-Rl and
CD95) are activated independently o f each other, it is esablished that they share a distal
proteolytic signal involving caspases (Kunstle et ah, 1997; Rouquet et ah, 1996a).
We have shown earlier that pretreatment o f BALB/c mice with Z-VAD-fmk (lOmg/kg)
15 minutes prior to AAP overdosing (500mg/kg) significantly protected against AAP-
induced liver injury (Figures 10-12), abolished histopathological changes (Figure 15),
and prevented the appearance o f TUNEL positive parenchymal cells (Figure 14).
As Z-VAD-fmk was developed and has been extensively used as a broad specificity
irreversible inhibitor o f caspases, we therefore decided to look for caspase activity in
AAP-induced liver injury. While the treatment o f BALB/c mice with anti-CD95 resulted
in the expected increase in Ac-DEVD-afc cleavage (representative o f caspase-3-like
activity), the cleavage o f Ac-DEVD-afc from AAP-treated mice showed a significant
inhibition o f such activity (Figures 18 and 19).
The cleavage o f the caspase-9 substrate Ac-LEHD-afc was similarly inhibited by AAP
treatment (Figure 20). Examination o f the time course o f the inhibition revealed that the
inhibition o f Ac-DEVD-afc (Figure 19) and Ac-LEHD-afc (Figure 20) occurred at 3hr
post dosing, which corresponds to the time when AAP-induced liver damage became
detectable (Figures 9 & 14).
On the other hand, measurements o f caspase-3 like activity (using Ac-DEVD-afc as a
substrate) showed that while anti-CD95 treatment resulted in a very significant rise (x l5
110
fold) in Ac-DEVD-afc cleavage, such activity was completely lacking in liver
homogenates from AAP-treated animals (Figure 18). The lack o f increase in activity was
not due to an endogenous inhibitory activity because the liver homogenates from AAP-
treated mice failed to decrease the DEVDase activity o f thymocyte apoptotic extract
(Figure 21) nor was any DEVDase activity released into the serum (Figure 22).
111
40
35
30 4<u W)
i 'a£ - S 25 4
■sI
2Ab£BaB
2 0 -
1 5 -
10
Iao
U
If)o\
8• i
{3}
Figure 18. Paracetamol-induced apoptosis does not involve the increase in caspase 3-
like activity. Mice were overdosed with AAP (500mg/kg, ip). After six hours, animals
were sacrificed and their livers snap-frozen. Homogenates were prepared as described under
Methods and were tested for caspase-3 like activity using Ac-DEVD-afc as a substrate.
Where indicated, animals were pretreated with Z-VAD-fmk (lOmg/kg in 2.5% DMSO in
PBS, iv) 15 minutes prior to dosing with AAP (500mg/kg, ip) or were injected with anti-
CD95 antibody (40pg/animal in PBS, ip) six hours before sacrifice. All data given as mean
± SEM. The numbers in parasthesis refer to the number o f animals in individual groups.
Significantly different from control (p<0.05).
112
0 1 2 3 4 65rSOOO
3.5--2500.S
ouAWDs -2000
2.5-.5Ii%
u
-1500
-1000
-5000.5-
2 3 4
Time (hours)
IH
<
Figure 19. Time-Course o f o f Ac-DEVD-afc cleavage activity (representative of
caspase-3 like activity) during paracetamol-induced liver injury. BALB/c mice
were injected with AAP (500mg/kg, ip) and were sacrificed at the indicated time
points. Each time point represents one animal. Hepatic tissue specimens and serum
samples were collected and processed for analysis o f Ac-DEVD-afc (A) and serum
ALT levels (A) as described in Methods.
113
4 i
3.5
*10 u AWD
1I0sa%CQ
1U
2.5
2
1.5
0.5
0 2 3 4
Time (hours)
3000
2500
2000
1500 HH
H
<1000
500
Figure 20. Time-Course o f inhibition o f Ac-LEHD-afc cleavage (a substrate for
caspase-9) during paracetamol-induced liver injury. BALB/c mice were injected
with AAP (500mg/kg, ip) and were sacrificed at the indicated time points. Each time
point represents one animal. Hepatic tissue specimens and serum samples were
collected and processed for analysis o f Ac-LEHD-afc (T ) and serum ALT levels (A)
as described in Methods.
114
I0 u AWD5•S10sa6
11I<«4-H0
1
1 8 0 i
160
140
120
100
176.9
165.58
uCQ
I5O
H
a0a
1o
u
obfi0V
101H
fi'fi
î
+
I01H
Figure 21. The failure of liver homogenates from AAP-treated animals to decrease
DEVDase activity o f thymocyte apoptotic-extract indicates the absence o f an
endogenous inhibitory activity in those homogenates. Thymocytes from male Wistar rats
incubated in the presence o f IpM dexamethasone were prepared as described under Methods.
BALB/c mice were overdosed with AAP (500mg/kg in PBS, ip) or with ip-PBS (Control).
After six hours, animals were sacrificed and their livers snap-frozen. Homogenates were
prepared as described under Methods and were tested for caspase-3 like activity using Ac-
DEVD-afc as a substrate. Each group consisted o f two animals.
115
1801
.S 160 b £S 140a.sI0aa&
1bfi
9
ob&
120
100
80
60
40
20
165.58
2.49 4.4
ufiu1
0>fi
.2fi fiu oo o "fi
S3K #o <H U <
Figure 22. Lack of DEVDase activity in serum from paracetamol-treated
BALB/c mice. Thymocytes from male Wistar rats incubated in the presence o f IpM
dexamethasone were prepared as described under Methods. BALB/c mice were
overdosed with AAP (500mg/kg in PBS, ip) or with ip-PBS (Control). Serum was
prepared for the measurement o f caspase-3 like activity using the substrate Ac-DEVD-
afc as described in Methods. After six hours, animals were sacrificed. Each group
consisted o f two animals.
116
4.2 Effector caspases and paracetamol toxicity
Caspases are a family o f at least 10 human cysteine proteases that participate in cytokine
maturation and in apoptotic signal transduction and execution mechanisms (Li et al.,
1997a). Caspases are synthesised as inactive zymogens composed o f three domains (an
N-terminal prodomain, the p20, and the plO domains) and whose activation requires
limited proteolysis or binding to co-factors. The mature enzyme is a heterotetramer
containing two p20/pl0 heterodimers and two ative sites (Eamshaw et al., 1999).
Based on their presumed biological function, caspases are often divided into three major
groups: (1) cytokine activators (like caspase-1), (2) initiators o f apoptosis (which is
further subdivided into intrinsic (caspase-9) and extrinsic activators (caspases -8 and -10),
and (3) executioners o f apoptosis (caspases -3, -6, and -7) (Cohen, 1997; Nicholson and
Thornberry, 1997; Salvesen and Dixit, 1997). Others divide caspases into two groups
based on the primary structure o f their NH2-terminal prodomain: long prodomain
“initiator” caspases which include caspases-2, -8, -9, and -10, and short prodomain
“effector” caspases which mainly include caspases-3 and -7 (Nicholson, 1999).
To transmit the apoptotic execution signal, caspase zymogens are sequentially activated
through either an intrinsic or an extrinsic pathway. The activation o f caspases at the apex
o f each pathway, the initiators, occurs by recruitment to specific adapter molecules
through homophilic interaction domains, and the activated initiators directly process the
executioner caspases to their catalytically active forms (Stennicke and Salvesen, 1998;
Stennicke and Salvesen, 2000).
117
We investigated the role o f caspases in AAP-mediated parenchymal cell apoptosis and
focused on the “effector” caspases-3 and -7, given their importance in liver apoptosis
(Jones et ah, 1998; Woo et ah, 1999; Zheng et ah, 2000; Zheng et ah, 1998).
Although no increase in Ac-DEVD-afc cleavage activity (reflecting the processing and
activation o f caspase-3) was observed in BALB/c mice treated with AAP (Figures 18 &
19), we further examined caspases-3 and -7 by western blot analysis to test whether the
lack o f activation was due to a lack o f processing or due to a post-processing inhibition.
Western blots from cytosolic (post-mitochondrial) fractions isolated from anti-CD95
antibody treated BALB/c mice showed the partial disappearance o f a band corresponding
to the size o f procaspase-3 (Figure 23 top) and this was also observed after AAP dosing.
However, this was not paralleled by the appearance o f the active fragments (pi 7 or p20)
in either treatment group even though the antibody used was able to recognize the large
fragments (Figure 23 top). The same observation was made using another anti-caspase-3
antibody that our lab had previously found to recognize pl7/p20 in mouse tissues (Jones
et ah, 1998).
In contrast, a clear loss o f the p29 form o f procaspase-7 with the appearance o f p i9 had
occurred after CD95 activation but not in AAP-exposed mice (Figure 23 bottom).
Therefore, it is clear that the classic execution caspases-3 and -7 do not play a detectable
role in carrying out AAP-induced damage whereas they do in the case o f CD95
engagement. However, caspases (the identity o f which is not known) are probably
involved in initiating AAP-induced liver injury.
118
Besides caspase-3 and caspase-7, the activation o f other caspases, especially caspase-9
and -8, was not determined. However, we did attempt to measure caspase-9 activation
but failed to see any processing after AAP or anti-CD95 suggesting that the method
lacked sufficient sensitivity. Likewise, there is a lack o f commercially available anti-
caspase-8 antibodies that reliably detect the processing o f the murine form o f this
caspase.
119
RUKu
o.oa
Icou
I'Sou I <
ITjo\s•is<
o\8
- Pro-caspase-3 p29
p17
01 soV
Iso
V
2cou I 3 3
ITiOs Os
Q P PU u U
• JmB a a< < <
Pro-caspase-7p29
p19
Figure 23. Processing of procaspases-3 and -7 in paracetamol-induced liver injury in
BALB/c mice. Animals were dosed with anti-CD95 (40(xg/animal, ip) or AAP (500mg/kg)
and after six hours, the animals were sacrificed and the livers removed for homogenisation
and isolation o f cytosolic fractions as described under Methods. Protein measurement was
carried out using Bio-Rad assay and each lane contained 50 micrograms. The proteins were
separated by SDS-PAGE and immunoblotted for caspases-3 (top) and caspase-7 (bottom).
Each lane represents a separate animal. In the blot for caspase-3, a positive control for the
detection o f pi 7 [from HeLa cells exposed to the human anti-CD95 antibody (CH-11, Kamiya
Biomedical Company, Seattle, WA)] was included.
120
Chapter 5
DNA fragmentation and paracetamol
121
5.1 Paracetamol overdosage causes loss of genomic DNA through
fragmenatation
It has previously been shown that, in male NIH swiss mice, Ca^^-dependent endonuclease
fragmentation o f DNA into oligonucleosomal-length fragments is a characteristic feature
o f paracetamol-induced hepatic cell death (Ray et al., 1993). Pretreatment o f these mice
with the Ca^^-calmodulin antagonist “chlorpromazine” or with the Ca^^-channel blocker
“verapamil” prevented both the increase in nuclear calcium and DNA damage (Ray et al.,
1993; Ray et al., 1996; Ray et al., 1990).
In our project, the process o f identifying DNA fragmentation on agarose gel
electrophoresis proved not to be straightforward. In early stages o f experiments, after
preparing 30% (w/v) liver homogenates in HB (Homogenisation Buffer: 0.25M Sucrose,
5mM Tris, pH 7.4) with 1% (w/v) SDS, DNA was extracted twice with phenol-
chlorophorm, preciptated once with ethanol/sodium acetate, and kept (at -20°C)
overnight. Next day, after centrifuging (at 2000 rpm), DNA pellet was resuspended in
TE-8 buffer (lOmM Tris-HCl, ImM EDTA, pH 8.0) and digested with DNase-free
RNase A (Ipg/ml) for 30 minutes at 37°C. DNA aliquots were then loaded onto a 1%
(w/v) agarose gel containing 0.2 pg/ml ethidium bromide and then separated by constant
voltage mode electrophoresis at 80 volts for 1-2 hours at room temperature. A Hind III
digest lambda DNA served as molecular size standards. Finally electrophoresis gels were
illuminated with 300nm light and a photographic record was made with instant film.
With the use o f the above mentioned protocol, the yield o f DNA was extremely low and a
DNA “smear” was produced (Figure 24).
122
s tU_© B
1 1 Q_©
u s < P.
i ©U >N
<<
Qgi<
k
m
a
i^ÈSSB
Figure 24. Early experiment showing a smear of DNA fragmentation.
“Control” mice received intraperitoneal PBS, “AAP” group were dosed with
intraperitoneal-AAP (500mg/kg), “z-VAD” group received intravenous z-VAD-fmk
(lOmg/kg, in 2.5% DMSO), and “AAP+zVAD” group were pretreated with z-VAD-
fmk 15 minutes prior to receiving AAP. Each lane represents a separate animal. All
animals were sacrificed six hours later.
123
Further attempts to detect DNA fragmentation were carried out (with minor modification)
as previously reported (Ray et al., 1993; Ray et al., 1992) and as outlined in “Materials
and Methods”.
In summary, 10% (w/v) liver homogenates o f frozen liver sections were homogenised in
LB (Lysis Buffer: O.IM Tris, lOmM EDTA, 0.5% (w/v) sodium N-lauroylsarcosine, pH
7.8) and were allowed to stand for 30 min at 4°C. Suspensions were digested with
200pg/ml o f proteinase K for 6 hours at 50°C on a shaking water bath. To remove
proteins associated with the DNA after dissolution o f liver tissue, DNA was extracted
three times with phenol/chloroform and precipitated with 100% ethanol and 2.5M sodium
acetate. After the third ethanol preciptation (overnight at -20°C), DNA was washed
(three times) with 66% ethanol and 2.5M sodium acetate, redissolved in TE-8 buffer
(lOmM Tris-HCl, ImM EDTA, pH 8.0) and digested with DNase-free Rnase A (Ipg/ml,
30 minutes at 37°C). DNA was redissolved in TE-8 for a final time and loaded onto a
1.6% (w/v) agarose gel containing 0.4|Lig/ml o f ethidium bromide. A Hind III digest o f 1-
DNA was used as a molecular size standard. Separation was achieved by constant
voltage mode electrophoresis at 10 volts overnight. Electrophoresis gels were
illuminated with UV light (300nm) and a photographic record was made with instant
film.
To examine the compatibility o f this new method with our system, an experiment was
designed to investigate the “Time-Course” o f AAP-induced effects on genomic DNA
fragmentation (Figure 25).
124
AAP-T reatment (Hours)
1 0 1 2 3 4 5 6
AAP-T reatment (Hours)
1 2 3 5 6
kbp
— 5.0
— 2.0
— 1.4
— 0.8
— 0.6
Figure 25. Time-Course of paracetamol-induced effects on the electrophoretic
behaviour of genomic DNA from livers of BALB/c mice. “Control” mice received
intraperitoneal PBS (0.5ml) and were sacrificed after six hours, whereas “AAP-treated
mice” were dosed with intraperitoneal-AAP (500mg/kg) and sacrificed after the
indicated number o f hours (1, 2, 3, 4, 5, and 6). Each lane represents a separate animal.
125
Figure 25 shows that AAP overdose produced marked degradation o f DNA (with a ladder
o f DNA fragments) between 3 and 6 hours.
Conjunct histopathological studies carried out by K Anwar (data not shown) confirmed
that AAP-induced hepatotoxicity did not appear until 3 hours after AAP treatment
(Anwar, 2001).
Based on the results shown in Figure 25, we concluded that this new protocol was more
likely to detect DNA fragmentation and, therefore, was adopted for all future experiments
with agarose gel electrophoresis.
5.2 Time-course for protection from paracetamol-induced
hepatotoxicity by Z-VAD-fmk
In order to identify the time-window through which intravenous Z-VAD-fmk (lOmg/kg,
dissolved in 2.5% DMSO in PBS) could still be protective to hepatocytes when
administered at different intervals after receiving intraperitoneal AAP (500mg/kg), we
designed a “Time-Course”experiment as illustrated in Figure 26.
Figure 26 shows that one single intravenous injection o f Z-VAD-fmk (lOmg/kg in 2.5%
DMSO in PBS) is protective against AAP-induced DNA damage and, consequently,
against liver injury (evidenced by normal or slightly raised serum enzymes as shown in
Figure 27). This protective effect is only achieved if Z-VAD-fmk was to be administered
simultaneously or up to 2 hours after overdosing with AAP.
126
On the other hand, loss o f the protective effect o f Z-VAD-fmk (after the 2-hour window)
was associated with severe liver injury as evidenced by DNA fragmentation (Figure 26)
and the significant perturbation in serum biochemistry (Figure 27).
127
Acetaminophen (AAP)
+Z-VAD-fmk at indicated time point
2-wSO
U
ux: upfl pC
I I
kbp
— 5.0
— 2.0
_ 1.4
_ 0.8
__ 0.6
Figure 26. Z-VAD-fmk prevents DNA fragmentation at different intervals after
paracetamol administration.
“Control” mice received intraperitoneal PBS (0.5ml). Intravenous Z-VAD-fmk
(lOmg/kg in 2.5% DMSO in PBS) was administered to the “AAP+Z-VAD-fmk”
group simultaneously (0 hr) or at the indicated time points (Ihr, 2hr, and 4hr) after
AAP. “AAP alone” group received intraperitoneal AAP (500mg/kg in PBS). Each
lane represents a separate animal. All animals were sacrificed after 6 hours.
128
1200
100C
i
Iao
U
kgo
X
k3O
X
% % %k kg g gO O o
X X X(N VO
Z-VAD-fmk (hours after AAP)
Figure 27. Z-VAD-fmk prevents the rise in serum ALT after paracetamol
intoxication.
“Control” mice received intraperitoneal PBS (0.5ml). Intravenous Z-VAD-fink (lOmg/kg
in 2.5% DMSO in PBS) was administered simultaneously (0 hr) or at the indicated time
points (Ihr, 2hr, 4hr, and 6hr) after AAP. All animals were sacrificed after 6 hours. ALT
activity in serum was measured using commercial kits (Roche) on a Cobas Bios auto
analyser according to the manufacturer’s instructions. Data given represents Mean ± SEM
o f %=5. *: Significantly different from control (p<0.05).
129
Chapter 6
Mitochondria and paracetamol
130
6.1 Mitochondrial cytochrome c and paracetamol hepatotoxicity
Mitochondria play an important role in the cell death induced by many drugs, including
hepatotoxicity from overdose o f AAP (Adams et al., 2001). Disruption o f mitochondrial
function is recognised to be an integral part o f AAP toxicity (Burcham and Harman,
1991; Esterline et al., 1989; Ramsay et al., 1989; Ray et al., 1999).
AAP toxicity has also been shown to cause ultrastructural changes in mitochondria
(swelling and fusion) and mitochondrial dysfunction (increased resting respiration,
decreased ADP-stimulated respiration, and decreased the respiratory control ratio; RCR)
occur early (10-30 minutes) after AAP adminisstration (Meyers et al., 1988; Ruepp et al.,
2002).
The detrimental effect o f AAP on mitochondria results in hepatic ATP depletion and the
formation o f ROS (Jaeschke, 1990; Tirmenstein and Nelson, 1990) and peroxynitrite
(HOONO) (Hinson et al., 1998; Knight and Jaeschke, 2002; Knight et al., 2001).
In apoptosis, release o f cytochrome c (13 kDa) from the mitochondrial intermembranous
space results in activation o f procaspase-9 in association with Apaf-1, which finally leads
to activation o f caspase-3. It is the activation o f the latter that is o f paramount
importance in the execution o f apoptosis both in in vitro and in vivo studies (Hampton et
ah, 1998; Kluck et al., 1997; Kroemer, 1999; Li et al., 1997b; Liu et al., 1996; Rodriguez
and Lazebnik, 1999; Yang et al., 1997; Zou et al., 1997).
131
Release o f cytochrome c from mitochondria in AAP overdose was reported by Adams et
al (Adams et al., 2001), Ferret et al (Ferret et al., 2001), and Knight and Jaeschki (Knight
and Jaeschke, 2002).
In order to investigate mitochondrial cytochrome c release associated with AAP-induced
hepatotoxicity, mitochondria were isolated as described under Methods.
We first needed to examine the possibility o f mitochondria contaminating the cytosolic
fraction (S-100) during preparation and to investigate if cytochrome c could have been
released into the cytosol o f control BALB/c mice receiving ip PBS on its own.
In blots from control animals, cytochrome c was detected in both mitochondrial and
cytosolic fractions, albeit to a much lesser extent in the latter (Figure 28). Co-blotting for
the inner mitochondrial membrane protein cytochrome c oxidase (COX) revealed the
presence o f mitochondria in the mitochondrial fractions only and therefore ruling out the
possibility o f any mitochondrial contamination o f the cytosol.
The rationale for focusing on mitochondrial cytochrome c (and probing it against COX),
rather than estimating cytosolic cytochrome c, was to avoid the risk o f underestimating
the release due to the rapid leak o f cytosolic proteins (e.g. as seen with ALT, AST, and
LDH) into the general circulation under in vivo conditions.
132
Mitoch
Cytosol
+ +
+ +
+ve control (Cyt c)
bx
m IT)fN (Noo O
COX
Cytc
Figure 28. Presence of mitochondria is confined to the mitochondrial fraction. No
mitochondrial contamination of the cytosol.
Two control BALB/c mice received PBS (0.5ml, ip) and were saerificed after six hours.
Livers were removed for homogenisation and separation into mitoehondrial and cytosolic
fractions were carried out as described under Methods. Proteins were separated by SDS-
PAGE and immunoblotted for cytochrome c (Cyt c: 13 kDa) and cytochrome c oxidase
subunit IV (COX: 17 kDa). Positive control o f cytochrome c was a gift from Dr Chow SC
(Centre for mechanism o f Human Toxicity, University o f Leicester, UK).
133
Recent reports by Nagai et al (Nagai et al., 2002) showed that in vitro hepatocytes
(cultured from C57BL/6 mice) treated with a cytotoxic dose o f AAP failed to release
cytochrome c from mitochondria and cell death was entirely necrotic. However, when
AAP was given concomitantly with TNF-a (but not either one alone), the mode o f cell
death was shifting towards apoptosis and was accompanied by cytochrome c release into
the cytosol and by an increase in caspase-8 and-3-like activities (Nagai et al., 2002).
To investigate the in vivo effect o f AAP toxicity on cytochrome c release from
mitochondria, BALB/c mice were overdosed with intraperitoneal AAP (or the positive
control; anti-CD95) as described under Methods
In our system, isolated mitochondria from AAP-treated mice contained less cytochrome c
compared to the controls (Figure 29). Similar results were observed in the positive
control group (treated with anti-CD95 antibody). Those results are in line with previous
in vivo studies implicating cytochrome c release in AAP toxicity (Adams et al., 2001;
Ferret et al., 2001; Knight and Jaeschke, 2002).
Co-blotting for the inner mitochondrial membrane protein cytochrome c oxidase (COX)
revealed that the loss o f cytochrome c was specific to the intermembrane resident
cytochrome c and was not caused by a general decrease in mitochondrial mass present in
the mitochondrial fraction (Figure 29).
Figure 29 also shows that the overall yield o f mitochondria from liver homogenates
prepared from AAP-exposed mice did not differ from that o f control animals, therefore
suggesting that no major change in mitochondrial density had occurred in response to the
hepatotoxic dose o f AAP.
134
a-CD95
AAP
+ + +
+ + +
< - COX
RatioCytc/C O X 1.7 1.3 1.4 0.7 0.4 0.3 0.1 0.7 0.3
Figure 29. Paracetamol induces cytochrome c release from liver mitochondria. BALB/c
mice were closed with AAP (500mg/kg, ip) or anti-CD95 antibody (40pg/animal, ip) and after
six hours, the animals were sacrificed and the livers removed for homogenisation and
isolation o f mitochondria as described under Methods. The mitochondrial proteins were
separated by SDS-PAGE and immunoblotted for cytochrome c (Cyt c) and cytochrome c
oxidase subunit IV (COX) (arrows). The ratios between Cyt c and COX were obtained by
density scanning o f the film using an MCID image analysis system. Each lane represents a
separate animal.
135
The pretreatment o f BALB/c mice with the broad spectrum casapse inhibitor Z-VAD-
fmk (lOmg/kg, iv) 15 minutes prior to injection o f AAP (500mg/kg, ip) only had minimal
effect on the extent o f cytochrome c release (Figure 30). The same was also observed in
anti-CD95 antibody-treated mice. Therefore we may conclude that the release o f
cytochrome c in this in vivo model o f AAP toxicity is essentially caspase-independent.
6.2 Release of cytochrome c from mitochondria correlates with the
release of adenylate kinase
As the release o f two mitochondrial proteins, cytochrome c and apoptosis-inducing factor
(AIF), into the soluble cytoplasm o f cells undergoing apoptosis is well established, it has
also been demonstrated that the intermembranous protein adenylate kinase isoenzyme 2
(AK2) was translocated into the cytosol concomitantly with cytochrome c in apoptotic
cells (Kohler et al., 1999; Single et al., 1998).
The next set o f experiments addressed the question whether the release o f cytochrome c
(Cyt c) from mitochondria was restricted to this protein or whether other intermembrane
space proteins o f higher molecular weight were also relocated into the cytosol. The
levels o f mitochondrial adenylate kinase (AK) were measured and correlated with the
activity o f COX as an indicator o f mitochondrial mass. As shown in Figure 31, the
results o f this set o f experiments support the notion that, in AAP overdose, the process o f
Cyt c release involves a non-selective change in outer membrane permeability.
136
a-CD95 - - - + + + + + +Z - V A D ..................................................... + + +
— — — ' "W* wag ' v <— c o x— — - Cytc
RatioCytc/COX 27.3 23.4 7.5 2.7 2.0 1.7 4.4 1.34 3.0
AAP - - - + + + + + +Z - V A D + + +
Cyfc/COX 1 1 1.3 1.2 2.1 2.4 2.0
COXCytc
Figure 30. Cytochrome c release from liver mitochondria after in vivo administration of
acetaminophen or anti-CD95 activation: Role of the pan-caspases inhibitor Z-VAD-fmk.
BALB/c mice were dosed with AAP (500mg/kg, ip) or anti-CD95 antibody (40pg/animal, ip)
and after six hours, the animals were sacrificed and the livers removed for homogenisation
and isolation o f mitochondria as described under Methods. Where indicated, animals were
pretreated with Z-VAD-fmk (lOmg/kg, iv) 15 minutes prior to dosing with AAP or anti-CD95
antibody. Mitochondrial proteins were separated by SDS-PAGE and immunoblotted for
cytochrome c (Cyt c) and cytochrome c oxidase subunit IV (COX). The ratios between Cyt c
and COX were obtained by density scanning o f the film using an MCID image analysis
system. Each lane represents a separate animal.
137
egk
%0
1
obo
U
0>soc:
Î
inG\
8I
<
Figure 31. Release of adenylate kinase from mitochondria by paracetamol and after
activation o f CD95. BALB/c mice were dosed with AAP (500mg/kg, ip) or anti-CD95
antibody (40pg/animal, ip) and after six hours, the animlas were sacrificed and the livers
removed for homogenisation and isolation o f mitochondria as described under Methods.
Mitochondrial activities o f adenylate kinase (AK) and cytochrome c oxidase (COX) were
assayed spectrophotometrically. Each bar represents the mean ratio o f AK/COX ± SEM
o f n=A. *: Significantly different from control (p<0.05). * * : Significantly different from
control (p<0.01).
138
6.3 Release of cytochrome c from mitochondria correlates with the loss
of succinate dehydrogenase activity
Succinate dehydrogenase (SDH) is an intramitochondrial enzyme o f the citrate cycle,
located in the inner mitochondrial membrane. It is a flavoprotein containing FAD and
plays a key role in the mechanism o f electronic transport and energy production. In
aerobic cells SDH catalyses the oxidation o f succinate to fumarate transferring the
electrons directly to ubiquinone (Munujos et al., 1993).
Although the exact contribution o f mitochondria in AAP-induced liver injury and cell
death is unclear, alterations to mitochondrial respiration with decrease in SDH activity
with AAP treatments have been reported both in vivo (Katyare and Satav, 1989) and in
vitro (Burcham and Harman, 1991).
Burcham and Harman (Burcham and Harman, 1991) showed that SDH (associated with
respiratory complex II in the inner mitochondrial membrane) was found to be very
sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited
to a lesser extent. They concluded that loss o f the ability to utilize succinate- and
NADH-linked substrates due to attack o f the respiratory chain by NAPQI causes a
disruption o f energy homeostasis in AAP hepatotoxicity.
Donnelly et al (Donnelly et al., 1994) also reported that inhibition o f mitochondrial
respiration preceded overt hepatic necrosis (as indicated by an elevation o f ALT activity)
after administration o f a hepatoxic dose o f AAP (750mg/kg) to fasted male CD-I mice.
139
However, loss o f SDH activity is not unique to AAP hepatotoxicity as it has also been
reported during ischaemia/reperfusion injuries (Benitez Bribiesca et al., 2000).
In our project, to investigate the effect o f in vivo AAP hepatoxicity on SDH activity,
SDH assay was determined by measuring the formation o f the red formazan due to
tétrazolium salt reduction, as outlined in Methods. As shown in figure 32, treatment o f
BALB/c mice with an overdose o f AAP has led to a significant drop in mitochondrial
SDH activity. Similar results were observed in anti-CD95 treated mice. This further
supports the direct involvement o f mitochondria in AAP hepatotoxicity.
140
21
Iao>a
I£
ÎTS
CQa1p
%
*1£A03k
'Va0■5O.tseW)Ss1'•a%
%01upGO0
1
18
15
12
IT)p o\
"ok
op 8
po < PÜ < <
Figure 32. Mitochondrial succinate dehydrogenase (SDH) activity after AAP and anti-
CD95 treatments. BALB/c mice were dosed with AAP (500mg/kg, ip) or anti-CD95
antibody (40pg/animal, ip) and after six hours, the animlas were sacrificed. The livers were
removed, homogenised, and mitochondria were isolated and assyed for SDH activities as
described under Methods. Each group consisted o f three animals. Data given represents
Mean ± SEM. Significantly different from control (p<0.01).
141
6.4 Role of pro-apoptotic members of the Bel-2 family in paracetamol-
induced hepatic injury
The role o f pro-apoptotic members o f the Bcl-2 family in the redistribution o f
cytochrome c (Cyt c) from mitochondria was investigated. As shown in figure 33, the
BH3-only 22 KDa protein Bid (called full-length or intact Bid) was found to undergo
cleavage (after both AAP- and anti-CD95-treatments) to a fragment corresponding to an
apparent molecular mass o f 15 KDa representing the COOH-terminal cleaved product
(called truncated Bid; t-Bid). After either treatment, both intact Bid and its truncated
form (t-Bid) were also found in the mitochondria, thus indicating translocation o f both
forms from the cytosol to the mitochondria.
Figure 33 also shows the density o f the band corresponding to t-Bid in the cytosolic
fraction was inversely related to that in the mitochondrial fraction in both treatment
groups. This is expected as full-length Bid is normally localised in the cytosol (Wang et
al., 1996b) and t-Bid is translocated to the mitochondria upon induction o f apoptosis (Li
et ah, 1998).
In western blot o f proteins, which are proteolytically processed during activation, one
expects to see a decrease in the proform if there is an increase in the cleavage product.
This is nicely shown for Bid cleavage in CD95-induced apoptosis (Figure 33 top).
However it is difficult to explain the increase in the proform (Bid) and a dramatic
increase in the cleaved form (t-Bid) in AAP-treated group (Figure 33 bottom).
142
We simply do not know the exact reason why on a protein to protein basis, there is no
detectable decrease in Bid levels after AAP. However, we suspect changes in Bid
turnover during AAP-induced injury.
It is worth noting that bands o f cytosolic t-Bid were very broad (and denser) in AAP-
treatment compared to those treated with anti-CD95, and the opposite was observed in
the mitochondrial fraction (wider and denser t-Bid in the anti-CD95 treatment compared
with AAP treatment). This may be related to the formation o f additional cleavage
products (like pl3 and p l l ) by caspase(s) that migrate closely with p i 5 (Gross et al.,
1999b) and this will overestimate the total amount o f Bid that was cleaved.
The pancaspase inhibitor Z-VAD-fmk was capable o f preventing the two processes
(cleavage o f intact Bid to t-Bid and the mitochondrial translocation o f the two forms)
after both (AAP- and anti-CD95-) treatments. This implied that cleavage o f Bid in AAP
treatment was caspase-dependent and suggested that the caspase(s) critical to the
initiation o f AAP-induced cell death is (are) located upstream o f Bid activation.
It is also difficult to explain why full-length Bid was identified in the mitochondrial
ft-action o f the negative control animals (without AAP or anti-CD95 treatment), and
moreover, how t-Bid was detected in the mitochondrial fraction in the negative controls
from the anti-CD95 treatment but not in the negative controls from the AAP experiment.
However, this is not utterly unexpected as other studies reported the presence o f intact
Bid in the mitochondrial fraction from control tissues (Desagher et al., 1999; Tafani et
al., 2002b). This behaviour o f Bid may be related to its lipid transfer activity as reported
by Esposti, et al (Esposti et al., 2001).
143
On the other hand, as shown in figure 34, overdosing BALB/c mice with AAP led to the
translocation o f Bax to the mitochondrial fraction, where it was found inserted in
mitochondria, primarily as a monomer although some dimer formation was also detected.
The pattern was similar in anti-CD95 antibody-treated animals with the difference that
after CD95 activation, the translocation o f Bax to the mitochondria and dimer formation
had ocurred to a greater extent.
At the same time as Z-VAD-fmk prevented processing o f Bid and its (and that o f t-Bid)
mitochondrial translocation, it failed to inhibit Bax translocation to the mitochondria.
This suggested that mitochondrial translocation o f Bax was not mediated by Bid and is,
therefore, caspase-independent and most likely involves other non-caspase factors
(Mikhailov et ah, 2001; Pan et al., 2001; Smaili et al., 2001; Tafani et al., 2002a).
144
a-CD95 Z-VAD -fmk
Cytosol
Mitochondria
Bid
t-B id
Bid
t-B id
AAPZ-VAD -fmk
Cytosol
Mitochondria
+ + + + + +- - - + + +
Bid
t-B id
Bid
t-B id
m
Figure 33. Paracetamol induces Bid processing and translocation to the mitochondria in
vivo. BALB/c mice were dosed with AAP (500mg/kg, ip) or anti-CD95 antibody (40|ig/animal,
ip) and after six hours, the animlas were saerificed and the livers removed for homogenisation and
isolation o f mitoehondria as deseribed under Methods. Where indicated, animals were pretreated
with Z-VAD-fmk (lOmg/kg, iv) 15 minutes prior to dosing with AAP or anti-CD95 antibody. The
cytosolic (post-mitochondrial fraction) and mitochondrial proteins were separated by SDS-PAGE
and immunoblotted for Bid. Each lane represents a separate animal.
145
a -CD95
Z-VADdimer
BaxCOX
VAPZ-VAD
dimer
BaxCOX
Figure 34. Paracetamol induces Bax translocation to mitochondria in vivo. BALB/c
mice were dosed with AAP (500mg/kg, ip) or anti-CD95 antibody (40pg/animal, ip) and
after six hours, the animals were sacrificed and the livers removed for homogenisation and
isolation o f mitochondria as described under Methods. Where indicated, animals were
pretreated with Z-VAD-fmk (lOmg/kg, iv) 15 minutes prior to dosing with AAP or anti-
CD95 antibody. Mitoehondrial proteins were separated by SDS-PAGE and immunoblotted
for Bax and cytochrome c oxidase (COX), the latter to ensure equal loading o f
mitoehondrial proteins. Each lane represents a separate animal.
146
Chapter 7
Discussion
147
7.1 Purpose of study
This project had two major aims: (1) The identification o f apoptosis in paracetamol-
induced liver injury and (2) the elucidation o f the mechanism and the role o f apoptosis in
paraeetamol-related hepatotoxicity.
The idea o f this project was bom after several laboratories have demonstrated that
systemic injection o f inhibitors o f caspases (Z-VAD-fmk and Ac-YVAD-cmk) not only
prevented in vivo hepatocyte apoptosis induced by injection o f anti-Fas antibody (or
TNF-a) but also completely protected the animals from the lethal effects o f such
substances (Kunstle et ah, 1997; Rodriguez et ah, 1996; Rouquet et ah, 1996b).
7.2 Background
Large amounts o f the widely used mild analgesic and antipyretic drug AAP may cause
acute liver failure and death in both human beings (Clark et ah, 1973; Davidson and
Eastham, 1966; Proudfoot and Wright, 1970) and experimental animals (Boyd and
Bereezky, 1966; Davis et ah, 1974; Dixon et ah, 1975; Mitchell et ah, 1973a).
The development o f liver damage following ingestion o f AAP falls into two phases
(Read, 1979). The first phase appears to be due to generation o f the reactive metabolite
NAPQI (Dahlin et ah, 1984; Mitchell et ah, 1973a), and can be effectively antagonised
by treatment with V-acetylcysteine (NAC) (Prescott et ah, 1979; Walker et ah, 1981).
148
Whereas in clinieal practice, the therapeutic oral dose o f AAP is 0 .5-lg every 4-6 hours
to a maximum o f 4g in a day, severe hepatic and renal damage can result from taking as
little as 150mg/kg (about lOg or 20 tablets) in one dose, which is only 2.5 times the
recommended maximum daily clinical dose (Laurence et al., 1997). Patients especially
at risk are those on enzyme-indueing drugs (e.g. carbamazepine, phénobarbital,
phenytoin, rifampicin, and alcohol) and those who are malnourished (in anorexia, in
alcoholism, or those who are HIV positive) to the extent that their livers and kidneys are
depleted o f glutathione by NAPQI (Moore et al., 1997).
The plasma concentration o f AAP is o f predictive value: if it lies above a semilogarithmic
graph joining points between 200mg/l (1.32 mmol/1) at 4 h after ingestion to 50mg/l (0.33
mmol/1) at 12 h, then serious hepatic damage is likely (Laurence et ah, 1997; Timbrell,
2000). However, plasma concentration measured earlier than 4 h is unreliable because o f
incomplete absorption (Laurence et ah, 1997).
If plasma levels at least 4 h post ingestion is above this “normal treatment line” and the
patient is presenting up to 24 h after ingestion, such patients should be given intravenous
NAC (prepared in 5% dextrose and given to a total o f around 300mg/kg over 20 hours, in
three divided doses) (BNF, March 2003). On the other hand, malnourished patients and
those on enzyme-inducing drugs should be treated if AAP level is above the “high risk
treatment line” (BNF, March 2003).
This phase o f acute toxicity is normally followed by a period o f apparent recovery.
However, if not treated early, and especially when the amount ingested is toxic, then
patient’s condition tends to deteriorate resulting, in the worst scenario, in liver failure and
death. The pathological mechanisms operating in this final phase o f damage are not fully
149
understood. The damage depends on the continued presence o f AAP in the blood stream,
and this is the best predictor o f likely clinical outcome (Insel, 1996). However treatment
with NAC has little benefit at this stage indicating that some factor distinct from AAP
metabolism may play a major role in the development o f liver damage.
It is generally accepted that the ultimate form o f hepatic damage caused by AAP is
necrosis (Adams et al., 2001; Gujral et al., 2002; Knight and Jaeschke, 2002; Pierce et
al., 2002). However, several reports have presented evidence for the occurrence o f
apoptosis in AAP-induced hepatic damage. For instance, AAP toxicity in rats is
accompanied by a small increase in the number o f isolated parenchymal cells with
apoptotic morphology (Dixon et al., 1975; Gujral et al., 2002; Ray and Jena, 2000; Ray et
ah, 1996), the formation o f oligonucleosomal-length DNA fragmentation and DNA
laddering (Ray et al., 1993; Ray et al., 1990), cleavage o f poly(ADP-ribose)polymerase
(PARP) (Zhang et al., 2000a), and the appearance o f TdT-catalysed dUTP-fluorescein
nick end-labeling (TUNEL) positive cells (Lawson et al., 1999).
More recently. Ferret and co-workers have shown that caspase-3 and -9 activities were
slightly increased in mice that were administered a hepatotoxic dose o f AAP (Ferret et
al., 2001), resulting in the in situ cleavage o f the caspase substrate PARP (Zhang et al.,
2000a).
Besides the possibility o f direct induction o f apoptosis by the cytotoxic metabolite o f
AAP, V-acetyl^-benzoquinone imine (NAPQI), additional factors and in particular,
cytokines such as TNF (released from non-parenchymal cells) and CD95 ligand have
been implicated in AAP-induced liver damage (Fiorucci et al., 2002; Gardner et al..
150
2002; Laskin et al., 1995; Zhang et al., 2000a). These are also well-established inducers
o f parenchymal cell apoptosis (Leist et ah, 1995a; Ogasawara et al., 1993).
Apoptosis is well known to play an important role in the aetiology o f several liver
diseases (Neuman, 2001; Patel et al., 1998). However, it is far from clear whether
apoptosis contributes to the induction o f acute liver failure by AAP.
7.3 Mode of cell death after paracetamol overdose in BALB/c mice:
Necrosis or Apoptosis?
It is well known that apoptosis and necrosis share common initial events and can no
longer be considered as completely separate forms o f cell death (Kass and Orrenius,
1999; Lemasters, 1999; Lemasters et al., 1998; Raffray and Cohen, 1997; Tsujimoto et
a l , 1997).
Even though the induction o f apoptosis in AAP-induced liver injury, as based on
morphological criteria was described over twenty-five years ago (Dixon et ah, 1975; Ray
et ah, 1993; Ray et ah, 1990; Zhang et ah, 2000a), the number o f cells with a truly
apoptotic morphology in the areas o f damage remains very low (Dixon et ah, 1975;
Gujral et ah, 2002). Likewise, the activation o f the apoptosis execution caspases-3 and -7
in response to AAP is moderate (Ferret et ah, 2001; Pierce et ah, 2002), i f not sometimes
absent (Lawson et ah, 1999) (This PhD). Consequently, the ultimate mechanism o f cell
death in AAP toxicity could be concluded as being necrotic (Gujral et ah, 2002).
151
Here, we report that the pan-caspase inhibitor Z-VAD-fmk prevented AAP-induced liver
damage. A similar protective effect was observed with Z-DEVD-cmk. As it is well
accepted that caspase inhibition does not prevent necrosis (oncosis) (Denecker et al.,
2001; Kalai et al., 2002), we therefore concluded that caspase inhibitors used in our study
(Z-VAD-fmk and Z-DEVD-cmk) do not prevent AAP-induced necrosis, and this has
recently been confirmed in vitro (Nagai et al., 2002). In the latter study they found that
AAP-induced glutathione depletion sensitises culture mouse hepatocytes to TNF-a-
induced apoptosis.
However, typical features o f necrosis were evident in cells surrounding central veins by
the third hour after AAP dosing o f BALB/c mice. Most likely, it is this un-interrupted
necrotic damage to hepatocytes that accounts for the fourfold increase in serum ALT in
AAP overdosed BALB/c mice, despite the presence o f caspase inhibitors.
At the dose o f AAP used in our project (500mg/kg), those transient necrotic areas
remained very small and were followed by the development o f large areas o f midzonal
damage with extensive haemorrhagic congestion. Several recent studies have reported
the importance o f such haemorrhagic damage in the development o f several forms o f
liver injury (Gao et al., 1998; Wanner et al., 1999).
From our data, it appears that preventing the development o f haemorrhagic damage in
midzonal areas may be critical to the hepatoprotective effects o f our caspase inhibitors in
AAP overdosing. We therefore proposed a model for AAP-induced liver injury whereby
the initial events leading to the injury actually involve an apoptotic pathway, as revealed
by the sensitivity to the caspase inhibitors. However apoptosis does not go to full
completion (only DNA fragmentation and nuclear pyknosis are observed) and
Ï52
degenerates into necrosis. A likely reason for the incompletion o f the apoptotic
programme and the switch from apoptosis to necrosis/oncosis or necro-apoptosis is the
loss o f cellular ATP, which is a well documented effect o f AAP intoxication in vivo
(Jaeschke, 1990; Knight and Jaeschke, 2002; Tirmenstein and Nelson, 1990) and in vitro
(Kamendulis and Corcoran, 1992) and results from the induction o f mitochondrial
dysfunction by AAP (Knight and Jaeschke, 2002; Meyers et ah, 1988; Ruepp et al.,
2002; Strubelt and Younes, 1992; Weis et al., 1992a).
Despite the fact that many reports have shown that cellular ATP is necessary for
apoptosis and its depletion causes an apoptogenic signal to become necrotic (Eguchi et
al., 1997; Kim et al., 2003; Leist et al., 1997b; Tsujimoto, 1997), it is not fully clear what
role(s) ATP has in hepatocyte apoptosis. For example, others have suggested that in the
liver, hepatocytes triggered to undergo apoptosis by TNF are actually very well capable
o f surviving in an ATP-depleted environment and without having to degenerate into
necrosis (Latta et al., 2000).
7.4 TUNEL assay and DNA fragmentation as markers of apoptosis in
paracetamol hepatotoxicity
Detection o f DNA fragments in situ using the TdT-catalysed dUTP-fluorescein nick end-
labeling (TUNEL) is increasingly applied to investigate active cell death (apoptosis).
However, when the specificity o f the assay was studied in well-defined models o f
apoptosis and necrosis as well as in postmortem autolysis in rat liver, it was found that a
similar TUNEL-positive reaction appeared in necrotic hepatocytes after a cytotoxic dose
153
o f carbon tetrachloride (CCI4) or N-nitrosomorpholine (NNM). Also, in insufficiently
fixed, autolytic livers TUNEL-positive nuclei were observed. Therefore, as DNA
fragmentation is common to different kinds o f cell death, its detection in situ (using
TUNEL assay) should not be considered a specific marker o f apoptosis (Grasl-Kraupp et
a l , 1995).
As it has been reported that the use o f laser scanning confocal microscopy is a very useful
tool in validating apoptosis (Hahn et al., 2001), it was therefore employed in examining
our histological sections stained with TUNEL to rule out non-specific damage.
In our study, TUNEL staining clearly confirmed the abundance o f fragmented DNA in
AAP-overdosed BALB/c, which is in agreement with previous studies (Ferret et al.,
2001; Gujral et al., 2002; Lawson et al., 1999). Our TUNEL analysis also showed that
the pancaspase inhibitor Z-VAD-fmk completely prevented the appearance o f apoptotic
cells after AAP administration and prevented the appearance o f liver damage. A similar
protective effect against AAP-induced liver damage was also observed with Z-DEVD-
cmk (Anwar, 2001). Both o f these results confirm that DNA fragmentation in AAP
toxicity is caspase-dependent.
We found that most TUNEL-positive hepatocytes were found in the midzonal area and
had nuclei that were highly condensed when examined by using the nucleic acid counter
stain propidium iodide (PI). Moreover, the distribution o f the TUNEL fluorescence was
restricted to the nuclear area and was not observed in the cytoplasm. This shows that
under our experimental conditions the TUNEL technique unambiguously identified
apoptotic cells rather than necrotic cells. This was further corroborated by transmission
electron microscopy (TEM) which showed that the hepatocytes in the congested zone
154
generally showed well-formed stacks o f rough endoplasmic reticulum with little evidence
for damage to mitochondria (unpublished data) (Anwar, 2001).
These findings strongly support the occurrence o f limited set o f apoptotic features in
AAP hepatotoxicity. This is in clear distinction to another study (Gujral et al., 2002)
where the TUNEL-positive cells were localised around the centrilobular areas and were
characterised by a generalised (but not nuclear) TUNEL staining o f the cells and features
typical o f necrosis. The difference between our study and the latter is most likely due to
differences in strain (C3HeB/FeJ versus BALB/c mice) and experimental conditions
(300mg/kg in fasted animals versus 500mg/kg with access to food and water ad libitum),
and this probably also explains why the latter laboratory did not see any protective effect
o f Z-VAD-fmk against AAP-induced hepatic injury in a previous study (Lawson et al.,
1999).
The absence o f execution caspase activation is not in contradiction with the occurrence o f
apoptotic nuclear condensation and DNA fragmentation [as detected by the TUNEL
reaction or as oligonucleosomal-length fragments when resolved by agarose gel
electrophoresis (Ray et al., 1990)].
Recently, Strasser and colleagues (Marsden et al., 2002) reported that the kinetics o f
apoptosis in caspase-9'^' or Apaf-T^' cells is virtually indistinguishable fi*om that observed
in wild-type cells. In these knockout cells, caspase-3 processing was completely absent
and only barely detectable levels o f active caspase-7 were reported.
Therefore, the classic pathway o f execution caspase activation downstream o f
mitochondrial cytochrome c release may be redundant for the generation o f some
155
apoptotic features. The lack o f caspase-3 activation would help explain why the PI-
counterstained nuclei o f the TUNEL-positive parenchymal cells in the AAP-treated livers
were highly condensed rather than typically fragmented as found after in vivo CD95
stimulation (Ogasawara et al., 1993) and resembled the abnormal apoptotic nuclear
morphology o f hepatocytes following CD95-activation in caspase-3'^' mice reported by
others (Zheng et al., 1998).
7.5 Initiation of paracetamol-induced apoptosis involves mitochondria:
Role of Bcl-2 family members
Bcl-2 family members play a key role in processes underlying programmed cell death or
apoptosis (Jacobson, 1997; Kroemer, 1997; Reed, 1997). It is increasingly appreciated
that proteins o f the Bcl-2 family are crucially involved in the control o f apoptotic
pathways (Chao and Korsmeyer, 1998; Vander Heiden and Thompson, 1999).
This family o f proteins is composed o f both proapoptotic (Bax, Bak, Bok, Bik, Blk, Bcl-
xs. Bad, Bid, Hrk, BNIP3, BimL, Bad, and EGL-1) and antiapoptotic (Bcl-2, B cI-xl, Bcl-
w, M cl-l, A l, NR-I3, BHRFl, LMW5-HL, 0RF16, KS-Bcl-2, E1B-19K, and CED-9)
molecules (Adams and Cory, 1998; Antonsson and Martinou, 2000). These proteins can
form homo- and hetero-dimers that involve amino acid sequences known as Bcl-2
homology (BH) domains. Although four BH domains (BH1-BH4) have been identified
(Kelekar and Thompson, 1998; Kroemer, 1997; Reed, 1997), it is the BH3 domain o f
proapoptotic members that is vitally important as it appears to be required for the
interaction between anti- and proapoptotic molecules (Chittenden et al., 1995).
156
The Bcl-2 family members Bax and Bid act as promoters o f apoptosis (Eskes et al., 1998;
Jurgensmeier et al., 1998; Li et al., 1998; Luo et al., 1998; Narita et al., 1998; Rosse et
al., 1998) and translocate to mitochondria to facilitate the release o f cytochrome c (and
other proteins resident in the intermembrane space) into the cytosol, where it binds to a
complex o f proteins to form the apoptosome (Bossy-Wetzel and Green, 1999; Eskes et
al., 2000; Gross et al., 1999b; Kluck et al., 1999; Vander Heiden and Thompson, 1999;
Zheng et ah, 2000). The apoptosome then promotes the self-cleavage o f procaspase
enzymes, which leads to the activation o f executioner caspases (Green, 2000). Emerging
evidence indicates that Bid is involved in various pathways o f apoptosis that interplay the
activation o f caspases with mitochondrial dysfunction (Green, 2000; Gross et al., 1999b;
Kudla et al., 2000; Luo et al., 1998; Wei et al., 2000; Yin et al., 1999; Zha et al., 2000).
Here we report that the induction o f the initial apoptotic response by AAP involved
mitochondria, most likely through the recruitment and activation o f the pro-apoptotic
Bcl-2 family members Bax and Bid and the release o f cytochrome c. The dimerization o f
Bax is an initial event in the integration o f Bax into the mitochondrial outer membrane
(Antonsson, 2001; Antonsson et al., 2001; Gross et al., 1998; Wolter et al., 1997), and
once integrated it is thought that Bax oligomerizes to form a cytochrome c-permeable
channel (Epand et ah, 2002; Pavlov et ah, 2001; Saito et ah, 2000).
It has also been shown that the BH3-only protein Bid facilitates Bax oligomerization and
insertion into the mitochondrial outer membrane, a process that is executed even more
efficiently by the C-terminal part o f cleaved Bid (called truncated Bid or t-Bid) (Eskes et
ah, 2000; Korsmeyer et ah, 2000; Kuwana et ah, 2002; Roucou et ah, 2002).
157
In this study we found that Bid was cleaved to a pi 5 truncated form o f similar apparent
molecular mass as t-Bid and was translocated to the mitochondrial fraction in response to
AAP treatment. The ability o f the pancaspase inhibitor Z-VAD-fmk to block Bid
proteolysis after AAP administration implicates caspase-8 and the participation o f a death
receptor in AAP-induced liver injury (Li et al., 1998; Luo et al., 1998), and suggests that
the formation o f t-Bid is mediated by caspases rather than by calpains (Chen et al., 2001)
or lysosomal proteases (Stoka et al., 2001).
Whether Bid cleavage in AAP hepatotoxicity occurred through an indirect activation o f
caspase-8 by downstream effector caspases or was mediated by caspase-8 through the
CD95 pathway (Fiorucci et al., 2002; Zhang et al., 2000a) or by TNF (Laskin et al.,
1995) remains to be elucidated.
Another feature observed in this study was that not only t-Bid but also full-length Bid
relocated to mitochondria in response to AAP and anti-CD95 antibody treatment. This
phenomenon has recently been observed in the presence o f physiological concentrations
o f phospholipids (such as phosphatidic acid and phosphatidylglycerol) and proposed a
major role for Bid in cellular lipid transfer between mitochondria and other intracellular
membranes, thereby explaining the dynamic relocation o f intact Bid (and t-Bid) to the
mitochondria (Esposti et al., 2001).
This may be related to the propensity o f t-Bid to bind to cardiolipin, which is a membrane
lipid unique to mitochondria (Lutter et al., 2000). The presence o f intact Bid in
mitochondria has also been confirmed in other studies (Desagher et al., 1999; Tafani et
a l , 2002b).
158
In our study, there were differences in the extent o f Bax translocation between AAP and
anti-CD95 treatments. In addition, the translocation o f Bax in response to anti-CD95
antibody treatment was nearly completely dependent on caspases (and possibly Bid
truncation) as evidenced by the ability o f the pan-caspase inhibitor Z-VAD-fmk to block
both Bid processing and Bax translocation. In the case o f AAP, Bax translocation to the
mitochondria was observed in agreement with a previous report (Adams et al., 2001).
However, Z-VAD-fmk, although inhibiting Bid proteolysis, failed to prevent Bax
translocation.
These findings suggest that CD95 death receptor mediated apoptosis in the liver proceeds
primarily via caspase-8-mediated Bid truncation, which is in agreement with the report
that Bid-deficient mice are nearly completely resistant to anti-CD95 antibody-induced
hepatocyte apoptosis in vivo (Yin et al., 1999). In contrast, additional factors must be
involved in AAP-induced Bax translocation to mitochondria. Such factors mediating Bax
translocation could include a perturbation o f intracellular Ca^ homeostasis (Pan et al.,
2001), ATP depletion (Mikhailov et al., 2001), a loss o f mitochondrial membrane
potential (Smaili et al., 2001), or changes in intracellular pH (Tafani et al., 2002a), all o f
which have previously been shown or are likely to occur and play a critical role in AAP
injury (Jaeschke, 1990; Kamendulis and Corcoran, 1992; Kass et al., 1992; Moore et al.,
1985; Weis eta l., 1992a).
At the same time that Adams et al (Adams et al., 2001) reported the crucial role played
by Bax as an early determinant o f AAP-mediated hepatotoxicity in mice, they also found
that overexpression o f the anti-apoptotic protein Bcl-2 not only failed to protect against
AAP toxicity but, unexpectedly, exacerbated the degree o f injury. The reason for this is
159
unclear, although the phenomenon appears similar to the apparently paradoxical
aggravation o f ischaemia/reperfusion-induced liver cell apoptosis and hepatic damage in
Bcl-2-overexpressing mice (Oshiro et ah, 2002).
In agreement with other reports (Adams et al., 2001; Ferret et al., 2001; Knight and
Jaeschke, 2002), AAP overdose in our project induced the release o f cytochrome c (Cyt
c) from mitochondria to the cytosol. The fact that Cyt c release into the cytosol occurred
even in the presence o f the pancaspase inhibitor Z-VAD-fmk suggests that the release in
AAP toxicity takes place in a caspase-independent manner.
It is known that cytochrome c translocation from mitochondria is a multistep process
involving an increase in outer membrane permeability, the diffusion o f a small pool o f
unbound cytochrome c and the dissociation o f the bulk o f the mitochondrial cytochrome
c from cardiolipin binding site followed by its release from mitochondria (Ott et al.,
2002). The peroxidation o f cardiolipin was recently reported after AAP administration to
mice, and this correlated with a loss o f mitochondrial cytochrome c (Ferret et al., 2001).
Interestingly, the in vitro induction o f necrosis by AAP may not trigger the release o f
cytochrome c from mitochondria (Nagai et al., 2002), the latter requiring an apoptotic
costimulus. However, the relevance o f these in vitro findings to the present in vivo study
remains to be determined.
The extent o f cytochrome c released from mitochondria in our study was essentially
identical after anti-CD95 antibody treatment or AAP administration, and accounted for
more than 50% o f the total mitochondrial cytochrome c content. Z-VAD-fmk only had a
minor inhibitory effect in the case o f CD95 but had no detectable effect on AAP-induced
160
cytochrome c release. In addition, AK, a protein o f twice the molecular weight o f
cytochrome c, was also released to a virtually identical degree.
Also, in this study and in agreement with previous reports (Burcham and Harman, 1991;
Katyare and Satav, 1989), we have demonstrated the loss o f succinate dehydrogenase
(SDH) activity in AAP-treated BALB/c mice (compared to saline-treated controls). This
further implicates NAPQI in disrupting mitochondrial respiration and compromising
cellular energy homeostatsis in paracetamol toxicity.
Although cytochrome c and AK release could have occurred in the absence o f inner
membrane permeability transition (MPT) (Jurgensmeier et al., 1998; Pavlov et al., 2001;
Waterhouse et al., 2001), several studies would support a model for the release o f
cytochrome c from mitochondria in the liver in situ implicating MPT in addition to Bax.
A-acetyl-^-benzoquinone imine (NAPQI), the reactive metabolite o f AAP, has previously
been shown to induce damage to mitochondria through its pro-oxidant activity and Ca^
mobilization and is capable o f inducing MPT in isolated mitochondria (Weis et al.,
1992a) and in isolated rat hepatocytes (Kass et al., 1992), whereas the MPT inhibitor
cyclosporin A was found to protect from AAP hepatotoxicity in vivo (Haouzi et al.,
2002).
As other reports have demonstrated that MPT (and outer mitochondrial membrane
damage) occurs in response to CD95 activation in hepatocytes in vitro (Hatano et ah,
2000) and in vivo (Feldmann et al., 2000) and in TNF-induced hepatocyte apoptosis in
vivo (Angermuller et al., 1999) and, as the pretreatment with cyclosporin A (a
permeability transition inhibitor) prevented or considerably delayed both anti-CD95
antibody-(Feldmann et al., 2000) and TNF-(Pastorino and Hoek, 2000) induced
Ï6Ï
mitochondrial outer membrane rupture, cytochrome c release, and apoptosis, we therefore
concluded that our observations fit a model whereby in vivo cytochrome c release after
AAP administration to BALB/c mice occurs in response to MPT.
7.6 Lack of execution caspase activity in paracetamol toxicity
Caspases are cysteine proteases, the active site o f which is highly conserved among all
caspases. Caspases require a reduced cysteine for their proteolytic activity (Hentze et al.,
2000; Hentze et al., 1999).
In our study, we were unable to correlate the protection by the caspase inhibitors (Z-
VAD-fmk and Z-DEVD-cmk) with an increase in caspase activity or the detection o f the
active form o f caspases-3 and -7. Using the caspase-3 and -9 peptide substrates, Ac-
DEVD-afc and Ac-LEHD-afc, a significant decrease in activity was measured when
serum levels o f ALT began to rise. Examination o f the time course o f the inhibition
revealed that the inhibition o f Ac-DEVD-afc and Ac-LEHD-afc occurred at 3hr post
dosing, which corresponds to the time when AAP-induced liver damage became
detectable on histopathology. This lack o f caspase activity was not due to release o f
caspases into the serum as DEVDase activity in serum from AAP-treated BALB/c mice
was negligible. Also, this caspase inactivity was not due to the presence o f endogenous
caspase inhibitors in the liver homogenates o f AAP-treated animals as homogenates from
those animals failed to suppress the DEVDase activity o f positive controls (thymocyte
apoptotic extract incubated with IpM dexamethasone for 6h).
162
Such lack o f caspase activation could have been due to oxidation or covalent adduct
formation with the caspases which are known to possess thiol group(s) (Lawson et ah,
1999). Consequently, this may inactivate the caspases (Nobel et al., 1997) or interfere
with their activation pathway (Hentze et al., 1999).
This could also be secondary to glutathione (GSH) depletion as recent reports (Hentze et
al., 2003; Hentze et al., 2002) have suggested that GSH depletion can block death
receptor-induced apoptosis at basically three levels within the signaling cascade: (1) the
conversion o f inactive procaspase-8 to active caspase-8; (2) the caspase-9-mediated
apoptosome pathway; and (3) the enzymatic activity o f the executioner caspase-3.
As loss o f GSH and modification o f protein sulfhydryl groups is well documented in
AAP toxicity (Moore et al., 1985; Tirmenstein and Nelson, 1990; Weis et al., 1992b), we
can therefore conclude that it is the delicate balance between the physiological
antioxidants (like GSH) and various oxidants which will determine whether caspase-
initiated apoptosis in AAP toxicity can proceed to completion.
Another likely reason for failure to activate caspases is depletion o f cellular ATP which is
characteristic o f AAP intoxication. As described earlier, AAP can disrupt mitochondrial
function (Knight and Jaeschke, 2002; Meyers et al., 1988; Ruepp et al., 2002; Strubelt
and Younes, 1992; Weis et al., 1992a) and result in cellular ATP depletion (Jaeschke,
1990; Knight and Jaeschke, 2002; Tirmenstein and Nelson, 1990).
Cellular ATP content is an apoptosis-related metabolic parameter that may be modified
selectively in the liver (Eguchi et al., 1997; Leist et al., 1997b). ATP depletion affects
apoptotic pathways in three fundamentally different ways. First, biochemical evidence
163
suggests that ATP, together with cytochrome c, is needed for the formation o f the
apoptosome (Li et al., 1997b; Liu et al., 1996). Accordingly ATP depletion will prevent
activation o f execution caspases at a step downstream o f cytochrome c release (Leist et
al., 1999a; Stridh et al., 1999). Such cells with damaged mitochondria are in most cases
still destined to die, but the absence o f caspase activity switches the mode o f cell death
from apoptosis to necrosis (Eguchi et al., 1997; Kim et al., 2003; Leist et al., 1997b;
Leist et ah, 1999a; Leist et al., 1999b; Stridh et al., 1999; Tsujimoto, 1997). Second, in
some models, ATP depletion may block apoptotic steps upstream o f cytochrome c release
(Leist et al., 1999b; Stridh et al., 1999). In such cases, both caspase activation and
mitochondrial damage are prevented, and cells may truly survive (Volbracht et al., 1999).
Finally, in cases where CD95 stimulation triggers a cascade o f proteolytic events that
directly lead to apoptosome-independent activation o f effector caspases, ATP depletion
may have no effect at all on caspase activation (Eguchi et al., 1999) or cell death (Ferrari
e ta l., 1998).
AAP is both a notorious producer o f reactive oxygen species (ROS) and a potent inducer
o f oxidative stress (Wendel, 1983; Wendel, 1984; Wendel et al., 1979; Wendel and
Hallbach, 1986; Wendel and Jaeschke, 1982; Wendel et al., 1982). Reactive nitrogen
species (particularly nitric oxide NO) may too be important in AAP hepatotoxicity
(Jaeschke et ah, 2003; James et ah, 2003). Thus, another reason for inability to activate
caspases may relate to the production o f ROS and nitrogen species, as both o f which can
block caspase activity.
As caspases are redox sensitive enzymes, they are regulated by ROS (Fadeel et al., 1998).
In the oxidative-stress model, generation o f ROS (namely hydrogen peroxide) through
164
redox cycling inhibits caspase activity, and caspase inactivation by this mechanism may
prevent cell death by apoptosis (Samali et al., 1999). Other reports suggest that,
depending on the degree o f the initial oxidative stress, caspases are either activated and
the cells die by apoptosis, or they remain inactive and therefore degenerate into necrosis
(Hampton and Orrenius, 1997).
By regulating the activity o f caspases, nitrogen species can also interfere with the
execution steps o f apoptosis. This may take place either upstream (i.e. processing o f
caspases-3 and -7) (Dimmeler et al., 1998) or downstream o f cytochrome c release
(Torok et al., 2002; Zech et al., 2003) or both (Kim et ah, 2002; Leist et al., 1999b; Li et
1999).
However, given that the execution caspases-3 and -7 were not activated under our
experimental conditions, it is clear that the apoptotic response to AAP was not properly
executed and most likely degenerated to give features o f premature secondary necrosis, a
conclusion that was supported by other reports (Adams et al., 2001; Pierce et al., 2002).
We also concluded that apoptosis has an important causal role in initiating the damage
but because o f the lack o f completion o f the process (possibly due to excessive loss o f
ATP) and its degeneration, the ultimate damage to the liver presents many features o f
necrosis, which is in clear agreement with the majority o f publications (Adams et al.,
2001; Gujral et ah, 2002; Knight and Jaeschke, 2002; Pierce et al., 2002).
The identity o f the caspase(s) that is (are) responsible for initiating AAP-induced liver
cell damage and was (were) targeted by the pancaspase inhibitor Z-VAD-fmk in this
study remains unclear.
165
Although the reactive haloketone moiety o f the peptide inhibitors has previously been
reported to interact with other proteases such as cathepsin B (Schotte et ah, 1999), we
found that the nonselective fmk-based peptide Z-FA-fmk failed to inhibit AAP-induced
liver injury. This confirms that the peptide inhibitors used in this study acted by targeting
the caspases involved in apoptosis.
Increasing the dose o f AAP to > 600 mg/kg resulted in the loss o f the protective effect o f
the caspase inhibitors used. Consequently, it was concluded that the dose window for Z-
VAD-fmk protection is relatively narrow. This suggests that the role o f apoptosis in
initiating key events that lead to liver damage in AAP toxicity is restricted to the lower
portion o f the hepatotoxic dose o f AAP, whereas at higher concentrations the primary
mechanism initiating cell death is necrosis.
As published reports on cases o f adult human AAP overdosage showed that the quantities
o f ingested AAP required to result in acute hepatic (and hepatorenal) failure ranged
between 14-142g (equivalent to 200-2000 mg/kg) (Prescott, 1996), and as the dose o f
AAP used in this study (500mg/kg) was well within this range, we therefore felt the
limitation o f apoptosis to this lower portion o f hepatotoxic dose o f AAP (as used in our
mouse-model) is o f great relevance to clinical cases o f human AAP toxicity.
Taken together our results show that caspases and apoptosis play an important causal role
in the mechanism o f AAP-induced hepatic injury by initiating the sequence o f events that
ultimately lead to liver necrosis.
166
7.7 Sinusoidal endothelial and Kupffer cells in paracetamol
hepatotoxicity: Role of hepatic congestion and cytokines
In our project, six hours after BALB/c mice were overdosed with AAP (500mg/kg), the
animals were hypothermic and the livers were grossly enlarged with massive
accumulation o f RBCs. The critical mechanism responsible for the congestion in the
liver appears to be the damage induced by AAP to the sinusoidal endothelial cells
(SECs). Such damage was documented (using TEM) and was in agreement with earlier
reports both in mice (Walker et ah, 1983) and patients (Bramley et al., 1991).
Hepatic congestion secondary to damaged SECs leads to the disruption o f hepatic
micro vascular perfusion which may then lead to acute liver injury and subsequent liver
failure (Gao et al., 1998; Takei et al., 1991; Thurman et al., 1988; Wanner et al., 1999).
Congestion may also be more critical in the development o f AAP-mediated hepatic injury
than the direct cytotoxic effects o f NAPQI on parenchymal cells (Dahlin et al., 1984;
Jollow et al., 1973; Mitchell et al., 1973a). The latter is typical o f the first phase o f
injury, and in this study centrilobular parenchymal cell necrosis was evident by 2-3 hours
post AAP administration but then regressed thereafter even though liver injury continued
to develop.
Prevention o f endothelial cell damage by the caspase inhibitors blocked the development
o f liver apoptosis, liver damage, and went on to protect from liver failure induced by
AAP. The critical role o f endothelial cell damage and ensuing congestion in liver failure
is also supported by recent work on the death receptor CD95 (Nishimura et al., 1997;
Wanner et al., 1999), on ischaemia-reperfusion-induced liver injury and concanavalin A-
167
induced hepatitis (Gao et ah, 1998; Knolle et al., 1996; Samarasinghe and Farrell, 1996;
Takei et al., 1995; Takei et al., 1991; Thurman et al., 1988).
The fact that no TUNEL-positive SECs were identified in sections obtained six hours
after AAP-dosing o f BALB/c mice suggests that endothelial cell apoptosis did not go to
completion, but rather was only sufficient to cause the formation o f large pores through
reorganization o f the cytoskeleton o f the SECs. However, a more recent re-investigation
o f the time-course o f AAP-induced hepatic damage has revealed the presence o f TUNEL-
positive SECs at four hours following AAP administration (Hinton R and Kass GEN,
unpublished results).
It is well known that SECs are exceedingly sensitive to damage during reperfusion
(Deaciuc et al., 1999; Gao et al., 1998; Samarasinghe and Farrell, 1996; Takei et al.,
1991; Thurman et al., 1988) and to TNF (Takei et al., 1995). Although direct activation
o f AAP by the SECS has been documented (DeLeve et al., 1997) and a further
contribution by parenchymal cell-produced NAPQI is also likely, it is still difficult to
establish the exact mechanism by which AAP is cytotoxic to SECs in vivo.
To investigate the role o f Kupffer cells in AAP-hepatotoxicity, we used gadolinium
chloride (GdCls) as a suppressor o f Kupffer cell function (Husztik et al., 1980; Laskin et
al., 1995). Pretreatment o f BALB/c mice with GdCls did not prevent the rise in serum
transaminases or the appearance o f damaged and congested areas. However TUNEL
staining confirmed that GdCfi-pretreatment prevents the formation o f apoptotic cells.
This lack o f protection (against hepatic injury) by GdClg rules out a Kupffer cell- or a
macrophage-derived component such as TNF or nitric oxide as major effectors o f hepatic
damage in AAP-induced injury. Conversely, the ability o f GdCls to prevent apoptosis o f
168
parenchymal cells implicates the direct involvement o f Kupffer cell-derived cytokines in
hepatocyte apoptosis.
Our results are in contrast with the study by Laskin and co-workers (Laskin et a l , 1995)
who showed that pretreatment o f Long Evans Hooded rats with GdCfi, completely
blocked AAP (800mg/kg)-induced hepatic necrosis, the increase in serum transaminases,
and liver damage. It is likely that species differences account for this difference in
response to AAP. In fact, it is known that SECs show marked strain differences in their
susceptibility to cytotoxic damage by AAP (DeLeve et al., 1997). The latter also
probably accounts for the lack o f cytoprotection by Z-VAD-fmk from AAP-induced
hepatic damage in fasted C3Heb/FeJ mice (Lawson et al., 1999) while in this study fed
BALB/c mice were used.
In this thesis, we report that Kupffer cell-derived cytokines are responsible for
parenchymal cell apoptosis in AAP-induced liver injury. However, inhibition o f
parenchymal cell apoptosis does not prevent hepatic injury and liver failure. In contrast,
damage to the sinusoidal liver endothelium appears to be the critical event that is
responsible for liver failure through hemorrhagic damage and shut down o f hepatic
microvascular perfusion.
169
7.8 CONCLUSION
In conclusion, our results show that the initiation o f mitochondrial pathway o f apoptosis
was shared by both CD95 and AAP. Yet, whereas anti-CD95 antibody treatment
activated the full caspase cascade as evidenced by the large increase in DEVDase
activity, the apoptotic pathway was only incompletely activated in response to AAP
treatment. Instead, it degenerated to induce premature secondary necrosis.
The development o f liver injury in AAP overdose was not directly related to parenchymal
cell apoptosis. Instead, damage to the sinusoidal endothelial cells appeared responsible
for liver failure through haemorrhagic damage and shut down o f hepatic microvascular
perfusion.
The protection provided by the caspase inhibitors highlights the importance o f caspases
(Figure 35) and apoptosis in initiating the events that lead to AAP-induced hepatic injury
and subsequent necrosis. However, further work is necessary to identify the caspase(s)
that initiate AAP-induced liver injury.
Even though the execution o f apoptosis may be carried out by other types o f proteases
whose identity remains to be established, the ability o f caspase inhibitors to block the
development o f liver failure should encourage further work into the possibility o f
targeting caspases as a therapeutic opportunity in AAP-induced liver injury.
170
K C
Œ 2D TNF
^ TNFRl
DISC
ca sp a se -8
Bid \ ?NAPQI
caspase-3
FTP??
caspase-9
Figure 35. Schematic representation of in vivo paracetamol-mediated apopotosis and
caspase activation. This pathway shows the working relationship between the paracetamol
metabolite NAPQI, TNF, caspases, Bid, Bax, and their convergence on the mitochondria and the
role played by the pancaspase inhibitor Z-VAD-fmk in modulating this response. Paracetamol
appears to activate apoptosis through both Kupffer cell (KC)-derived cytokine (TNF) release
causing death receptor-mediated apoptosis o f parenchymal cells (Pathway I) and a direct
activation pathway o f the mitochondria (Pathway II). The latter may play a predominant role in
apoptosis o f SECs and may be responsible for the hepatic congestion described in this thesis.
171
Chapter 8
References
172
Adams, J. M. and Cory, S. (1998). The Bcl-2 protein family: arbiters o f cell survival. Science 281, 1322-6.
Adams, M. L., Pierce, R. H., Vail, M. E., White, C. C., Tonge, R. P., Kavanagh, T. J., Fausto, N., Nelson, S. D. and Bruschi, S. A. (2001). Enhanced acetaminophen hepatotoxicity in transgenic mice overexpressing BCL-2. Molecular Pharmacology 60,907-15.
Afford, S. and Randhawa, S. (2000). Apoptosis. Mol Pathol 53, 55-63.Ahmad, M., Srinivasula, S. M., Wang, L., Talanian, R. V., Litwack, G., Femandes-
Alnemri, T. and Alnemri, E. S. (1997). CRADD, a novel human apoptotic adaptor molecule for caspase-2, and FasL/tumor necrosis factor receptor-interacting protein RIP. Cancer Research 57,615-9.
Allen, R. T., Hunter, W. J. and Agrawal, D. K. (1997). Morphological and biochemical characterization and analysis o f apoptosis. Journal o f Pharmacological and Toxicological Methods 37,215-28.
Angermuller, S., Schumann, J., Fahimi, H. D. and Tiegs, G. (1999). Ultrastructura! alterations o f mitochondria in pre-apoptotic and apoptotic hepatocytes o f TNF alpha-treated galactosamine-sensitized mice. Annals o f the New York Academy o f Sciences 887, 12-7.
Ankarcrona, M., Dypbukt, J. M., Bonfoco, E., Zhivotovsky, B., Orrenius, S., Lipton, S. A. and Nicotera, P. (1995). Glutamate-induced neuronal death: a succession o f necrosis or apoptosis depending on mitochondrial function. Neuron 15, 961-73.
Antonsson, B. (2001). Bax and other pro-apoptotic Bcl-2 family "killer-proteins" and their victim the mitochondrion. Cell and Tissue Research 306, 347-61.
Antonsson, B. and Martinou, J. C. (2000). The Bcl-2 protein family. Experimental Cell Research 256,50-7.
Antonsson, B., Montessuit, S., Lauper, S., Eskes, R. and Martinou, J. C. (2000). Bax oligomerization is required for channel-forming activity in liposomes and to trigger cytochrome c release from mitochondria. Biochemical Journal 345 Pt 2, 271-8.
Antonsson, B., Montessuit, S., Sanchez, B. and Martinou, J. C. (2001). Bax is present as a high molecular weight oligomer/complex in the mitochondrial membrane o f apoptotic cells. The Journal o f Biological Chemistry 276,11615-23.
Anwar, K. (2001). Role o f apoptosis (programmed cell death) in acute liver failure (PhD Thesis). In School o f Biomedical and Life Sciences (SBLS): University o f Surrey, United Kingdom.
Arthur, M. J., Bentley, I. S., Tanner, A. R., Saunders, P. K., Millward Sadler, G. H. and Wright, R. (1985). Oxygen-derived free radicals promote hepatic injury in the rat. Gastroenterology 89, 1114-22.
Ashkenazi, A., Pai, R. C., Fong, S., Leung, S., Lawrence, D. A., Marsters, S. A., Blackie, C., Chang, L., McMurtrey, A. E., Hebert, A., DeForge, L., Koumenis, I. L., Lewis, D., Harris, L., Bussiere, J., Koeppen, H., Shahrokh, Z. and Schwall, R. H. (1999). Safety and antitumor activity o f recombinant soluble Apo2 ligand. Journal o f Clinical Investigation 104,155-62.
Aupeix, K., Hugel, B., Martin, T., Bischoff, P., Lill, H., Pasquali, J. L. and Freyssinet, J. M. (1997). The significance o f shed membrane particles during programmed cell death in vitro, and in vivo, in HIV-1 infection. The Journal o f Clinical Investigation 99, 1546-54.
173
Balaban, R. S., Mootha, V. K. and Arai, A. (1996). Spectroscopic determination o f cytochrome c oxidase content in tissues containing myoglobin or hemoglobin. Analytical Biochemistry 237,274-8.
Barile, M., Valenti, D., Hobbs, G. A., Abruzzese, M. P., Keilbaugh, S. A., Passarella, S., Quagliariello, E. and Simpson, M. V. (1994). Mechanisms o f toxicity o f 3'-azido- 3'-deoxythymidine. Its interaction with adenylate kinase. Biochemical Pharmacology 48, 1405-12.
Bedossa, P. and Paradis, V. (1995). Transforming growth factor-beta (TGF-beta): a key- role in liver fibrogenesis. Journal ofHepatology 22, 37-42.
Beg, A. A. and Baltimore, D. (1996). An essential role for NF-kappaB in preventing TNF-alpha-induced cell death [see comments]. Science 274, 782-4.
Benedetti, A., Brunelli, E., Risicato, R., Cilluffo, T., Jezequel, A. M. and Orlandi, F. (1988). Subcellular changes and apoptosis induced by ethanol in rat liver. Journal ofHepatology^., 137-43.
Benitez Bribiesca, L., Gomez Camarillo, M., Castellanos Juarez, E., Mravko, E. and SanchezSuarez, P. (2000). Morphologic, biochemical and molecular mitochondrial changes during reperfusion phase following brief renal ischemia. Annals o f the New York Academy o f Sciences 926, 165-79.
Benoist, C. and Mathis, D. (1997). Cell death mediators in autoimmune diabetes—no shortage o f suspects. Cell 89,1-3.
Bemuau, D., Feldmann, G., Degott, C. and Gisselbrecht, C. (1981). Ultrastructural lesions o f bile ducts in primary biliary cirrhosis. A comparison with the lesions observed in graft versus host disease. Human Pathology 12, 782-93.
Bertin, J., Armstrong, R. C., Ottilie, S., Martin, D. A., Wang, Y., Banks, S., Wang, G. H., Senkevich, T. G., Alnemri, E. S., Moss, B., Lenardo, M. J., Tomaselli, K. J. and Cohen, J. I. (1997). Death effector domain-containing herpesvirus and poxvirus proteins inhibit both Fas- and TNFRl-induced apoptosis. Proceedings o f the National Academy o f Sciences o f the United States o f America 94, 1172-6.
Beuers, U., Boyer, J. L. and Paumgartner, G. (1998). Ursodeoxycholic acid in cholestasis: potential mechanisms o f action and therapeutic applications. Hepatology 2S, 1449-53.
Bcutler, B. and Cerami, A. (1988). Tumor necrosis, cachexia, shock, and inflammation: a common mediator. Annual Review o f Biochemistry 57, 505-18.
Biava, C. and Mukhlova, M. M. (1965). Electron microscopic observation on councilman-like acidophilic bodies and other forms o f acidophilic changes in human liver cells. American Journal o f Pathology 46, 775-802.
Bissell, D. M., Wang, S. S., Jamagin, W. R. and Roll, F. J. (1995). Cell-specific expression o f transforming growth factor-beta in rat liver. Evidence for autocrine regulation o f hepatocyte proliferation. Journal o f Clinical Investigation 96, 447- 55.
BNF. (March 2003). British National Formulary 45, pp. 22-23: British Medical Association (BMA) and Royal Pharmaceutical Society o f Great Britain.
Bodmer, J. L., Holler, N., Reynard, S., Vinciguerra, P., Schneider, P., Juo, P., Blenis, J. and Tschopp, J. (2000). TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nature Cell Biology 2,241-3.
Boise, L. H., Gonzalez Garcia, M., Postema, C. E., Ding, L., Lindsten, T., Turka, L. A., Mao, X., Nunez, G. and Thompson, C. B. (1993). bcl-x, a bcl-2-related gene that functions as a dominant regulator o f apoptotic cell death. Cell 74, 597-608.
174
Boldin, M. P., Goncharov, T. M., Goltsev, Y. V. and Wallach, D. (1996). Involvement o f MACH, a novel MORTl/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85, 803-15.
Bonfoco, E., Krainc, D., Ankarcrona, M., Nicotera, P. and Lipton, S. A. (1995). Apoptosis and Necrosis: 2 distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or Nitric oxide superoxide in cortical cell-cultures. Proceedings o f the National Academy o f Sciences o f the United States o f America 92, 7162-7166.
Boobis, A. R., Seddon, C. E., Nasseri-Sina, P. and Davies, D. S. (1990). Evidence for a direct role o f intracellular calcium in paracetamol toxicity. Biochemical Pharmacology 1277-81.
Boobis, A. R., Tee, L. B., Hampden, C. E. and Davies, D. S. (1986). Freshly isolated hepatocytes as a model for studying the toxicity o f paracetamol. Food and Chemical Toxicology 24, 731-6.
Borner, C., Martinou, L, Mattmann, C., Irmler, M., Schaerer, E., Martinou, J. C. and Tschopp, J. (1994). The protein bcl-2 alpha does not require membrane attachment, but two conserved domains to suppress apoptosis. The Journal o f Cell Biology 126, 1059-68.
Bossy-Wetzel, E. and Green, D. R. (1999). Caspases induce cytochrome c release from mitochondria by activating cytosolic factors. Journal o f Biological Chemistry 274,17484-90.
Botla, R., Spivey, J. R., Aguilar, H., Bronk, S. F. and Gores, G. J. (1995). Ursodeoxycholate (UDCA) inhibits the mitochondrial membrane permeability transition induced by glycochenodeoxycholate: a mechanism o f UDCA cytoprotection. Journal o f Pharmacology and Experimental Therapeutics 272, 930-8.
Boyd, E. M. and Bereczky, G. M. (1966). Liver necrosis from paracetamol. British Journal o f Pharmacology 26,606-14.
Boyer, C. S., Moore, G., A. and Moldeus, P. (1993). Submitochondrial localization o f the NAD+ glycohydrolase. The Journal o f Biological Chemistry 208,4016-4020.
Bramley, P. N., Rathbone, B. J., Forbes, M. A., Cooper, E. H. and Losowsky, M. S. (1991). Serum hyaluronate as a marker o f hepatic derangement in acute liver damage. Journal ofHepatology 13, 8-13.
Bredesen, D. E. (1995). Neural apoptosis. Annals o f Neurology 38, 839-51.Buckley, C. D., Pilling, D., Henriquez, N. V., Parsonage, G., Threlfall, K.,
Scheel Toellner, D., Simmons, D. L., Akbar, A. N., Lord, J. M. and Salmon, M. (1999). RGD peptides induce apoptosis by direct caspase-3 activation. Nature 397, 534-9.
Budihardjo, L, Oliver, H., Lutter, M., Luo, X. and Wang, X. (1999). Biochemical pathways o f caspase activation during apoptosis. Annu Rev Cell Dev Biol 15 ,269- 90.
Buendia, B., Santa Maria, A. and Courvalin, J. C. (1999). Caspase-dependent proteolysis o f integral and peripheral proteins o f nuclear membranes and nuclear pore complex proteins during apoptosis. Journal o f Cell Science 112 ( Pt 11), 1743-53.
Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R. and Cox, D. W. (1993). The Wilson disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene. Nature Genetics 5, 327-37.
175
Bump, N. J., Hackett, M., Hugunin, M., Seshagiri, S., Brady, K., Chen, P., Ferenz, C., Franklin, S., Ghayur, T. and Li, P. (1995). Inhibition o f ICE family proteases by baculovirus antiapoptotic protein p35. Science 269, 1885-8.
Burcham, P. C. and Harman, A. W. (1991). Acetaminophen toxicity results in site- specific mitochondrial damage in isolated mouse hepatocytes. Journal o f Biological Chemistry 266,5049-54.
Buttke, T. M. and Sandstrom, P. A. (1994). Oxidative stress as a mediator o f apoptosis. Immunology Today 15, 7-10.
Cain, K., Inayat-Hussain, S. H., Couet, C. and Cohen, G. M. (1996). A cleavage-site- directed inhibitor o f interleukin-1 beta-converting enzyme-like proteases inhibits apoptosis in primary cultures o f rat hepatocytes. Biochemical Journal 314 ( Pt 1), 27-32.
Carr, B. I., Hayashi, L, Branum, E. L. and Moses, H. L. (1986). Inhibition o f DNA synthesis in rat hepatocytes by platelet-derived type beta transforming growth factor. Cancer Research 46,2330-4.
Cascales, M., Alvarez, A., Gasco, P., Femandez-Simon, L., Sanz, N. and Bosca, L.(1994). Cocaine-induced liver injury in mice elicits specific changes in DNA ploidy and induces programmed death o f hepatocytes. Hepatology 20,992-1001.
Castilla, A., Prieto, J. and Fausto, N. (1991). Transforming growth factors beta 1 and alpha in chronic liver disease. Effects o f interferon alfa therapy. New England Journal o f Medicine 324,933-40.
Cecconi, F. (1999). Apafl and the apoptotic machinery. Cell Death Differ 6,1087-98.Cecconi, F., Alvarez Bolado, G., Meyer, B. L, Roth, K. A. and Gruss, P. (1998). Apafl
(CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727-37.
Chai, J., Du, C., Wu, J. W., Kyin, S., Wang, X. and Shi, Y. (2000). Structural and biochemical basis o f apoptotic activation by Smac/DIABLO. Nature 406, 855-62.
Chao, D. T. and Korsmeyer, S. J. (1998). BCL-2 family: regulators o f cell death. Annual Review o f Immunology 16, 395-419.
Chau, B. N., Cheng, E. H., Kerr, D. A. and Hardwick, J. M. (2000). Aven, a novel inhibitor o f caspase activation, binds Bcl-xL and Apaf-1. Mo/ Cell 6 ,31-40.
Chaudhaiy, P. M., Eby, M., Jasmin, A., Bookwalter, A., Murray, J. and Hood, L. (1997). Death receptor 5, a new member o f the TNFR family, and DR4 induce FADD- dependent apoptosis and activate the NF-kappaB pathway. Immunity 7, 821-30.
Chen, M., He, H., Zhan, S., Krajewski, S., Reed, J. C. and Gottlieb, R. A. (2001). Bid is cleaved by calpain to an active fragment in vitro and during myocardial ischemia/reperfusion. The Journal o f Biological Chemistry 276,30724-8.
Chiba, T., Takahashi, S., Sato, N., Ishii, S. and Kikuchi, K. (1996). Fas-mediated apoptosis is modulated by intracellular glutathione in human T cells. European Journal o f Immunology 26,1164-9.
Chirillo, P., Pagano, S., Natoli, G., Puri, P. L., Burgio, V. L., Balsano, C. and Levrero, M. (1997). The hepatitis B virus X gene induces p53-mediated programmed cell death. Proceedings o f the National Academy o f Sciences o f the United States o f America 94, 8162-7.
Chittenden, T., Flemington, C., Houghton, A. B., Ebb, R. G., Gallo, G. J., Elangovan, B., Chinnadurai, G. and Lutz, R. J. (1995). A conserved domain in Bak, distinct from BHl and BH2, mediates cell death and protein binding functions. Embo Journal 14, 5589-96.
176
Choi, K. S., Lim, I. K., Brady, J. N. and Kim, S. J. (1998). ICE-like protease (caspase) is involved in transforming growth factor beta 1-mediated apoptosis in FaO rat hepatoma cell line. Hepatology 27,415-21.
Chow, S. C., Weis, M., Kass, G. E. N., Holmstrom, T. H., Eriksson, J. E. and Orrenius, S.(1995). Involvement o f multiple proteases during Fas-mediated apoptosis in T- lymphocytes. FEBSLetters 364, 134-138.
Chu, Z. L., McKinsey, T. A., Liu, L., Gentry, J. J., Malim, M. H. and Ballard, D. W. (1997). Suppression o f tumor necrosis factor-induced cell death by inhibitor o f apoptosis C-IAP2 is under NF-kappaB control. Proceedings o f the National Academy o f Sciences o f the United States o f America 94, 10057-62.
Cikala, M., Wilm, B., Hobmayer, E., Bottger, A. and David, C. N. (1999). Identification o f caspases and apoptosis in the simple metazoan Hydra. Current Biology 9, 959- 62.
Clark, R., Borirakchanyavat, V., Davidson, A. R., Thompson, R. P., Widdop, B., Goulding, R. and Williams, R. (1973). Hepatic damage and death from overdose o f paracetamol. Lancet 1,66-70.
Cohen, G. M. (1997). Caspases: the executioners o f apoptosis. BiochemicalJournal 326 ( Pt 1), 1-16.
Cohen, G. M., Sun, X. M., Snowden, R. T., Dinsdale, D. and Skilleter, D. N. (1992a). Key morphological features o f apoptosis may occur in the absence o f intemucleosomal DNA fragmentation. Biochemical Journal 286 ( Pt 2), 331-4.
Cohen, J. J., Duke, R. C., Fadok, V. A. and Sellins, K. S. (1992b). Apoptosis and programmed cell death in immunity. Annual Review o f Immunology 10,267-93.
Colell, A., Garcia-Ruiz, C., Miranda, M., Ardite, E., Mari, M., Morales, A., Corrales, F., Kaplowitz, N. and Femandez-Checa, J. C. (1998). Selective glutathione depletion o f mitochondria by ethanol sensitizes hepatocytes to tumor necrosis factor. Gastroenterology 115,1541-51.
Coles, B., Wilson, I., Wardman, P., Hinson, J. A., Nelson, S. D. and Ketterer, B. (1988). The spontaneous and enzymatic reaction o f N-acetyl-p-benzoquinonimine with glutathione: a stopped-fiow kinetic study. Archives o f Biochemistry and Biophysics 264,253-60.
Collins, R. J., Harmon, B. V., Gobe, G. C. and Kerr, J. F. (1992). Intemucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. International Journal o f Radiation Biology 61,451-3.
Crompton, M. (1999). The mitochondrial permeability transition pore and its role in cell death. Biochemical Journal 341 ( Pt 2), 233-49.
Cryns, V. and Yuan, J. (1998). Proteases to die for [published erratum appears in Genes Dev 1999 Feb 1;13(3):371]. Genes and Development 12, 1551-70.
Dahlin, D. C., Miwa, G. T., Lu, A. Y. and Nelson, S. D. (1984). N-acetyl-p- benzoquinone imine: a cytochrome P-450-mediated oxidation product o f acetaminophen. Proceedings o f the National Academy o f Sciences o f the United States o f America 81, 1327-31.
Darmon, A. J., Nicholson, D. W. and Bleackley, R. C. (1995). Activation o f the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature 377,446-8.
Date, M., Matsuzaki, K., Matsushita, M., Sakitani, K., Shibano, K., Okajima, A., Yamamoto, C., Ogata, N., Okumura, T., Seki, T., Kubota, Y., Kan, M., McKeehan, W. L. and Inoue, K. (1998). Differential expression o f transforming
177
growth factor-beta and its receptors in hepatocytes and nonparenchymal cells o f rat liver after CC14 administration. Journal ofHepatology 28, 572-81.
Davidson, D. G. and Eastham, W. N. (1966). Acute liver necrosis following overdose o f paracetamol. British M edicalJournal 5512,497-9.
Davis, D. C., Potter, W. Z., Jollow, D. J. and Mitchell, J. R. (1974). Species differences in hepatic glutathione depletion, covalent binding and hepatic necrosis after acetaminophen. Life Sciences 14,2099-109.
De Vos, K., Goossens, V., Boone, E., Vercammen, D., Vancompemolle, K., Vandenabeele, P., Haegeman, G., Piers, W. and Grooten, J. (1998). The 55-kDa tumor necrosis factor receptor induces elustering o f mitochondria through its membrane-proximal region. Journal o f Biological Chemistry 273, 9673-80.
Deaciuc, I. V., D'Souza, N. B., Sarphie, T. G., Schmidt, J., Hill, D. B. and McClain, C. J. (1999). Effects o f exogenous superoxide anion and nitrie oxide on the scavenging function and electron microscopic appearance o f the sinusoidal endothelium in the isolated, perfused rat liver. Journal ofHepatology 30,213-21.
Deaciuc, I. V., D'Souza, N. B. and Spitzer, J. J. (1995). Tumor necrosis factor-alpha cell- surface receptors o f liver parenchymal and nonparenchymal cells during acute and chronic alcohol administration to rats. Alcoholism, Clinical and Experimental Research 19,332-8.
Debbas, M. and White, E. (1993). Wild-type p53 mediates apoptosis by E lA , which is inhibited by E lB . Genes & Development 7, 546-54.
Decoster, E., Vanhaesebroeck, B., Vandenabeele, P., Grooten, J. and Piers, W. (1995). Generation and biological characterization o f membrane-bound, uncleavable murine tumor necrosis factor. The Journal o f Biological Chemistry 270, 18473-8.
Degli Esposti, M. (1999). To die or not to die—the quest o f the TRAIL receptors. Journal o f Leukocyte Biology 65,535-42.
DeLeve, L. D., Wang, X., Kaplowitz, N., Shulman, H. M., Bart, J. A. and van der Hoek, A. (1997). Sinusoidal endothelial cells as a target for acetaminophen toxicity. Direct action versus requirement for hepatocyte activation in different mouse strains. Biochemical Pharmacology 53, 1339-45.
Denecker, G., Vercammen, D., Steemans, M., Vanden Berghe, T., Brouckaert, G., Van Loo, G., Zhivotovsky, B., Piers, W., Grooten, J., Declercq, W. and Vandenabeele, P. (2001). Death receptor-induced apoptotic and necrotic cell death: differential role o f caspases and mitochondria. Cell Death and Differentiation 8, 829-40.
Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B. and Martinou, J. C. (1999). Bid-induced conformational change o f Bax is responsible for mitochondrial cytochrome c release during apoptosis. Journal o f Cell Biology 144, 891-901.
Desbarats, J., Duke, R. C. and Newell, M. K. (1998). Newly discovered role for Pas ligand in the cell-cycle arrest o f CD4+ T cells. Nature Medicine 4,1377-82.
Deveraux, Q. L., Roy, N., Stennicke, H. R., Van Arsdale, T., Zhou, Q., Srinivasula, S. M., Alnemri, E. S., Salvesen, G. S. and Reed, J. C. (1998). lAPs block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition o f distinct caspases. Embo Journal 17,2215-23.
Deveraux, Q. L., Stennicke, H. R., Salvesen, G. S. and Reed, J. C. (1999). Endogenous inhibitors o f caspases. Journal o f Clinical Immunology 19,388-98.
178
Deveraux, Q. L., Takahashi, R., Salvesen, G. S. and Reed, J. C. (1997). X-linked lAP is a direct inhibitor o f cell-death proteases. Nature 388,300-4.
Diehl, A. M., Yin, M., Fleckenstein, J., Yang, S. Q., Lin, H. Z., Brenner, D. A., Westwick, J., Bagby, G. and Nelson, S. (1994). Tumor necrosis factor-alpha induces c-jun during the regenerative response to liver injury. American Journal o f Physiology 267, G552-61.
Dimmeler, S., Haendeler, J., Sause, A. and Zeiher, A. M. (1998). Nitric oxide inhibits APO-l/Fas-mediated cell death. Cell Growth & Differentiation : the Molecular Biology Journal o f the American Association For Cancer Research 9,415-22.
Dixon, M. P., Dixon, B., Aparicio, S. R. and Loney, D. P. (1975). Experimental paracetamol-induced hepatic necrosis: a light- and electron-microscope, and histochemical study. Journal o f Pathology 116,17-29.
Dong, Z., Saikumar, P., Weinberg, J. M. and Yenkatachalam, M. A. (1997). Internucleosomal DNA cleavage triggered by plasma membrane damage during necrotic cell death. Involvement o f serine but not cysteine proteases. American Journal o f Pathology 151,1205-13.
Donnelly, P. J., Walker, R. M. and Racz, W. J. (1994). Inhibition o f mitochondrial respiration in vivo is an early event in acetaminophen-induced hepatotoxicity. Archives o f Toxicology 68,110-8.
Drappa, J., Vaishnaw, A. K., Sullivan, K. E., Chu, J. L. and Elkon, K. B. (1996). Pas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity. The New England Journal o f Medicine 335,1643-9.
Du, C., Pang, M., Li, Y., Li, L. and Wang, X. (2000). Smae, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating lAP inhibition. Cell 102,33-42.
Duan, H. and Dixit, V. M. (1997). RAIDD is a new 'death' adaptor moleeule. Nature 385, 86-9.
Duke, R. C., Chervenak, R. and Cohen, J. J. (1983). Endogenous endonuclease-induced DNA fragmentation: an early event in cell-mediated cytolysis. Proceedings o f the National Academy o f Sciences o f the United States o f America 80, 6361-5.
Eamshaw, W. C., Martins, L. M. and Kaufmann, S. H. (1999). Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annual Review o f Biochemistry 68, 383-424.
Edwards, M. J., Keller, B. J., Kauffman, P. C. and Thurman, R. G. (1993). The involvement o f Kupffer cells in carbon tetrachloride toxicity. Toxicology and Applied Pharmacology 119,275-9.
Eguchi, Y., Shimizu, S. and Tsujimoto, Y. (1997). Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Research 57, 1835-40.
Eguchi, Y., Srinivasan, A., Tomaselli, K. J., Shimizu, S. and Tsujimoto, Y. (1999). ATP- dependent steps in apoptotic signal transduction. Cancer Research 59,2174-81.
elSisi, A. E., Earnest, D. L. and Sipes, I. G. (1993). Vitamin A potentiation o f earbon tetrachloride hepatotoxicity: role o f liver macrophages and active oxygen species. Toxicology and Applied Pharmacology 119,295-301.
Emery, J. G., McDonnell, P., Burke, M. B., Deen, K. C., Lyn, S., Silverman, C., Dul, E., Appelbaum, E. R., Eichman, C., DiPrinzio, R., Dodds, R. A., James, I. E., Rosenberg, M., Lee, J. C. and Young, P. R. (1998). Osteoprotegerin is a receptor for the cytotoxic ligand TRAIL. Journal o f Biological Chemistry 273, 14363-7.
179
Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A. and Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD [see comments] [published erratum appears in Nature 1998 May 28;393(6683):396]. Nature 391,43-50.
Epand, R. P., Martinou, J. C., Montessuit, S., Epand, R. M. and Yip, C. M. (2002). Direct evidence for membrane pore formation by the apoptotic protein Bax. Biochemical and Biophysical Research Communications 298, 744-9.
Eskes, R., Antonsson, B., Osen-Sand, A., Montessuit, S., Richter, C., Sadoul, R., Mazzei, G., Nichols, A. and Martinou, J. C. (1998). Bax-induced cytochrome C release from mitochondria is independent o f the permeability transition pore but highly dependent on Mg2+ ions. Journal o f Cell Biology 143,217-24.
Eskes, R., Desagher, S., Antonsson, B. and Martinou, J. C. (2000). Bid induces the oligomerization and insertion o f Bax into the outer mitochondrial membrane. Molecular and Cellular Biology 20,929-35.
Esposti, M. D., Erler, J. T., Hickman, J. A. and Dive, C. (2001). Bid, a widely expressed proapoptotic protein o f the Bcl-2 family, displays lipid transfer activity. Molecular and Cellular Biology 21,7268-76.
Esterline, R. L., Ray, S. D. and Ji, S. (1989). Reversible and irreversible inhibition o f hepatic mitochondrial respiration by acetaminophen and its toxic metabolite, N- acetyl-p-benzoquinoneimine (NAPQI). Biochemical Pharmacology 2387-90.
Padeel, B., Ahlin, A., Henter, J. I., Orrenius, S. and Hampton, M. B. (1998). Involvement o f caspases in neutrophil apoptosis: regulation by reactive oxygen species. Blood 92,4808-18.
Padeel, B., Orrenius, S. and Zhivotovsky, B. (1999). Apoptosis in human disease: a new skin for the old ceremony? Biochemical and Biophysical Research Communications 266,699-717.
Pallon, J. P. and Simandl, B. K. (1978). Evidence o f a role for cell death in the disappearance o f the embryonic human tail. The American Journal o f Anatomy 152,111-29.
Pang, J. W., Shen, W. W., Meager, A. and Lau, J. Y. (1996). Activation o f the tumor necrosis factor-alpha system in the liver in chronic hepatitis B virus infection. American Journal o f Gastroenterology 91, 748-53.
Paubion, W. A., Guicciardi, M. E., Miyoshi, H., Bronk, S. P., Roberts, P. J., Svingen, P. A., Kaufmann, S. H. and Gores, G. J. (1999). Toxic bile salts induce rodent hepatocyte apoptosis via direct activation o f Pas. Journal o f Clinical Investigation 103, 137-45.
Peldmann, G. (1997). Liver apoptosis. Journal ofHepatology 26 Suppl 2 ,1-11.Peldmann, G., Haouzi, D., Moreau, A., Durand-Schneider, A. M., Bringuier, A., Berson,
A., Mansouri, A., Pau, D. and Pessayre, D. (2000). Opening o f the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Pas-mediated hepatic apoptosis in mice. Hepatology 31, 674-83.
Perluga, J. and Allison, A. C. (1978). Role o f mononuclear infiltrating cells in pathogenesis o f hepatitis. Lancet 2, 610-1.
Perrari, D., Stepczynska, A., Los, M., Wesselborg, S. and Schulze_Osthoff, K. (1998). Differential regulation and ATP requirement for caspase-8 and caspase-3 activation during CD95- and anticancer drug-indueed apoptosis. The Journal o f Experimental Medicine 188, 979-84.
180
Ferret, P. J., Hammoud, R., Tulliez, M., Tran, A., Trebeden, H., Jaffray, P., Malassagne,B., Calmus, Y., Weill, B. and Batteux, F. (2001). Detoxifieation o f reactive oxygen species by a nonpeptidyl mimic o f superoxide dismutase cures acetaminophen-induced acute liver failure in the mouse. Hepatology (Baltimore, M /.; 33,1173-80.
Finucane, D. M., Bossy-Wetzel, E., Waterhouse, N. J., Cotter, T. G. and Green, D. R.(1999). Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. Journal o f Biological Chemistry 274, 2225-33.
Fiorucci, S., Antonelli, E., Mencarelli, A., Palazzetti, B., Alvarez Miller, L., Muscara, M., del Soldato, P., Sanpaolo, L., Wallace, J. L. and Morelli, A. (2002). A NO- releasing derivative o f aeetaminophen spares the liver by acting at several checkpoints in the Fas pathway. British Journal o f Pharmacology 135, 589-99.
Friedlander, R. M., Brown, R. H., Gagliardini, V., Wang, J. and Yuan, J. (1997). Inhibition o f ICE slows ALS in mice. Nature 388,31.
Fromenty, B. and Pessayre, D. (1995). Inhibition o f mitochondrial beta-oxidation as a mechanism o f hepatotoxicity. Pharmacology and Therapeutics 67, 101-54.
Fujita, T., Ishido, S., Muramatsu, S., Itoh, M. and Hotta, H. (1996). Suppression o f actinomycin D-induced apoptosis by the NS3 protein o f hepatitis C virus. Biochemical and Biophysical Research Communications 229, 825-31.
Galle, P. R., Hofmann, W. J., Walczak, H., Schaller, H., Otto, G., Stremmel, W., Krammer, P. H. and Runkel, L. (1995). Involvement o f the CD95 (APO-l/Fas) receptor and ligand in liver damage. Journal o f Experimental Medicine 182, 1223-30.
Gantner, F., Leist, M., Lohse, A. W., Germann, P. G. and Tiegs, G. (1995). Concanavalin A-induced T-cell-mediated hepatie injury in mice: the role o f tumor necrosis idicXor. Hepatology 2 1 ,190-8.
Gao, W., Bentley, R. C., Madden, J. F. and Clavien, P. A. (1998). Apoptosis o f sinusoidal endothelial cells is a critical mechanism o f preservation injury in rat liver transplantation. Hepatology 27, 1652-60.
Garcia Martinez, V., Macias, D., Ganan, Y., Garcia Lobo, J. M., Francia, M. V., Femandez Teran, M. A. and Hurle, J. M. (1993). Intemueleosomal DNA fragmentation and programmed cell death (apoptosis) in the interdigital tissue o f the embryonic chick leg bud. Journal o f Cell Science 106 ( Ft 1), 201-8.
Garcia-Ruiz, C., Morales, A., Ballesta, A., Rodes, J., Kaplowitz, N. and Femandez- Checa, J. C. (1994). Effect o f chronic ethanol feeding on glutathione and functional integrity o f mitochondria in periportal and perivenous rat hepatocytes. Journal o f Clinical Investigation 94, 193-201.
Gardner, C. R., Laskin, J. D., Dambach, D. M., Saeco, M., Durham, S. K., Bruno, M. K., Cohen, S. D., Gordon, M. K., Gerecke, D. R., Zhou, P. and Laskin, D. L. (2002). Reduced hepatotoxicity o f acetaminophen in mice lacking inducible nitric oxide synthase: potential role o f tumor necrosis factor-alpha and interleukin-10. Toxicology and Applied Pharmacology 184,27-36.
Gavrieli, Y., Sherman, Y. and Ben-Sasson, S. A. (1992). Identification o f programmed cell death in situ via specific labeling o f nuclear DNA fragmentation. Journal o f Cell Biology 119 ,493-501.
Glueksmann, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol Rev Camb Philos Soc 26, 59-86.
181
Goldin, R. D., Hunt, N. C., Clark, J. and Wickramasinghe, S. N. (1993). Apoptotic bodies in a murine model o f alcoholic liver disease: reversibility o f ethanol-induced changes. Journal o f Pathology 171, 73-6.
Goldin, R. D., Ratnayaka, I. D., Breach, C. S., Brown, I. N. and Wickramasinghe, S. N.(1996). Role o f macrophages in acetaminophen (paracetamol)-induced hepatotoxicity. Journal o f Pathology 179,432-5.
Gonzalez-Garcia, M., Perez-Ballestero, R., Ding, L., Duan, L., Boise, L. H., Thompson,C. B. and Nunez, G. (1994). bcl-XL is the major bcl-x mRNA form expressed during murine development and its product localizes to mitochondria. Development 120,3033-42.
Gomall, A. G., Bardwill, C. J. and David, M. M. (1949). Determination o f serum proteins by means o f the biuret reaetion. Journal o f Biological Chemistry 177, 751-766.
Grande, J. P. (1997). Role o f transforming growth factor-beta in tissue injury and repair. Proceedings o f the Society For Experimental Biology and Medicine 214,27-40.
Graper, L. (1914). Eine neue Anschauung uber physiologische Zellausschaltung. Arch Zellforsch 12, 373-394.
Grasl-Kraupp, B., Ruttkay-Nedecky, B., Koudelka, H., Bukowska, K., Bursch, W. and Sehulte-Hermann, R. (1995). In situ detection o f fragmented DNA (TUNEL assay) fails to discriminate among apoptosis, necrosis, and autolytic cell death: a cautionary note. Hepatology 21,1465-8.
Green, D. R. (1998). Apoptotic pathways: the roads to ruin. Cell 94, 695-8.Green, D. R. (2000). Apoptotic pathways: paper wraps stone blunts scissors. Cell 102, 1-
4.Green, D. R. and Reed, J. C. (1998). Mitochondria and apoptosis. Science 281, 1309-12.Gregus, Z., Madhu, C. and Klaassen, C. D. (1988). Species variation in toxication and
detoxication o f acetaminophen in vivo: a comparative study o f biliary and urinary excretion o f acetaminophen metabolites. The Journal o f Pharmacology and Experimental Therapeutics 244,91-9.
Gressner, A. M., Polzar, B., Lahme, B. and Mannherz, H. G. (1996). Induction o f rat liver parenchymal cell apoptosis by hepatic myofibroblasts via transforming growth factor beta. Hepatology 23, 571-81.
Griffiths, S. D., Clarke, A. R., Healy, L. E., Ross, G., Ford, A. M., Hooper, M. L., Wyllie, A. H. and Greaves, M. (1997). Absence o f p53 permits propagation o f mutant cells following genotoxic damage. Oncogene 14, 523-31.
Gross, A., Jockel, J., Wei, M. C. and Korsmeyer, S. J. (1998). Enforced dimerization o f BAX results in its translocation, mitochondrial dysfunction and apoptosis. Embo Journal 17, 3878-85.
Gross, A., McDonnell, J. M. and Korsmeyer, S. J. (1999a). BCL-2 family members and the mitochondria in apoptosis. Genes and Development 13, 1899-911.
Gross, A., Yin, X. M., Wang, K., Wei, M. C., Jockel, J., Milliman, C., Erdjument- Bromage, H., Tempst, P. and Korsmeyer, S. J. (1999b). Caspase cleaved BID targets mitochondria and is required for cytochrome c release, while BCL-XL prevents this release but not tumor necrosis factor-Rl/Fas death. Journal o f Biological Chemistry 274, 1156-63.
Guicciardi, M. E. and Gores, G. J. (1998). Is ursodeoxycholate an antiapoptotic drug? Hepatology 28, 1721-3.
182
Guilhot, S., Miller, T., Comman, G. and Isom, H. C. (1996). Apoptosis induced by tumor necrosis factor-alpha in rat hepatocyte cell lines expressing hepatitis B virus. American Journal o f Pathology 148, 801-14.
Gujral, J. S., Knight, T. R., Farhood, A., Bajt, M. L. and Jaeschke, H. (2002). Mode of cell death after acetaminophen overdose in mice; apoptosis or oncotic necrosis? Toxicological Sciences : An Official Journal o f the Society o f Toxicology 67, 322- 8 .
Gunawardhana, L., Mobley, S. A. and Sipes, 1. G. (1993). Modulation o f 1,2- dichlorobenzene hepatotoxicity in the Fischer-344 rat by a scavenger o f superoxide anions and an inhibitor o f Kupffer cells. Toxicology and Applied Pharmacology 119,205-13.
Hackenbroek, C. R. (1968). Chemical and physical fixation o f isolated mitochondria in low-energy and high-energy states. Proceedings o f the National Academy o f Sciences o f the United States o f America 61, 598-605.
Hahn, Y. S., Soguero, C. and Cruise, M. (2001). Towards a reliable parameter o f liver damage in hepatitis C: TUNEL versus caspase activation. Hepatology (Baltimore, M /.; 34, 840-1.
Hakem, R., Hakem, A., Duncan, G. S., Henderson, J. T., Woo, M., Soengas, M. S., Elia, A., de la Pompa, J. L., Kagi, D., Khoo, W., Potter, J., Yoshida, R., Kaufman, S.A., Lowe, S. W., Penninger, J. M. and Mak, T. W. (1998). Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339-52.
Hamada, E., Nishida, T., Uchiyama, Y., Nakamura, J., Isahara, K., Kazuo, H., Huang, T. P., Momoi, T., Ito, T. and Matsuda, H. (1999). Activation o f Kupffer cells and caspase-3 involved in rat hepatocyte apoptosis induced by endotoxin. Journal o f Hepatology 'ill, 807-18.
Hampton, M. B. and Orrenius, S. (1997). Dual regulation o f caspase activity by hydrogen peroxide: implications for apoptosis. Febs Letters 414, 552-6.
Hampton, M. B., Zhivotovsky, B., Slater, A. F., Burgess, D. H. and Orrenius, S. (1998). Importance o f the redox state o f cytochrome c during caspase activation in cytosolic extracts. Biochemical Journal 329 ( Pt 1), 95-9.
Hanabuchi, S., Koyanagi, M., Kawasaki, A., Shinohara, N., Matsuzawa, A., Nishimura, Y., Kobayashi, Y., Yonehara, S., Yagita, H. and Okumura, K. (1994). Fas and its ligand in a general mechanism o f T -cell-mediated cytotoxicity. Proceedings o f the National Academy o f Sciences o f the United States o f America 91,4930-4.
Haouzi, D., Cohen, I., Vieira, H. L., Poncet, D., Boya, P., Castedo, M., Vadrot, N., Belzacq, A. S., Fau, D., Brenner, C., Feldmann, G. and Kroemer, G. (2002). Mitochondrial permeability transition as a novel principle o f hepatorenal toxicity in vivo. 7, 395-405.
Harada, K., Ozaki, S., Gershwin, M. E. and Nakanuma, Y. (1997). Enhanced apoptosis relates to bile duct loss in primary biliary cirrhosis. Hepatology 26, 1399-405.
Harper, N., Farrow, S. N., Kaptein, A., Cohen, G. M. and MacFarlane, M. (2001). Modulation o f tumor necrosis factor apoptosis-inducing ligand- induced NF- kappa B activation by inhibition o f apical caspases. The Journal o f Biological Chemistry 276, 34743-52.
Hatano, E., Bradham, C. A., Stark, A., limuro, Y., Lemasters, J. J. and Brenner, D. A. (2000). The mitochondrial permeability transition augments Fas-induced apoptosis in mouse hepatocytes. Journal o f Biological Chemistry 275, 11814-23.
183
Hayes, M. P., Berrebi, G. A. and Henkart, P. A. (1989). Induction o f target cell DNA release by the cytotoxic T lymphocyte granule protease granzyme A. The Journal o f Experimental Medicine 170, 933-46.
Healy, J. I. and Goodnow, C. C. (1998). Positive versus negative signaling by lymphocyte antigen XQCtpXoxs. Annual Review o f Immunology 16, 645-70.
Heldin, C. H., Miyazono, K. and ten Dijke, P. (1997). TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 390,465-71.
Heller, R. A. and Kronke, M. (1994). Tumor necrosis factor receptor-mediated signaling pathways. Journal o f Cell Biology 126, 5-9.
Henderson, S., Huen, D., Rowe, M., Dawson, C., Johnson, G. and Riekinson, A. (1993). Epstein-Barr virus-coded BHRFl protein, a viral homologue o f Bcl-2, protects human B cells from programmed cell death. Proceedings o f the National Academy o f Sciences o f the United States o f America 90, 8479-83.
Hengartner, M. O. (2000). The biochemistry o f apoptosis. Nature 407, 770-6.Hentze, H., Gantner, F., Kolb, S. A. and Wendel, A. (2000). Depletion o f hepatic
glutathione prevents death receptor-dependent apoptotic and necrotic liver injury in mice. American Journal o f Pathology 156,2045-56.
Hentze, H., Kunstle, G., Volbracht, C., Ertel, W. and Wendel, A. (1999). CD95-Mediated murine hepatic apoptosis requires an intact glutathione status. Hepatology (Baltimore, Md.) 30,177-85.
Hentze, H., Latta, M., Kunstle, G., Lucas, R. and Wendel, A. (2003). Redox control o f hepatic cell death. Toxicology Letters 139,111-8.
Hentze, H., Schmitz, L, Latta, M., Krueger, A., Krammer, P. H. and Wendel, A. (2002). Glutathione dependence o f caspase-8 activation at the death-inducing signaling complex. The Journal o f Biological Chemistry 277, 5588-95.
Higaki, K., Yano, H. and Kojiro, M. (1996). Fas antigen expression and its relationship with apoptosis in human hepatocellular earcinoma and noncancerous tissues. American Journal o f Pathology 149,429-37.
Higuchi, H., Kurose, I., Kato, S., Miura, S. and Ishii, H. (1996). Ethanol-induced apoptosis and oxidative stress in hepatoeytes. Alcoholism, Clinical and Experimental Research 20, 340A-346A.
Hinson, J. A., Pike, S. L., Pumford, N. R. and Mayeux, P. R. (1998). Nitrotyrosine- protein adducts in hepatic centrilobular areas following toxic doses o f aeetaminophen in mice. Chemical Research in Toxicology 11,604-7.
Hinson, J. A., Pohl, L. R., Monks, T. J. and Gillette, J. R. (1981). Acetaminophen- indueed hepatotoxicity. Life Sciences 29, 107-16.
Hinton, R. H. and Grasso, P. (2000). Hepatotoxicity. In General and Applied Toxicology (2" edition; Volume 2) (Editors: B. Ballantyne, T. C. Marrs and T. Syversen), pp. 853-865: Macmillan Reference Ltd.
Hsu, H., Shu, H. B., Pan, M. G. and Goeddel, D. V. (1996). TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84,299-308.
Hsu, H., Xiong, J. and Goeddel, D. V. (1995). The TNF receptor 1-associated protein TRADD signals cell death and NF-kappa B activation. Cell 81,495-504.
Hu, W. H., Johnson, H. and Shu, H. B. (1999). Tumor necrosis factor-related apoptosis- indueing ligand receptors signal NF-kappaB and JNK activation and apoptosis through distinct pathways. The Journal o f Biological Chemistry 274,30603-10.
184
Hu, Y., Ding, L., Spencer, D. M. and Nunez, G. (1998). WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation. Journal o f Biological Chemistry 273,33489-94.
Hug, H., Strand, S., Grambihler, A., Galle, J., Hack, V., Stremmel, W., Krammer, P. H. and Galle, P. R. (1997). Reactive oxygen intermediates are involved in the induction o f CD95 ligand mRNA expression by cytostatic drugs in hepatoma cells. Journal o f Biological Chemistry 272,28191-3.
Hunter, J. J. and Parslow, T. G. (1996). A peptide sequence from Bax that converts Bcl-2 into an activator o f apoptosis. The Journal o f Biological Chemistry 271, 8521-4.
Husztik, E., Lazar, G. and Parducz, A. (1980). Electron microscopic study o f Kupffer-cell phagocytosis blockade induced by gadolinium chloride. British Journal o f Experimental Pathology 61, 624-30.
Inayat-Hussain, S. H., Couet, C., Cohen, G. M. and Cain, K. (1997).Processing/activation o f CPP32-like proteases is involved in transforming growth factor beta 1-induced apoptosis in rat hepatocytes. Hepatology 25, 1516-26.
Insel, P. A. (1996). Analgesic-antipyretic and antiinflammatory agents and drugsemployed in the treatment o f gout. In Goodman and Gilman's ThePharmacological Basis o f Therapeutics (ed. J. G. Hardman, L. E. Limbird and A.G. Gilman), pp. 617-657: McGraw-Hill Professional.
Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Bums, K., Mattmann, C., Rimoldi, D., French, L. E. and Tschopp, J. (1997). Inhibition o f death receptor signals by cellular FLIP [see comments]. Nature 388, 190-5.
Jacobson, M. D. (1996). Reactive oxygen species and programmed cell death. Trends in Biochemical Sciences 21, 83-6.
Jacobson, M. D. (1997). Apoptosis: Bcl-2-related proteins get connected. Current Biology : C b l , R277-81.
Jacobson, M. D., Bume, J. F., King, M. P., Miyashita, T., Reed, J. C. and Raff, M. C. (1993). Bcl-2 blocks apoptosis in cells lacking mitochondrial DNA. Nature 361, 365-9.
Jacobson, M. D., Weil, M. and Raff, M. C. (1997). Programmed cell death in animal development. Cell 88,347-54.
Jaesehke, H. (1990). Glutathione disulfide formation and oxidant stress during acetaminophen-induced hepatotoxieity in mice in vivo: the protective effect o f allopurinol. The Journal o f Pharmacology and Experimental Therapeutics 255, 935-41.
Jaeschke, H., Knight, T. R. and Bajt, M. L. (2003). The role o f oxidant stress and reactive nitrogen species in aeetaminophen hepatotoxicity. Toxicology Letters 144, 279- 88 .
James, L. P., Mayeux, P. R. and Hinson, J. A. (2003). Acetaminophen-induced hepatotoxicity. Drug Metabolism and Disposition: the Biological Fate o f Chemicals 1499-506.
Jenner, P. and Olanow, C. W. (1996). Oxidative stress and the pathogenesis o f Parkinson’s disease. Neurology 47, S I61-70.
Jo, M., Kim, T. H., Seol, D. W., Esplen, J. E., Dorko, K., Billiar, T. R. and Strom, S. C.(2000). Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand [see comments]. Nature Medicine 6, 564- 7.
185
Jollow, D. J., Mitchell, J. R., Potter, W. Z., Davis, D. C., Gillette, J. R. and Brodie, B. B.(1973). Acetaminophen-induced hepatic necrosis. II. Role o f covalent binding in vivo. Journal o f Pharmacology and Experimental Therapeutics 187, 195-202.
Jollow, D. J., Thorgeirsson, S. S., Potter, W. Z., Hashimoto, M. and Mitchell, J. R.(1974). Acetaminophen-induced hepatic necrosis. VI. Metabolic disposition o f toxic and nontoxic doses o f acetaminophen. Pharmacology 12,251-71.
Jones, E. Y., Stuart, D. I. and Walker, N. P. (1989). Structure o f tumour necrosis factor. Nature 338,225-8.
Jones, R. A., Johnson, V. L., Buck, N. R., Dobrota, M., Hinton, R. H., Chow, S. C. and Kass, G. E. (1998). Fas-mediated apoptosis in mouse hepatoeytes involves the processing and activation o f caspases. Hepatology 27, 1632-42.
Juo, P., Kuo, C. J., Yuan, J. and Blenis, J. (1998). Essential requirement for caspase- 8/FLICE in the initiation o f the Fas-induced apoptotic cascade. Current Biology 8, 1001 - 8 .
Jurgensmeier, J. M., Xie, Z., Deveraux, Q., Ellerby, L., Bredesen, D. and Reed, J. C.(1998). Bax directly induces release o f cytochrome c from isolated mitochondria. Proceedings o f the National Academy o f Sciences o f the United States ofAmerica 95,4997-5002.
Kagi, D., Vignaux, F., Ledermann, B., Burki, K., Depraetere, V., Nagata, S., Hengartner, H. and Golstein, P. (1994). Fas and perforin pathways as major mechanisms o f T cell-mediated cytotoxicity. Science 265, 528-30.
Kaipia, A. and Hsueh, A. J. (1997). Regulation o f ovarian follicle atresia. Annual Review o f Physiology 59, 349-63.
Kalai, M., Van Loo, G., Vanden Berghe, T., Meeus, A., Burm, W., Saelens, X. and Vandenabeele, P. (2002). Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA. Cell Death and Differentiation 9, 981-94.
Kamendulis, L. M. and Coreoran, G. B. (1992). Independence and additivity o f cultured hepatocyte killing by Ca2+ overload and ATP depletion. Toxicology Letters 63, 277-87.
Kane, R. E., Li, A. P. and Kaminski, D. R. (1995). Sulfation and glucuronidation o f acetaminophen by human hepatocytes cultured on Matrigel and type 1 collagen reproduces conjugation in vivo. Drug Metabolism and Disposition 23,303-7.
Kass, G. E., Juedes, M. J. and Orrenius, S. (1992). Cyclosporin A protects hepatocytes against prooxidant-induced cell killing. A study on the role o f mitochondrial Ca2+ cycling in cytotoxicity. Biochemical Pharmacology 44, 1995-2003.
Kass, G. E. and Orrenius, S. (1999). Calcium signaling and cytotoxicity. Environmental Health Perspectives 107 Suppl 1,25-35.
Katyare, S. S. and Satav, J. G. (1989). Impaired mitochondrial oxidative energy metabolism following paraeetamol-induced hepatotoxicity in the rat. British Journal o f Pharmacology 96, 51-8.
Kawahara, H., Matsuda, Y. and Takase, S. (1994). Is apoptosis involved in alcoholic hepatitis? ^/co/ïo/ and Alcoholism (Oxford, Oxfordshire) 29 Suppl 1,113-8.
Kelekar, A. and Thompson, C. B. (1998). Bcl-2-family proteins: the role o f the BH3 domain in apoptosis. Trends in Cell Biology 8,324-30.
Kerr, J. F. (1971). Shrinkage necrosis: a distinct mode o f cellular death. The Journal o f Pathology 105, 13-20.
186
Kerr, J. F. and Searle, J. (1972a). The digestion o f cellular fragments within phagolysosomes in carcinoma cells. Journal o f Pathology 108, 55-8.
Kerr, J. F. and Searle, J. (1972b). A suggested explanation for the paradoxically slow growth rate o f basal-cell careinomas that contain numerous mitotic figures. The Journal o f Pathology 107,41-4.
Kerr, J. F., Wyllie, A. H. and Currie, A. R. (1972). Apoptosis: a basic biological phenomenon with wide-ranging implieations in tissue kinetics. British Journal o f Cancer 26,239-257.
Kim, H., Lee, H. and Yun, Y. (1998). X-gene product o f hepatitis B virus induces apoptosis in liver cells. Journal o f Biological Chemistry 273,381-5.
Kim, H. J., Rozman, P. and Klaassen, C. D. (1995). Acetaminophen does not decrease hepatic 3 '-phosphoadenosine 5'-phosphosulfate in mice. Journal o f Pharmacology and Experimental Therapeutics 275,1506-11.
Kim, J. S., He, L. and Lemasters, J. J. (2003). Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochemical and Biophysical Research Communications 304,463-70.
Kim, P. K., Kwon, Y. G., Chung, H. T. and Kim, Y. M. (2002). Regulation o f caspases by nitric oxide. Annals o f the New York Academy o f Sciences 962,42-52.
Kim, T. W., Pettingell, W. H., Jung, Y. K., Kovacs, D. M. and Tanzi, R. E. (1997). Alternative cleavage o f Alzheimer-associated presenilins during apoptosis by a caspase-3 family protease. Science 277,373-6.
Kischkel, F. C., Lawrence, D. A., Chuntharapai, A., Schow, P., Kim, K. J. and Ashkenazi, A. (2000). Apo2L/TRAIL-dependent recruitment o f endogenous F ADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611-20.
Klion, F. M. and Schaffner, F. (1966). The ultrastructure o f acidophilic "Councilmanlike" bodies in the liver. American Journal o f Pathology 48, 755-67.
Kluck, R. M., Bossy-Wetzel, E., Green, D. R. and Newmeyer, D. D. (1997). The release o f cytochrome c from mitochondria: a primary site for Bcl-2 regulation o f apoptosis [see comments]. Science 275,1132-6.
Kluck, R. M., Esposti, M. D., Perkins, G., Renken, C., Kuwana, T., Bossy-Wetzel, E., Goldberg, M., Allen, T., Barber, M. J., Green, D. R. and Newmeyer, D. D.(1999). The pro-apoptotic proteins. Bid and Bax, cause a limited permeabilization o f the mitochondrial outer membrane that is enhanced by cytosol. Journal o f Cell Biology 147, 809-22.
Knight, T. R. and Jaeschke, H. (2002). Acetaminophen-induced inhibition o f Fas receptor-mediated liver cell apoptosis: mitochondrial dysfunction versus glutathione depletion. Toxicology and Applied Pharmacology 181,133-41.
Knight, T. R., Kurtz, A., Bajt, M. L., Hinson, J. A. and Jaeschke, H. (2001). Vascular and hepatocellular peroxynitrite formation during acetaminophen toxicity: role o f mitochondrial oxidant stress. Toxicological Sciences : An Official Jourhal o f the Society o f Toxicology 62,212-20.
Knolle, P. A., Gerken, G., Loser, E., Dienes, H. P., Gantner, F., Tiegs, G., Meyer zum Buschenfelde, K. H. and Lohse, A. W. (1996). Role o f sinusoidal endothelial cells o f the liver in concanavalin A-induced hepatic injury in mice. Hepatology 24, 824-9.
Knudson, C. M. and Korsmeyer, S. J. (1997). Bcl-2 and Bax function independently to regulate cell death. Nature Genetics 16, 358-63.
187
Koga, H., Sakisaka, S., Ohishi, M., Sata, M. and Tanikawa, K. (1997). Nuclear DNA fragmentation and expression o f Bcl-2 in primary biliary cirrhosis. Hepatology 25,1077-84.
Kohler, C., Gahm, A., Noma, T., Nakazawa, A., Orrenius, S. and Zhivotovsky, B. (1999). Release o f adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis. Febs Letters 447, 10-2.
Korsmeyer, S. J., Wei, M. C., Saito, M., Weiler, S., Oh, K. J. and Schlesinger, P. H.(2000). Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release o f cytochrome c. Cell Death and Differentiation!, 1166-73.
Koseki, T., Inohara, N., Chen, S. and Nunez, G. (1998). ARC, an inhibitor o f apoptosis expressed in skeletal muscle and heart that interacts selectively with caspases. Proceedings o f the National Academy o f Sciences o f the United States ofAm erica 95,5156-60.
Kostic, V., Jackson Lewis, V., de Bilbao, P., Dubois Dauphin, M. and Przedborski, S.(1997). Bcl-2: prolonging life in a transgenic mouse model o f familial amyotrophic lateral sclerosis. Science 277,559-62.
Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T. J., Kirschner, M. W., Koths, K., Kwiatkowski, D. J. and Williams, L. T. (1997). Caspase-3-generated fragment o f gelsolin: effector o f morphological change in apoptosis. Science 278,294-8.
Kriegler, M., Perez, C., DeFay, K., Albert, I. and Lu, S. D. (1988). A novel form o f TNF/cachectin is a cell surface cytotoxic transmembrane protein: ramifieations for the complex physiology o f TNF. Cell 53,45-53.
Kroemer, G. (1997). The proto-oncogene Bcl-2 and its role in regulating apoptosis [published erratum appears in Nat Med 1997 Aug;3(8):934]. Nature Medicine 3, 614-20.
Kroemer, G. (1999). Cytochrome c. Current Biology 9, R468.Kroemer, G. and Reed, J. C. (2000). Mitochondrial control o f cell death. Nature Medicine
6,513-9.Kuang, A. A., Diehl, G. E., Zhang, J. and Winoto, A. (2000). F ADD is required for DR4-
and DR5-mediated apoptosis: lack o f trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts. The Journal o f Biological Chemistry 275,25065-8.
Kudla, G., Montessuit, S., Eskes, R., Berrier, C., Martinou, J. C., Ghazi, A. and Antonsson, B. (2000). The destabilization o f lipid membranes induced by the C- terminal fragment o f caspase 8-cleaved bid is inhibited by the N-terminal fragment. Journal o f Biological Chemistry 275,22713-8.
Kuida, K., Haydar, T. F., Kuan, C. Y., Gu, Y., Taya, C., Karasuyama, H., Su, M. S., Rakic, P. and Flavell, R. A. (1998). Reduced apoptosis and cytochrome c- mediated caspase activation in mice lacking caspase 9. Cell 94, 325-37.
Kunstle, G., Hentze, H., Germann, P. G., Tiegs, G., Meergans, T. and Wendel, A. (1999). Concanavalin A hepatotoxicity in mice: tumor necrosis factor-mediated organ failure independent o f easpase-3-like protease activation. Hepatology 30, 1241-51.
Kunstle, G., Leist, M., Uhlig, S., Revesz, L., Feifel, R., MacKenzie, A. and Wendel, A.(1997). ICE-protease inhibitors block murine liver injury and apoptosis caused by CD95 or by TNF-alpha. Immunology Letters 55, 5-10.
188
Kurosawa, H., Que, F. G., Roberts, L. R., Fesmier, P. J. and Gores, G. J. (1997). Hepatocytes in the bile duct-ligated rat express Bcl-2. American Journal o f Physiology 272, G1587-93.
Kurose, L, Higuchi, H., Miura, S., Saito, H., Watanabe, N., Hokari, R., Hirokawa, M., Takaishi, M., Zeki, S., Nakamura, T., Ebinuma, H., Kato, S. and Ishii, H. (1997). Oxidative stress-mediated apoptosis o f hepatoeytes exposed to acute ethanol intoxication. Hepatology 25,368-78.
Kuwana, T., Mackey, M. R., Perkins, G., Ellisman, M. H., Latterich, M., Schneiter, R., Green, D. R. and Newmeyer, D. D. (2002). Bid, Bax, and lipids cooperate to form supramoleeular openings in the outer mitochondrial membrane. Cell 111, 331-42.
Lacronique, V., Mignon, A., Fabre, M., Viollet, B., Rouquet, N., Molina, T., Porteu, A., Henrion, A., Bouscary, D., Varlet, P., Joulin, V. and Kahn, A. (1996). Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nature Medicine 2, 80-6.
Larrick, J. W. and Wright, S. C. (1990). Cytotoxic mechanism o f tumor necrosis factor- alpha. Faseh Journal 4,3215-23.
Laskin, D. L., Gardner, C. R., Price, V. F. and Jollow, D. J. (1995). Modulation o f macrophage funetioning abrogates the acute hepatotoxicity o f acetaminophen. Hepatology (Baltimore, Md.) 21,1045-50.
Laskin, D. L. and Pilaro, A. M. (1986). Potential role o f activated macrophages in acetaminophen hepatotoxicity. I. Isolation and characterization o f activated macrophages from rat liver. Toxicology and Applied Pharmacology 86,204-15.
Laskin, D. L., Pilaro, A. M. and Ji, S. (1986). Potential role o f activated macrophages in acetaminophen hepatotoxicity. II. Mechanism o f macrophage accumulation and activation. Toxicology and Applied Pharmacology 86,216-26.
Latta, M., Kunstle, G., Leist, M. and Wendel, A. (2000). Metabolic depletion o f ATP by fructose inversely controls CD95- and tumor necrosis factor receptor 1-mediated hepatie apoptosis. The Journal o f Experimental Medicine 191, 1975-85.
Laurence, D. R., Bennett, P. N. and Brown, M. J. (1997). Inflammation, arthritis and nonsteroidal anti-inflammatory drugs (NSAIDs): Paracetamol. In Clinical Pharmacology, pp. 253-255: Churchill Livingstone.
Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A. and Jaeschke, H. (1998). Parenchymal cell apoptosis as a signal for sinusoidal sequestration and transendothelial migration o f neutrophils in murine models o f endotoxin and Fas- antibody-induced liver injury [see comments]. Hepatology 28, 761-7.
Lawson, J. A., Fisher, M. A., Simmons, C. A., Farhood, A. and Jaeschke, H. (1999). Inhibition o f Fas receptor (CD95)-induced hepatic caspase activation and apoptosis by acetaminophen in mice. Toxicology and Applied Pharmacology 156, 179-86.
Lazebnik, Y. A., Takahashi, A., Moir, R. D., Goldman, R. D., Poirier, G. G., Kaufmann, S. H. and Eamshaw, W. C. (1995). Studies o f the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proceedings o f the National Academy o f Sciences o f the United States o f America 92, 9042-6.
Lee, S. H., Shin, M. S., Lee, J. Y., Park, W. S., Kim, S. Y., Jang, J. J., Dong, S. M., Na, E. Y., Kim, C. S., Kim, S. H. and Yoo, N. J. (1999). In vivo expression o f soluble Fas and FAP-1: possible mechanisms o f Fas resistance in humanhepatoblastomas. Journal o f Pathology 188,207-12.
189
Leist, M., Gantner, F., Bohlinger, L, Germann, P. G., Tiegs, G. and Wendel, A. (1994). Murine hepatocyte apoptosis induced in vitro and in vivo by TNF-alpha requires transcriptional arrest. Journal o f Immunology 153, 1778-88.
Leist, M., Gantner, F., Bohlinger, L, Tiegs, G., Germann, P. G. and Wendel, A. (1995a). Tumor necrosis factor-induced hepatocyte apoptosis precedes liver failure in experimental murine shock models. American Journal o f Pathology 146, 1220-34.
Leist, M., Gantner, F., Jilg, S. and Wendel, A. (1995b). Aetivation o f the 55 kDa TNF receptor is necessary and sufficient for TNF-induced liver failure, hepatocyte apoptosis, and nitrite release. Journal o f Immunology 154, 1307-16.
Leist, M., Gantner, F., Kunstle, G. and Wendel, A. (1998). Cytokine-mediated hepatic apoptosis. Reviews o f Physiology Biochemistry and Pharmacology 133, 109-55.
Leist, M., Gantner, F., Naumann, H., Bluethmann, H., Vogt, K., Brigelius-Flohe, R., Nicotera, P., Volk, H. D. and Wendel, A. (1997a). Tumor necrosis factor-induced apoptosis during the poisoning o f mice with hepatotoxins. Gastroenterology 112, 923-34.
Leist, M., Single, B., Castoldi, A. F., Kuhnle, S. and Nicotera, P. (1997b). Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis. Journal o f Experimental Medicine 185,1481-6.
Leist, M., Single, B., Naumann, H., Fava, E., Simon, B., Kuhnle, S. and Nicotera, P. (1999a). Inhibition o f mitochondrial ATP generation by nitric oxide switches apoptosis to necrosis. Experimental Cell Research 249,396-403.
Leist, M., Single, B., Naumann, H., Fava, E., Simon, B., Kuhnle, S. and Nicotera, P. (1999b). Nitric oxide inhibits execution o f apoptosis at two distinct ATP- dependent steps upstream and downstream o f mitochondrial cytochrome c release. Biochemical and Biophysical Research Communications 258,215-21.
Lemasters, J. J. (1999). Mechanisms o f hepatic toxicity-V. Necrapoptosis and the mitochondrial permeability transition: shared pathways to necrosis and apoptosis. American Journal o f Physiology 276, G1-6.
Lemasters, J. J., Nieminen, A. L., Qian, T., Trost, L. C., Elmore, S. P., Nishimura, Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A. and Herman, B.(1998). The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochimica Et Biophysica Acta 1366,177-96.
LeSage, G. D., Benedetti, A., Glaser, S., Marucci, L., Tretjak, Z., Caligiuri, A., Rodgers, R., Phinizy, J. L., Baiocchi, L., Francis, H., Lasater, J., Ugili, L. and Alpini, G. (1999a). Acute carbon tetrachloride feeding selectively damages large, but not small, cholangiocytes from normal rat liver. Hepatology 29, 307-19.
LeSage, G. D., Glaser, S. S., Marucei, L., Benedetti, A., Phinizy, J. L., Rodgers, R., Caligiuri, A., Papa, E., Tretjak, Z., Jezequel, A. M., Holcomb, L. A. and Alpini,G. (1999b). Acute carbon tetrachloride feeding induces damage o f large but not small cholangiocytes from BDL rat liver. American Journal o f Physiology 276, G1289-301.
Li, H., Zhu, H., Xu, C. J. and Yuan, J. (1998). Cleavage o f BID by caspase 8 mediates the mitoehondrial damage in the Fas pathway o f apoptosis. Cell 94,491-501.
Li, J., Billiar, T. R., Talanian, R. V. and Kim, Y. M. (1997a). Nitric oxide reversibly inhibits seven members o f the caspase family via S-nitrosylation. Biochemical and Biophysical Research Communications 240,419-24.
190
Li, J., Bombeck, C. A., Yang, S., Kim, Y. M. and Billiar, T. R. (1999). Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes. The Journal o f Biological Chemistry 274, 17325-33.
Li, P., Nijhawan, D., Budihardjo, L, Srinivasula, S. M., Ahmad, M., Alnemri, E. S. and Wang, X. (1997b). Cytochrome c and dATP-dependent formation o f Apaf- l/caspase-9 eomplex initiates an apoptotic protease cascade. Cell 91,479-89.
Lim, H. L., Nelson, D. R. and Fang, J. W. S. (1994). The tumor necrosis factor-alpha system in chronic hepatitis C. Hepatology 20 ,251A.
Lin, Y., Devin, A., Rodriguez, Y. and Liu, Z. G. (1999). Cleavage o f the death domain kinase RIP by caspase-8 prompts TNF-induced apoptosis. Genes & Development 13,2514-26.
Liu, J., Sato, C. and Marumo, F. (1991). Characterization o f the acetaminophen- glutathione conjugation reaction by liver microsomes: species difference in the effeets o f acetone. Toxicology Letters 56,269-74.
Liu, X., Kim, C. N., Yang, J., Jemmerson, R. and Wang, X. (1996). Induction o f apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. C e//86, 147-57.
Lockshin, R. A. and Williams, C. M. (1964). Programmed eell death II: Endocrine potentiation o f the breakdown o f the intersegmental muscles o f silk moths. J Insect Physiol 10, 643-649.
Loeffler, M. and Kroemer, G. (2000). The mitochondrion in cell death control: certainties and incognita. Experimental Cell Research 256,19-26.
Lorenzo, H. K., Susin, S. A., Penninger, J. and Kroemer, G. (1999). Apoptosis indueing factor (AIF): a phylogenetically old, caspase-independent effector o f cell death. Cell Death Differ 6,516-24.
Louis, H., Le Moine, O., Peny, M. O., Quertinmont, E., Fokan, D., Goldman, M. and Deviere, J. (1997). Production and role o f interleukin-10 in eoncanavalin A- induced hepatitis in mice. Hepatology 25,1382-9.
Lowe, S. W., Jacks, T., Housman, D. E. and Ruley, H. E. (1994). Abrogation o f oncogene-associated apoptosis allows transformation o f p53-deficient cells. Proceedings o f the National Academy o f Sciences o f the United States ofAm erica 91,2026-30.
Loweth, A. C., Williams, G. T., James, R. F., Searpello, J. H. and Morgan, N. G. (1998). Human islets o f Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1 beta and Fas ligation. Diabetes 47, 727-32.
Lowin, B., Hahne, M., Mattmann, C. and Tschopp, J. (1994). Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370, 650-2.
Luo, K. X., Zhu, Y. F., Zhang, L. X., He, H. T., Wang, X. S. and Zhang, L. (1997). In situ investigation o f Fas/FasL expression in chronic hepatitis B infection and related liver diseases. Journal o f Viral Hepatitis 4 ,303-7.
Luo, X., Budihardjo, I., Zou, H., Slaughter, C. and Wang, X. (1998). Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation o f cell surface death receptors. Cell 94,481-90.
Lutter, M., Fang, M., Luo, X., Nishijima, M., Xie, X. and Wang, X. (2000). Cardiolipin provides specifieity for targeting o f tBid to mitochondria. Nature Cell Biology 2, 754-61.
191
Lyons, S. K. and Clarke, A. R. (1997). Apoptosis and carcinogenesis. British M edical Bulletin 53, 554-69.
MacFarlane, M. (2003). TRAIL-induced signalling and apoptosis. Toxicology Letters 139, 89-97.
MacFarlane, M., Cohen, G. M. and Dickens, M. (2000). JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis o f T- cells: differential requirements for caspase activation. The Biochemical Journal 348 Pt 1,93-101.
Majno, G. and Joris, I. (1995). Apoptosis, oncosis, and necrosis. An overview o f cell death [see comments]. American Journal o f Pathology 146, 3-15.
Mancini, M., Anderson, B. O., Caldwell, E., Sedghinasab, M., Paty, P. B. and Hockenbery, D. M. (1997). Mitochondrial proliferation and paradoxical membrane depolarization during terminal differentiation and apoptosis in a human colon carcinoma cell line. Journal o f Cell Biology 138,449-69.
Mansouri, A., Gaou, I., Fromenty, B., Berson, A., Letteron, P., Degott, C., Erlinger, S. and Pessayre, D. (1997). Premature oxidative aging o f hepatic mitochondrial DNA in Wilson's disease. Gastroenterology 113,599-605.
Marsden, V. S., O Connor, L., O Reilly, L. A., Silke, J., Metcalf, D., Ekert, P. G., Huang, D. C., Cecconi, F., Kuida, K., Tomaselli, K. J., Roy, S., Nicholson, D. W., Vaux, D. L., Bouillet, P., Adams, J. M. and Strasser, A. (2002). Apoptosis initiated by Bcl-2-regulated caspase activation independently o f the cytochrome c/Apaf-1 /caspase-9 apoptosome. Nature 419, 634-7.
Martinon, F., Holler, N., Richard, C. and Tschopp, J. (2000). Activation o f a pro- apoptotic amplification loop through inhibition o f NF-kappaB-dependent survival signals by caspase-mediated inactivation o f RIP. Febs Letters 468, 134-6.
Martinou, L, Desagher, S., Eskes, R., Antonsson, B., Andre, E., Fakan, S. and Martinou, J. C. (1999). The release o f cytochrome c from mitochondria during apoptosis o f NGF-deprived sympathetic neurons is a reversible event. Journal o f Cell Biology 144, 883-9.
Massague, J. (1990). The transforming growth factor-beta family. Annual Review o f Cell Biology 6,597-641.
Massague, J., Hata, A. and Lui, F. (1997). Signalling through the Smad pathway. Trends C e llB io ll, 187-192.
McClain, C., Hill, D., Schmidt, J. and Diehl, A. M. (1993). Cytokines and alcoholic liver disease. Seminars in Liver Disease 13, 170-82.
McDonnell, J. M., Fushman, D., Milliman, C. L., Korsmeyer, S. J. and Cowbum, D.(1999). Solution strueture o f the proapoptotic molecule BID: a structural basis for apoptotic agonists and antagonists. Cell 96, 625-34.
McKenna, S. L. and Cotter, T. G. (1997). Functional aspects o f apoptosis in hematopoiesis and consequences o f failure. Advances in Cancer Research 71, 121-64.
Meredith, T. J. and Vale, J. A. (1984). Epidemiology o f analgesic overdose in England and Wales. Human Toxicology 3 Suppl, 61S-74S.
Meyers, L. L., Beierschmitt, W. P., Khairallah, E. A. and Cohen, S. D. (1988). Acetaminophen-induced inhibition o f hepatic mitochondrial respiration in mice. Toxicology and Applied Pharmacology 93, 378-87.
192
Mikhailov, V., Mikhailova, M., Pulkrabek, D. J., Dong, Z., Yenkatachalam, M. A. and Saikumar, P. (2001), Bcl-2 prevents Bax oligomerization in the mitochondrial outer membrane. The Journal o f Biological Chemistry 276,18361-74.
Miro, O., Cardellach, P., Barrientos, A., Casademont, J., Rotig, A. and Rustin, P. (1998). Cytochrome c oxidase assay in minute amounts o f human skeletal muscle using single wavelength spectrophotometers. Journal o f Neuroscience Methods 80 ,107- 11.
Mitamura, S., Ikawa, H., Mizuno, N., Kaziro, Y. and Itoh, H. (1998). Cytosolic nuclease activated by caspase-3 and inhibited by DFF-45. Biochemical and Biophysical Research Communications 243,480-4.
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Davis, D. C., Gillette, J. R. and Brodie, B. B. (1973a). Acetaminophen-induced hepatie necrosis. I. Role o f drug metabolism. Journal o f Pharmacology and Experimental Therapeutics 187, 185-94.
Mitchell, J. R., Jollow, D. J., Potter, W. Z., Gillette, J. R. and Brodie, B. B. (1973b). Acetaminophen-induced hepatic necrosis. IV. Protective role o f glutathione. Journal o f Pharmacology and Experimental Therapeutics 187,211-7.
Miyoshi, H., Rust, C., Roberts, P. J., Burgart, L. J. and Gores, G. J. (1999). Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas [see comments]. Gastroenterology 117,669-77.
Mizuhara, H., O'Neill, E., Seki, N., Ogawa, T., Kusunoki, C., Otsuka, K., Satoh, S., Niwa, M., Senoh, H. and Fujiwara, H. (1994). T cell activation-associated hepatic injury: mediation by tumor necrosis factors and protection by interleukin 6. Journal o f Experimental Medicine 179, 1529-37.
Montel, A. H., Bochan, M. R., Hobbs, J. A., Lynch, D. H. and Brahmi, Z. (1995). Fas involvement in cytotoxicity mediated by human NK cells. Cellular Immunology 166,236-46.
Moore, K., Ramrakha, P. K. and Punit, S. (1997). Oxford Handbook o f Acute Medicine, pp. 664-665: Oxford University Press.
Moore, M., Thor, H., Moore, G., Nelson, S., Moldeus, P. and Orrenius, S. (1985). The toxicity o f acetaminophen and N-acetyl-p-benzoquinone imine in isolated hepatocytes is associated with thiol depletion and increased cytosolic Ca2+. Journal o f Biological Chemistry 1611, 13035-40.
Muller, M., Strand, S., Hug, H., Heinemann, E. M., Walczak, H., Hoftnann, W. J., Stremmel, W., Krammer, P. H. and Galle, P. R. (1997). Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-l/Fas) receptor/ligand system and involves activation o f wild-type p53. Journal o f Clinical Investigation 99, 403-13.
Munujos, P., Coll Canti, J., Gonzalez Sastre, F. and Gella, F. J. (1993). Assay o f succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor. Analytical Biochemistry 212, 506-9.
Murawaki, Y., Ikuta, Y., Nishimura, Y., Koda, M. and Kawasaki, H. (1996). Serum markers for fibrosis and plasma transforming growth faetor-beta 1 in patients with hepatocellular carcinoma in comparison with patients with liver cirrhosis. Journal o f Gastroenterology and Hepatology 11,443-50.
Murgia, M., Pizzo, P., Sandona, D., Zanovello, P., Rizzuto, R. and Di Virgilio, F. (1992). Mitochondrial DNA is not fragmented during apoptosis. The Journal o f Biological Chemistry 267,10939-41.
193
Muriel, P., Garciapina, T., Perez-Alvarez, V. and Mourelle, M. (1992). Silymarin protects against paracetamol-induced lipid peroxidation and liver damage. Journal o f Applied Toxicology 12,439-42.
Muschen, M., Warskulat, U., Douillard, P., Gilbert, E. and Haussinger, D. (1998). Regulation o f CD95 (APO-l/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells. Hepatology 27,200-8.
Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scafifidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E. and Dixit, V. M. (1996). FLICE, a novel FADD-homologous ICE/CED-3- like protease, is recruited to the CD95 (Fas/APO-1) death—inducing signaling complex. Cell 85, 817-27.
Muzio, M., Stockwell, B. R., Stennicke, H. R., Salvesen, G. S. and Dixit, V. M. (1998). An induced proximity model for caspase-8 activation. Journal o f Biological Chemistry 273,2926-30.
Nagai, H., Matsumaru, K., Feng, G. and Kaplowitz, N. (2002). Reduced glutathione depletion causes necrosis and sensitization to tumor necrosis factor-alpha-induced apoptosis in cultured mouse hepatocytes. Hepatology (Baltimore, Md.) 36, 55-64.
Nagata, S. (1997). Apoptosis by death factor. Cell 88,355-65.Nagata, S. (2000). Apoptotic DNA fragmentation. Experimental Cell Research 256, 12-8.Nagata, S. and Golstein, P. (1995). The Fas death faetor. Science 267, 1449-56.Nakamoto, Y., Guidotti, L. G., Pasquetto, V., Schreiber, R. D. and Chisari, F. V. (1997).
Differential target cell sensitivity to CTL-aetivated death pathways in hepatitis B virus transgenie mice. Journal o f Immunology 158, 5692-7.
Nanji, A. A. (1998). Apoptosis and alcoholic liver disease. Seminars in Liver Disease 18, 187-90.
Nanji, A. A., Zhao, S., Sadrzadeh, S. M. and Waxman, D. J. (1994). Use o f reverse transcription-polymerase chain reaction to evaluate in vivo cytokine gene expression in rats fed ethanol for long periods. Hepatology 19, 1483-7.
Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J., Matsuda, H. and Tsujimoto, Y. (1998). Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proceedings o f the National Academy o f Sciences o f the United States ofAm erica 95, 14681-6.
Nelson, D. R., Gonzalez Peralta, R. P., Qian, K., Xu, Y., Marousis, C. G., Davis, G. L. and Lau, J. Y. (1997). Transforming growth factor-beta 1 in chronic hepatitis C. Journal o f Viral Hepatitis 4 ,29-35.
Nelson, S. D. (1990). Moleeular meehanisms o f the hepatotoxicity caused by aeetaminophen. Seminars in Liver Disease 10,267-78.
Nelson, S. D. (1995). Mechanisms o f the formation and disposition o f reactive metabolites that can cause acute liver injury. Drug Metabolism Reviews 27, 147- 77.
Neuman, M. G. (2001). Apoptosis in diseases o f the liver. Critical Reviews in Clinical Laboratory Sciences 109-66.
Neuman, M. G., Angulo, P. and Jorgensen, R. A. (1999a). Serum Fas ligand in patients with primary biliary cirrhisis reflects severity o f the disease: effect o f treatment with ursodeoxycholic acid. Hepatology 3 0 ,470A.
194
Neuman, M. G., Angulo, P. and Shear, N. H. (1999b). Relationship between serum cytokine level, degree o f liver damage and ursodeoxycholate in patients with primary biliary cirrhosis. J Hepatol 30, 156.
Neuman, M. G., Shear, N. H., Bellentani, S. and Tiribelli, C. (1998). Role o f cytokines in ethanol-induced cytotoxicity in vitro in Hep G2 eells. Gastroenterology 115,157- 66.
Neuman, M. G., Shear, N. H., Cameron, R. G., Katz, G. and Tiribelli, C. (1999c). Ethanol-induced apoptosis in vitro. Clinical Biochemistry'il , 547-55.
Newmeyer, D. D., Farschon, D. M. and Reed, J. C. (1994). Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria [see comments]. Cell 79,353-64.
Ng, F. W., Nguyen, M., Kwan, T., Branton, P. E., Nicholson, D. W., Cromlish, J. A. and Shore, G. C. (1997). p28 Bap31, a Bcl-2/Bcl-XL- and procaspase-8-associated protein in the endoplasmic reticulum. Journal o f Cell Biology 139, 327-38.
Nicholson, D. W. (1999). Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ 6, 1028-42.
Nicholson, D. W. (2000). From bench to clinic with apoptosis-based therapeutic agents. Nature 407, 810-6.
Nicholson, D. W. and Thomberry, N. A. (1997). Caspases: killer proteases. Trends in Biochemical Sciences 1 1 ,299-306.
Nicotera, P., Leist, M. and Ferrando May, E. (1998). Intracellular ATP, a switch in the decision between apoptosis and necrosis. Toxicology Letters 102-103, 139-42.
Nishimura, Y., Hirabayashi, Y., Matsuzaki, Y., Musette, P., Ishii, A., Nakauchi, H., Inoue, T. and Yonehara, S. (1997). In vivo analysis o f Fas antigen-mediated apoptosis: effects o f agonistic anti-mouse Fas mAh on thymus, spleen and liver. International Immunology 9, 307-16.
Nobel, C. S., Kimland, M., Nicholson, D. W., Orrenius, S. and Slater, A. F. (1997). Disulfiram is a potent inhibitor o f proteases o f the caspase family. Chemical Research in Toxicology 10,1319-24.
Oberhammer, F., Bursch, W., Parzefall, W., Breit, P., Erber, E., Stadler, M. and Schulte Hermann, R. (1991). Effect o f transforming growth factor beta on cell death o f cultured rat hepatocytes. Cancer Research 51,2478-85.
Oberhammer, F., Bursch, W., Tiefenbacher, R., Froschl, G., Pavelka, M., Purchio, T. and Sehulte-Hermann, R. (1993). Apoptosis is induced by transforming growth factor- beta 1 within 5 hours in regressing liver without significant fragmentation o f the DNA. Hepatology 18, 1238-46.
Oberhammer, F., Fritsch, G., Pavelka, M., Froschl, G., Tiefenbacher, R., Purchio, T. and Sehulte-Hermann, R. (1992a). Induction o f apoptosis in cultured hepatoeytes and in the regressing liver by transforming growth factor-beta 1 occurs without activation o f an endonuclease. Toxicology Letters 64-65 Spec No, 701-4.
Oberhammer, F. A., Pavelka, M., Sharma, S., Tiefenbacher, R., Purchio, A. F., Bursch, W. and Sehulte-Hermann, R. (1992b). Induction o f apoptosis in cultured hepatocytes and in regressing liver by transforming growth factor beta 1. Proceedings o f the National Academy o f Sciences o f the United States ofAm erica 89, 5408-12.
Ogasawara, J., Watanabe-Fukunaga, R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S. (1993). Lethal effect o f the anti-
195
Fas antibody in mice [published erratum appears in Nature 1993 Oct 7;365(6446):568]. Nature 364, 806-9.
Olivetti, G., Abbi, R., Quaini, F., Kajstura, J., Cheng, W., Nitahara, J. A., Quaini, E., Di Loreto, C., Beltrami, C. A., Krajewski, S., Reed, J. C. and Anversa, P. (1997). Apoptosis in the failing human heart. The New England Journal o f Medicine 336, 1131-41.
Olivetti, G., Quaini, F., Sala, R., Lagrasta, C., Corradi, D., Bonacina, E., Gambert, S. R., Cigola, E. and Anversa, P. (1996). Acute myocardial infarction in humans is associated with activation o f programmed myocyte cell death in the surviving portion o f the heart. Journal o f Molecular and Cellular Cardiology 28,2005-16.
Oltvai, Z. N., Milliman, C. L. and Korsmeyer, S. J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed eell death. Cell 74, 609-19.
Oppenheim, R. W. (1991). Cell death during development o f the nervous system. Annual Review o f Neuroscience 14,453-501.
Orlinick, J. R. and Chao, M. V. (1998). TNF-related ligands and their receptors. Cellular Signalling 10, 543-51.
Oshiro, T., Shiraishi, M. and Muto, Y. (2002). Adenovirus mediated gene transfer o f antiapoptotic protein in hepatic ischemia-reperfusion injury: the paradoxical effect o f Bcl-2 expression in the reperfused liver. The Journal o f Surgical Research 103, 30-6.
Otsuki, Y., Misaki, O., Sugimoto, O., Ito, Y., Tsujimoto, Y. and Akao, Y. (1994). Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle. Lancet 344,28-9.
Ott, M., Robertson, J. D., Gogvadze, V., Zhivotovsky, B. and Orrenius, S. (2002). Cytochrome c release from mitochondria proceeds by a two-step process. Proceedings o f the National Academy o f Sciences o f the United States ofAm erica 99, 1259-63.
Ozdemirler, G., Aykac, G., Uysal, M. and Oz, H. (1994). Liver lipid peroxidation and glutathione-related defence enzyme systems in mice treated with paracetamol. Journal o f Applied Toxicology 14,297-9.
Pan, G., Ni, J., Wei, Y. F., Yu, G., Gentz, R. and Dixit, V. M. (1997). An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277, 815-8.
Pan, G., Ni, J., Yu, G., Wei, Y. F. and Dixit, V. M. (1998). TRUNDD, a new member o f the TRAIL receptor family that antagonizes TRAIL signalling. Fehs Letters 424, 41-5.
Pan, Z., Bhat, M. B., Nieminen, A. L. and Ma, J. (2001). Synergistic movements o f Ca(2+) and Bax in cells undergoing apoptosis. The Journal o f Biological Chemistry 276,32257-63.
Passarella, S., Ostuni, A., Atlante, A. and Quagliariello, E. (1988). Increase in the ADP/ATP exchange in rat liver mitochondria irradiated in vitro by helium-neon laser. Biochemical and Biophysical Research Communications 156, 978-86.
Pastorino, J. G. and Hoek, J. B. (2000). Ethanol potentiates tumor necrosis factor-alpha cytotoxicity in hepatoma cells and primary rat hepatocytes by promoting induction o f the mitochondrial permeability transition. Hepatology 31,1141-52.
196
Pastorino, J. G., Marcineviciute, A., Cahill, A. and Hoek, J. B. (1999). Potentiation by chronic ethanol treatment o f the mitochondrial permeability transition. Biochemical and Biophysical Research Communications 265,405-9.
Patel, T., Bronk, S. F. and Gores, G. J. (1994a). Increases o f intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes. Journal o f Clinical Investigation 94,2183-92.
Patel, T., Bronk, S. F. and Gores, G. J. (1994b). Increases o f intracellular magnesium promote glycodeoxycholate-induced apoptosis in rat hepatocytes. Journal o f Clinical Investigation 94,2183-92.
Patel, T. and Gores, G. J. (1995). Apoptosis and hepatobiliary disease. Hepatology 21, 1725-41.
Patel, T., Roberts, L. R., Jones, B. A. and Gores, G. J. (1998). Dysregulation o f apoptosis as a mechanism o f liver disease: an overview. Seminars in Liver Disease 18, 105- 14.
Pavlov, E. V., Priault, M., Pietkiewicz, D., Cheng, E. H., Antonsson, B., Manon, S., Korsmeyer, S. J., Mannella, C. A. and Kinnally, K. W. (2001). A novel, high conductance channel o f mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. The Journal o f Cell Biology 155, 725-31.
Pennington, R. J. (1961). Bioehemistry o f Dystrophic Muscle: Mitochondrial Succinate- Tetrazolium Reductase and Adenosine Triphosphatase. Biochemical Journal 80, 649-654.
Perez, C., Albert, I., DeFay, K., Zachariades, N., Gooding, L. and Kriegler, M. (1990). A nonsecretable cell surface mutant o f tumor necrosis factor (TNF) kills by cell-to- cell contact. Cell 63,251-8.
Perkins, C., Kim, C. N., Fang, G. and Bhalla, K. N. (1998). Overexpression o f Apaf-1 promotes apoptosis o f untreated and paclitaxel- or etoposide-treated HL-60 cells. Cancer Research 58,4561-6.
Perkins, C. L., Fang, G., Kim, C. N. and Bhalla, K. N. (2000). The role o f Apaf-1, caspase-9, and bid proteins in etoposide- or paclitaxel-induced mitochondrial events during apoptosis. Cancer Research 60, 1645-53.
Peter, M. E., Heufelder, A. E. and Hengartner, M. O. (1997). Advances in apoptosis research. Proceedings o f the National Academy o f Sciences o f the United States o f America 94, 12736-7.
Pierce, R. H., Franklin, C. C., Campbell, J. S., Tonge, R. P., Chen, W., Fausto, N., Nelson, S. D. and Bruschi, S. A. (2002). Cell culture model for acetaminophen- induced hepatocyte death in vivo. Biochemical Pharmacology 64,413-24.
Pinkoski, M. J., Hobman, M., Heibein, J. A., Tomaselli, K., Li, F., Seth, P., Froelich, C. J. and Bleackley, R. C. (1998). Entry and trafficking o f granzyme B in target cells during granzyme B-perforin-mediated apoptosis. Blood 92, 1044-54.
Pitti, R. M., Marsters, S. A., Ruppert, S., Donahue, C. J., Moore, A. and Ashkenazi, A. (1996). Induction o f apoptosis by Apo-2 ligand, a new member o f the tumor necrosis factor cytokine family. Journal o f Biological Chemistry 271, 12687-90.
Poupon, R., Chretien, Y., Poupon, R. E., Ballet, F., Calmus, Y. and Damis, F. (1987). Is ursodeoxycholic acid an effective treatment for primary biliary cirrhosis? Lancet 1, 834-6.
Prescott, L. F. (1996). Paracetamol: A Critical Bibliographic Review: Taylor and Francis.
197
Prescott, L. F., Illingworth, R. N., Critchley, J. A., Stewart, M. J., Adam, R. D. and Proudfoot, A. T. (1979). Intravenous N-acetylcystine: the treatment o f choice for paracetamol poisoning. British MedicalJournal 2,1097-100.
Prescott, L. F., Roscoe, P., Wright, N. and Brown, S. S. (1971). Plasma-paracetamol half- life and hepatic necrosis in patients with paraeetamol overdosage. Lancet 1, 519- 22.
Proudfoot, A. T. and Wright, N. (1970). Acute paracetamol poisoning. British Medical Journal 3, 557-8.
Przybocki, J. M., Reuhl, K. R., Thurman, R. G. and Kauffman, F. C. (1992). Involvement o f nonparenchymal cells in oxygen-dependent hepatic injury by allyl alcohol. Toxicology and Applied Pharmacology 115, 57-63.
Puisieux, A., Ji, J., Guillot, C., Legros, Y., Soussi, T., Isselbacher, K. and Ozturk, M. (1995). p53-mediated cellular response to DNA damage in cells with replicative hepatitis B virus. Proceedings o f the National Academy o f Sciences o f the United States o f America 92,1342-6.
Que, F. and Gores, G. J. (1997). Apoptosis and the gastrointestinal system. Advances in Pharmacology 41,409-28.
Que, F. G., Phan, V. A., Phan, V. H., LaRusso, N. F. and Gores, G. J. (1999). GUDC inhibits cytochrome c release from human cholangiocyte mitochondria. Journal o f Surgical Research 83, 100-5.
Raff, M. C. (1992). Social controls on cell survival and cell death. Nature 356, 397-400.Raffray, M. and Cohen, G. M. (1997). Apoptosis and necrosis in toxicology: a continuum
or distinct modes o f cell death? Pharmacology & Therapeutics 75, 153-77.Ramsay, R. R., Rashed, M. S. and Nelson, S. D. (1989). In vitro effects o f acetaminophen
metabolites and analogs on the respiration o f mouse liver mitochondria. Archives o f Biochemistry and Biophysics 273,449-57.
Rano, T. A., Timkey, T., Peterson, E. P., Rotonda, J., Nicholson, D. W., Becker, J. W., Chapman, K. T. and Thomberry, N. A. (1997). A combinatorial approach for determining protease specificities: application to interleukin-1 beta converting enzyme (ICE). Chemistry and Biology 4, 149-55.
Rao, L., Perez, D. and White, E. (1996). Lamin proteolysis facilitates nuclear events during apoptosis. Journal o f Cell Biology 135, 1441-55.
Ray, C. A., Black, R. A., Kronheim, S. R., Greenstreet, T. A., Sleath, P. R., Salvesen, G. S. and Pickup, D. J. (1992a). Viral inhibition o f inflammation: cowpox vims encodes an inhibitor o f the interleukin-1 beta converting enzyme. Cell 69, 597- 604.
Ray, S. D. and Jena, N. (2000). A hepatotoxic dose o f acetaminophen modulates expression o f BCL-2, BCL-X(L), and BCL-X(S) during apoptotic and necrotic death o f mouse liver cells in vivo. Archives o f Toxicology 73, 594-606.
Ray, S. D., Kamendulis, L. M., Gurule, M. W., Yorkin, R. D. and Corcoran, G. B. (1993). Ca2+ antagonists inhibit DNA fragmentation and toxic cell death induced by acetaminophen. Faseh Journal 7,453-63.
Ray, S. D., Kumar, M. A. and Bagchi, D. (1999). A novel proanthocyanidin IH636 grape seed extract increases in vivo Bcl-XL expression and prevents acetaminophen- induced programmed and unprogrammed cell death in mouse liver. Archives o f Biochemistry and Biophysics 369,42-58.
Ray, S. D., Mumaw, V. R., Raje, R. R. and Fariss, M. W. (1996). Protection o f acetaminophen-induced hepatocellular apoptosis and necrosis by cholesteryl
198
hemisuccinate pretreatment. Journal o f Pharmacology and Experimental Therapeutics 219, 1470-83.
Ray, S. D., Sorge, C. L., Kamendulis, L. M. and Corcoran, G. B. (1992b). Ca(-H-)- activated DNA fragmentation and dimethylnitrosamine-induced hepatic necrosis: effects o f Ca(++)-endonuclease and poly(ADP-ribose) polymerase inhibitors in mice. Journal o f Pharmacology and Experimental Therapeutics 2 6 3 ,387-94.
Ray, S. D., Sorge, C. L., Raucy, J. L. and Coreoran, G. B. (1990). Early loss o f large genomic DNA in vivo with accumulation o f Ca2+ in the nucleus during acetaminophen-induced liver injury. Toxicology and Applied Pharmacology 106,346-51.
Ray, S. D., Sorge, C. L., Tavaeoli, A., Raucy, J. L. and Corcoran, G. B. (1991). Extensive alteration o f genomic DNA and rise in nuclear Ca2+ in vivo early after hepatotoxic acetaminophen overdose in mice. Advances in Experimental Medicine and Biology 283, 699-705.
Read, A. E. (1979). The Liver and Drugs. In Liver and Biliary Diseases (ed. R. Wright, K. G. Alberti and S. Karran), pp. 822-847: W.B. Saunders Co Ltd.
Reed, J. C. (1997). Double identity for proteins o f the Bel-2 family. Nature 387, 773-6.Reed, J. C., Jurgensmeier, J. M. and Matsuyama, S. (1998). Bcl-2 family proteins and
mitochondria. Biochimica Et Biophysica Acta 1366,127-37.Regnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z. and Rothe, M. (1997).
Identification and characterization o f an IkappaB kinase. Cell 90, 373-83.Rivero, M., Crespo, J. and Casafront, F. (1998). Fulminant hepatitis by HBV: Role o f Fas
system. Hepatology 28 (2 suppl), 482A.Rizzino, A. (1988). Transforming growth factor-beta: multiple effeets on cell
differentiation and extracellular matrices. Developmental Biology 130,411-22.Roberts, L. R., Kurosawa, H., Bronk, S. F., Fesmier, P. J., Agellon, L. B., Leung, W. Y.,
Mao, F. and Gores, G. J. (1997). Cathepsin B contributes to bile salt-induced apoptosis o f rat hepatocytes. Gastroenterology 113,1714-26.
Robertson, J. D. and Orrenius, S. (2000). Molecular mechanisms o f apoptosis induced by cytotoxic chemicals. Crc Critical Reviews in Toxicology 30, 609-627.
Rodrigues, C. M., Fan, G., Ma, X., Kren, B. T. and Steer, C. J. (1998). A novel role for ursodeoxycholie acid in inhibiting apoptosis by modulating mitochondrial membrane perturbation. Journal o f Clinical Investigation 101,2790-9.
Rodrigues, C. M., Ma, X., Linehan-Stieers, C., Fan, G., Kren, B. T. and Steer, C. J.(1999). Ursodeoxycholic acid prevents cytochrome c release in apoptosis by inhibiting mitochondrial membrane depolarization and channel formation. Cell Death Differ 6, 842-54.
Rodriguez, L, Matsuura, K., Ody, C., Nagata, S. and Vassalli, P. (1996). Systemic injection o f a tripeptide inhibits the intracellular activation o f CPP32-like proteases in vivo and fully protects mice against Fas-mediated fulminant liver destruction and death. Journal o f Experimental Medicine 184,2067-72.
Rodriguez, J. and Lazebnik, Y. (1999). Caspase-9 and APAF-1 form an active holoenzyme. Genes and Development 13, 3179-84.
Rosse, T., Olivier, R., Monney, L., Rager, M., Conus, S., Fellay, L, Jansen, B. and Borner, C. (1998). Bcl-2 prolongs cell survival after Bax-induced release o f cytochrome c [see comments]. Nature 391,496-9.
199
Roucou, X., Montessuit, S., Antonsson, B. and Martinou, J. C. (2002). Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein. The Biochemical Journal 368, 915-21.
Roulston, A., Mareellus, R. C. and Branton, P. E. (1999). Viruses and apoptosis. Annual Review o f Microbiology 53, 577-628.
Rouquet, N., Carlier, K., Briand, P., Wiels, J. and Joulin, V. (1996a). Multiple pathways o f Fas-induced apoptosis in primary culture o f hepatocytes. Biochemical and Biophysical Research Communications 229,27-35.
Rouquet, N., Pages, J. C., Molina, T., Briand, P. and Joulin, V. (1996b). ICE inhibitor YVADcmk is a potent therapeutic agent against in vivo liver apoptosis. Current Biology 6, W92-5.
Rubin, R. A., Kowalski, T. E., Khandelwal, M. and Malet, P. F. (1994). Ursodiol for hepatobiliary disorders. Annals o f Internal Medicine 121,207-18.
Rudel, T. and Bokoch, G. M. (1997). Membrane and morphological changes in apoptotic cells regulated by caspase-mediated activation o f PAK2. Science 276, 1571-4.
Ruepp, S. U., Tonge, R. P., Shaw, J., Wallis, N. and Pognan, F. (2002). Genomics and proteomics analysis o f acetaminophen toxicity in mouse liver. Toxicological Sciences : An Official Journal o f the Society o f Toxicology 65, 135-50.
Russell, W. E., Coffey, R. J., Ouellette, A. J. and Moses, H. L. (1988). Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat. Proceedings o f the National Academy o f Sciences o f the United States o f America 85, 5126-30.
Rust, C. and Gores, G. J. (2000). Apoptosis and liver disease. American Journal o f Medicine 108, 567-74.
Sachs, L. and Lotem, J. (1993). Control o f programmed cell death in normal and leukemic cells: new implications for therapy. Blood 82,15-21.
Sadrzadeh, S. M., Nanji, A. A. and Priee, P. L. (1994). The oral iron chelator, 1,2- dimethyl-3 -hydroxypyrid-4-one reduces hepatic-free iron, lipid peroxidation and fat accumulation in chronically ethanol-fed rats. Journal o f Pharmacology and Experimental Therapeutics 269,632-6.
Saito, M., Korsmeyer, S. J. and Schlesinger, P. H. (2000). BAX-dependent transport o f eytochrome c reeonstituted in pure liposomes. Nature Cell Biology 2, 553-5.
Sakahira, H., Enari, M. and Nagata, S. (1998). Cleavage o f CAD inhibitor in CAD activation and DNA degradation during apoptosis [see comments]. Nature 391, 96-9.
Saleh, A., Srinivasula, S. M., Acharya, S., Fishel, R. and Alnemri, E. S. (1999). Cytochrome c and dATP-mediated oligomerization o f Apaf-1 is a prerequisite for procaspase-9 activation. Journal o f Biological Chemistry 274, 17941-5.
Salvesen, G. S. and Dixit, V. M. (1997). Caspases: intracellular signaling by proteolysis. C e//91,443-6.
Samali, A., Nordgren, H., Zhivotovsky, B., Peterson, E. and Orrenius, S. (1999). A comparative study o f apoptosis and necrosis in HepG2 cells: oxidant-induced caspase inactivation leads to neerosis. Biochemical and Biophysical Research Communications 255, 6-11.
Samarasinghe, D. A. and Farrell, G. C. (1996). The central role o f sinusoidal endothelial cells in hepatic hypoxia-reoxygenation injury in the rat. Hepatology 24, 1230-7.
Sanderson, N., Factor, V., Nagy, P., Kopp, J., Kondaiah, P., Wakefield, L., Roberts, A.B., Spom, M. B. and Thorgeirsson, S. S. (1995). Hepatie expression o f mature
200
transforming growth factor beta 1 in transgenic mice results in multiple tissue lesions. Proceedings o f the National Academy o f Sciences o f the United States o f America 92,2572-6.
Sasatomi, K., Noguchi, K., Sakisaka, S., Sata, M. and Tanikawa, K. (1998). Abnormal accumulation o f endotoxin in biliary epithelial cells in primary biliary cirrhosis and primary sclerosing cholangitis. Journal ofHepatology 29,409-16.
Sattler, M., Liang, H., Nettesheim, D., Meadows, R. P., Harlan, J. E., Eberstadt, M., Yoon, H. S., Shuker, S. B., Chang, B. S., Minn, A. J., Thompson, C. B. and Fesik,S. W. (1997). Structure o f Bcl-xL-Bak peptide complex: recognition between regulators o f apoptosis. Science 275, 983-6.
Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H. and Peter, M. E. (1998). Two CD95 (APO-l/Fas) signaling pathways. Embo Journal 17, 1675-87.
Schneider, P., Thome, M., Bums, K., Bodmer, J. L., Hofinann, K., Kataoka, T., Holler, N. and Tschopp, J. (1997). TRAIL receptors 1 (DR4) and 2 (DR5) signal FADD- dependent apoptosis and activate NF-kappaB. Immunity 7, 831-6.
Schotte, P., Declercq, W., Van Huffel, S., Vandenabeele, P. and Beyaert, R. (1999). Nonspecific effects o f methyl ketone peptide inhibitors o f caspases. Febs Letters 442, 117-21.
Schwall, R. H., Robbins, K., Jardieu, P., Chang, L., Lai, C. and Terrell, T. G. (1993). Activin induces cell death in hepatocytes in vivo and in vitro. Hepatology 18,347-56.
Sedlak, T. W., Oltvai, Z. N., Yang, E., Wang, K., Boise, L. H., Thompson, C. B. and Korsmeyer, S. J. (1995). Multiple Bcl-2 family members demonstrate selective dimerizations with Bax. Proceedings o f the National Academy o f Sciences o f the United States o f America 92, 7834-8.
Seeff, L. B., Cuccherini, B. A., Zimmerman, H. J., Adler, E. and Benjamin, S. B. (1986). Acetaminophen hepatotoxieity in alcoholics. A therapeutic misadventure. Annals o f Internal Medicine 104,399-404.
Seshagiri, S. and Miller, L. K. (1997). Baculovims inhibitors o f apoptosis (lAPs) block activation o f Sf-caspase-1. Proceedings o f the National Academy o f Sciences o f the United States o f America 94, 13606-11.
Shear, N. H., Malkiewicz, 1. M., Klein, D., Koren, G., Randor, S. and Neuman, M. G.(1995). Acetaminophen-induced toxicity to human epidermoid cell line A431 and hepatoblastoma cell line Hep G2, in vitro, is diminished by silymarin. Skin Pharmacology 8,279-91.
Sheridan, J. P., Marsters, S. A., Pitti, R. M., Gurney, A., Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C. L., Baker, K., Wood, W. I., Goddard, A. D., Godowski, P. and Ashkenazi, A. (1997). Control o f TRAIL-induced apoptosis by a family o f signaling and deeoy receptors. Science 277, 818-21.
Sheron, N., Lau, J., Daniels, H., Goka, J., Eddleston, A., Alexander, G. J. and Williams, R. (1991). Increased production o f tumour necrosis factor alpha in chronic hepatitis B virus infection. Journal ofHepatology 12,241-5.
Sheron, N., Lau, J. Y., Daniels, H. M., Webster, J., Eddleston, A. L., Alexander, G. J. and Williams, R. (1990). Tumour necrosis factor to treat chronic hepatitis B virus infection. Lancet 336,321-2.
Shi, Y. and Massague, J. (2003). Mechanisms o f TGF-beta signaling from cell membrane to the nucleus. Cell 113, 685-700.
201
Shimizu, S., Narita, M. and Tsujimoto, Y. (1999). Bcl-2 family proteins regulate the release o f apoptogenic cytochrome c by the mitochondrial channel VDAC [see comments]. Nature 399,483-7.
Shirai, Y., Kawata, S., Tamura, S., Ito, N., Tsushima, H., Takaishi, K., Kiso, S. and Matsuzawa, Y. (1994). Plasma transforming growth factor-beta 1 in patients with hepatocellular carcinoma. Comparison with chronic liver diseases. Cancer 73, 2275-9.
Shiratori, Y., Kawase, T., Shiina, S., Okano, K., Sugimoto, T., Teraoka, H., Matano, S., Matsumoto, K. and Kamii, K. (1988). Modulation o f hepatotoxicity by macrophages in the liver. Hepatology (Baltimore, Md.) 8, 815-21.
Shiratori, Y., Takikawa, H., Kawase, T. and Sugimoto, T. (1986). Superoxide anion generating eapacity and lysosomal enzyme activities o f Kupffer cells in galactosamine induced hepatitis. Gastroenterologia Japonica 21, 135-44.
Shresta, S., Pham, C. T., Thomas, D. A., Graubert, T. A. and Ley, T. J. (1998). How do cytotoxic lymphocytes kill their targets? Current Opinion in Immunology 10, 581- 7.
Shu, H. B., Halpin, D. R. and Goeddel, D. V. (1997). Casper is a F ADD- and caspase- related inducer o f apoptosis. Immunity 6, 751-63.
Single, B., Leist, M. and Nicotera, P. (1998). Simultaneous release o f adenylate kinase and cytochrome c in cell death. 5, 1001-3.
Skalka, M., Matyasova, J. and Cejkova, M. (1976). DNA in chromatin o f irradiated lymphoid tissues degrades in vivo into regular fragments. FEBS Letters 12, 271- 274.
Slee, E. A., Zhu, H., Chow, S. C., MacFarlane, M., Nicholson, D. W. and Cohen, G. M.(1996). Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z- VAD.FMK) inhibits apoptosis by blocking the proeessing o f CPP32. Biochemical Journal 315 ( Pt 1), 21-4.
Slomiany, A., Piotrowski, E., Grabska, M., Piotrowski, J. and Slomiany, B. L. (1999). Chronic ethanol-initiated apoptosis in hepatocytes is induced by changes in membrane biogenesis and intracellular transport. Alcoholism, Clinical and Experimental Research 23,334-43.
Smaili, S. S., Hsu, Y. T., Sanders, K. M., Russell, J. T. and Youle, R. J. (2001). Bax translocation to mitochondria subsequent to a rapid loss o f mitochondrial membrane potential. Cell Death and Differentiation 8,909-20.
Smith, C. A., Farrah, T. and Goodwin, R. G. (1994). The TNF receptor superfamily o f cellular and viral proteins: activation, costimulation, and death. Cell 76, 959-62.
Smith, R. A. and Baglioni, C. (1987). The active form o f tumor necrosis factor is a trimer. The Journal o f Biological Chemistry 262,6951-4,
Spengler, U., Zachoval, R., Gallati, H., Jung, M. C., Hoffmann, R., Riethmuller, G. and Pape, G. (1996). Serum levels and in situ expression o f TNF-alpha and TNF- alpha binding proteins in inflammatory liver diseases. Cytokine 8, 864-72.
Spivey, J. R., Bronk, S. F. and Gores, G. J. (1993). Glycochenodeoxycholate-induced lethal hepatocellular injury in rat hepatocytes. Role o f ATP depletion and cytosolic free caleium. Journal o f Clinical Investigation 92,17-24.
Sprick, M. R., Weigand, M. A., Rieser, E., Rauch, C. T., Juo, P., Blenis, J., Krammer, P. H. and Walczak, H. (2000). FADD/MORTl and caspase-8 are recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated by TRAIL receptor 2. Immunity 1 2 ,599-609.
202
Stahelin, B. J., Marti, U., Zimmermann, H. and Reichen, J. (1999). The interaction of Bcl-2 and Bax regulates apoptosis in biliary epithelial cells o f rats with obstructive jaundice. Virchows Archiv 434,333-9.
Steller, H. (1995). Mechanisms and genes o f cellular suicide. Science 267, 1445-9.Stennicke, H. R. and Salvesen, G. S. (1998). Properties o f the caspases. Biochimica Et
Biophysica Acta 1387, 17-31.Stennicke, H. R. and Salvesen, G. S. (2000). Caspases - controlling intracellular signals
by protease zymogen activation. Biochimica Et Biophysica Acta 1477,299-306.Stoka, V., Turk, B., Schendel, S. L., Kim, T. H., Cirman, T., Snipas, S. J., Ellerby, L. M.,
Bredesen, D., Freeze, H., Abrahamson, M., Bromme, D., Krajewski, S., Reed, J.C., Yin, X. M., Turk, V. and Salvesen, G. S. (2001). Lysosomal protease pathways to apoptosis. Cleavage o f bid, not pro-caspases, is the most likely route. The Journal o f Biological Chemistry 276,3149-57.
Strand, S., Hofinann, W. J., Grambihler, A., Hug, H., Volkmann, M., Otto, G., Wesch,H., Mariani, S. M., Hack, V., Stremmel, W., Krammer, P. H. and Galle, P. R.(1998). Hepatic failure and liver cell damage in acute Wilson’s disease involve CD95 (APO-l/Fas) mediated apoptosis. Nature Medicine 4, 588-93.
Strand, S., Hofmann, W. J., Hug, H., Muller, M., Otto, G., Strand, D., Mariani, S. M., Stremmel, W., Krammer, P. H. and Galle, P. R. (1996a). Lymphocyte apoptosis induced by CD95 (APO-l/Fas) ligand-expressing tumor cells—a mechanism o f immune evasion? Nature Medicine 2 ,1361-6.
Strand, S., Hofmann, W. J., Hug, H., Muller, M., Otto, G., Strand, D., Mariani, S. M., Stremmel, W., Krammer, P. H. and Galle, P. R. (1996b). Lymphocyte apoptosis induced by CD95 (APO-l/Fas) ligand-expressing tumor cells—a mechanism o f immune evasion? [see comments]. Nature Medicine 2 ,1361-6.
Stremmel, W., Meyerrose, K. W., Niederau, C., Hefter, H., Kreuzpaintner, G. and Strohmeyer, G. (1991). Wilson disease: clinical presentation, treatment, and survived. Annals o f Internal Medicine 115, 720-6.
Stridh, H., Fava, E., Single, B., Nicotera, P., Orrenius, S. and Leist, M. (1999). Tributyltin-induced apoptosis requires glycolytic adenosine trisphosphate production. Chemical Research in Toxicology 12, 874-82.
Strubelt, O. and Younes, M. (1992). The toxicological relevance o f paracetamol-induced inhibition o f hepatic respiration and ATP depletion. Biochemical Pharmacology 44, 163-70.
Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Brenner, C., Larochette, N., Prévost, M. C., Alzari, P. M. and Kroemer, G. (1999a). Mitochondrial release o f caspase-2 and -9 during the apoptotic process. Journal o f Experimental Medicine 189,381-94.
Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, L, Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R., Siderovski, D. P., Penninger, J. M. and Kroemer, G. (1999b). Molecular characterization o f mitochondrial apoptosis-inducing factor [see comments]. Nature 397,441-6.
Susin, S. A., Zamzami, N., Castedo, M., Daugas, E., Wang, H. G., Geley, S., Fassy, F., Reed, J. C. and Kroemer, G. (1997a). The central executioner o f apoptosis: multiple connections between protease activation and mitochondria in Fas/APO- 1/CD95- and ceramide-induced apoptosis. Journal o f Experimental Medicine 186, 25-37.
203
Susin, S. A., Zamzami, N., Castedo, M., Hirsch, T., Marchetti, P., Macho, A., Daugas, E., Geuskens, M. and Kroemer, G. (1996). Bcl-2 inhibits the mitochondrial release o f an apoptogenic protease. Journal o f Experimental Medicine 184, 1331-41.
Susin, S. A., Zamzami, N., Larochette, N., Dallaporta, B., Marzo, L, Brenner, G., Hirsch, T., Petit, P. X., Geuskens, M. and Kroemer, G. (1997b). A cytofluorometric assay o f nuclear apoptosis induced in a cell-free system: application to ceramide- induced apoptosis. Experimental Cell Research 236,397-403.
Tabibzadeh, S. (1996). The signals and molecular pathways involved in human menstruation, a unique process o f tissue destruction and remodelling. Molecular Human Reproduction 2 ,77-92.
Tafani, M., Cohn, J. A., Karpinich, N. O., Rothman, R. J., Russo, M. A. and Farber, J. L. (2002a). Regulation o f intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha. The Journal o f Biological Chemistry 277,49569-76.
Tafani, M., Karpinich, N. O., Hurster, K. A., Pastorino, J. G., Schneider, T., Russo, M. A. and Farber, J. L. (2002b). Cytochrome c release upon Fas receptor activation depends on translocation o f fiill-length bid and the induction o f the mitochondrial permeability transition. The Journal o f Biological Chemistry 277, 10073-82.
Takahashi, R., Deveraux, Q., Tamm, L, Welsh, K., Assa-Munt, N., Salvesen, G. S. and Reed, J. C. (1998). A single BIR domain o f XIAP sufficient for inhibiting caspases. Journal o f Biological Chemistry 273, 7787-90.
Takei, Y., Kawano, S., Nishimura, Y., Goto, M., Hagai, H., Chen, S. S., Omae, A., Fusamoto, H., Kamada, T., Ikeda, K. and al, e. (1995). Apoptosis: a new mechanism o f endothelial and Kupffer cell killing. Journal o f Gastroenterology andHepatology 10 Suppl 1, S65-7.
Takei, Y., Marzi, L, Gao, W. S., Gores, G. J., Lemasters, J. J. and Thurman, R. G. (1991). Leukocyte adhesion and cell death following orthotopic liver transplantation in the rat. Transplantation 51,959-65.
Tanzi, R. E., Petrukhin, K., Chernov, L, Pellequer, J. L., Wasco, W., Ross, B., Romano,D. M., Parano, E., Pavone, L. and Brzustowicz, L. M. (1993). The Wilson disease gene is a copper transporting ATPase with homology to the Menkes disease gene. Nature Genetics 5,344-50.
Tata, J. R. (1966). Requirement for RNA and protein synthesis for induced regression o f the tadpole tail in organ culture. Developmental Biology 13, 77-94.
Terradillos, O., Pollicino, T., Lecoeur, H., Tripodi, M., Gougeon, M. L., Tiollais, P. and Buendia, M. A. (1998). p53-independent apoptotic effects o f the hepatitis B virus HBx protein in vivo and in vitro. Oncogene 17,2115-23.
Tewari, M., Beidler, D. R. and Dixit, V. M. (1995). CrmA-inhibitable cleavage o f the 70- kDa protein component o f the U1 small nuclear ribonucleoprotein during Fas- and tumor necrosis factor-induced apoptosis. The Journal o f Biological Chemistry 270, 18738-41.
Thomas, B. H., Zeitz, W. and Coldwell, B. B. (1974). Effect o f aspirin on biotransformation o f 14C-acetaminophen in rats. Journal o f Pharmaceutical Sciences 63,1367-70.
Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Bums, K., Bodmer, J. L., Schroter, M., Scaffidi, C., Krammer, P.H., Peter, M. E. and Tschopp, J. (1997). Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386, 517-21.
204
Thompson, C. B. (1995). Apoptosis in the pathogenesis and treatment o f disease. Science 267, 1456-62.
Thorgeirsson, S. S., Teramoto, T. and Factor, V. M. (1998). Dysregulation o f apoptosis in hepatocellular carcinoma. Seminars in Liver Disease 18,115-22.
Thornberry, N. A. and Lazebnik, Y. (1998). Caspases: enemies within. Science 281, 1312-6.
Thornberry, N. A., Rano, T. A., Peterson, E. P., Rasper, D. M., Timkey, T., Garcia Calvo, M., Houtzager, V. M., Nordstrom, P. A., Roy, S., Vaillancourt, J. P., Chapman, K. T. and Nicholson, D. W. (1997). A combinatorial approach defines specificities o f members o f the caspase family and granzyme B. Functional relationships established for key mediators o f apoptosis. Journal o f Biological Chemistry 272, 17907-11.
Thurman, R. G., Marzi, I., Seitz, G., Thies, J., Lemasters, J. J. and Zimmerman, F. (1988). Hepatic reperfusion injury following orthotopic liver transplantation in the rat. Transplantation 46, 502-6.
Tiegs, G., Hentschel, J. and Wendel, A. (1992). A T cell-dependent experimental liver injury in mice inducible by concanavalin A. Journal o f Clinical Investigation 90, 196-203.
Timbrell, J. (2000). Tissue lesions: liver necrosis: Paracetamol. In Priciples o f Biochemical Toxicology, pp. 272-278: Taylor and Francis.
Tirmenstein, M. A. and Nelson, S. D. (1990). Acetaminophen-induced oxidation o f protein thiols. Contribution o f impaired thiol-metabolizing enzymes and the breakdown o f adenine nucleotides. Journal o f Biological Chemistry 265,3059-65.
Torok, N. J., Higuchi, H., Bronk, S. and Gores, G. J. (2002). Nitric oxide inhibits apoptosis downstream o f cytochrome C release by nitrosylating caspase 9. Cancer Research 62, 1648-53.
Tracey, K. J. and Cerami, A. (1993). Tumor necrosis factor, other cytokines and disease. Annual Review o f Cell Biology 9,317-43.
Tran-Thi, T. A., Decker, K. and Baeuerle, P. A. (1995). Differential activation o f transcription factors NF-kappa B and AP-1 in rat liver macrophages. Hepatology 22, 613-9.
Trump, B. F. and Berezesky, I. K. (1995). Calcium-mediated cell injury and cell death. Faseh Journal 9 ,219-28.
Tsujimoto, Y. (1997). Apoptosis and necrosis: intracellular ATP level as a determinant for cell death modes. Cell Death D iff4,429-434.
Tsujimoto, Y., Shimizu, S., Eguchi, Y., Kamiike, W. and Matsuda, H. (1997). Bcl-2 and Bcl-xL block apoptosis as well as necrosis: possible involvement o f common mediators in apoptotic and necrotic signal transduction pathways. Leukemia 11 Suppl 3,380-2.
Uren, A. G., Coulson, E. J. and Vaux, D. L. (1998). Conservation o f baculovirus inhibitor o f apoptosis repeat proteins (BIRPs) in viruses, nematodes, vertebrates and yeasts. Trends in Biochemical Sciences 23, 159-62.
Uyama, O., Matsuyama, T., Michishita, H., Nakamura, H. and Sugita, M. (1992). Protective effects o f human recombinant superoxide dismutase on transient ischemic injury o f CAl neurons in gerbils. Stroke; a Journal o f Cerebral Circulation 23, 75-81.
205
Van Antwerp, D. J., Martin, S. J., Kafri, T., Green, D. R. and Verma, I. M. (1996). Suppression o f TNF-alpha-induced apoptosis by NF-kappaB [see comments]. Science 274, 787-9.
Vander Heiden, M. G. and Thompson, C. B. (1999). Bcl-2 proteins: regulators o f apoptosis or o f mitochondrial homeostasis? Nature Cell Biology 1, E209-16.
Varfolomeev, E. E., Schuchmann, M., Luria, V., Chiannilkulchai, N., Beckmann, J. S., Mett, I. L., Rebrikov, D., Brodianski, V. M., Kemper, O. C., Kollet, O., Lapidot, T., Soffer, D., Sobe, T., Avraham, K. B., Goncharov, T., Holtmann, H., Lonai, P. and Wallach, D. (1998). Targeted disruption o f the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apol, and DR3 and is lethal prenatally. Immunity 9,267-76.
Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J. and Vaux, D. L. (2000). Identification o f DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing lAP proteins. Cell 102,43-53.
Volbracht, C., Leist, M. and Nicotera, P. (1999). ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation. Molecular Medicine (Cambridge, Mass.) 5 ,477-89.
Walczak, H., Miller, R. E., Ariail, K., Gliniak, B., Griffith, T. S., Kubin, M., Chin, W., Jones, J., Woodward, A., Le, T., Smith, C., Smolak, P., Goodwin, R. G., Rauch,C. T., Schuh, J. C. and Lynch, D. H. (1999). Tumoricidal activity o f tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nature Medicine 5, 157- 63.
Walker, R. M., Massey, T. E., McElligott, T. F. and Racz, W. J. (1981). Acetaminophen- induced hypothermia, hepatic congestion, and modification by N-acetylcysteine in mice. Toxicology and Applied Pharmacology 59,500-7.
Walker, R. M., Racz, W. J. and McElligott, T. F. (1983). Scanning electron microscopic examination o f acetaminophen-induced hepatotoxicity and congestion in mice. American Journal o f Pathology 113, 321-30.
Walker, R. M., Racz, W. J. and McElligott, T. F. (1985). Acetaminophen-induced hepatotoxic congestion in mice. Hepatology 5,233-40.
Wang, C. Y., Mayo, M. W. and Baldwin, A. S. J. (1996a). TNF- and cancer therapy- induced apoptosis: potentiation by inhibition o f NF-kappaB [see comments]. Science 274, 784-7.
Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V. and Baldwin, A. S. J.(1998). NF-kappaB antiapoptosis: induction o f TRAFl and TRAF2 and c-IAPl and C-IAP2 to suppress caspase-8 activation. Science 281, 1680-3.
Wang, K., Yin, X. M., Chao, D. T., Milliman, C. L. and Korsmeyer, S. J. (1996b). BID: a novel BH3 domain-only death agonist. Genes and Development 10,2859-69.
Wang, T. H. and Wang, H. S. (1996). p53, apoptosis and human cancers. Journal o f the Formosan M edical Association 95, 509-22.
Wang, T. H. and Wang, H. S. (1999). Apoptosis: (1). Overview and clinical significance. Journal o f the Formosan M edical Association 98,381-93.
Wang, X. W., Gibson, M. K., Vermeulen, W., Yeh, H., Forrester, K., Sturzbecher, H. W., Hoei)makers, J. H. and Harris, C. C. (1995). Abrogation o f p53-induced apoptosis by the hepatitis B virus X gene. Cancer Research 55,6012-6.
Wanner, G. A., Mica, L., Wanner-Schmid, E., Kolb, S. A., Hentze, H., Trentz, O. and Ertel, W. (1999). Inhibition o f caspase activity prevents CD95-mediated hepatic
206
microvascular perfusion failure and restores Kupffer cell clearance capacity. Faseb Journal 13,1239-48.
Waterhouse, N. J., Goldstein, J. C., von Ahsen, O., Schuler, M., Newmeyer, D. D. and Green, D. R. (2001). Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. The Journal o f Cell Biology 153, 319-28.
Wei, M. C., Lindsten, T., Mootha, V. K., Weiler, S., Gross, A., Ashiya, M., Thompson,C. B. and Korsmeyer, S. J. (2000). tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c. Genes & Development 14,2060-71.
Weis, M., Kass, G. E., Orrenius, S. and Moldeus, P. (1992a). N-acetyl-p-benzoquinone imine induces Ca2+ release from mitochondria by stimulating pyridine nucleotide hydrolysis. The Journal o f Biological Chemistry 267, 804-9.
Weis, M., Morgenstem, R., Cotgreave, I. A., Nelson, S. D. and Moldeus, P. (1992b). N- acetyl-p-benzoquinone imine-induced protein thiol modification in isolated rat hepatocytes. Biochemical Pharmacology 43, 1493-505.
Wendel, A. (1983). Hepatic lipid peroxidation: caused by acute drug intoxication, prevented by liposomal glutathione. International Journal o f Clinical Pharmacology Research 3 ,443-7.
Wendel, A. (1984). [The significance o f lipid peroxidation in drug-induced liver damage]. Zeitschrift Fur Gastroenterologie 22, 9-15.
Wendel, A., Feuerstein, S. and Konz, K. H. (1979). Acute paracetamol intoxication o f starved mice leads to lipid peroxidation in vivo. Biochemical Pharmacology 28, 2051-5.
Wendel, A. and Hallbach, J. (1986). Quantitative assessment o f the binding o f acetaminophen metabolites to mouse liver microsomal phospholipid. Biochemical Pharmacology 35,385-9.
Wendel, A. and Jaeschke, H. (1982). Drug-induced lipid peroxidation in mice—III. Glutathione content o f liver, kidney and spleen after intravenous administration o f free and liposomally entrapped glutathione. Biochemical Pharmacology 21, 3607- II.
Wendel, A., Jaeschke, H. and Gloger, M. (1982). Drug-induced lipid peroxidation in mice—II. Protection against paracetamol-induced liver necrosis by intravenous liposomally entrapped glutathione. Biochemical Pharmacology 31,3601-5.
Whitehouse, L. W., Paul, C. J., Wong, L. T. and Thomas, B. H. (1977). Effect o f aspirin on a subtoxic dose o f I4C-acetaminophen in mice. Journal o f Pharmaceutical Sciences 66, 1399-403.
Whyte, M. and Evan, G. (1995). Apoptosis. The last cut is the deepest. Nature 376,17-8.Wiley, S. R., Schooley, K., Smolak, P. J., Din, W. S., Huang, C. P., Nicholl, J. K.,
Sutherland, G. R., Smith, T. D., Rauch, C. and Smith, C. A. (1995). Identification and characterization o f a new member o f the TNF family that induces apoptosis. Immunity 3,673-82.
Williams, A. J., Coakley, J. and Christodoulou, J. (1998). Automated analysis o f mitochondrial enzymes in cultured skin fibroblasts. Analytical Biochemistry 259, 176-80.
Wilson, M. R. (1998). Apoptosis: unmasking the executioner. Cell Death Differ 5, 646-52.
207
Wolf, B. B, and Green, D. R. (1999). Suicidal tendencies: apoptotic cell death by caspase family proteinases. Journal o f Biological Chemistry 274,20049-52.
Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G. and Youle, R. J.(1997). Movement o f Bax from the cytosol to mitochondria during apoptosis. Journal o f Cell Biology 139, 1281-92.
Woo, M., Hakem, A., Elia, A. J., Hakem, R., Duncan, G. S., Patterson, B. J. and Mak, T. W. (1999). In vivo evidence that caspase-3 is required for Fas-mediated apoptosis o f hepatocytes. Journal o f Immunology 163,4909-16.
Wyllie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284,555-6.
Wyllie, A. H. (1997a). Apoptosis and carcinogenesis. European Journal o f Cell Biology 73, 189-97.
Wyllie, A. H. (1997b). Apoptosis: an overview. British Medical Bulletin 53,451-65.Wyllie, A. H., Morris, R. G., Smith, A. L. and Dunlop, D. (1984). Chromatin cleavage in
apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. Journal o f Pathology 142,67-77.
Xu, Y., Jones, B. E., Neufeld, D. S. and Czaja, M. J. (1998). Glutathione modulates rat and mouse hepatocyte sensitivity to tumor necrosis factor toxicity. Gastroenterology 115,1229-37.
Xue, D. and Horvitz, H. R. (1995). Inhibition o f the Caenorhabditis elegans cell-death protease CED-3 by a CED-3 cleavage site in baculovirus p35 protein. Nature 377, 248-51.
Yacoub, L. K., Fogt, F., Griniuviene, B. and Nanji, A. A. (1995). Apoptosis and bcl-2 protein expression in experimental alcoholic liver disease in the rat. Alcoholism, Clinical and Experimental Research 19, 854-9.
Yamada, T. and Ohyama, H. (1988). Radiation-induced interphase death o f rat thymocytes is internally programmed (apoptosis). International Journal o f Radiation Biology and Related Studies in Physics, Chemistry, and Medicine 53, 65-75.
Yamada, T., Ohyama, H., Kinjo, Y. and Watanabe, M. (1981). Evidence for the intemucleosomal breakage o f chromatin in rat thymocytes irradiated in vitro. Radiation Research 85, 544-53.
Yamada, Y., Kirillova, I., Peschon, J. J. and Fausto, N. (1997). Initiation o f liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor. Proceedings o f the National Academy o f Sciences o f the United States o f America 94, 1441-6.
Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. L, Jones, D. P. and Wang, X. (1997). Prevention o f apoptosis by Bcl-2: release o f cytochrome c from mitochondria blocked [see comments]. Science 275,1129-32.
Yang, J. C. and Cortopassi, G. A. (1998). Induction o f the mitochondrial permeability transition causes release o f the apoptogenic factor cytochrome c. Free Radical Biology and Medicine 24,624-31.
Yang, X., Chang, H. Y. and Baltimore, D. (1998). Essential role o f CED-4 oligomerization in CED-3 activation and apoptosis. Science 281, 1355-7.
Yeh, W. C., Hakem, R., Woo, M. and Mak, T. W. (1999). Gene targeting in the analysis o f mammalian apoptosis and TNF receptor superfamily signaling. Immunological Reviews 169,283-302.
208
Yin, X. M., Oltvai, Z. N. and Korsmeyer, S. J. (1994). BH l and BH2 domains o f Bcl-2 are required for inhibition o f apoptosis and heterodimerization with Bax [see comments]. Nature 369,321-3.
Yin, X. M., Wang, K., Gross, A., Zhao, Y., Zinkel, S., Klocke, B., Roth, K. A. and Korsmeyer, S. J. (1999). Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400, 886-91.
Yoo, Y. D., Ueda, H., Park, K., Flanders, K. C., Lee, Y. L, Jay, G. and Kim, S. J. (1996). Regulation o f transforming growth factor-beta 1 expression by the hepatitis B virus (HBV) X transactivator. Role in HBV pathogenesis. Journal o f Clinical Investigation 97, 388-95.
Yoshida, H., Kong, Y. Y., Yoshida, R., Elia, A. J., Hakem, A., Hakem, R., Penninger, J. M. and Mak, T. W. (1998). Apafl is required for mitochondrial pathways o f apoptosis and brain development. Cell 94, 739-50.
Young, L. S., Dawson, C. W. and Eliopoulos, A. G. (1997). Viruses and apoptosis. British M edical Bulletin 53, 509-21.
Yuan, J. and Yankner, B. A. (2000). Apoptosis in the nervous system. Nature 407, 802-9.Zech, B., Kohl, R., von Knethen, A. and Brune, B. (2003). Nitric oxide donors inhibit
formation o f the Apaf-l/caspase-9 apoptosome and activation o f caspases. The Biochemical Journal 371, 1055-64.
Zha, J., Harada, H., Osipov, K., Jockel, J., Waksman, G. and Korsmeyer, S. J. (1997). BH3 domain o f BAD is required for heterodimerization with BCL-XL and pro- apoptotic activity. The Journal o f Biological Chemistry 272,24101-4.
Zha, J., Weiler, S., Oh, K. J., Wei, M. C. and Korsmeyer, S. J. (2000). Posttranslational N-myristoylation o f BID as a molecular switch for targeting mitochondria and apoptosis. Science 290, 1761-5.
Zhang, H., Cook, J., Nickel, J., Yu, R., Stecker, K., Myers, K. and Dean, N. M. (2000a). Reduction o f liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nature Biotechnology 18, 862-7.
Zhang, H., Xu, Q., Krajewski, S., Krajewska, M., Xie, Z., Fuess, S., Kitada, S., Godzik, A. and Reed, J. C. (2000b). BAR: An apoptosis regulator at the intersection o f caspases and Bcl-2 family proteins. Proceedings o f the National Academy o f Sciences o f the United States o f America 97,2597-602.
Zheng, T. S., Hunot, S., Kuida, K., Momoi, T., Srinivasan, A., Nicholson, D. W., Lazebnik, Y. and Flavell, R. A. (2000). Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation. Nature Medicine 6, 1241-1247.
Zheng, T. S., Schlosser, S. F., Dao, T., Hingorani, R., Crispe, I. N., Boyer, J. L. and Flavell, R. A. (1998). Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo. Proceedings o f the National Academy o f Sciences o f the United States o f America 95,13618-23.
Zhivotovsky, B. D., Zvonareva, N. B. and Hanson, K. P. (1981). Characteristics o f rat thymus chromatin degradation products after whole-body x-irradiation. International Journal o f Radiation Biology and Related Studies in Physics, Chemistry, and Medicine 39,437-40.
Zhu, N., Khoshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C. and Lai, M. M. (1998). Hepatitis C virus core protein binds to the cytoplasmic domain o f tumor necrosis factor (TNF) receptor 1 and enhances TNF-induced apoptosis. Journal o f Virology 72, 3691-7.
209
Zhuang, J. and Cohen, G. M. (1998). Release o f mitochondrial cytochrome c is upstream o f caspase activation in chemical-induced apoptosis in human monocytic tumour cells. Toxicology Letters 102-103,121-9.
Zhuang, J., Dinsdale, D. and Cohen, G. M. (1998). Apoptosis, in human monocytic THP.l cells, results in the release o f cytochrome c from mitochondria prior to their ultracondensation, formation o f outer membrane discontinuities and reduction in inner membrane potential. Cell Death Differ 5, 953-62.
Zimmerman, B. T., Crawford, G. D., Dahl, R., Simon, F. R. and Mapoles, J. E. (1995). Mechanisms o f acetaldehyde-mediated growth inhibition: delayed cell cycle progression and induction o f apoptosis. Alcoholism, Clinical and Experimental Research 19,434-40.
Zou, H., Henzel, W. J., Liu, X., Lutschg, A. and Wang, X. (1997). Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation o f caspase-3. Cell 90,405-13.
Zou, H., Li, Y., Liu, X. and Wang, X. (1999). An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. Journal o f Biological Chemistry 274, 11549-56.
Zwacka, R. M., Zhang, Y., Zhou, W., Halldorson, J. and Engelhardt, J. F. (1998). Ischemia/reperfusion injury in the liver o f BALB/c mice activates AP-1 and nuclear factor kappaB independently o f IkappaB degradation. Hepatology 28, 1022-30.
210
MATERIAL REDACTED AT REQUEST OF UNIVERSITY
Reproduced with permission of copyright owner. Further reproduction prohibited without permission.
top related