Synthesis,Characterization And Identification Of New DNA Adducts In Vitro And In Vivo After Exposure To Divinyl Sulfone

It’s well known that SM(Sulfur Mustard,2,2’-dichlorodiethyl sulfide)is a banned chemical warfare agent that is controlled by the Organisation for the Prohibition of Chemical Weapons(OPCW).SM is one of the most hazardous chemical warfare agents(CWAs),and can injure skin,eyes,respiratory system,etc.SM still poses a threat to public safety for its facile synthesis,hypertoxicity,difficult therapy.DNA adduct is one of the most important biomarkers.Until now,the detailed toxicological mechanism of SM is still not very clear because SM has high reactive capability and complicated damage mechanism.Among them,formation of DNA covalent adducts maybe the most important and commonly mode of DNA damage.Our preliminary results showed that SM would be oxidized to mustard sulfoxide(SMO).And then,SMO could be further oxidized to mustard sulfone(SMO2)in vivo.SMO2 was extremely unstable,which would spontaneously convert into divinyl sulfone(DVS).In previous research,It was found that DVS was highly toxic compound.More importantly,DVS had been demonstrated to retain the vesicant property like SM and possess high reactive activity,which could cause respiratory and allergic reactions.Consequently,it was necessary to conduct the detailed investigation of the DNA damage of DVS.However,there was no intensive research of the toxic mechanism of DVS,including DVS-DNA adducts.In this article,we aimed to study the important oxidative metabolic product DVS,focusing on its reactivity and adduct formation to ative molecules in vivo including glutathione,purines,nucleotide and DNA,and to clarify the molecular basis of the oxidative damage and DNA leisure induced by DVS.Based on these studies,the role of DVS on serial toxic effects during SM intoxication would be demonstrated and determined,and the question of whether a secondary toxic damage caused by metholites of SM would be clarified,which will provide new insight into the SM toxicity and be helpful for the prevention and trearment of the SM poisoning.In present paper,we designed and optimized synthetic routes for preparation of series of new adducts.The structure,purity and exact addition site of adducts were characterized using HPLC-MS/MS and NMR.Furthermore,we developed the HPLC-MS/MS for determination of DNA adducts in vivo or vitro based on the synthesized compounds.Lastly,the type,concentration and distribution of DNA-adducts in cells and mouses exposed by DVS produced by DVS or DVS-GSH were investigated in detail.The results will play an important role in clarify the damage mechanism of SM.This dissertation consists of five chapters.The first chapter is introduction.In this chapter,we firstly summarized the physical and chemical properties of SM.Multiple metabolic pathways including oxidative hydrolysis pathway and binding pathway were reviewed.More attentions were paid to DVS,which is an important oxidative products orignating from SM.The detailed introduction about the toxicity and damage of DVS was introduced.Lastly,the objectives,contents and new insights of this dissertation were briefly outlined at the end of this chapter.The second chapter was about the synthesis and identification of standards.In this chapter,DVS standard was prepared via "series oxidation" and dechlorination using SM as the starting material.Then,five novel DVS-purine and GSH-DVS-purine adduct standards were obtained through optimization of the synthetic routes and substrates.The structure identification were performed with 1HNMR GC/MS,LC/MS and NMR.In the third chapter,the reactive capability of DVS with purine,nucloside and DNA in vitro was investigated under phisological condition.The detection methods for DVS-DNA adduct were developed based on the synthesized standards.The DNA-adducts were determined and identified in cells after exposure to DVS based on the developed methods.The results showed that five DVS-DNA adducts can be determined in aqueous solution,including N3-HESVA,N7-HESVG,N3-AHESEHG-N7,N7-GHESEHG-N7,N3-AHESEHA-N3.However,only four not five adducts were detected in cells experiments.The results in this chapter indicated that DVS has DNA damage similar to SM.In fourth chapter,the characterization and tissue distribution of different DVS-DNA adducts in vivo were studied.The model for SD rats exposed to DVS through intravenous injection was establied.The abudance of DVS-DNA adducts in some key tissues include liver,spleen,lung,kidney and brain were determined at interval.The results showd that only four not five adducts were detected in vivo,which is compatible with cell experiments.Compared with SM,the content and type of DVS-DNA adducts in the tissues is significant difference,and the duration time is longer than SM-DNA adducts,some of DNA adducts could exist in vivo for serveral days.However,the distribution of DVS-DNA adducts in tissues were not significant.In chapter five,the covalent addition products of DVS-GSH,which is a phase two metabolites of DVS,with DNA is studied.The results showed that DVS has better reactivity with GSH in aquesous sotluion under physilogical condition.The main products is monoadduct DVS-GSH not DVS-GSH-DVS if the reaction time is controlled within 30 minutes under the condition of ice bath.Moreover,DVS-GSH can also react with adenine or guanine exist in the DNA chains to produce two novel adducts,such as GSH-DVS-Ade and GSH-DVS-Gua.The abudance of GSH-DVS-Ade is higher than GSH-DVS-Gua in the mixtures of DVS and DNA.Therefore,the damage effect of this phase two metabolites should be paid more attention.It’s worthy to carry out further study about the detailed toxic mechanism of DVS-GSH.In present paper,we have completed the preparation of series DVS-DNA adducts,establishment of the LC-MS/MS method of DVS-DNA adducts,identification of DVS-DNA adducts in cells and animals,and investigated the DNA damage effect of DVS-GSH.