Exploring the impact of lysis conditions on the alkaline standard and Fpg-modified in vivo comet assay

The comet assay is a method used to detect DNA lesions. The protocol involves cell lysis and electrophoresis of the DNA once the cells are embedded in agarose. In this study, we investigated how lysis duration and pH affect Wistar rat tissues. A single oral dose of 200 mg/kg methyl methanesulfonate (MMS) to induce DNA strand breaks (SBs), or 5 mg/kg MMS or 400 mg/kg potassium bromate (KBrO₃) to induce Fpg-sensitive sites, was administered to male Wistar rats (n = 3 rats/group). The negative control rats received saline solution. After 3 h, the rats were sacrificed and different tissues were analysed after necropsy or snap-frozen in liquid nitrogen before analysis. The tested duration of lysis was from no lysis to overnight (pH 10). Different pH (i.e. 7 and 10) were tested in longer lysis (up to 1 week) in the case of the standard assay and in 1-hour lysis in the case of the Fpg- modified assay. No significant differences in SBs were found among the lysis lengths, including no lysis, but, after applying longer lysis in frozen samples, up to 1 -week, an increase in SBs was seen in the samples from treated rats. A neutral pH seems to slightly increase the % DNA in tail obtained. In the case of using Fpg a 5-min lysis was needed to detect Fpg-sensitive sites. The detection of Fpg-sensitive sites in MMS-treated rats was time and pH dependent. On the contrary, there were no differences in KBrO₃ detected lesions.