Overnight lysis improves the efficiency of detection of DNA damage in the alkaline comet assay

The ability to detect DNA damage using the alkaline comet assay depends on pH, lysis time and temperature during lysis, However, it is not known wheth er different lysis conditions identify different types of DNA damage or sim ply measure the same damage with different efficiencies. Results support th e latter interpretation for radiation, but not for the alkylating agent MNN G. For X-ray-induced damage, cells showed the same amount of damage, regard less of lysis pH (12.3 compared to >13), However, increasing the duration o f lysis at 5 degreesC from 1 h to more than 6 h increased the amount of DNA damage detected by almost twofold. Another twofold increase in apparent da mage was observed by conducting lysis at room temperature (22 degreesC) for 6 h, hut at the expense of a higher background level of DNA damage. The ox ygen enhancement ratio and the rate of rejoining of single-strand breaks af ter irradiation were similar regardless of pH and lysis time, consistent wi th more efficient detection of strand breaks rather than detection of damag e to the DNA bases. Conversely, after MNNG treatment, DNA damage was depend ent on both lysis time and pH. With the higher-pH lysis, there was a reduct ion in the ratio of oxidative base damage to strand breaks as revealed usin g treatment with endonuclease III and formami-dopyrimidine glycosylase. The refore, our current results support the hypothesis that the increased sensi tivity of longer lysis at higher pH for detecting radiation-induced DNA dam age is due primarily to an increase in efficiency for detecting strand brea ks, probably by allowing more time for DNA unwinding and diffusion before e lectrophoresis. (C) 2001 by Radiation Research Society.