The investigation of the clotrimazole induced apoptosis mechanism in saccharomyces cerevisiae

Clotrimazole is an antifungal drug whose potential antineoplastic usage has been taken into consideration recently. In the scope of this thesis, it was primarily aimed to highlight the cytotoxic and apoptotic effects of clotrimazole on eukaryotic model Saccharomyces cerevisiae BY4742 yeast cells. This organism has been gradually become a preferred and popular research tool, especially in exploring human disease over the years because it can be easily handled and genetically modified. To examine cytotoxic effect of clotrimazole on yeast cells, FUN-1 viability test was used. The apoptotic character of death was determined by Annexin V – PI and DAPI - TUNEL double stainings which indicate phosphatidylserine externalization and DNA fragmentation, respectively. The variations in mitochondrial membrane potentials in BY4742 cells treated with clotrimazole were investigated in a concentration and time dependent manner. The dependency of clotrimazole induced apoptotic death to certain mitochondrial proteins including yeast caspase, apoptosis inducing factor, endonuclease G and HtrA-like serine protease Nma111p was highlighted with the use of knockout mutant strains. Oxidant and antioxidant status together with the resulting damage in S. cerevisiae BY4742 yeast cells exposed to clotrimazole were researched. For this aim, the variations in intracellular reactive nitrogen and reactive oxygen levels and the activities of NADH and NADPH oxidases were measured. Among antioxidant defense system, the activities of superoxide dismutase, catalase, and glutathione related enzymes and the levels of reduced – oxidized glutathione were determined. Finally, as markers of clotrimazole induced oxidative stress conditions, malondialdehyde and protein carbonyl levels were investigated. These findings can be summarized as clotrimazole induced apoptotic cell death in yeast mainly depends on metacaspase acivity. On the other hand, although significant inductions have been observed in antioxidant defence system, it could not protect cells from clotrimazole based stress conditions.