Study On The DNA Damage Property And Its Mechanism Of 6aromatic Amines In Cultured Cells

Purpose:The purpose of present study was to evaluate the genotoxicity property of six aromatic amines which was widely used in industrial production using phosphorylated histone H2AX(γ-H2AX)as an indicator in cultured cells,and to clarify the mechanism behind the genotoxicity,so that provide valuable evidence in evaluation of the carcinogenicity and the role in causing occupational bladder cancer.method:In this study,the detection of γ-H2AX mainly used Western blotting.In addition,immunofluorescence staining and flow cytometry were also used to detect formation of the γ-H2AX.In the detection of DNA double-strand breaks experment,the biased sinusoidal field gel electrophoresis was used.In cell survival rate measurement,the trypan blue staining method and the Alamar blue cell proliferation and toxicity detection reagent were used.In the intracellular reactive oxygen species level measurement,the DCFH-DA active oxygen fluorescent probe and the flow cytometry or microplate reader was used.When measuring the intracellular caspase-3 activity,Ac-Asp-Asn-Leu-Asp-MCA detection reagent was used and the fluorescence intensity was detected by a microplate reader.Results:1.The production of γ-H2AX can be observed in 1T1 cells after treatment with all aromatic amine compounds.However,there are significant differences in the amount of gH2AX formation after six aromatic amine exposure.DMA and OCA generated the strongest formation of γ-H2AX among them,while ANL induced the weakest production of γ-H2AX among the six chemical substances.The over all order is as follows: OCA = DMA> OT>ANS> PT = ANL.2.The production of γ-H2AX generated by 6 kinds of aromatic amine compounds in1T1 cells all in a dose-response manner.3.In the biased sinusoidal field gel electrophoresis experiment,the formation of DNA double-strand breaks can be clearly observed in 1T1 cells treated with OCA,DMA and OT,while the formation of DNA double-strand breaks cannot be observed in the cells treated with the other three aromatic amines.4.The survival rate of 1T1 cells treated with six aromatic amines for 4 h did not significantly decrease compared to the control in all the tested concentration(cell survival was in the 90% above).5.DMA can genrate the formation of γ-H2AX in WRL-68 and 1T1 cells in a dose-(1-10 m M)and time-(0.5-6 h)dependent manner.6.DMA can induce DNA double-strand breaks formation in WRL cells and 1T1 cells in a dose-response maner after 4 h treatment.7.The results of immunofluorescence staining showed that γ-H2AX was indeed formed in the 1T1 cell nucleus after DMA exposure,and the site of γ-H2AX generation was consistent with that of 53Bp1.8.After treatment with the same conditions as the dose-response study,the survival rate of WRL cells and 1T1 cells did not decrease significantly compared with the control,and there was no obvious activation of intracellular caspase-3 was observed.9.In the results of flow cytometry,the increase of in tracellular reactive oxygen species in 1T1 cells generated by DMA is in accordance with the dose-response manner,and in the presence of inhibitors of cytochrome P450 enzymes 1-aminobenzotriazole(ABT),disulfiram(DSF)and the antioxidant N-acetylcysteine(NAC),the amount of reactive oxygen species decreased in a dose-dependent manner.10.The production of γ-H2AX is also significantly inhibited in the presence of antioxidant NAC,but not completely.While the γ-H2AX induced by H2O2 was almost completely suppressed by the antioxidants NAC.Conclusions:In the first chapter,all the 6 aromatic amine compounds can generates the formation of γ-H2AX,and the cell survival rate does not significantly change after treatment under the same conditions.These indicating that the production of γ-H2AX is not caused by cell apoptosis.The biased sinusoidal field gel electrophoresis also clearly detected the formation of DNA double-strand breaks,indicating that the formation of γ-H2AX was indeed occurred after DNA damage by the aromatic amines.These all showed that six aromatic amines possesse the genotoxicity.However,there are significant differences in the amount of gH2AX formation after the treatment with the six aromatic amines,indicating that their genotoxicity is clearly different.In second chapter,DMA generated γ-H2AX,DNA doublestrand break,and immunofluorescence focus formation in cultured human urothelial cells and liver cells,indicating that it is genotoxic.DMA did not decrease the cell survival,and not increase the caspase-3 activity,but induced DNA double-strand break formation,indicating that DMA induce the formation of γ-H2AX through DNA double-strand break,not cell apoptosis.The production of intracellular reactive oxygen species and γ-H2AX is significantly reduced in the presence of CYP2E1 enzyme inhibitors,indicating that the generation of γ-H2AX caused by DMA is mainly mediated by ROS produced by CYP2E1 metabolism.Finally,γ-H2AX caused by DMA is not completely inhibited in the presence of antioxidants,while γ-H2AX caused by H2O2 is completely inhibited by antioxidants,indicating that there are other pathways that mediate DMA genotoxicity in addition to the ROS pathway.