The Role And Mechanism Of Oxidative Damage In Cytotoxicity Induced By Mustard Gas In Human Skin

Mustard gas,with its chemical name sulfur dichloride diethyl ether,belongs to erosive chemical warfare agent.Pure sulfur mustard was synthesized by Germany Meyer for the first time in 1886.Sulfur mustard was large-scale used in the First World War,the Second World War and the Iran-Iraq war.As a result,it is one of the most effective chemical warfare agents tested by war and called "the king of toxic agent".Mustard gas is also the main component of Japanese ACWs and the crime culprit of Qiqihar gas event occurred on August 4,2003.Mustard gas is characterized by simplicity of synthesis process,effortlessness of recovery when in wartime and the memace to terrorist event.However,sulfur mustard is difficult to prevent and cure,and the current clinical treatment is limited to symptomatic treatment putting SM skin poisoning as burns.There is no specific and effective detoxification method.The skin is both the primary toxic target organ and the first barrier to the body defense system.It is divided into epidermis and dermis,mainly composed with keratinocytes and fibroblasts respectively.Oxidative stress is considered to be one of the starting and key eps of SM toxicity with its mechanism not clear yet.Based on the above views,it is considered theoretical significant and practical valuable that establish the in vitro model to study the toxic characteristics and the toxic mechanism of SM on the skin cells.Overview The chapter1 preface was divided into three parts,namely,mustard gas introduction,mustard gas research progress of oxidative stress mechanism in domestic and overseas and the purpose,content,innovation of this project.Firstly,we introduced the historical background,physicochemical properties,biotoxicity and toxicity mechanism of mustard gas.Secondly,we introduced generation of oxidative stress and antioxidant system.Finally,the purpose,content,innovation of this paper was described.The chapter2 was about the study on the toxic effects and oxidative stress mechanism of SM to human foreskin fibroblasts.The chapter3 was about the study on the toxic effects and oxidative stress mechanism of SM to hacat cells.The experimental design ideas were described in chapter 3 foreword section,including the selection of oxidative stress indicators and introduction of antioxidant pathway and oxidative stress-related apoptotic signaling pathway.Finally,scientific problems were put forward.Objective This study attended to study the toxic characteristics and oxidative stress mechanism of the mustard gas base on the humanized in vitro model.Finally,we explored the role of oxidative stress in the skin toxicity of mustard gas any futher.Methods HFFs were separated from skin by trypsin digestion method.In this way,we obtaind pure and stabled HFFs and built the fibroblasts model.The proliferation inhibition effect of SM on the HFFs was detected by CCK-8 assay.The content of reactive oxygen species in cells were detected by DCFH-DA fluorescent probe staining method.The enzyme activity of SOD in the cells was tested by WST-8 method.MDA kit was used to mesure the content of MDA in HFFs.The dose-response relationship of apoptotic rate was obtained through Annexin-PI fluorescence staining method.The expression of Nrf2,caspase-3,cytochrome C and caspase-9 were measured by Western Blot assay.Results The proliferation of HFFs was inhibited in a dose-dependent manner after treatment with sulphur mustard for 24h(IC50 value=315?M).The intracellular ROS level was significantly higher than that of the control group.The ROS level induced by SM were increased in a dose-dependent manner.The activity of SOD decreased with the increase of the concentration of mustard gas.The content of MDA in the cells increased with the concentration of mustard gas.And the apoptotic rate of fibroblasts increased with the increase of the concentration of mustard gas.After treatment with ROS scavenger NAC,the cell viability of cells was significantly higher than that of the SM treatment group.The proliferation of hacat cells was inhibited in a dose-dependent manner after treatment with sulphur mustard for 24h(IC50 value=112?M).The intracellular ROS level was significantly higher than that of the control group.The ROS level induced by SM were increased in a dose-dependent manner.The enzyme activity of SOD increased with the increase of the concentration of mustard gas.The content of MDA in the hacat cells increased with the concentration of mustard gas.And the apoptotic rate of hacat cells increased with the increase of the concentration of mustard gas.The expression of Nrf2 in the hacat cells increased at the concentration of 25?M and 100?M.The expression of Caspase-3 in the hacat cells decreased with the increasing concentration of mustard gas.The intracellular Cleaved Caspase-3 appeared at the concentration of 25?M and 100?M,and cytochrome C increased significantly at the highest concentration.Conclusions SM had significant cytotoxic effect on fibroblasts in dose-dependent manner.Different concentrations of mustard gas induced oxidative stress of fibroblasts,and the antioxidant capacity of cells was weakened.As a result,lipid peroxidation occurred.The degree of oxidative damage was positively correlated with the concentration of mustard gas.SM could induce the apoptosis of fibroblasts.NAC could reduce the cytotoxic effect of SM.SM had significant cytotoxic effect on hacat cells in dose-dependent manner.Different concentrations of mustard gas induced oxidative stress of hacat cells followed by lipid peroxidation.The degree of oxidative damage was positively correlated with the concentration of SM.SM could induce the apoptosis of hacat cells.The Nrf2 pathway was involved in the anti-oxidation process of the hacat cells against SM.SM induced cell apoptosis through mitochondria pathway.