The Role Of Oxidative Stress Induced By Antimony In The Representative Cells Of Respiratory System

ObjectiveAntimony is a kind of heavy metal and has been widely used in the manufacture of pigments,ceramics,fireworks,glass,flame retardants and brake pads.With the rapid development of mining,smelting and processing of Antimony industry,a large amount of Antimony has been released into the atmosphere,thus resulting increased occupational exposure of Antimony.Therefore,the health hazards of occupation exposure to antimony are getting more and more concerns.Generally,the Antimony in atmosphere environment mainly enters the body through the respiratory tract,which can cause irritation of the upper respiratory tract and lung injury.However,the current research mainly investigates its health effect on the respiratory system through epidemiological approach,and few studies have been conducted to reveal its cellular and molecular mechanism.In this thesis,we used Antimony Chloride(SbCl3)as a representative substance of Antimony to evaluate the effect of SbCl3 exposure on celluar injury in A549 cells and BEAS-2B cells,two typical cell models when investigating respiratory toxicology,and to explore the role and origin of oxidative stress in SbCl3-treated cells.The aim of this study was to elucidate the biological effect of SbCl3 and its underlying mechanism,thus providing a novel protective strategy for respiratory damage induced by Antimony exposure.MethodsFirstly,CCK-8 was used to measure cell viability,PI staining was combined with flow cytometry to analyze cell cycle,Annexin-V staining was combined with flow cytometry to detect cell apoptosis,DCFH-DA probe was incubated with cells to determine the ROS level.As the main sources of intracellular ROS are from the cytoplasmic NADPH oxidase(NOX)family or mitochondria,we furthermore adopted DHE probe incubated with cell to detect the cytoplasmic ROS and MitoSOX staining to detect mitochondrial ROS,as well as real-time quantitative PCR and Western blot to analyze the expression of related proteins/enzymes.Finally,the NADPH oxidase inhibitor DPI and the mitochondrial ROS scavenger Mito-TEMPO were pretreated with the cells and the recovery of the cell biological effects initated by Antimony was evaluated.Results1.The toxic effect of Antimony on A549 cells(1)CCK-8 analysis showed that SbCl3 significantly decreased A549 cells viability but not apoptosis after 24h treatment.PI staining and revealed that cell cycle of G2/M phase was significantly arrested.Moreover,Western bloting showed that the cell cycle related protein CyclinA、CyclinB and protein kinase CDK1、CDK2 were down-regulated.(2)DCFH-DA assay showed that SbCl3 increased A549 cells ROS level,while Anti-oxidant NAC pretreatment could significantly reduce cell cycle arrest and recovered cell viability.(3)DHE staining revealed that no significant change of A549 cells cytoplasmic ROS was found,quantitative PCR and Western blot showed that mRNA and protein levels of NOX1,NOX2,NOX5 were not significantly,and cell viability did not show significant recovery after NADPH oxidase inhibitor DPI treated;Meanwhile,MitoSOX staining showed mitochondrial ROS level was increased,and mitochondrial ROS specific scavenger Mito-TEMPO could reduce cell cycle arrest and rescue cell cycle related protein CyclinA、CyclinB and protein kinase CDK1、CDK2 expression as well as cell viability.2.The toxic effect of Antimony on A549 cells(1)CCK-8 analysis showed that the SbCl3 decreased BEAS-2B cell viability after 24h treatment and increased cell apoptosis rate significantly.(2)DCFH-DA assay showed that SbCl3 caused increase of BEAS-2B cell ROS level,and NAC pretreatment could significantly reduce cell apoptosis rate and recover cell viability.(3)DHE staining revealed that BEAS-2B cell cytoplasmic ROS was increased,and quantitative PCR and Western blot revealed that mRNA and protein levels of NOX1,NOX2,NOX5 were up-regulated.DPI treatment partly recovered cell viability;Meanwhile,MitoSOX staining showed mitochondrial mtROS level was also increased.Mito-TEMPO treatment partly recovered cell viability.DPI and Mito-TEMPO co-treated could significantly reduce BEAS-2B cell apoptosis rate and rescue cell viability.Conclusions(1)Antimony exposure induces ROS generation in A549 cells,which causes cell cycle arrest in G2/M phase and cell viability decreation.(2)Mitochondrial ROS scavenger can significantly reduce Antimony-induced A549 cell cycle arrest and recover cell viability,while NADPH oxidase inhibitor has failed to recover this effect,suggesting that mitochondrial ROS may play a crucial role in the injury of A549 cells induced by antimony.(3)Antimony exposure induces ROS generation in BEAS-2B cells,decreases cell viability and induces cell apoptosis.(4)Mitochondrial ROS scavenger and NADPH oxidase inhibitor pretreatment can inhibit BEAS-2B cell injury caused by Antimony exposure,suggesting that both mitochondria and NADPH oxidase are main sources of oxidative stress initated by Antimony.In conclusion,oxidative stress is one of the important mechanisms of Antimony toxicity.The intervention of different sources of oxidative stress and regulation of intracellular ROS generation can help to protect respiratory system from Antimony-induced adverse effect.