Susceptibility factors in paracetamol-induced acute liver failure

281 Pages·2015·45.3 MB·English

by  Possamai, Lucia

Susceptibility factors in paracetamol-induced acute
liver failure

Lucia Anne Possamai

Imperial College London

Department of Hepatology

2015

Thesis submitted for the degree of Doctor of Philosophy
Imperial College London

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Declaration of Originality

I declare that the work in this thesis is my own and all else is appropriately referenced.

Copyright Declaration

The copyright of this thesis rests with the author and is made available under a Creative
Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy,
distribute or transmit the thesis on the condition that they attribute it, that they do not use it
for commercial purposes and that they do not alter, transform or build upon it. For any reuse
or redistribution, researchers must make clear to others the licence terms of this work.

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Dedication

To my parents, Anne and Paul Possamai

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Acknowledgements
I am grateful to a number of people for their support and help throughout the period of my
research studies. It is a pleasure to have to opportunity to thank them for their contribution.
Firstly, I am enormously grateful for the support and inspiration of my primary supervisor
Professor Mark Thursz. It has been a great pleasure and privilege to work in his research
group under his guidance. His encouragement and leadership have enriched my research
experience beyond measure.
I would like to thank Professor Roger Cox, at MRC Harwell for the warm welcome he
extended to me as an external PhD student, his thoughtful advice and generously given time.
Dr Quentin Anstee gave me invaluable help during the first years of my PhD and offered me
vital guidance in my application for a MRC Fellowship. I am extremely grateful to him for this
help and for his on going mentorship from afar. I would like to thank Dr Harry Antoniades for
his supervision and advice in the later stages of my PhD. I have been lucky to experience his
infectious enthusiasm for research and am very grateful for his time and supervision.
There are a number of people who have offered me assistance, practical help and tutorship
during my research, enabling me to master new skills. I am enormously grateful to Dr I. Jane
Cox for introducing me to 1H-NMR and for her patient encouragement. I thank Dr Horace
Williams for his time in teaching me the basics of multi-variate statistical analysis for
metabonomics. Dr Simon Yona at UCL offered important help in teaching me the principles
of isolation of hepatic immune cells from murine liver and I thank him.
In the department of Hepatology at Imperial College Dr Fouzia Saddiq, Dr Suzanne Knapp
and Dr Wafa Khamri have all generously offered help and guidance with laboratory
techniques. I am very grateful to them for their kindness and patience.
Members of the Liver Group at MRC Harwell, Dr Danilo Concas, Bishan Wu and Carl
Hassett helped me with the mouse work at the beginning of the project. I am very grateful for
their assistance and teaching. The animals technicians and ward managers at the Mary

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Lyons Centre, MRC Harwell were an important source of training, advice and help. I am
grateful to them all, but in particular Michelle Stewart and Mark Harrison.
The staff at Central Biomedical Services at Imperial College, Gareth Wild in particular, have
also helped me greatly in the care of mice and as a source of guidance.
I thank Professor Robert Goldin for his advice and expertise on the evaluation of liver injury
in murine liver tissue.
I have had the privilege of joining a department with fantastic co-workers and am immensely
grateful to them for their encouragement. Sharing a research office with dedicated
colleagues has enriched my PhD experience. In particular, I would like to thank Dr Gemma
Petts who has assisted me with my histology processing and mouse studies and Dr Nikhil
Vergis for being a source of lively debate and contrary opinion. They have both offered me
support and coffee when needed and are great friends.
I would like to thank my husband, Chris Valerio, for his unwavering and patient support and
my children Alex and Rosa born during this project and without whom I would have finished it
far sooner. My extended family have also provided enormous encouragement and practical
support, particularly in the later stages and I am very grateful to them.
Finally I would like to acknowledge and thank the Medical Research Council UK, for the
wonderful opportunity they have given me to undertake a period of funded research.

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Abstract
Paracetamol is a popular antipyretic and analgesic medication. It is a known hepatotoxin in
overdose and is the commonest cause of acute liver failure in the UK. There is significant
variability in inter-individual susceptibility to the hepatotoxic effects of paracetamol, which is
incompletely understood. This thesis describes work done in murine models of paracetamol-
induced acute liver failure with the aim of identifying causes of variable susceptibility and
understanding the immune response to liver injury.
A quantitative trait locus (QTL) mapping approach was taken using a murine strain
susceptible to paracetamol hepatotoxicity (C3H/HeH) crossed with a relatively resistant strain
(C57BL/6). Novel QTLs on murine chromosome 17 and 18 were identified that associated
with response to paracetamol. Within the loci a number of candidate genes were identified.
A survey of 10 inbred mouse strains for their response to paracetamol was conducted and
highlighted the large variability within each strain. It was hypothesised that this variability
might be due to differences in intestinal microbiota. A study of the role of intestinal microbiota
in paracetamol-induced liver failure was conducted, by comparing response in germ free
(GF) mice and conventional (CV) controls. This demonstrated that the presence of intestinal
microbiota influenced the sulphonation:glucuronidation ratio during paracetamol metabolism.
Although the extent of liver injury as assessed by necrosis and liver enzyme elevation was
the same in GF and CV mice, there was evidence of a protective effect of a sterile intestine.
Finally a reverse genetics approach was taken to study the influence of the gene Slpi in the
secondary immune response to liver injury. It was shown that absence of SLPI protected
mice against peak liver injury.
The work presented in this thesis highlights some potential sources of variability in response
to paracetamol and a number of targets that with further research could have therapeutic
potential.

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Publications arising from this work
Papers
Possamai LA, McPhail MJ, Khamri W, Wu B, Concas D, Harrison M, Williams R, Cox RD,
Cox IJ, Anstee QM, Thursz MR. The role of intestinal microbiota in murine models of
acetaminophen-induced hepatotoxicity. Liver Int. 2014 Sep 22. doi:10.1111/liv.12689.
PubMed PMID: 25244648.

Possamai LA, Khamri W, Triantafyllou E, Wendon JA, Thursz MR, Antoniades CG. Could
targeting secretory leukocyte protease inhibitor be an effective therapeutic option to prevent
infections in acute liver failure? Immunotherapy. 2014 Jun;6(6):667-9. PubMed PMID:
25041028.

Possamai LA, Thursz MR, Wendon JA, Antoniades CG. Modulation of
monocyte/macrophage function: a therapeutic strategy in the treatment of acute liver failure.
J Hepatol. 2014 Aug;61(2):439-45. doi: 10.1016/j.jhep.2014.03.031. Epub 2014 Apr 2.
PubMed PMID: 24703954.

Abstracts
Anstee QM, Possamai LA, Concas D, Wu B, Hassett C, Cox RD, Thomas HC, Thursz MR,
Novel quantitative trait locus for acetaminophen- induced hepatotoxicity identified on
chromosome 18. J Hepatol. 2010 Vol. 52, S311

Possamai LA, Harrison M, Cox RD, Williams R, Anstee QM, Thursz MR, Cox IJ
1H-NMR metabolic profiling in germ-free and conventionally housed mouse models of
acetaminophen-induced hepatotoxicity. J Hepatol. 2012 Vol. 56, S529

Possamai LA, Petts G, Khamri W, Antoniades CG, Thursz MR. The role of hepatic secretory
leucocyte protease inhibitor (SLPI) in a murine acute liver failure model. J Hepatol 2014, Vol.
60, Issue 1, S118

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Table of Contents
Declaration of Originality ................................................................................................... 2

Copyright Declaration ........................................................................................................ 2

Dedication ........................................................................................................................... 3

Acknowledgements ............................................................................................................ 4

Abstract ............................................................................................................................... 6

Publications arising from this work .................................................................................. 7

Table of Contents ............................................................................................................... 8

1
  INTRODUCTION .............................................................................................................. 22

1.1
  Paracetamol: from discovery to the nation’s favorite painkiller ......................... 22

1.2
  The clinical syndrome of paracetamol-induced acute liver failure ..................... 23

1.2.1
  Definition ............................................................................................................. 23

1.2.2
  Epidemiology ....................................................................................................... 24

1.2.3
  Clinical progression ............................................................................................. 25

1.2.4
  Treatment ............................................................................................................ 28

1.3
  Hepatic metabolism of paracetamol ...................................................................... 29

1.4
  Hepatocyte injury and cell death in paracetamol toxicity .................................... 31

1.4.1
  Covalent bonding & protein adduct formation ..................................................... 31

1.4.2
  Oxidative stress and protein nitration .................................................................. 31

1.4.3
  The mitochondrial permeability transition ............................................................ 32

1.4.4
  The role of apoptosis and necrosis ..................................................................... 33

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1.5
  The immune response in paracetamol-induced acute liver failure ..................... 37

1.5.1
  Damage associated molecular pattern (DAMP) signalling .................................. 37

1.5.2
  Intracellular signalling and inflammasome activation .......................................... 40

1.5.3
  The role of monocytes and macrophages ........................................................... 42

1.5.4
  The role of neutrophils ........................................................................................ 46

1.6
  Animal models of paracetamol-induced acute liver failure ................................. 51

1.7
  Variability in response to the hepatotoxic effects of paracetamol ..................... 51

1.8
  Genetic susceptibility factors ................................................................................. 52

1.8.1
  Metabolic phase .................................................................................................. 52

1.8.2
  Cell death & immune activation phase ................................................................ 53

1.9
  Environmental susceptibility .................................................................................. 54

1.10
  Summary and aims ................................................................................................ 55

2
  GENETIC SUSCEPTIBILITY FACTORS: QTL MAPPING FOR PARACETAMOL
SUSCEPTIBILITY .................................................................................................................. 58

2.1
  Background and aims ............................................................................................. 58

2.1.1
  Quantitative traits ................................................................................................ 58

2.1.2
  Inbred strains of laboratory mice ......................................................................... 59

2.1.3
  QTL mapping ...................................................................................................... 59

2.2
  Materials and methods ............................................................................................ 61

2.2.1
  Animal Maintenance ............................................................................................ 61

2.2.2
  Breeding strategy for QTL mapping .................................................................... 61

2.2.3
  Phenotyping procedure for QTL mapping ........................................................... 61

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2.2.4
  Biochemistry analysis .......................................................................................... 64

2.2.5
  DNA extraction .................................................................................................... 64

2.2.6
  SNP Genotyping ................................................................................................. 65

2.2.7
  Data ..................................................................................................................... 65

2.2.8
  Mapping .............................................................................................................. 66

2.2.9
  Single QTL genome scans .................................................................................. 66

2.2.10
  Two-QTL scan ................................................................................................... 67

2.2.11
  Multiple QTL analysis using Bayesian interval mapping ................................... 67

2.2.12
  Candidate gene identification ............................................................................ 67

2.4
  Results ...................................................................................................................... 69

2.4.1
  Phenotyping ........................................................................................................ 69

2.4.2
  Mapping – descriptive data ................................................................................. 69

2.4.3
  Single-QTL scan ALT at 24 hours ....................................................................... 71

2.4.4
  Single-QTL scan glucose at 24 hours ................................................................. 75

2.4.5
  Single-QTL scan bilirubin at 24 hours ................................................................. 76

2.4.6
  Two dimensional QTL scanning .......................................................................... 77

2.4.7
  Multiple QTL model ............................................................................................. 78

2.4.8
  Candidate gene identification .............................................................................. 83

2.6
  Discussion ................................................................................................................ 92

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