Study Of The Proliferative Effect Of Microcystin On Primary Rat Hepatocytes And The Possible Mechanisms Involved

In recent years, the phenomena of algal blooms occur frequently worldwide, bringing about serious threat to human health. Microcystin (MC), a class of biological toxicon and monocyclic heptapeptide compound, is produced by blue-green algae, and more than 80 kinds of isomers have been found. MC is stable and difficult to degrade, and cannot be completely eliminated from drinking water by applying conventional water treatment technology. In addition, MC can also enter the body through the food chain in the form of biological enrichment. The harm of MC to human health through drinking water or food has attracted widespread attention. The biologically toxic target organ of MC is liver. At present, it has been suggested that, MC's dualistic toxicity is dose-dependent, i.e., high dose of MC causes programmed cell death or apoptosis, while low dose of MC promotes cell proliferation or tumor formation. The hazards of MC were mainly from drinking water or food through the long-term low dose exposure, which was correlated with the high incidence of liver cancer showed by more and more epidemiological data. Therefore, MC's toxicity, especially its potential chronic tumor promoting toxicity has become one of the research focuses. Current studies on MC's toxic mechanisms are focused on its molecular apoptosis-inducing mechanism, but its tumor-promoting mechanisms are rare at home and abroad. Animal studies showed that, MC was a tumor-promoting agent rather than initiator, and uncontrolled cell proliferation was important in tumor formation, therefore, the study on the proliferation- promoting effect of MC is essential to clarify its mechanisms of tumor promotion.Our subject based on the current research status of MC and the problems, select the most toxic, fruitful and harmful MC-LR as our research object, and employ primary rat hepatocytes, on which the cell toxicity is studied from cellular and molecular biology perspective. Firstly, we systematically studied the specific hepatotoxicity characteristics of MC, and clearly understood the apoptosis-inducing and proliferation-promoting dualistic toxicity of MC-LR and its dose range; then based on that, we further analyzed the mechanism of the proliferation promotion of MC-LR, to provide experimental and theoretical basis for the illumination of the relationship between MC and high incidence of liver cancer and provide clues for the further study of the molecular mechanisms of tumor promotion simultaneously.Our study established primary rat hepatocytes model by collagenase perfusion method, then by monitoring the characteristic zymogram of liver damage-alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the culture medium and analyzing the characteristic surface marker of hepatocytes, confirmed that the integrity of the isolated hepatocytes was good and the purity was nearly 100%. So a suitable experimental model for research was fundamentally established for the subsequent observations of experimental phenomena and detections of experimental indicators.By exposure to 10-5～10-12 mol/L gradient of concentration of MC-LR, to observe its toxicity features during a longer time period (24 h-72 h), found that the dualistic toxicity of MC-LR on primary rat hepatocytes is dose-dependent, i.e.,μM level of MC-LR caused cytotoxic effect, manifested as the deterioration of cell morphology, the significant decrease of cell viability, and apparent increase of apoptosis rate, while nM～pM level of MC-LR promoted cell proliferation, manifested as the increase of cell viability, the gain of the proportion of S phase cells in cell cycle, and the increase of DNA synthesis rate.Further studies found that the phosphorylation status of MAPK family members, intracellular ROS levels and the antioxidant systems were related to the proliferative effect of MC-LR on primary rat hepatocytes. Specifically performed as:the phosphorylation status of MAPK family was closely related to cell proliferation of low dose (10-9～10-12 mol/L) MC-LR, i.e., in the active time (48 h or less), ERK1/2 phosphorylation activity was significantly increased, while JNK1/2 and p38 phosphorylation activity inhibited; after exposure to 10-9～10-12mol/L concentration of MC-LR, ROS generation was mildly increased, and there was a big overlap in the time interval (48 h or less) between the mild and repeated stimulation of low-level ROS and the proliferative effects of MC-LR, which is consistent with the results of Cell Cycle and MAPK.