Research On DNA Damage Effects Of Sulfur Mustard And Its Metabolites In Skin Cells

Sulfur mustard(SM)is a typical erosive chemical agent.Since the first use in Germany in 1917,the armies of different countries have followed suit,SM caused serious casualties in several wars.Exposure to SM can cause acute toxicity to the skin,eyes and respiratory system because of its highly chemical reactivty.Due to the larger area,skin tissue is the primary target and important effect organ of SM,and percutaneous exposure is the most common route of exposure to SM.Since its first synthesis,the research on the mechanism of SM has lasted over one century,and there are many hypotheses such as DNA damage,PARP activation,inflammatory reaction,glutathione depletion,proteolytic enzyme activation,apoptosis and calcium homeostasis.But DNA alkylation damage is generally considered to be the basis of biotoxicity and genotoxicity of SM.According to the report,a large number of SMO and other metabolites will produced after SM exposure,and SMO will be oxidized to produce SMO2,which is not stable and further turn into highly toxic divinyl sulfone(DVS).So what is the toxic effect of the large number of sulfone metabolites,and whether can lead to DNA damage is currently unclear.Objective: To study the cytotoxicity and DNA damage effects of SM and DVS by using Haca T cells as a toxicity evaluation model.The aim of this study is to compare the toxicity differences of SM and DVS,and to reveal the mode of DNA damage,providing experimental basis to elucidate the toxicological mechanism of SM.Methods: SM was prepared at a rate of 6.25?M,12.5?M,25?M,50?M,100?M,200?M,300?M,400?M,DVS was prepared into 3.125?M,6.25?M,12.5?M,25?M,50?M,100?M,200?M,400?M.The CCK-8 kit and LDH kit were used to detect the survival rate and LDH leakage rate of Haca T cells treated with SM and DVS for 24 hours respectively.The morphological changes of Haca T cells exposed to SM and DVS for 24 hours were observed by inverted microscopy.Apoptosis rate of cells exposed to SM and DVS respectively for 24 hours was detected by Annexin V-FITC Apoptosis Detection Kit.The DNA damage of cells exposed to SM and DVS respectively for 24 hours was evaluated by single cell gel electrophoresis.Fluorescence quantitative RT-PCR was used to detect the expression of DNA damage repair genes RAD51?XPC?XPD?AAG?APEX1?MGMT?ABH3?KU70 and KU80 of cells which exposed to SM and DVS for 6 hours,12 hours and 24 hours respectively;The expression of DNA damage repair related proteins ?H2AX,PARP,p53 and phosphorylated p53 of cells exposed to SM and DVS respectively for 24 hours was detected by western blot.Results and discussion: The results of the cytotoxicity test showed that different concentrations of SM inhibited the proliferation of Haca T cells in a dose-dependent manner,and the half inhibitory concentration IC50 value was 121 ?M;LDH release is an important indicator of cell membrane integrity.The LDH leakage rate of cells treated with SM increased first and then decreased.With the increase of the dose,the damage of the cells was more and more serious,leading to the increase of LDH leakage rate.When more than a certain dose,a large number of cell death,LDH leakage rate would decreased gradually.It was found that the number of floating cells increased gradually with the increase of the dose,and the results were consistent with the toxicity test results of CCK-8.The results of cytotoxicity of DVS showed that the cell viability decreased gradually in a dose-dependent manner with the increase of the dose in the range of 0~25?M.However,at 25?M to 50?M,the cell viability was significantly increased,and then decreased gradually in the dose range of 50-400?M.Observation of cell morphology found that in the 0~25?M dose range,with the exposure concentration increases,the cells become smaller,and the dead cells float in the culture medium;but in the range of 50~400?M,microscopic observation found in the culture medium almost no floating dead cells,cells become rounded,and have obvious vacuoles.LDH leakage rate also increased first and then decreased,and finally increased again.The reasons of this phenomena may be: 1.DVS as a sulfone compound,at a certain dose,has a similar effect as DMSO that can maintain cell membrane stable;2.The mechanism of the reaction in the range of 0~25?M and 50~400?M may be different.At low doses,DVS causes the cell membrane to rupture.When a certain threshold is reached,DVS acts like a fixative,causing cell death,but still maintaining the integrity of the cell membrane.As for the specific mechanism of toxicity research remains to be further explored.Annexin is a class of phospholipid-binding proteins widely distributed in the cytoplasm of eukaryotic cells.Fluorescent probe FITC-labeled Annexin V can detect the important features of phosphatidylserine eversion of the cell apoptosis simply and directly.The results showed that SM could cause apoptosis in a dose-dependent manner obviously.About 50% of the cells were apoptotic at 100?M.The experimental results were consistent with the cytotoxicity test results.The apoptosis rate of cells treated with DVS increased gradually from 0?M to 25?M,but then decreased from 25?M to 50?M,and at last increased from 50?M to 400?M.The results were consistent with CCK-8 cytotoxicity test results.The reason may be that Annexin V-FITC cell apoptosis detection kit is mainly through the cell membrane eversion and cell membrane rupture for fluorescent staining.According to the previous test results,in the range of 0~25?M,DVS can cause cell membrane rupture,so the apoptosis rate gradually increased.However,at 50?M,microscopic observation showed that the cells still maintained relatively complete cell morphology,so the apoptosis rate decreased.Single cell gel electrophoresis test is a commonly used method to observe DNA breakage in cells.The results showed that SM and DVS could cause obvious DNA breakage at low dose,and as the dose increase,the degree of DNA damage increased and produced significant comet tailing.And at the same dose,the damage caused by SM was significantly greater than that of DVS.The results showed that both SM and DVS could cause DNA damage and had some genetic toxicity.The expression of DNA damage repair related genes were detected by fluorescence quantitative RT-PCR.The results showed that the expression of DNA damage related genes were increased in different degrees after 24 hours of SM exposure,and the expressions of RAD51 and ABH3 were the most obviously increased,for about 6 times and 5 times respectively,and the other genes were not more than 3 times.At time of 12 hours after SM exposure,the gene expression also increased to varying degrees,but the level was less than 24 hours.At time of 6 hours,gene expression was almost unchanged.The experimental results showed that DNA damage caused by SM may be mainly repaired by direct removal and homologous recombination.After 24 hours of DVS exposure,the expression of DNA damage related gene was significantly decreased,and there was also a decrease at time of 12 hours,but there was no change at time of 6 hours.The possible reason may be the toxic principle of SM and DVS is different.Toxic reaction principle of SM is substitution reaction,removal of chlorine,combined with the DNA nucleophilic sites,generate a variety of hydrolysis products;The principle of DVS toxicity is addition reaction,double bond breakage,combined with the material to produce a variety of products.?H2AX,PARP and p53 are important proteins in the cells and play an important role in the process of DNA damage repair.In this study,the expression of these proteins was detected by Western blot.The results showed that the expression of ?H2AX increased slightly and the PARP 116 KD protein decreased gradually with the increase of the dose,p53 and pho-p53 increased first and then decreased.The results showed that p53 and PARP could play an important role in cells after the DNA damage caused by SM in the low dose range.When the dose was increased,DNA damage was difficult to be repaired,p53 and PARP initiate apoptotic signaling pathways and promote cell death.The expression of ?H2AX and PARP did not change after 24 hours of DVS exposure,p53 increased first and then decreased with the increase of dose,while pho-p53 was not detected.Cell DNA damage repair process is a complex pathway system,is a result involved of multiple proteins,multiple pathways together,so further study is needed to fully reveal the SM and DVS DNA damage repair process.Conclusions: Through the study of this experiment,we draw the following conclusions: SM and DVS have obvious cytotoxic effect on Haca T cells,which can induce apoptosis.Meanwhile,the cytotoxicity of DVS is greater than SM.SM and DVS have a certain genetic toxicity,can cause DNA breakage damage,SM DNA toxicity is greater than DVS.The DNA damage of Haca T cells induced by SM was mainly repaired by homologous recombination and direct removal,however,the expression of the repair related genes caused by the DVS exposure is decreased.?H2AX,PARP and p53 were involved in the DNA damage repair process induced by SM,while DVS did not cause significant changes in these proteins expression.