Researches On The Mechanism Of DNA Double Strand Break Damage And Repair Induced By 1,2:3,4-diepoxybutane

Background:1,3-butadiene(BD)is an important industrial material for the synthesis of rubber,plastics and other products in petrochemical and manufacturing industries.BD has been listed as one of the top 40 most important chemicals by the United States because of the wide range of application and a high exposure to professional populations.In addition,it can be also found in automobile exhaust,cigarette smoke and edible oil fume.BD is one of the 9 kinds of priority pollutants in the indoor air.Therefore,BD is not only an occupational exposure pollutant,but also a major environmental pollutant.The hazardous effects of BD on huaman health have been expanded from the professional population to the general population.BD can induce the formation of multiple organ tumors in rodents,and population studies have also shown positive results for the onset of leukemia,thus BD is identified as a human carcinogen(group 1)by IARC and EPA.The carcinogenic effects of BD mainly derived from its epoxidation products generated by in vivo metabolism: Epoxy-1,2-3-butene(EB),1,2-dihydroxy-3,4-epoxybutane(EBD)and 1,2:3,4-diepoxybutane(DEB).All of above epoxidation products can form adducts with DNA,thus resulting in the genetic damage.And the toxicology of DEB is the strongest.Therefore,most of previous studies reagrding the mechanism of BD carcinogenesis focused on elucidating the genetic damage.Previous studies have shown that BD and its metabolites can induce double strand break(DSB)of DNA and chromosome damage,but the repair mechanism of BD-induced DSB remains unclear.In our previous study,we found that BD exposure was associated with an increase in the proportion of nucleoplasmic bridge(NPB).NPB is a marker of chromosome damage and an important step of carcinogenesis in cell.The study of the formation mechanism of NPB induced by BD will provide an important basis for elucidating the carcinogenic process of BD,preventing and reducing chromosomal damage and the incidence rate of human cancers.It has been suggested that the formation of NPB may be related to the process that a dicentric chromosome is formed because of the incorrect repair of the DNA double strand break by the non-homologous end joining(NHEJ),and pulled to the two daughter nucleus in the anaphase of cell division.These results suggested that after DNA double strand break,the main repair pathways,namely HR and NHEJ repair pathways,might be affected by BD and its metabolites induced,which raises the error repair efficiency,thus generating NPB.Therefore,the present study amied to investigate the potential effects of DEB,the active metabolite of BD,on the DNA double strand break repair pathway upon DNA strand breaks and chromosome damage in human lymphoblastoid cells,and the study also verified the roles of the repair pathways in the formation of DEB-induced NPB.These results would provide the basis of an more in-depth study for the mechanism of BD-induced genetic damage.Content:1.Researches on the cytotoxic effect of DEB on TK6 cellsWe first established the DEB-treated human lymphoblastoid cells(TK6 cells)model in the present study.To evaluate the effects of DEB on TK6 cells viability and proliferation,the MTS and EdU methods were respectively used.Parallel,the changes of cell cycle and apoptosis were detected by PI staing and Annexin V-FITC/PI staining using flow cytometry.2.Detection of cytogenetic damage induced by DEBWe used the cytokinesis block micronucleus test(CBMNT)to analyze DEB-induced chromosomes damage(micronucleus rate,nucleoplasmic bridge rate,nuclear buds rate and mitotic index).The expression of DNA double strand break marker(gamma-H2AX)was detected by Western blot and immunofluorescence technique.3.Researches on effects of DEB on the repair of DNA double strand breakWe used the MCF-7 cells containing HR repair efficiency test substrate to evaluate the change of HR repair efficiency after DEB treatment.We extracted total protein of the cells after DEB treatment,labeled the linear plasmid using the 32 P,and investigated the changes of NHEJ repair efficiency in Cell-free system.Furtherly,the expressions of key protein molecules involved in DNA damage repair pathway such as ATM,p-ATM,gamma-H2 AX,Ku80,DKA-PKcs,XLF,DNA-ligase,IV BRCA1,and so on,were detected by Western blot,Slot blot or immunofluorescence.4.The role of key NHEJ pathway genes in chromosome damage induced by DEB.Furthermore,the chemical inhibitor of Nu7026 was applied to inhibit the expression of protein DNA-PKcs,the transfected ShRNA was applied to inhibit the expression of protein Ku80.Then we analyze the changes of chromosome damage with CBMNT,and evaluate the effect of NHEJ repair pathway in DEB-induced DNA double strand break damage and NPB formation.Results:1.DEB induces cell proliferation inhibition,cell cycle arrest and apoptosis in TK6 cellsWe found that DEB could inhibit the activity of TK6 cells and decrease the synthesis of DNA in a dose-dependent manner.Further results from flow cytometry showed that DEB could inhibit the progression of TK6 cells cycle and cells division was arrested in G2/M phase.Moreover,DEB could induce apoptosis of TK6 cells,and the number of apoptotic cells was significantly increased after DEB treatment for 48 h(P < 0.01 or 0.001).2.DEB induced chromosome damage and DNA double strand break in TK6 cellsThe results of CBMNT showed that exposure to DEB increased the rate of micronucleus,nucleoplasmic bridge and nuclear buds in TK6 cells,induced chromosome damage,decreased the mitotic index and inhibited cell proliferation.In addition,the expression of DNA double strand break marker gamma-H2 AX protein increased first and then decreased with prolonged time.But the expression of gamma-H2 AX always maintained at a high level after 10 ?M DEB treatment.Immunofluorescence assay also confirmed that the fluorescence intensity of gamma-H2 AX significantly increased in those cells trested by 10 ?M DEB.These results suggest that DEB caused DNA double strand break and chromosome damage in TK6 cells.3.Effect of DEB on HR and NHEJ repair efficiency and expression of related genesWe detected the effects of DEB-induced HR repair efficiency using the cell model containing p DR-GFP substrate.The results showed that the proportion of fluorescence positive cells reflecting HR repair efficiency decreased significantly with DEB treatment for 24 h at a dose-dependent manner.It suggested that DEB reduced the efficiency of HR.The result of in vitro NHEJ repair assay showed that with the increase of DEB concentration,the joint product of the linear plasmid increased significantly,which suggested that DEB improved the NHEJ repair efficiency.In addition,the detection results of proteins in DSB repair pathway showed that the expression of BRCA1 protein in the HR repair pathway was significantly reduced with DEB treatment for 24 h.At the same time,the expression of the NHEJ pathway proteins,including DNA-PKcs,XLF and DNA-ligaseIV,increased significantly at 4 h or 24 h after DEB treatment.However,we didn't find the change of the core protein of NHEJ repair pathway Ku80 using the detection of western blot.But the results of immunofluorescence and Slot blot showed that after the cells were treated with DEB for 4 h,Ku80 protein formed focus,the content of Ku80 protein combined with DNA significantly increased at a dose-dependent manner.These results suggested that DEB improved the efficiency of NHEJ repair pathway and promoted the expression of related genes or the combination with DNA.Meanwhile,DEB reduced the efficiency of HR repair pathway and related gene expression4.NHEJ pathway is involved in DEB-induced NPB formationThe expressions of DNA-PKcs and Ku80 proteins were inhibited by chemical inhibitor and sh RNA respectively,and the role of NHEJ pathway in DEB-induced chromosome damage and NPB formation was observed by CBMNT.The results showed that compared with those of the group of only DEB treatment,the rates of micronucleus and nuclear buds were significantly increased and the rate of NPB was significantly decreased in DEB-treated TK-6 cells with the inhibition of DNA-PKcs expression,.At the same time,the transfection of Ku80 sh RNA showed similar results.These results suggested that the inhibition of DNA-PKcs or Ku80 increased rates of the micronucleus and nuclear buds induced by DEB,but decreased the rate of NPB.These results also confrimed that the NHEJ pathway may be involved in the formation of NPB induced by DEB.Conclusion:We found that DEB induced DNA double strand break and chromosome damage including micronuclei,nuclear buds and nucleoplasmic bridge in TK6 cells,and caused cell proliferation inhibition,cell cycle arrest and apoptosis.Furthermore,we found that DEB inhibited HR repair pathway and activated NHEJ repair pathway in the process of DNA double strand breaks repair.And the NHEJ repair pathway might be involved in the formation of DEB-induced nuclear cytoplasmic bridge.