Chromatographic analysis of Microcystis aeruginosa toxic peptides and evaluation of effects on epithelial cells

Due to the increase in worlds' population, especially safety and quality of drinking water resources have become very important in recent years worldwide. The entity of cyanobacteria in water, and identification of their toxic components have become primary research subjects, since these data must be achieved to avoid their toxic or fatal effects on human and all living organisms. Furthermore, toxic substances and their mechanisms of action must be fully elucidated to develop efficient strategies for the prevention or treatment of pathological processes arising from cyanobacterial contamination. Studies with Microcystis aeruginosa, a cyanobacterium living in almost all freshwater sources in all over the World, have revealed that this species has higher toxicity than other algae species. This toxicity threatens the lives of all living beings, especially humans and animals. The toxic components enter the plant circulation system through the absorption by the plants during the irrigation, and accordingly participate to food chain by not only the use of contaminated water, but also the consumption of the plants irrigated with this water. Many peptides with high hepatotoxic activity have been described in Microcystis aeruginosa. While these toxic peptides are generally retained in the cell, they are also released from the cell due to cell lysis, or by active transport systems. Dietary toxic peptides are transported to the liver by organic anion transport proteins and inhibit protein phosphatase 1 and protein phosfatase 2A enzymes, resulting in an increase of intracellular phosphoprotein, and associated intrahepatic bleeding, cell necrosis and tumor formation in the liver. The aim of this study was to evaluate the cytotoxic effects of Microcystis aeruginosa extract, which contains toxic peptides and proteins, and its chromatographically separated fractions on human epithelial cells. In the first step of the study, total extract was prepared from the Microcystis aeruginosa cells in the culture. The extract was then separated into 8 fractions by high performance liquid chromatography (HPLC) method. Different concentrations of total extract and fractions introduced to ECV304 cells on mid-log phase for 48 hours, and their concentration-dependent effects on cell viability were statistically evaluated. In addition, the molecular weights of the polypeptides in total extract and each fraction were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Total algal extract showed the highest toxic effect (~70%), as expected. Effects of fractions on cell proliferation varied. Some fractions (1, 2, 4, 5 and 6 no.) possessed cytotoxic activity (55%, 40%, 40%, 35% and 30%, respectively) while especially lower concentrations of some others (3, 7 and 8 no.), on the contrary, exerted proliferative effect (80%-5%). This thesis is the first study reporting the separation of toxic peptides in Microcystis aeruginosa extract by HPLC and the individual cytotoxic effect of each fraction. Since the highest cytotoxic activity on the epithelial cells was detected in total algal extract, the polypeptides found in different fractions seem to contribute to the cytotoxicity synergistically. Electrophoretic analyses revealed that total extract and fractions contained common or different polypeptides in a wide range of molecular weight (5-300 kDa). These results are important for the detection of toxic molecules targetted in the prevention and treatment of Microcystis aeruginosa-induced toxicity. The HPLC procedure optimized in this study may be a guide for future studies to analyze the toxic peptides of Microcystis aeruginosa and other algae. In future studies, the use of total proteins that are precipitated from total algal extract along with reference toxic peptides rather than this extract itself may produce better results for the identification of toxic peptides in Microcystis aeruginosa. Furthermore, peptides-specific 1- and 2-dimensional PAGE systems might be used to visualize the toxic peptides in the fractions.