Effect of ochratoxin A on toxicity and gene expression inhepatocytes and kidney tubulus cells : role of dietaryantioxidants

The mycotoxin ochratoxin A (OTA) exhibits a wide range of toxic effects in animals and humans including hepato-, nephro-, neuro-, and genotoxicity. The toxicity mechanisms of OTA are not yet fully understood. It has been suggested that inhibition of protein synthesis, disturbance of mitochondrial function, and oxidative stress are involved in OTA mediated toxicity. The studies presented in this thesis aimed to counteract OTA cytotoxicity in HepG2 cells by various antioxidants such as vitamin E and dietary flavonoids, for example epigallocatechin gallate and quercetin. Furthermore, the effects of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO), modulators of intracellular glutathione (GSH) levels, on OTA cytotoxicity were investigated. Neither vitamin E nor flavonoids protected HepG2 cells from OTA induced cell death. This lack of protection by antioxidants might be due to an insufficient access of the antioxidants to the intracellular sites of reactive oxygen species (ROS) production and/or suggests a minor role for ROS in OTA mediated hepatotoxicity. In agreement, neither NAC nor BSO affected OTA induced cytotoxicity, suggesting that GSH may not be pivotally involved in the protection from acute OTA toxicity in HepG2 cells. A second aim of this thesis was to study the gene modulatory activity of OTA in HepG2 cells using Affymetrix GeneChip® technology. OTA reduced mRNA levels of genes encoding for proteins that are centrally involved in energy metabolism, such as phosphoglycerate kinase 1, isocitrate dehydrogenase 3 (NAD+) beta, and uncoupling protein 2. All mitochondrial genes affected by OTA were at least twofold down regulated. A particularly strong downregulation was observed for mRNA and protein levels of the insulin-like growth factor binding protein 1, which has been demonstrated to counteract p53 mediated apoptosis in mitochondria. Overall, the data presented in this thesis suggest that OTA may mediate its cytotoxicity in liver cells partly by lowering mRNA levels of genes crucial for energy homeostasis