In vitro cell-based cytotoxicity studies in toxicology

During the drug discovery and development studies, one of the conditions leading to great time, cost and labor loss is to figure out that the toxicity of the candidate compound in the later stages of the process, and that all work done so far has been taken off the edge. For this reason, the pharmaceutical industry and regulatory agencies express that in vitro rapid screening tests to be conducted in the early stages of drug discovery and development studies and the investigation of the cytotoxicity of substances will prevent, in part, these losses. Based on this information and suggestion, in this thesis project, probable cytotoxic effects of two flavonoids (apigenin) and biflavonoid (amentoflavon) were investigated by 3 different methods in Chinese hamster over (CHO) cells which are healthy cell lines. These; "Trypan Blue Experiment" based on the staining of dead cells and counting under the microscope, "MTT Experiment", which is indicative of the metabolic activity of the cell, and Lactate Dehydrogenase Enzyme (LDH) Extraction Test, as a measure of membrane damage / integrity. Firstly, partial validations of the methods were carried out in the presence of a known cytotoxic reference substance. Then the cytotoxic potentials of the materials were investigated by all three methods. One of these substances; amentoflavone, has been studied in recent years to have some beneficial biological effects, but there is no data on the toxicity potential of the substance. Therefore, our aim in this study is to elucidate possible cytotoxic effects of amentoflavone and to elucidate the mechanism of the possible cytotoxic effect of amentoflavone with these tests, which are determined by three different mechanisms. Our findings show that the trypan blue assay has high standard deviations and the MTT test results are more accurate in all three methods. Amentoflavone' s cytotoxicity was found the same in trypan blue, LDH and MTT assays and we suggests that this substance causes cell death by cell membrane damage at 100 μM concentration. No correlation was observed in the trypan blue and LDH assays used to determine cell viability due to membrane damage of apigenin, but the cytotoxic concentration in the MTT assay was found to be 33.3 μM. Therefore, the cytotoxic effect of apigenin is thought to be caused by mitochondrial damage. In addition, MTT assay has been shown to be an effective method for early detection of cell damage because, it is based on mitochondrial enzyme activity in determining cytotoxicity.