Toxicogenomic approaches for the prediction of hepatotoxicity in vitro [Elektronische Ressource] / presented by Jens Hrach

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Dissertation submitted to the Combined faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-biologist Jens Hrach Born in: Gross-Gerau, Germany Oral-examination: Toxicogenomic approaches for the prediction of hepatotoxicity in vitro Referees: Prof. Dr. Thomas Holstein PD Dr. Suat Özbek INDEX ACKNOWLEDGEMENTS.................................................................................. 2 ABBREVIATIONS ............................................................................................. 3 SUMMARY......................................................................................................... 5 ZUSAMMENFASSUNG..................................................................................... 7 1 INTRODUCTION ......................................................................................... 9 1.1 Endeavors of modern toxicology..................................................................9 1.2 The liver morphology and its cell types .....................................................10 1.3 Hepatocytes and xenobiotic metabolism...................................................12 1.

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Dissertation

submitted to the
Combined faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences



























presented by

Diplom-biologist Jens Hrach
Born in: Gross-Gerau, Germany

Oral-examination:













































Toxicogenomic approaches
for the prediction of hepatotoxicity in vitro






















Referees: Prof. Dr. Thomas Holstein
PD Dr. Suat Özbek
INDEX
ACKNOWLEDGEMENTS.................................................................................. 2
ABBREVIATIONS ............................................................................................. 3
SUMMARY......................................................................................................... 5
ZUSAMMENFASSUNG..................................................................................... 7
1 INTRODUCTION ......................................................................................... 9
1.1 Endeavors of modern toxicology..................................................................9
1.2 The liver morphology and its cell types .....................................................10
1.3 Hepatocytes and xenobiotic metabolism...................................................12
1.4 Hepatotoxicity...............................................................................................18
1.5 In vitro liver models......................................................................................19
1.6 Endpoints for the analysis of hepatocyte cultures ...................................27
1.7 Toxicogenomics ...........................................................................................28
1.8 Techniques for global gene expression analysis......................................31
1.9 Toxicoproteomics.........................................................................................35
1.10 Aim of this work............................................................................................37
2 MATERIALS AND METHODS .................................................................. 38
2.1 Materials ........................................................................................................38
2.1.1 Chemicals and reagents.......................................................................................... 38
2.1.2 Technical equipment and auxiliary material............................................................ 40
2.1.3 Kits........................................................................................................................... 42
2.1.4 Software .................................................................................................................. 42
2.1.5 Culture media and supplements ............................................................................. 43
2.1.6 Buffers and solutions............................................................................................... 43
2.1.6.1 Perfusion buffers for rat liver perfusion ........................................................... 43
2.1.6.2 Buffers for SELDI-TOF-MS 44
2.1.6.3 Buffers for protein-preparation adn immunodetection..................................... 44
2.1.6.4 Buffers and solutions for Illumina BeadChip arrays ........................................ 45
®2.1.6.5 Buffers and solutions for Affymetrix Gene Chips ........................................... 45
2.2 Methods.........................................................................................................47
2.2.1 Cell culture .............................................................................................................. 47
2.2.1.1 Isolation of primary rat hepatocytes ................................................................ 47
2.2.1.2 Trypan Blue exclusion test .............................................................................. 48
2.2.1.3 Preparation of culture dishes........................................................................... 48 INDEX
2.2.1.4 Plating of cells..................................................................................................49
2.2.1.5 Culture of FaO and HepG2-cells .....................................................................50
2.2.1.6 Suspension culture ..........................................................................................50
2.2.1.7 Precision cut liver slices ..................................................................................50
2.2.1.8 Isolation of primary human hepatocytes..........................................................51
2.2.1.9 HepaRG cells...................................................................................................51
2.2.2 Rat in vivo study ......................................................................................................51
2.2.3 Biochemical methods and cell viability assays........................................................52
®2.2.3.1 CellTiter-Glo Luminescent cell viability assay................................................ 52
2.2.3.2 WST-1-assay53
2.2.3.3 LDH release.....................................................................................................53
2.2.3.4 Cytochrome P450 isoform induction and activity ............................................55
2.2.3.5 Canalicular transporter activity ........................................................................56
2.2.4 Molecular biological methods ..................................................................................56
2.2.4.1 Isolation of RNA and proteins..........................................................................56
2.2.4.2 Quantification and quality check of nucleic acids............................................57
2.2.4.3 TaqMan® Low Density Arrays (TLDA) ............................................................58
2.2.4.4 Processing of RNA for Illumina and Affymetrix Chips .....................................61
2.2.5 Microarray data analysis..........................................................................................66
2.2.5.1 Data extraction and quality control from Illumina BeadChip arrays ................ 66
2.2.5.2 y control from Affymetrix arrays.............................. 67
2.2.6 Protein separation by SDS polyacrylamide gel electrophoresis (SDS-PAGE) ....... 68
2.2.7 Protein detection by western blot analysis and immune detection.......................... 69
2.2.8 SELDI-TOF analysis................................................................................................70
3 RESULTS AND DISCUSSIONS................................................................73
3.1 Comparison of different global gene expression platforms.................... 73
3.1.1 Results of the platform comparison study ...............................................................76
3.1.1.1 Experimental layout .........................................................................................76
3.1.1.2 Intraplatform comparability ..............................................................................78
3.1.1.3 Interplatform 80
3.1.1.4 Biological interpretation ...................................................................................85
3.1.2 Conclusions of the platform comparison study........................................................97
3.2 Establishment of a longer term cell culture of primary rat and human
hepatocytes........................................................................................................... 100
3.2.1 Morphological and functional characterization of primary rat hepatocytes ...........101
3.2.1.1 Morphological examinations..........................................................................101
3.2.1.2 CYP inducibility..............................................................................................104
3.2.1.3 Canalicular transport .....................................................................................107
3.2.1.4 Conclusions of the morphological and functional data..................................108 INDEX
3.3 Global expression studies with different human and rat cell culture
systems ..................................................................................................................110
3.3.1 Initial changes introduced by the process of perfusion......................................... 114
3.3.1.1 Primary rat hepatocytes ................................................................................ 114
3.3.1.2 Primary human hepatocytes.......................................................................... 116
3.3.2 Temporal changes in global gene expression....................................................... 119
3.3.3 Analysis of protein expression with SELDI-TOF ................................................... 124
3.3.4 Gene expression in established cell lines used as reference ............................... 127
3.3.5 Changes of gene expression early in culture - Cellular adaptation processes in
primary hepatocytes.......................................................................................................... 129
3.3.5.1 Liver slices..................................................................................................... 134
3.3.6 Molecular mechanisms affected over time in culture ............................................ 137
3.3.6.1 Overview of the affected mechanisms in rat hepatocytes............................. 137
3.3.6.2 Response to wounding, oxidative stress and immune response.................. 139
3.3.6.3 ECM, cytoskeleton and tissue remodelling ................................................... 141
3.3.6.4 Metabolic competence .................................................................................. 142
3.3.6.5 Intracellular signalling and transcription factors ............................................ 144
3.3.6.6 Affected mechanisms in human hepatocytes................................................ 146
3.3.7 Confirmation of the microarray results with TaqMan PCR.................................... 148
3.3.8 Conclusions from the characterization of primary hepatocytes in culture............. 150
3.4 Development of an in vitro liver toxicity prediction model based on
longer term primary hepatocyte culture..............................................................155
3.4.1 Introduction to the in vitro prediction model .......................................................... 155
3.4.2 Short description of the test compounds............................................................... 155
3.4.3 Experimental setup and dose finding.................................................................... 159
3.4.4 Data Analysis and establishment of an in vitro prediction model for hepatotoxicity
………................................................................................................................................ 163
3.4.5 Analysis of the top ranked genes of the prediction model .................................... 169
3.5 Insights into the mechanisms of action for selected compounds.........171
3.5.1 EMD X ................................................................................................................... 171
3.5.2 AAP ....................................................................................................................... 174
3.5.3 Dex ........................................................................................................................ 177
4 CONCLUDING REMARKS AND FUTURE PERSPECTIVES................. 179
5 REFERENCES ........................................................................................ 183
APPENDIX..................................................................................................... 201


ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS

I want to thank all the people who contributed directly or indirectly to this work!

A special thanks to my thesis adviser Prof. Dr. Thomas Holstein. He escorted me
throughout my studies and raised my general interest in biological sciences with his
enthusiasm and knowledge of the molecular processes in living organisms.

I am deeply grateful to Dr. Phil Hewitt for giving me the opportunity to do my PhD work
in the laboratories of the institute for toxicology at Merck-Serono and for all his support
and scientific advice throughout their duration. He encouraged me to work
independently; discussions with him were an important factor in focusing my interest
into the field of toxicogenomics and beyond.

I would also like to thank Dr. Stefan Müller for his interest, the helpful support and
useful suggestions throughout my work. He has always been open for discussions and
put my work into the right context.

For their willingness to serve as referee for my thesis, I would like to thank PD Dr. Suat
Özbek and Prof. Ursula Kummer. Another thanks to Dr. Peter-Jürgen Kramer and Prof.
Dr. Hans Harleman, heads of the institute of toxicology, for the interest in my PhD work
and their helpful comments. Both of them permitted me not only to perform my studies
in their institute but also enabled me to attend advanced trainings courses and
scientific conferences.

I would like to thank all the people at Merck, especially, all the members of our group
who contributed to this work by discussions, help and friendship. Dr. Anja von
Heydebreck and Dr. Eike Staub for bioinformatical support, Jörg Hiller for solving
(nearly) all my computer-problems, Klaudia Clement, Bettina von Eiff, Yvonne Walter,
Margret Kling and Melanie Kühnl for all their support in the lab, Gregor Tuschl, Nadine
Zidek, Julia Pieh, and all other previous, current and upcoming PhD-Students for
making this an unforgettable part of my live.

Most of all, I want to thank my whole family for all their support and love throughout the
years. Without their help in every possible way, this work wouldn’t have been possible.
A big hug and thank you to my wife, for all her grateful understanding, for encouraging
me and for giving me the best time of my life.
2ABBREVIATIONS
ABBREVIATIONS
% Percentage
[ ] Concentration
+/- FCS With or without the addition of fetal calf serum
°C Centigrad
µl Micro litre
ALDH Aldehyde dehydrogenase
AN Accession Number
BNF Beta-naftoflavon
bp Basepair
BROD Benzyloxyresorufin O-debenzylase
BSA Bovine Serum Albumin
Carboxi-DCFDA 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate
CHAPS 3-[(3-Cholamidopropyl)-dimethylammonio}-1-
propanesulfonate
CO Carbonic acid 2
Da Dalton
Dex Dexametasone
DMSO Dimethyl sulfoxide
(c)DNA (complementary) Desoxy ribonucleic acid
DTT Dithiothreitol
ECVAM European Centre for the Validation of Alternative Methods
EDTA Ethylenediaminetetraacetic acid
EROD 7-ethoxyresorufin-O-deethylase
FBS Fetal Bovine Serum
FC Fresh cells (hepatocytes directly after perfusion)
FDA Food and Drug Administration
g Gram
GLP Good Laboratory Practice
GSH Glutathione
h Hour
H&E Hematoxylin and eosin stain
HepaRG Human hepatoma cell line
HepG2 Human hepe
Hz Hertz (cycles per second)
i.p. intraperitoneal
ITS Insulin, Transferrin, Selenit
IVT In vitro transcription reaction
k Kilo
3ABBREVIATIONS
kilodaltons kDa
l Litre
LDH Lactate dehydrogenase
M Molarity
mA Mili-Ampere
min Minute
ML Monolayer culture
mm millimetre
mRNA Messenger Ribonucleic acid
MTD Maximum tolerated dose
MW Molecular Weight
nm Nanometre
NRU Neutral Red Uptake
OD Optical density
ON Over night
PAGE Polyacrylamide gel electrophoresis
PB Phenobarbital
PB1 Perfusion Buffer 1
PB2 Perfusion Buffer 2
PBS Phosphate Buffered Saline
PCA Principal Components Analysis
PCR Polymerase Chain Reaction
PL Plastic culture
rcf Relative centrifugal force
REACH Registration, Evaluation and Authorization of Chemicals
RMA Robust multi-array average
(c) RNA (complementary) Ribonucleic acid
rpm rounds per minute
rRNA Ribosomal Ribonucleic acid
RT Room temperature
SDS Sodium dodecyl sulphate
sec Second
SELDI-TOF Surface-enhanced laser desorption/ionization – time
of flight
SOM Self Organizing Map
Susp. Suspension
SW Sandwich culture
TLDA TaqMan Low Density Array
WB Washing buffer


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