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

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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

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1 1 Q_©

u s < P.

i ©U >N

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k

m

a

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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

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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

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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

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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..

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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).

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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

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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

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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

Π2D 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

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