Cl^-/Dopamine/Cu²⁺ Induced Oxidative Plasmid Dna Damage

Fenton-like reactions are a class of reactions in which metal ions such as Cu²⁺produce ROS or metal oxygen complexes with H₂O₂,which can oxidize a variety of biological macromolecules and cause biological damage.The latest research of our research group shows that Cl^-,as a macroelement present in large quantities inside and outside cells,can accelerate the Fenton-like reaction under the H₂O₂/Cu²⁺system and produce more reactive oxygen species.DA is an important neurotransmitter in the human body with strong antioxidant capacity,but it can also show pro-oxidation in the presence of Cu²⁺,participate in Fenton-like reactions,and induce DNA oxidative damage,while whether Cl^-plays a role in DNA oxidative damage under the DA/Cu²⁺system has not been reported.In this project,the effect of Cl^-on DNA oxidative damage of DNA under the DA/Cu²⁺system was systematically investigated by taking the plasmid DNA of Escherichia coli p MD-xyn-11A as the research object,analyzing the damage type,and exploring the possible damage mechanism,so as to elucidate the role of Cl^-in the oxidative damage of DNA by free radicals.The main findings are as follows:1.The damage of Cl^-to DNA under the DA/Cu²⁺system was investigated.The results of DNA configuration changes showed that the degree of DNA damage was positively correlated with Cl^-concentration,and the DNA damage rate at 0.1 mmo/L Na Cl was 1.3times that of the control.At the same time,the degree of DNA damage was also positively correlated with DA concentration,PB concentration and p H value.2.The type of ROS generated in the reaction was identified.Different radical probes were selected for detection,and it was found that the response values of DCFH,APF,HPF,SOSG,XTT and NBT all increased with the increase of Cl^-concentration,and the relative intensities of each probe at Cl^-concentration of 0.2 mol/L were 1.4,1.85,1.45,1.56,1.1and 1.2 times of the control,respectively,and the results showed that Cl^-could accelerate the formation of·OH,¹O₂and·O₂^-in the reaction,in which·OH was dominant.In order to further verify the ROS produced,a quencher was added to the reaction,and it was found that DMSO,Na N₃,and SOD all showed effective inhibitory effects on DNA damage,and the DNA damage rates at 5%DMSO and 10%Na N₃were 1.8 and 1.84 times that of the control,respectively,indicating that the reaction generated·OH,¹O₂,and·O₂-.3.The specific damage sites of DNA oxidative damage in the reaction system were investigated.By restriction enzyme-specific digestion reaction,DNA oxidative damage base sites were found to be guanine（G-G）.And HPLC quantitative analysis showed that the damage signature product 8-OHd G was generated in the reaction.4.The effects of some common antioxidants on DNA damage in the Cl^-/DA/Cu²⁺system were investigated.High concentrations of GSH（>20μmol/L）,GSSG,melatonin and VE showed protective effects against DNA damage.In contrast,VC,low concentration GSH（<20μmol/L）,tea polyphenols,β-carotene and anthocyanins all accelerated the oxidative damage of DNA and showed pro-oxidation.In conclusion,Cl^-can accelerate the oxidative damage of DA/Cu²⁺plasmid DNA,and this study explains the damage effect of Cl^-/DA/Cu²⁺system on DNA at the chemical level,highlighting the potential toxicological effect of Cl^-,and has important reference value in the biomedical field to explain the damage of high-salt diet to cells and organisms.