The Role And Mechanism Of Cl~- In Copper-Induced DNA Oxidative Damage

Studies have shown that Cu（Ⅱ）can generate free radicals through the Fenton-like reaction,causing cell damage including base modification,DNA-protein cross-linking,DNA single-strand or double-strand break,and further Cause various diseases.Chloride ion（Cl^-）is the most abundant anion in the human body.It has the positive effects of maintaining electrolyte balance,sterilization and antisepsis,and also affects the p H of cells and causes autoimmune diseases.A recent study by the research team found that Cl^-can accelerate the Fenton reaction of Cu（Ⅱ）and produce more reactive oxygen species（ROSs）.The effect of this reaction on intracellular polysaccharides,proteins,DNA and other biological macromolecules is still unknown.In this study,p MD19-T-xyn plasmid DNA（3370 bp）and human embryonic kidney cell（HEK293T）genomic DNA were used as the research object.The effects of Cl^-on copper-induced DNA damage were systematically investigated.The damage types were analyzed and the possible damage mechanisms were discussed.This study has important guiding significance for the pathogenesis of biological processes such as cancer and aging,diagnosis of diseases,and drug selection,and provides a theoretical basis for the nature and details of related diseases.The main findings are as follows:1.Using copper-induced DNA damage as a control（reaction system:p MD19-T-xyn plasmid DNA 40 ng/μL,10 mmol/L p H 7.0 PB buffer,0.1 mmol/L H₂O₂,20μmol/L Cu（Ⅱ）,reaction at 37°C for 60 min）,the difference was investigated.Damage to plasmid DNA by anions(Cl^-/NO^3-/SO4^2-/Cl O^4-/PO₄^3-)and halogen anions（F^-/Br^-/I^-）.The results of gel electrophoresis showed that only Cl^-caused significant DNA damage at the same ion concentration（0.1 mol/L）,PO₄^3-and I^-had protective effects on DNA damage,and DNA damage rate increased with increasing Cl^-concentration（about 1.8 times）.The damage of plasmid DNA by different cationic chlorides（Li Cl/Na Cl/k Cl/Rb Cl/Cs Cl/Mg Cl₂/Ca Cl₂）was investigated.The gel electrophoresis results showed that at the same Cl^-concentration（0.1 mol/L）,DNA formed a precipitate in Ca Cl₂,and the others caused significant DNA damage.Some human metal ionic compounds（K（Ⅰ）/Rb（Ⅰ）/Mg（Ⅱ）/Ca（Ⅱ）/Mn（Ⅱ）/Ni（Ⅱ）/Zn（Ⅱ）/Pb（Ⅱ）/Cr（Ⅲ）/Go（Ⅲ）/Al（Ⅲ）/Fe（Ⅲ）/Cu（Ⅱ）/Fe（Ⅱ））were selected to participate in the oxidative damage of plasmid DNA in H₂O₂.It was found that only Cu（Ⅱ）and Fe（Ⅱ）caused oxidative damage of plasmid DNA and Cl^-only accelerated the oxidative damage of plasmid DNA caused by Cu（Ⅱ）.2.Oxidative damage of plasmid DNA induced by Cu（Ⅱ）of Vc and DA under Cl^-action（reaction system:p MD19-T-xyn plasmid DNA 40 ng/μL,10 mmol/L p H 7.0 PB buffer,20μmol/L Cu（Ⅱ）,Reaction at 37°C for 60 min）.It was found that both Vc and DA caused oxidative damage of plasmid DNA,and the damage rate increased with the increase of Cl^-concentration.The degradation rate of 0.1 mol/L Cl^-medium DNA in 5μmol/L Vc was 1.59 times that of 5 mmol/L;the degradation rate of Covalent closed circular DNA in0.1 mol/L Cl^-in 1μmol/L DA was 2.36 times that of 5 mmol/L.Further studies found that metal ion compounds（K（Ⅰ）/Rb（Ⅰ）/Mg（Ⅱ）/Ca（Ⅱ）/Mn（Ⅱ）/Ni（Ⅱ）/Zn（Ⅱ）/Pb（Ⅱ）/Cr（Ⅲ）/Go（Ⅲ）/Al（Ⅲ）/Fe（Ⅲ）/Cu（Ⅱ）/Fe（Ⅱ））induced oxidative damage of plasmid DNA induced by Vc and DA,respectively.In the study,only Cu（Ⅱ）and Fe（Ⅱ）caused oxidative damage of plasmid DNA,and Cl^-only accelerated the oxidative damage of plasmid DNA caused by Cu（Ⅱ）.3.Take copper-induced DNA damage as control（reaction system:human embryonic kidney cell（HEK293T）genomic DNA 370 ng/μL,10 mmol/L p H 7.0 PB buffer,0.76mmol/L H₂O₂,20μmol/L Cu（Ⅱ）,reaction at 37°C for 60 min）,investigate the effect of Cl^-on the damage of genomic DNA.After digestion with Nth and Fpg,gel electrophoresis results showed that the higher the Cl^-concentration,the more genomic DNA damage sites.Quantitative analysis by HPLC showed that 8-OHd G production was about 50 times that of the control group under 0.1 mol/L Cl^-conditions,and an 8-OHd G was produced on average about 9.8×10⁵ bases.4.The protective effects of different free radical quenchers on DNA oxidative damage were investigated.Copper-induced DNA damage under Cl^-action as a control（reaction system:p MD19-T-xyn plasmid DNA 40 ng/μL,10 mmol/L p H 7.0 PB buffer,0.1 mmol/L H₂O₂,20μmol/L Cu（Ⅱ）,0.1 mol/L Cl^-,37°C reaction for 60 min）.DMSO,Na N₃,propanol,and mannitol were found to have protective effects.The ratio of Covalent closed circular DNA in 0.5%DMSO,10%Na N₃,10%propanol,and 140 mmol/L mannitol was1.77,1.33,1.77,and 1.67 times of the control group,respectively.Taurine has no protective effect.It was indicated that Cl^-accelerates the formation of free radicals such as hydroxyl radical（·OH）and singlet oxygen（1O₂）in Cu（Ⅱ）-induced DNA oxidative damage,and promotes oxidative damage of DNA.