Toxicity Study Of Spinetoram In Human Hepatoma HepG2 Cells

As one of the best-selling botanical pesticide,Spinetoram was through AA green food standard and widely used in comprehensive management of crop and storage pest and animal parasite control.With the emergence of Spinosad toxicity events,people with a suspected about the safety of Spinetoram,which is the derivative of Spinosad.According to the health risk assessment by the US Environmental Protection Agency(EPA),Spinetoram is mainly concentrated in a variety of animal models and the results considerd safety.However,there are less toxicity risk assessment was detected in cellular levels.In this study,human HepG2 cells were selected as in vitro cell models to evaluate the toxicity of Spinetoram to human hepatocellular cells.Cell viability and inhibit cell proliferation were detected by MTT and colony formation experiments.DNA damage was confirmed by Alkaline single-cell gel electrophoresis and yH2AX immunofluorescence experiments.The accumulation of reactive oxygen species in the mitochondria caused oxidative damage and induced the production of 8hydroxydeoxyguanosine.DNA damage repair protein PARP,which caused DNA repair to be blocked or repair failure was cleavage and the cleavage of PARP mainly comes from the activation of Caspase-3 protein during apoptosis,which also indicated that damaged cells may be repaired through the apoptotic pathway.Cell apoptosis was confirmed by Hoechst 33258 staining and flow-through Annexin-FITC/PI double staining.The opening of mitochondrial permeability transition pore,the decrease of mitochondrial membrane potential,the release of cytochrome C,the activation of caspase-3/9 and the decrease of Bcl-2/Bax value further confirmed that Spinetaram caused apoptosis through mitochondrial signal transduction pathway.It was confirmed Spinetram could induce autophagy of HepG2 cells by MDC staining,transmission electron microscopy and Ad-mCherry-GFP-LC3B transfection experiments.The occurrence of autophagy was further confirmed by the detection of the expressions of the autophagy marker proteins LC3,p62 and Beclin-1.The mitochondria was wrapped by autophago lysosomes through Lyso-Tracker Red and Mito-Tracker Green fluorescence double labeling method,and the decrease of intracellular ATP content indicated mitochondrial autophagy.Eventually,the phosphorylation level of the AMPK protein was increased,the phosphorylation level of the mTOR protein and the phosphorylation level of the mTOR downstream effector protein p70s6k were decreased.The above results demonstrated that Spinetarm caused autophagy in HepG2 cells and activated the AMPK/mTOR signaling pathway.