The influence of temperature during alkaline treatment and electrophoresison results obtained with the comet assay

The alkaline comet assay (single-cell gel electrophoresis) is becoming esta blished as a genotoxicity test with manyfold applications in vitro and in v ivo. While the underlying principles are identical, various modifications o f the method are in use which clearly affect the sensitivity and resolving power of the assay. One variable of potential importance that has been disr egarded until now is temperature during alkaline treatment and electrophore sis. We therefore performed comet assay experiments with human blood and V7 9 Chinese hamster cells using two different temperatures (4 and 20 degrees C, i.e. room temperature) during alkaline treatment and electrophoresis. DN A damage was induced by the two standard mutagens gamma irradiation and met hyl methanesulfonate (MMS). The results clearly indicate significant differ ences in the detection of background and mutagen-induced DNA damage at thes e two temperatures. Under otherwise identical test conditions (including th e duration of alkaline treatment and electrophoresis), increased temperatur e during alkaline treatment and electrophoresis strongly enhances DNA migra tion. Our findings suggest that the comet assay should be performed under s trictly controlled and reproducible temperature conditions. In any case the temperature during alkaline treatment and electrophoresis should be stated in a publication to allow for a critical evaluation of results obtained wi th the comet assay. (C) 1999 Elsevier Science Ireland Ltd. All rights reser ved.