Mass Spectrometry Analysis Of Histone Phosphorylation Modification Reveals Genotoxicity And Modes Of Action For Chemicals

Genotoxic chemicals are those that can damage DNA directly or indirectly,cause gene mutation or mutagenesis in vivo,characterized by the possibility and tendency of carcinogenesis.Genotoxic chemicals can cause damage to human genetic material at relatively low concentrations.Because of the potential serious harm of genotoxicity to humans,the delusive mechanism of genetic damage caused by genotoxic chemicals,and the low ability to control genotoxicity of chemicals,it is urgent to establish a rapid and effective method for detecting,evaluating genotoxicity.The current genotoxicity testing methods have shifted from traditional animal in vivo experiments to in vitro testing methods.A wide range of in vitro genotoxicity evaluation methods including Ames test,mouse lymphoma assay(MLA),chromosome aberration assay(CA),micronucleus assay(MN),etc.have been developed,but keep the challenges derived from low throughput and false positive.In addition,the accurate quantification of genotoxicity cannot be achieved.The existence of a series of bottleneck problems has greatly limited the further popularization and application of the above technologies.More importantly,the current technologies cannot discriminate efficiently the two main types of genotoxic chemicals(i.e.clastogens and aneugens).Therefore,it is urgent to find new ways to establish a method for genotoxicity testing that meet the above requirements.This thesis focuses on the research of new methods for genotoxicity testing based on the common effect target molecules y-H2AX and p-H3 in DNA damage repair.At present,the research methods of y-H2AX and p-H3 at home and abroad are mainly limited to immunological methods,such as immunoblotting and enzyme-linked immunosorbent assay.Although the immunoassays can provide good sensitivity,their specificity is limited by poor batch-to-batch reproducibility as well as some cross-reactivity derived from antibodies,and their accurate quantification is still a tough task.Therefore,it is unable to quantify the specific processes of DNA damage,repair and transcription.The development of soft ionization-based biomass spectrometry has promoted the progress of proteomics.In recent years,glycosylation and phosphorylation proteomics have developed rapidly.Mass spectrometry(MS)technology makes the identification and quantification of protein modification sites more extensive and accurate.Therefore,this thesis takes y-H2AX and p-H3 as the starting points for research,applies the idea of combining effect-oriented analysis and spectroscopy techniques.This paper focuses on classic genotoxic model compounds and susceptible cell lines with their own metabolic drug enzyme system.Based on highly sensitive and highly specific liquid chromatography tandem mass spectrometry(LC-MS/MS),the work intends to develop a new genotoxic screening strategy to simultaneously detect H2AX-S139 phosphorylation site,H3-S10 phosphorylation site,and unmodified peptides.By quantitatively monitoring the dynamic dose-dependent relationship and time-course relationship of ?-H2AX/H2AX as well as p-H3/H3 in model genotoxic compounds-exposed cells,genotoxic equivalent factor and evaluation model can be constructed.In addition,by exploring the profiles of y-H2AX and p-H3 in chemical exposed cells,genotoxic chemicals with different modes of action can be easily discriminated,leading to a clear judgment among clastogens,aneugens and cytotoxic compounds.Furthermore,the carcinogenic risk can be evaluated based on detection results.The new established in vitro strategy for genotoxicity detection will provide technical support for solving practical problems such as drug genotoxic impurity control and clinical chemotherapy drug evaluation,and have important significance and application prospects in many research fields.This thesis is divided into five chapters.The first chapter is the introduction.In this part,the definition and characteristic of genotoxic chemicals,two major types of genotoxic chemicals and genotoxic impurities were summarized.The development of in vitro evaluation methods of genotoxicity and the progress of protein quantification methods based on MS technology in recent years were described.Finally,we put forward the purpose,research contents and innovation points of this thesis.In the second chapter,we developed a new genotoxic screening strategy based on the use of the common effect target molecules y-H2AX(clastogenicity biomarker)and p-H3(aneugenicity biomarker)in combination with cell lines with distinct biotransformation properties.A highly sensitive and highly specific stable isotope dilution-liquid chromatography-tandem mass spectrometry(ID-LC-MS/MS)was established to detect H2AX-S139 phosphorylation site,H3-S10 phosphorylation site,and unmodified peptides simultaneously,and validation was performed.Results showed that the established method has good sensitivity,linear range,accuracy and precision.Based on the established ID-LC-MS/MS method,we quantitatively monitor time-course relationship of y-H2AX/H2AX and p-H3/H3 in two classical genotoxic chemicals with different modes of action exposed cells(HepG2,HeLa),respectively.Results showed that the clastogen camptothecin and the aneugen colchicine had different effects on the expression levels of y-H2AX and p-H3 in the cells.After camptothecin induced DSB s,cells initiate DNA repair and DNA transcription is almost suppressed.This chapter preliminarily achieves quantitative assessment of genotoxic chemicals with different modes of action and dynamical monitoring of DNA damage/repair and transcription processes.The third chapter is MS analysis reveals different orchestration and dynamic processes of two major types of genotoxic chemicals on y-H2AX and p-H3.In this chapter,we expanded the number of test compounds and applied the established method to monitor dose-dependent relationship and time-course relationship of y-H2AX/H2AX and p-H3/H3.We find that clastogens significantly induce an increase in y-H2AX and a decrease in p-H3;aneugens have no obvious effect on y-H2AX,whereas induce either an increase or a decrease in p-H3.In addition,the specific profiles of clastogens and aneugens affecting DNA damage may be dynamically observed,which in turn provides insights into the processes involving DNA damage repair as well as transcription.Taken together,these results suggest that robust MS analysis of y-H2AX and p-H3 can not only quantitatively differentiate mechanistic information of clastogens and aneugens,but also dynamically present the detail profiles of DNA damage and repair processes.The fourth chapter is that monitoring cellular y-H2AX reveals a potential in carcinogenicity evaluation for genotoxic chemicals.With an established ID-LC-MS/MS method to detect H2AX and y-H2AX,we quantitatively and dynamically monitored the relationships between the percentage of ?-2AX to total H2AX(R?/T)and different genotoxic carcinogens(GCs)in two human cell lines(HepG2 and HeLa)owing different biotransformation capabilities.Dose-dependent response of GCs towards genotoxic effort,as R?/T among minimum effective concentrations,were well observed.Comparing with high content analysis to offer end-point results,ID-LC-MS/MS provided a feasibility to dynamically monitoring the y-H2AX changes.We unraveled a positive association between the carcinogenic levels of GCs and the R?/T values in the time-effect curves describing DNA damage potential and repair capacity.The fifth chapter is the application of genotoxicity evaluation method in vitro.In this chapter,we firstly monitored the dose-effect and time-effect relationships between Aristolochic Acid(AA)and ?-H2AX in two human cell lines(HepG2 and HeLa)based on the established ID-LC-MS/MS method for simultaneous determination of peptides derived from H2AX and ?-2AX.Dose-effect relationship revealed that ?-H2AX in cells exposed to 1,10,100 ?M of(Aristolochic Acid ?,AA?)and(Aristolochic Acid ?,AA?)in a dose-dependent manner,and the elevated level of y-H2AX formed by AAI was relatively higher than that formed by AA?.Time-effect curves of y-H2AX in cells exposed to 100 ?M of AAI and AA? demonstrated an obvious difference in the later period,i.e.,the proportion of y-H2AX had been on the rise in AAI exposed cells,while the level of y-H2AX appeared a falling trend in AA? exposed cells.Additionally,we preliminarily explored the changes of DNA repair-associated proteins(ERCC1 and p53)expression levels in HepG2 cells exposed to 100 ?M of AAI and AAII by applying western blot analysis,and observed AA?/? have opposite effects on the expression levels of ERCC1 and p53,which further help to understand the potential molecular mechanisms for their genotoxicity.Overall,our data demonstrated that AAI is more genotoxic risk than AAII in two cancer cell lines that we analyzed,which probably enhance our understanding of the relative carcinogenicity of AAI and AA?.Finally,based on the established ID-LC-MS/MS method for simultaneous determination of peptides derived from H2AX and y-H2AX,we investigated the expression of ?-H2AX in valsartan drug substances on two test cells(HepG2 and HeLa).Compared with the 0.1%DMSO control group,valsartan does not change the R?/T value,that is,valsartan itself does not affect the cell y-H2AX expression level HepG2 and HeLa;while valsartan solution containing different concentrations of N-Nitrosodimethylamine(NDMA)significantly increased R?/T value of HepG2 cells.According to the criteria for genotoxicity evaluation,NDMA showed genotoxicity in HepG2 cells with corresponding drug metabolism enzyme system.