Toxicity Effect Of Silver Nanoparticles On SMMC-7721 Cells And Preliminary Investigation Of Its Mechanism

With the increasing number of applications for commercial silver nanoparticles?AgNPs?products,the risk for human and ennvironment exposure to AgNPs are also increasing.However,there was no accurate conclusion about the biological safety of AgNPs now.In this work,SMMC-7721 cells were used as the research model,and the traditional toxicological methods were used to study the biotoxic effects of AgNPs on cells based on AgNO₃ as a positive control.To further study the perturbation of AgNPs on intracellular metabolites and related metabolic pathways from the perspective of metabolomics,and analyzed their effects on cell biological activity and function.The purpose is to reveal the potential mechanism of AgNPs cytoxic effects,which is of great significance for the application and safety evaluation of metal nanomaterials.First,the morphology of AgNPs were observed by transmission electron microscopy,the particle sizes were identified by nanometer particle size analyzer,the stability and dispersibility of AgNPs,were characterized by ultraviolet-visible spectrophotometer.The characterization results showed that the particle sizes of AgNPs were relatively uniform,with an average particle size of 22.49±4.73 nm,and the particle shapes were approximately spherical,which can be stably dispersed in DMEM medium.Secondly,the cellar viability was measured in SMMC-7721 cells by the MTT assay,the results showed that decreased viability in SMMC-7721 cells in response to AgNPs and AgNO₃ exposure was dose-dependent.After 24 h of exposure to AgNPs and AgNO₃,the IC₅₀values for cells were 170.88±18.5 mg/L and 30.43±4.33 mg/L by SPSS calculation,respectively.Morphological observations indicated dose-related alterations in the exposed SMMC-7721 cells.Cells in the negative control group showed normal shuttle shapes and they grew by adhering to the wall of culture plate.After 24 h of exposure to AgNPs and AgNO₃,the cells became round,cytoplasm concentrated and detached from the culture plate.Under the action of high concentration of AgNPs,there were black particles on the surface and inside of the cells.In addition,the effects of AgNPs and AgNO₃ on oxidative stress-related indicators?GSH,SOD,CAT?,cell cycle and apoptosis were studied.The measurement results showed that the exposure of AgNPs and AgNO₃ would induced oxidative stress in the cells,resulting in the decrease of intracellular antioxidant enzymes and GSH.The effects of the two materials on cell cycle and apoptosis were detected by PI staining method and Annexin V/PI double staining method using flow cytometry.The results showed that AgNPs and AgNO₃exposed to SMMC-7721 cells can caused cell G₂/M phase arrest,early apoptosis of cells,and there was a certain dose-dependent.From which we can seen that AgNPs and AgNO₃ had similar toxic effects on SMMC-7721 cells,and it was speculated that the biological toxicity of AgNPs may be related to Ag⁺.Finally,using the non-targeted metabolomics technology based on LC-MS,the differentially expressed endogenous metabolites were determined and related metabolic pathways were analyzed in SMMC-7721 cells exposed to AgNPs and AgNO₃.Metabolomics data showed that AgNPs and AgNO₃ can caused abnormal metabolism of nicotinamide adenine dinucleotide,taurine,phosphatidylcholine,and 5-phosphate ribose.In addition,AgNPs can caused abnormal metabolism of glucuronic acid,N-acetyl sphingosine,and galactose sulfate ceramide.AgNO₃ can specifically affected the metabolism of?R?-4'-phosphate-L-cysteamide and arachidonic acid when it acted on cells.Metabolic pathway analysis found that AgNPs and AgNO₃ both affected taurine and low taurine metabolism,niacin and nicotinamide metabolism,and pentose phosphate metabolism pathways,and most of these pathways were involved in energy metabolism and oxidative stress in cells.In addition,AgNPs would also disrupted the metabolism of sphingolipids and pathway of pentose and glucuronate conversion.AgNO₃ would affected the biosynthesis of pantothenic acid and CoA,arachidonic acid metabolism,which might be related to the cytotoxicity difference caused by AgNO₃ and AgNPs.Combining the results of traditional cytotoxicity and metabolomics,it could be speculated that the mechanism of AgNPs toxicity to cells is mainly generated through oxidative damage and related pathways,and Ag⁺dissociated by AgNPs particles might play a major role in it.