TY - THES T1 - High throughput toxicity, physiological and metabolic studies for the characterization of hepatocytes and human embryonic stem cell derived hepatocyte-like cells A1 - Beckers,Simone Y1 - 2011/05/06 N2 - A dynamic respiration assay based on luminescence decay time detection of oxygen for high throughput toxicological assessment is presented. The method applies 24-well plates (OxoDishes). Dissolved oxygen concentration is measured by a SensorDishReader reading the oxygen sensor optodes immobilized in the centre of each well. This method allows LC50 calculations and recording of toxicokinetic profiles. Adherent primary rat hepatocytes and Hep G2 cell line were exposed to known toxic compounds. The novel assay showed to be robust, flexible and an improvement to current methods. Three human embryonic stem cell (hESC) lines, which have been directed towards hepatocyte-like cells were characterized and compared to Hep G2 cells and primary human hepatocytes for evaluation of their application for predictive toxicity testing and drug metabolism studies. In multi-well plate formats, repeatedly cells were identified to be hepatocyte-like by morphologic evaluation. Gene expression of liver specific genes and hepatic lineage markers were evaluated. The cells showed functional hepatic characteristics, such as albumin secretion, glycogen storage and urea synthesis. Phase I and phase II metabolism of midazolam, phenacetin and diclofenac was detected for the respective metabolites and the toxicity to diclofenac was confirmed by a toxicodynamics study. The characterization results described here provide a unique overview of the functionality of hESC derived hepatocytes. Multiple physiological and 13C-labeling studies on hESC derived epatocytes-like cells and primary human hepatocytes, exposed to sub-toxic diclofenac concentrations were performed to identify metabolic pathways. In addition, their response to drug treatment was evaluated. Glycolysis, TCA cycle, amino acid degradation, albumin synthesis and pyruvate (re)cycling were considered for a stoichiometric metabolic flux model. MFA analysis revealed influence of sub-toxic diclofenac concentrations on the oxidative phosphorylation pathway. KW - Toxizität KW - High-throughput screening KW - Leber KW - Metabolismus CY - Saarbrücken PB - Universitäts- und Landesbibliothek AD - Postfach 151141, 66041 Saarbrücken UR - http://scidok.sulb.uni-saarland.de/volltexte/2011/3665 ER -