New approaches to improve prediction of drug-induced liver injury Dissertation zur Erlangung des naturwissenschafltichen Doktorgrades der Julius-Maximilians-Universität Würzburg vorgelegt von Melanie Adler aus Greiz Würzburg 2012 Eingereicht am 02.02.2012 bei der Fakultät für Chemie und Pharmazie Gutachter der schriftlichen Arbeit 1. Gutachter PD. Dr. Angela Mally 2. Gutachter Prof. Dr. Ulrike Holzgrabe 1. Prüfer PD Dr. Angela Mally 2. Prüfer Prof. Dr. Ulrike Holzgrabe 3. Prüfer Prof. Dr. Wolfgang Dekant des öffentlichen Promotionskolloquiums Datum des öffentlichen Promotionskolloquiums: 27.02.2012 Doktorurkunde ausgehändigt am ………………………………………………. Für meine Eltern "Wer immer tut, was er schon kann, bleibt immer das, was er schon ist." Henry Ford INDEX INDEX Index .............................................................................................................. I Abbreviations ............................................................................................... V 1 Introduction .......................................................................................... 1 1.1 The liver as a critical target organ of drug toxicity ................................ 1 1.1.1 Liver morphology and function ............................................................................ 3 1.1.2 Xenobiotic metabolism ........................................................................................ 4 1.1.3 Types and mechanisms of drug-induced liver injury ........................................... 6 1.2 Methods for detection of hepatotoxicity ................................................10 1.2.1 Primary hepatocytes as an in vitro model to study hepatotoxicity ..................... 10 1.2.2 Traditional approaches ...................................................................................... 11 1.2.3 Application of omics technologies ..................................................................... 12 1.2.3.1 Novel biomarker candidates for hepatotoxicity ........................................................ 13 1.2.3.2 Mechanistic understanding of hepatotoxicity ........................................................... 15 1.2.4 RNA interference as an investigative tool in mechanistic toxicology................. 17 1.2.4.1 Mechanism of siRNA-mediated gene silencing ....................................................... 18 1.3 Aims of this work .....................................................................................21 2 Materials and Methods ...................................................................... 22 2.1 Materials ...................................................................................................22 2.1.1 Technical equipment ......................................................................................... 22 2.1.2 Chemicals and reagents ................................................................................... 23 2.1.3 Culture media and supplements ....................................................................... 25 2.1.4 Kits .................................................................................................................... 25 2.1.5 Buffers and solutions ......................................................................................... 26 2.1.6 Antibodies ......................................................................................................... 28 2.1.7 Oligonucleotides ................................................................................................ 29 2.1.8 Software ............................................................................................................ 30 2.2 In vivo studies ..........................................................................................30 2.2.1 Standardized study design of the InnoMed Predictive Toxicology Project ........ 30 2.2.2 Clinical examination .......................................................................................... 32 2.2.3 Construction of tissue microarrays .................................................................... 33 2.3 In vitro studies .........................................................................................33 2.3.1 Isolation of primary rat hepatocytes .................................................................. 34 2.3.2 Trypan Blue exclusion test ................................................................................ 35 2.3.3 Culture conditions of hepatocytes ..................................................................... 36 I INDEX 2.3.4 Transfection of siRNA duplex ........................................................................... 36 2.3.5 Treatment with compound BAY16 .................................................................... 37 2.3.6 WST-1 assay .................................................................................................... 38 2.3.7 CellTiter-Glo® Luminescent cell viability assay ................................................. 39 2.4 Molecular biological methods ............................................................... 40 2.4.1 Isolation of total RNA from tissues .................................................................... 40 2.4.2 Isolation of total RNA from rat hepatocytes ...................................................... 41 2.4.3 RNA quantification and quality control .............................................................. 41 2.4.4 cDNA synthesis and qRT- PCR using SYBR® Green ....................................... 42 2.4.5 cDNA synthesis and qRT- PCR using hydrolysis probes ................................. 45 2.4.6 Microarray experiments .................................................................................... 46 2.4.6.1 Preparation of labeled aRNA ................................................................................... 48 2.4.6.2 Hybridization and microarray processing ................................................................. 49 2.4.6.3 Data extraction and analysis .................................................................................... 50 2.5 Biochemical methods ............................................................................. 51 2.5.1 Protein preparation from tissue ......................................................................... 51 2.5.2 Protein preparation from rat hepatocytes .......................................................... 52 2.5.3 Protein separation by SDS polyacrylamid gel electrophoresis ......................... 52 2.5.4 Protein detection by western blot analysis and immune detection ................... 53 2.5.5 Immunofluorescence ........................................................................................ 55 2.5.6 Immunohistochemical analyses ........................................................................ 55 2.5.7 Quantification of proteins by ELISA .................................................................. 57 2.5.7.1 Development and validation of the rat NGAL ELISA ............................................... 58 2.5.7.2 Thiostatin ELISA ....................................................................................................... 59 2.5.7.3 Clusterin ELISA ........................................................................................................ 60 2.5.8 PON1 activity assay .......................................................................................... 60 3 Assessment of candidate biomarkers of drug induced liver injury in preclinical toxicity studies ................................................. 61 3.1 Background ............................................................................................. 61 3.2 Results ..................................................................................................... 62 3.2.1 Biomarker responses to BAY16 in relation to traditional endpoints of toxicity .. 62 3.2.1.1 Clinicopathological and histopathological observations ........................................... 62 3.2.1.2 Gene expression of biomarkers ............................................................................... 65 3.2.1.3 Detection and quantification of biomarkers in serum and urine ............................... 67 3.2.1.4 Immunolocalization of putative biomarkers .............................................................. 70 3.2.1.5 Summary .................................................................................................................. 72 3.2.2 Biomarker responses to EMD335823 in relation to traditional endpoints of toxicity ............................................................................................................... 73 3.2.2.1 Histopathological and clinicopathological observations ........................................... 73 II INDEX 3.2.2.2 Gene expression of biomarkers ............................................................................... 76 3.2.2.3 Detection and quantification of biomarkers in serum and urine ............................... 77 3.2.2.4 Immunolocalization of putative biomarkers .............................................................. 79 3.2.2.5 Summary .................................................................................................................. 80 3.2.3 Biomarker responses to BI-3 in relation to traditional endpoints of toxicity ....... 81 3.2.3.1 Clinicopathological and histopathological observations ........................................... 81 3.2.3.2 Gene expression of biomarkers ..............................................................................