Influence of experimental conditions on data variability in the liver comet assay

The comet assay has increasingly been used for regulatory genotoxicity testing in recent years. While the technique of the comet assay per se on a first glance seems to be relatively “simple”, the experimental execution of the assay such as organ and slide preparation, electrophoresis or comet scoring can have a strong impact on the results of the assay. It has been demonstrated that some technical parameters, e.g. electrophoresis or scoring of comets can have a strong impact on the assay, but little is known how initial steps, from tissue sampling during necropsy up to slide preparation, can influence the quality of the results. Therefore, we investigated which of the multitude of steps in processing the liver in the comet assay are most critical and have an impact on DNA migration. Among the eight parameters assessed, two (temperature and storage time) were chosen to determine how those steps impact the sensitivity of the assay in animals treated with ethyl methanesulfonate. A single oral dose of EMS was administered at dose levels of 50, 100 and 200 mg/kg, 3 h prior to necropsy. The results showed that sample cooling emerged as the predominant influence factor, whereas variations in other elements of the procedure (e.g. size of the liver piece sampled, time needed to proceed the liver tissue post-mortem, agarose temperature or time of lysis) seem to be of little relevance. Storing of liver samples of up to 6 hours under cooled conditions did not cause an increase in tail intensity. In contrast, storing the tissue in H buffer at room temperature, a considerable time-dependent increase in the comet parameters was observed. Comet assay parameters were substantially higher at all dose levels including the vehicle control. Likewise, while a dose response was still apparent, the sensitivity was much lower when expressed as fold change versus the concurrent negative controls