Identification Of Novel Crosslinked DNA Adducts At Abasic Sites In Cells And Their Application Research

The integrity of DNA is very important to organisms.Both exogenous factors and internal factors in the organism often cause DNA damage.Genotoxic chemicals can directly or indirectly cause cell DNA damage,can hinder genome replication and transcription,and have the potential threat of inducing serious diseases such as cancer and malformations.Due to its widespread existence in our daily life and environment,it is very necessary to build a screening and prevention system for genotoxic chemicals.At present,mass spectrometry（MS）has been widely used in the research of DNA damage by virtue of its advantages.However,in the face of the huge number and variety of genotoxic chemicals in life,the above research strategies cannot achieve complete screening,so it is urgent to find DNA damage common biomarkers to be used in the screening of genotoxic chemicals and in the building of toxicity testing methods.This thesis mainly focuses on the potential and"common"biomarker of DNA damage,abasic（Apurinic/Apyrimidinic/abasic,AP）sites,taking its crosslink adduct as the quantitative research object,to carry out the confirmation and idetification of AP sites crosslink adducts in cells and research on their application.The generation of AP sites is related to many types of DNA damage such as base mismatches,DNA adducts,and base deamination,making AP sites one of the common forms of DNA damage.At present,it has been applied to the research of various types of DNA damage,such as DNA alkylation,oxidative stress and ionizing radiation.Due to the nonselectivity limitations of the quantitative method and the structural instability of the AP sites itself,the accurate quantification of the AP sites remains challenging.Recently,studies have found that under Saline conditions,AP sites can react with bases on its complementary strand（AP-base）to form crosslinks on the DNA duplex,which is stable and difficult to repair,which suggests that AP-base crosslink plays an important role in DNA alkylation damage and warrants further investigation.However,up to now,the detailed situation of AP-base crosslink in vitro and in vivo is still unknown.Therefore,this thesis takes AP-base adducts as the starting points for research and carries out the confirmation research of AP-base crosslink by high-resolution mass spectrometry（LC-HRMS）screening technology.Based on the highly sensitive and specific liquid chromatography-mass spectrometry（LC-MS/MS）method,we aim to quantitatively monitoring of the dynamic basal levels of AP-base adducts and its proportions of isomers,and to study its influencing factors.Taking genotoxic compounds with different and classic mode of action as the research object,we aim to study the dose-response relationship,time-response relationship and damage-repair changes with AP-base adducts with the theoretical idea of combining biology,analytical chemistry and quantitative technology-toxicology,and provide technical methods and foudational datas for constructing new principles and new methods in vitro testing that can monitor DNA damage and repair processes through AP-base adducts,laying the foundation for further genotoxic substance screening and toxicity testing methods.This thesis is divided into the following five chapters:The first chapter is the introduction.In this part,the characteristics and deficiencies of the current genotoxic substance screening and toxicity testing methods were briefly summarized,and the structural characteristics,quantitative methods and biological progress,AP-base Crosslinks of AP sites in recent years were mainly described.We clarified that AP-base adducts is a potential and ideal DNA damage biomarker,and finally proposed the research goals,research contents and key problems to be solved ungently of this paper in terms of AP-base adducts.The second chapter is the synthesis and characterization of standards.Taking d R and deoxyadenosine monohydrate（d A）as starting materials,the two directly underwent an addition reaction,and the AP-d A adducts were directly and efficiently obtained.The active groups of d R and deoxyguanosine monohydrate（d G）were derivatized and protected,respectively,followed by a step of"oxime formation"reaction.Then the reaction mixture was directly removed the protecting groups or was reducted and removed the protecting groups,and finally the AP-d G and reduced AP-d G(AP-d G_red)adducts were obtained.On this basis,by optimizing the reaction time,separation and purification processes,the yield of AP-d G and AP-d G_red adducts has been improved.The structure identification of above standards was performed with LC-MS/MS,LC-HRMS and NMR.A total of three adducts were synthesized:AP-d A,AP-d G and AP-d Gred adducts,and the purity was higher than 96%,96%and 92%respectively,all of which met the testing requirements.The third chapter describes the identification of AP-base adducts in cells and the construction of quantitative method for AP-base adducts.Two common DNA hydrolysis methods including acid hydrolysis and enzymatic hydrolysis were comparatively used to pretreat the sample from in vitro incubation solution.The produced DNA adducts were screened and quantitatively detected by mass spectrometry.Results showed that the acid hydrolysis was more efficient and suitable for the quantitative analysis of structure-known DNA adducts by mass spectrometry.For enzymatic hydrolysis method,it could produce various adduct forms,such as base-and/or nucleoside-adducts,which is suitable for screening and identification of structure-unknown adducts.The enzymatic hydrolysis method（Group III）was determined to be used in the screening and identification of AP-base adducts.The cell samples after enzymolysis were screened by high-resolution mass spectrometry（LC-HRMS）screening technology.It was for the first time that we found AP sites can form three adducts with nucleobases in cells,namely AP-d A,AP-d G and AP-d C crosslink adducts.In the multiple reaction monitoring mode（MRM）,a highly sensitive and highly specific liquid chromatography tandem mass spectrometry（LC-MS/MS）method for the detection of AP-base adducts in cells was established,and its methodological content-selectivity,linear range,matrix effect,accuracy and precision were studied,and the results showed that the established method meets the testing requirements.In the fourth chapter is the study on the distribution characteristics of AP-base adducts in different cell lines.The basal levels of AP-base adducts and its proportions of all isomers at a series of time points in different cell lines after passage were determined,and the possible influencing factors about basal levels and proportions of AP-base were also studied.Taking the potential and common DNA damage biomarker,AP-base adduct,as the starting point in DNA damage and repair,the AP-base content at a series of time points the three different cell lines,two types of adherent cells（Hep G2 and L02）and one suspension cell（AHH1）,after passage,and the proportion of each isomer were detected and analyzed based on the established LC-MS/MS（MRM）method.The experimental results showed that the basal levels of AP-base showed a time-dependent change with different cultured times after cell passage,and there was no significant difference under the AP-base time-response profiles curve（AUC）value of the three cell lines.By studying the different concentrations of trypsin and the different content of trypsin during cell passage,the results showed that the above two factors had no significant importance on the time-response profiles of AP-base content after cells passaging.It is inferred that it may be related to cell replication and cell cycle.The study also found that the proportions of the four isomers of AP-d A and AP-d G adducts at a series of points after the passage of the cells were significantly different from those of the solution.Two factors,may be related to the AP-base isomer content,that the different p H matrix solutions and the different time of enzymatic digestion of cell DNA samples added with standards were studied.The results showed that the above two factors had no significant importance on the proportions of AP-base isomer after cells passage,and it is inferred that the change of the proportions of AP-base isomers may be related to the intracellular biotransformation.The fifth chapter was to detect and analyze the influce of AP-base contents in cells after exposure to toxic chemicals.Based on the current exposure experiment of alkylated compounds,the dose-response relationship revealed that SM induced an increase in the level of AP-base adducts in cells;the time-response relationship revealed that when cells are exposed to high doses of SM compounds for 1 h,the adduct content decreased and shows a significant dose-dependent relationship and when the exposure time is prolonged at 24 h,the content of adducts showed an increasing trend.Damage repair experiments about SM exposure suggested that AP-base adducts could be used to indicate and evaluate the processes of DNA damage and repair.