Effects Of DNA Repair Inhibitors On Comet Assay

Comet assay detects DNA single-strand breaks of single cell.Because the time that breaks lasts is very short during DNA repair process,if we add DNA repair inhibitors in the assay,the sensitivity and repetition may be enhanced.The research firstly uses 24-well plastic culture dishes in comet assay, applies hydroxyurea,cytosine arabinoside and 2',3'-dideoxythymidine 5'-triphosphate simultaneously, explores the reasonable concentrations value of the three inhibitors,and investigates the effects of repair inhibitors on comet assay in V79 cells and primary mouse hepatocytes under such concentrations. Meanwhile,the effects are researched that cytosine arabinoside and 2',3'-dideoxythymidine 5'-triphosphate block repair of different DNA damages.Before cells exposure to genotoxic substance,the amount of cells in every well is about 1 × 10⁵² × 10⁵.The cells are well-distributed. The three inhibitors applying simultaneously under the concentrations of 2mmol/L hydroxyurea,0.1mmol/L cytosine arabinoside,20umol/L2',3'-dideoxythymidine 5'-triphosphate or lmmol/L hydroxyurea,0.5mmol/L cytosine arabinoside,20μmol/L 2',3'-dideoxythymidine 5'-triphosphate don't damage DNA,but can significantly add the tail length value of comets,When V79 cells exposure to 0.01mmol/L K₂Cr₂O₇. The three inhibitors under such concentrations significantly enhance the sensitivity of comet assay in V79 cellsand primary mouse hepatocytes in different degrees,when cells exposure to organic extracts from drinking water, K2Cr2Oy,B(a)P and benzidine, respectively. Meanwhile,the repetition will also be enhanced in themry.DNA single strand breaks and large molecular DNA adducts are shaped when V79 cells exposure to different genotoxic substance.Before the lesions are repaired, cytosine arabinoside and 2',3'-dideoxythymidine 5'-triphosphate are added to culture medium, respectively.The results show that cytosine arabinoside completely prevents the repair process of large molecular DNA adducts,while 2',3'-dideoxythymidine 5'-triphosphate can't. Both cytosine arabinoside and 2',3'-dideoxythymidine 5'-triphosphate block repair of DNA single strand breaks ,the effects of cytosine arabinoside are more significant.We can learn about the sorts of DNA lesions that new genotoxic substances cause through observing the effects of cytosine arabinoside and 2',3'-dideoxythymidine 5'-triphosphate on repair process.Draw a conclusion, the modified comet assay in 24-well plastic culture dishes,applying three DNA repair inhibitors simultaneously,is convenience, quickness , little sample,high sensitivity, high repetition,and adapt to detect large amount of specimens. Meanwhile, We can learn about the sorts of DNA lesions that new genotoxic substances cause through observing the effects of cytosine arabinoside and 2',3'-dideoxythymidine 5'-triphosphate on repair process.