The Mechanisms Of Multi-Locus Deletion Mutation Induced By Silver

With the breakthrough of nanotechnology,silver shows unique physical,chemical,and biochemical properties at the nanometer scale.Inevitably,the growing application of AgNPs has caused the releasement of the particles in the environment and the released AgNPs greatly increased the potential risk of occupational and public exposure.For the concern of environmental safety and human health,the in-depth study on the mechanisms of AgNP toxicity and the evaluation of their potential risk are two main issues to be addressed urgently at present.The main purpose of our study is to explore the mechanism of multi-locus deletion mutation induced by different sizes of AgNPs at the subcelluar level of human-hamster hybrid(AL)cells.First,the effects of different internalization pathways of AgNPs on genotoxicity were explored.The transmission electron microscope(TEM)combined with cell section technique were used to study the distribution of AgNPs with different particle size in cells.And the results showed that the 5nm AgNPs entered the cell mainly through the liquid phase,the 25nm AgNPs were internalized mainly through the endocytosis mediated by caveolin protein,and the 75nm AgNPs were mainly through energy-dependent endocytosis.Through the detection of micronucleus formation rates sand CD59 gene mutation rates,we found that there was a size negative and time-dose positive manner in genotoxicity induced by AgNPs.And the CD59 gene mutation spectrum indicated that AgNPs could induce multi-locus deletion mutation.Second,to explore the effects of AgNPs on the structure and function of lysosome and mitochondria,and to study the contribution of lysosome and mitochondrial dysfunction to AgNPs-induced gene deletion mutation.It was found that AgNPs induced size-dependent damage to the structure and function of mitochondria and lysosomes.Using ROS scavengers N-Acetyl-L-cysteine(NAC)and Mito-TEMPO could reduce the genotoxicity of 5nm particle size AgNPs and eliminate the genotoxicity of 25nm size AgNPs.On the other hand,the weak response of mitochondrial DNA deletion-ρ0AL cells to AgNP-induced CD59 gene mutation and micronucleus formation revealed the important role of mitochondria in AgNPs-induced genotoxicity.At the same time,we found that,small particle size AgNPs could cause structural damage of lysosome osmotic expansion and stress response.on the other hand,it could affect the function of proton pump H+-ATPase on lysosome membrane,induce the functional damage of lysosomal alkalization and decrease of internal enzyme activity;and the re-acidification of lysosome could reduce the genotoxicity induced by AgNPs to some extent.Finally,at the level of autophagy,it was found that although AgNPs could induce particle size-dependent autophagy,5nm particle size AgNPs induced autophagy flux blocking and autophagy dysfunction by inhibiting the fusion of autophagosomes and lysosomes;and the reactivation of autophagy reduced the genotoxicity induced by 5nm particle size AgNPs.In summary,the internalization pathway of AgNPs,the interaction between mitochondria and lysosomes and autophagy are all involved in AgNP-induced gene mutations.On the one hand,AgNPs induces particle size-dependent lysosome and mitochondrial damage;on the other hand,autophagy induced by AgNPs can not remove the accumulation of damaged organelles and ROS,which aggravates DNA damage,chromosome aberration and CD59 gene mutation caused by AgNPs.This study revealed the mechanism of AgNPs-induced multi-locus deletion mutation,and our data provide clear evidence to illustrate the role of subcellular targets in the genotoxicity of AgNPs in mammalian cells,which laid the basis for better understanding the health risk of AgNPs and their related products.