Cytotoxicity And Molecular Mechanism Of Avermectin

Avermectin(AVM)has been widely used in agriculture and animal husbandry based on its broad spectrum of effective anthelmintic activity and specificity targets.In recent years,a larger number of studies showed non-target organisms have adverse reactions to AVM in environment,whereas the cytotoxic effects and molecular mechanism of AVM on human cells have not been well characterized.In this study,both the cytotoxic effects and molecular mechanism of AVM on insect Sf9 cells,human HeLa and HepG2 cells in vitro.We clarify that AVM inhibited the viability of Sf9,HeLa,HepG2 cells.We have used alkaline comet assay and yH2AX foci formation to detect DNA damage.As expected,we found AVM caused DNA double-strand breaks in HeLa cells,as measured by significance of comet assay parameters(e.g.,tail DNA)and increases of yH2AX foci in HeLa cells.Moreover,established assays of cytotoxicity were performed to characterize the mechanism of AVM toxicity on insect Sf9 and human HeLa,HepG2 cells.We demonstrated that AVM-induced DNA damage of cells were mediated by generated ROS.In the case of AVM-induced DNA damage,it was found that the damaged base of the DNA chain was associated with purine or formamide pyrimidine;8-oxodGs were generated and accumulated during the process of DNA damage,and they caused the change in spatial structure of DNA strand,potentially causing mutations and inducing carcinogenesis.Avermectin caused DNA repair protein PARP cleaved into two polypeptides,in which 24 kD polypeptide sustainedly bind with DNA.The binding would result in DNA being unable to be repaired and cause cell death.PARP cleavage is mainly derived from the activation of caspase3 during apoptosis,which indicates that AVM causes DNA damage and then initiate inhibition of cell proliferation or cause cell death through apoptosis.AVM was proved to be able to induce apoptosis by Hoechst staining,AO/EB staining,Annexin V-FITC/PI double staining and flow cytometry.The results demonstrated the collapse of mitochondrial membrane potential,and up-regulating the expression level of Bax/Bcl-2 resulted in a release of cytochrome c into cytosol as well as the activation of caspase-9/-3.After caspase 3 activity was inhibited by caspase-specific inhibitor,the rate of apoptosis was found decreased,indicating that AVM-induced apoptosis is a caspase-dependent pathway.Using the transmission electron microscopy and the fluorescence staining,a typical autophagic bubble structure was observed in the cells induced by AVM,which was confirmed by further studies on the autophagy marker proteins LC3?/?,Beclinl,p62.Through the detection of ATP content and the phosphorylation of mTOR,AMPK and p70s6k in autophagy signal transduction pathway,it was found that through injuring mitochondria AVM could reduce the ATP content to induceAMPK/MTOR-mediated autophagy signaling pathway,which in turn affects the growth,proliferation and survival of cells by influencing the downstream phosphorylation level of p70s6k.Therefore,study on toxic effect and mechanism of avermectin to insect and human cells will supply important clues for working out their toxicological effects and potential risks to human health,and will also provide a deep-level effective evaluation strategy for increasingly popular pesticide safety issues.