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

Hrach

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2009
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 11 Mo

Extrait

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.........