Drug induced liver injury is a frequent cause of adverse drug reactions and for withdrawal of drugs from the market. It is therefore important to be able to detect the hepatotoxic potential of a new compound already early in drug development and also to identify susceptibility factors which may be associated with the development of liver injury in patients. This is only possible when the mechanisms of adverse drug reactions are known. Acquaintance of the toxicological mechanisms enables us to predict potential susceptibility factors and also biomarkers for drug-associated liver injury. In this grant application, I propose two separate projects in this area. In part A, hepatic toxicity of tyrosine kinase inhibitors (TKI), a drug class which has become very important for cancer treatment, is going to be investigated. So far, liver injury from asymptomatic elevation of transaminases up to liver failure has been described in patients treated with TKIs, but mechanistic studies are rare. I propose to start with a broad approach using established toxicological methods in order not to miss potential mechanisms. Since TKIs have been associated with mitochondrial toxicity in heart and skeletal muscle, a focus on impairment of mitochondrial function will be made after the broad approach. I plan to study first the toxicological effects of different TKIs in established cell models (HepG2 cells, HepaRG cells, primary human hepatocytes) in order to characterize toxicological mechanisms in vitro. After having obtained information about the mechanism, potential susceptibility factors (e.g. impaired mitochondrial function in case of mitochondrial toxicity or enzyme induction in case of toxicity associated with metabolites) can be postulated. Such susceptibility factors can then be tested in a second in vitro step by genetic or pharmacological modification of the cells used to assess the mechanisms of toxicity. In a further step, these factors will also be tested in suitable animal models in vivo. In part B, I propose to focus on inhibition of hepatic mitochondrial fatty acid metabolism, which, according to my experience, is an important toxicological mechanism of many drugs affecting the liver. Mitochondrial fatty acid metabolism is a complex metabolic pathway, starting with the activation of fatty acids and conversion of the acyl-CoA formed to the respective acylcarnitine, which is transported into the mitochondrial matrix, reconverted to the acylcarnitine and finally β-oxidized to form acetyl-CoA. According to the complexity of this pathway, mitochondrial fatty acid oxidation is susceptible for derangements by chemical compounds such as drugs. We will first study test compounds which are known inhibitors of hepatic fatty acid metabolism in the above mentioned in vitro test systems in order to show that this type of toxicity can be demonstrated in cell cultures in vitro. This will also allow us the screen for biomarkers with a targeted metabolite approach (acylcarnitines) as well as a non-targeted metabolomic approach. We will then test a number of drugs known to be hepatotoxic using our cellular test systems and biomarkers for impaired mitochondrial fatty acid metabolism. In these studies, we also plan to investigate the effects of drugs and/or accumulating fatty acid metabolism intermediates on the expression of key enzymes and on the regulation of mitochondrial fatty acid metabolism. Finally, similar to part A, susceptibility factors can be postulated and tested in genetically or pharmacologically altered cells and in experimental animals. If successful, the proposed studies will not only enlarge our knowledge regarding mechanisms of adverse drug reactions, but also generate new test systems and reveal new biomarkers for hepatotoxic drugs. Furthermore, they challenge the current thinking about drug-associated idiosyncrasy with potentially large consequences for drug development and drug treatment of patients.