Chapter 17: Application of the Comet Assay in Nanotoxicology

The comet assay is an excellent and widely applied method for investigating genotoxicity of engineered nanomaterials (NMs). A great challenge with NMs is interference between the NM and the test assay, leading to a risk of obtaining unreliable results. Here, we describe both the standard alkaline version of the comet assay in vitro and the modified version that allows detection of oxidized DNA lesions by applying lesion-specific endonucleases (e.g. formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing NM genotoxicity, as well as basic methodological considerations, such as the importance of carrying out physico–chemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design—including treatment conditions, cell number, cell culture plate format and volume of treatment medium on the plate—is crucial and can have an impact on results, especially when testing NMs. Toxicity of NMs depends greatly upon physio–chemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the high-throughput miniaturized version of the Comet assay is essential. Protocols are provided for both standard 2-gel system and 12-gel format slides.