| Nanomaterial,which is a kind of novel material with wide application,developed rapidly in recent years and appeared in our daily life more often than before.In which zinc oxide nanoparticle is one of nanomaterials people engaged most in their life due to its wide application,well-established production process and success in commercialization.Evaluation of the impact of zinc oxide nanoparticles on cellular proteome using proteomic method could help us understand the overall kinetic changes in cellular response to metal oxide nanoparticles and is of great significance in studying the mechanism of cellular homeostasis and nanoparticle toxicity.In this work,A549 cell line was used as cell model.CCK-8 method and flow cytometry was used to determine the cytotoxicity of zinc oxide nanoparticle at different concentration.Then we investigated the cellular proteome response to zinc oxide nanoparticles at sub-cytotoxic concentration after its exposure to cells after different period of time using iTRAQ,which is a high through-put proteomic method.Western blot was applied to further study proteins we interested in.Intracellular oxidative stress was evaluated through determination of intracellular ROS level using fluorescent probe DCFH-DA and detection of glutathione/oxidized glutathione.In the cell viability test,we measured the cytotoxicity of zinc oxide nanoparticles at different concentration and compared it with the cytotoxicity of zinc sulfate.As a result,we found that though cytotoxicity of zinc oxide nanoparticles seemed to have similarity to zinc ions,it differed from cytotoxicity of zinc ions significantly.Through measurement of cellular proteome using iTRAQ,much more differentially expressed proteins were observed after cells being treated with zinc oxide nanoparticles for 9 hours than 24 hours.Also,most of these proteins expressed in the pattern that showed a significant decrease after exposure to zinc oxide nanoparticles and then increased at 24 hours,which reveals the importance of exposure time in zinc oxide nanoparticles toxicity mechanism studies.Further bioinformatics analyses were conducted in order to understand the signal transduction in cells stimulated by zinc oxide nanoparticles.Intracellular ROS and glutathione determination showed that high level of oxidative stress was presented in cell after treatment with zinc oxide nanoparticles for 9 hours,and thus can lead to the significance of particle toxicity in zinc oxide nanoparticles toxicity mechanism.It can be observed from western blot analysis that the expression of NF-?B p65 and PNPase both rose significantly after 9 hours of exposure,which further proved the presence of particle toxicity,and the elevation after 9 hours of exposure and then reduction of expression of HSP90 to untreated level after 24 hours of exposure revealed that the cells had made response to the exogenous stimulation and managed to maintain the intracellular homeostasis.According to the experiment results,a deduction was made that toxicity of nanoparticles consists both of particle toxicity and ion toxicity.When exposed to nanoparticles at low concentration,cells will suffer from a complex toxicity from both particles and ions in a short period.Cells will soon make response and the expression of proteins will be regulated to engage the stimuli,and the pressure induced by particles will first be relieved or eliminated,while toxicity caused by ions dissolved from nanoparticle can cost more time to suppress.As a result,long time treatment may conceal the toxicity induced by particles and in turn highlight the toxicity of metal ions.The result we made reveal the importance of exposure time in the study of nanoparticles toxicity and would provide a new perspective for studying toxicity mechanism of nanoparticles. |
PDF Full Text Request