Study On The Potential Toxicity And Molecular Mechanism Of Tebufenozide On Human Cells

Tebufenozide,a new kind of non-steroidal insect growth regulator,is one of the most widely used insecticides in agriculture.It is known that it has the characteristics of high efficiency,specific target and environmental friendliness and has no teratogenic and mutagenic effects on mammals.It is effective to control Lepidoptera pests by interfering the production of natural ecdysone leading to early maturity and incomplete molting,finally reaching the effect of pest control.In recent years,food safety incidents caused by pesticide pollution have been frequently reported.Although each pesticide variety was subjected to rigorous toxicity assessment before listed,it still can not completely eliminate the serious threat to human health.Therefore,the re-evaluation of pesticide safety is in line with the people's strong concern for food safety.At present,there are few reports on the toxicity of tebufenozide to human cells.In this study,human HeLa cells were used as test models which were widely used in the study of cellular toxicology to research the potential toxicity of tebufenozide.In this study,we systematically investigated the mechanisms of DNA damage,cell cycle arrest and apoptosis,using cell biology and modern molecular biology techniques.The clone formation experiment showed that the proliferation of HeLa cells inhibited by tebufenozide in a concentration-dependent manner.The Immunofluorescence assay(IFA)showed that tebufenozide induced HeLa nucleus to form yH2AX foci in a dose-dependent manner.It is suggested that tebufenozide triggers DNA double-strand breaks of HeLa cells.The analysis of cell cycle and agarose gel electrophoresis indicated that there was G1/S cycle arrest in HeLa cells induced by tebufenozide.Moreover,the activity of Cyclin E and CDK 2 was inhibited by activating p21 gene,and the cell cycle could not change from G1 phase to S phase.Besides,western blotting assay showed that the expression of p53 and Bax were up-regulated with the down-regulation of Bcl-2 and PARP was cleaved.There is evidence that p53-mediated apoptosis controls mitochondrial pathway by regulating Bax/Bcl-2 protein with the inhibition of DNA damage repair,resulting in apoptosis.And the apoptosis of HeLa cells induced by tebufenozide could cause the damage of mitochondrial structure and function including the change of mitochondrial permeability,decrease of mitochondrial number and ATP content.Finally,the toxicity test of mice indicated that tebufenozide triggered the damage of liver and kidney of mice and apoptosis with the generation of oxidative damage.The results of this study indicate that tebufenozide has potential toxic effects on human cells.It induces cell cycle arrest and apoptosis by activating p53 to mediate mitochondrial pathway regulated by Bax/Bcl-2 proteins.However,the present experimental data can not confirm whether there are other toxic mechanisms.