Molecular mechanisms of drug-induced hepatic steatosis

Steatosis of the liver is defined by an excess accumulation of intracellular triglycerides within the hepatocytes. Nonalcoholic fatty liver disease ranges from simple steatosis to nonalcoholic steatohepatitis. It is a serious health problem worldwide, reported to affect 15 – 30 % of the population in developed countries. Inordinate accumulation of fat is damaging to the liver, because high levels of triglycerides, free fatty acids, or intermediates of lipid metabolism are extremely problematic for a cell. A further problem is that nonalcoholic fatty liver disease can be a risk factor for developing adverse reactions to drugs, interfering with hepatic lipid metabolism, leading to idiosyncratic drug-induced liver injury. Drug-induced liver injury has been the major cause of drugs failing market-approval or for later withdrawal from the market. An improved preclinical detectability of such adverse reactions would therefore be highly appreciated by the industry, as well as by patients.
The present work consists of three projects investigating molecular mechanisms of drug-induced hepatic steatosis in vitro, and is emphasizing on the application of an optimized method to measure the acylcarnitine pattern of cells, treated with toxicants. We focused on the measurement of acylcarnitines, because they are known to reflect the cellular acyl-CoA pattern, allowing us to make interpretations on the specific location of inhibition by a substance. Furthermore, the liquid chromatography-tandem mass spectrometry method, used for this determination, allows for a fast and economic workup and analysis of a high number of samples, applicable for high-throughput screenings. Additionally, the small sample volume needed for the analysis allows this assay to be linked together with many other 96-well format assays. An important finding in our study was that the acylcarnitine method repeatedly turned out to be a more sensitive approach to identify drugs inhibiting fatty acid oxidation, as the older, well-established methods, such as radio-enzymatic determination of β-oxidation inhibition, or lipid accumulation experiments.
In the first paper, we aimed to establish and optimize the semi-quantitative acylcarnitine measurement with three specific and well-characterized inhibitors of fatty acid oxidation. We compared, as well as supplemented, the results of the new method with older, well-established methods. In a second step of the study, we applied the same methods on three compounds, of which adverse reactions are not well understood, to obtain new insights about their steatogenic mechanisms.
In the two following papers, investigating the catechol-O-methyl-transferase inhibitors tolcapone and entacapone, we aimed to expand the knowledge of tolcapone-associated steatosis and liver toxicity observed in clinics. Entacapone was included as well, because of its structural similarity. We studied in detail effects on lipid metabolism, as well as on their actions on mitochondrial respiration and cell death, for a comprehensive toxicological study about the two drugs.