Abstract: Purpose: To investigate DNA damage of human lymphocytes after treated with H2O2. Methods: Comet assay was employed to assess DNA damage of human lymphocytes treated with either various concentrations of H2 O2 (0、10、20、40、80 μmol/L) for 20 minutes or 20 μmol/L H2O2 for different time (0、10、20、30 minutes). To quantify the DNA damage, three different comet parameters were evaluated: the tail length, comet length and tail moment. Laser scanning confocal microscope (LSCM) was also introduced to investigate the 3-D distribution of DNA within the comet. Four fluorescent dyes including propidium iodide (PI), ethidium bromide (EB), acridine orange (AO) and Hochest33258 were compared. Results: The tail moments (-±s) were 0.001±0.002、0.31±0.35、1.23±0.96、2.82±1.38、3.52±0.96(P<0.01), respectively, after treatment with various concentrations of H2O2(0、10、20、40、80 μmol/L); the tail moments(-±s) were 0.05±0.09、0.22±0.30、1.62±0.93、1.89±1.09 (P<0.01), respectively, after treatment for different time(0、10、20、30 minutes ). Tail length and comet length also exhibited significant increases (P<0.01) with the increased dose and extended time. The 3-D reconstruction made by LSCM gave a more distinct image of the comet compared with the fluorescence microscope. PI and EB were better dyes in terms of the definition of the image. Conclusion: H2O2 induces dose-dependent and time-dependent cytotoxic effect in terms of DNA damage on human lymphocytes. The LSCM provides substantial and measurable improvement in the definition of the“comet". PI and EB are suitable for comet assay.

Key words: comet assay, lymphocyte, DNA damage, laser scanning confocal microscope