Shortening of alkaline DNA unwinding time does not interfere with detecting DNA damage to mouse and human spermatozoa in the comet

22 23 The Comet assay was performed on mouse and human spermatozoa to 24 examine the effect of alkaline DNA unwinding time. The spermatozoa were 25 treated in vitro with the DNA-damaging agents, methyl methanesulfonate 26 (MMS) or hydrogen peroxide (H2O2), and then embedded in agarose gel on 27 glass slides. The slides were immersed in alkaline solution (> pH 13) for 1, 5, 28 10, and 20 min, and then subjected to the electrophoresis under neutral 29 conditions. In mouse spermatozoa, comet tails seen in solvent controls 30 became brighter and longer as the alkaline DNA unwinding time increased. 31 However, in the MMS-treated mouse spermatozoa, a smaller difference in 32 the damage from that in the solvent control was seen with time within a dose. 33 DNA damage induced by H2O2 could also be detected accurately after alkali 34 treatment for 1 to 20 min. In human spermatozoa, DNA damage induced by 35 MMS and H2O2 could be detected in a dose-dependent manner after alkali 36 treatment for 1 min. The ability of the comet assay to detect DNA damage 37 was not adversely affected by the short period (1 min) of the alkaline DNA 38 unwinding time. 39 40