Study Of The Involved Molecular Mechanisms Of Microcystin Induced Proliferation On Primary Rat Hepatocytes

With the global epidemic of algal blooms, the chance of human exposure to microcystins was greatly increase, also, algae toxins studies have attracted more and more attention. Microcystin (MC), a class of biological toxicon and monocyclic heptapeptide compound, is produced by blue-green algae, and more than90kinds of isomers have been found. A large number of studies have shown that drinking water contaminated with low concentrations of MC is related with high incidence of tumors, such as primary liver cancer, colorectal cancer and gastrointestinal tumors. At present, it has been suggested that MC dualistic toxicity is dose-dependent, high dose of MC cause programmed cell death or apoptosis,while low dose of MC promotes cell proliferation or tumor formation. The hazard of MC were mainly from drinking water or food through the long-term low dose expose, 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 toxic mechanism are focused on. Animal studies showed that, MC was a tumor-promoting agent rather than initiator, and uncontrolled cell proliferation was important in tumor formation, there, the study on proliferation-promoting effect of MC is essential to clarify its mechanisms of tumor promotion. Earlier study showed (1×10-5～1×10-6mol/L) of MC-LR cause cytotoxic effect, manifested as deterioration of cell morphology, while(1×10～91×10-12mol/L) 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 in crease of DNA synthesis rate. Based on this, this study continue using the primary rat hepatocyte as the research model, further explore cell proliferation effect of low doses of MC-LR, in earlier and more time points observe the effect of MC induce cell proliferation and the possible molecular mechanism, from the Angle of cell biology and molecular biology to explore the changes of cell cycle and cyclins and MAPK signal pathway witch closely related to cell proliferation when primary rat hepatocyte cell expose to low-dose MC. Provide experimental basis and theoretical basis for clarifying the MC on the relationship between the high incidence of liver cancer.Study the effect of primary rat hepatocye expose to (1×10-9～1×10112mol/L) of MC from3h to24h,we found that the cell proliferation occour to12h and the effect most obvious in1×10-11mol/L group,shown as cell live rate increase and rising rates of cell cycle in S phase cells.In order to further research the mechanism of cell proliferation-induce of MC-LR, we observe the expression of target molecules from3h to24h. Expose to MC-LR, it can be found that both cyclin D1and cyclin E have increased expression in their mRNA and protein level in3h-6h. cyclin A have high level in6h-12h. The high level of Phosphorylation activity of MAPK family is also observed in3h-6h. Additionally, the subunits of MAPK downstream transcription factor(AP-1) c-fos enhance their activity in all mRNA, protein and phosphorylation activity level in3h-6h, the c-fos and its phosphorylation high level keep Till12h, while the mRNA level is inconsistent with them, shows a downward changes. In24h group all the change is invisible. The enhance of c-jun protein and its phosphorylation activity continuous to and the mRNA level can be found3h-24h. All various molecular effect the most apparent in1×10-10or1×10-11mol/L dose group, and overlap with cell proliferation effect on dose groups, and before cell proliferation. Experimental result indicates high level of cyclin expression, MAPK family protein phosphorylation activity and AP-1activity may be involve of the effect of induce cell proliferation of MC-LR.