Alkaline Comet Assay to Assess Genotoxicity in Zebrafish Larvae

The zebrafish (Danio rerio) is a model organism widely used in several research fields due to its characteristics and numerous advantages, such as optical embryo transparency, fully sequenced genome, orthologous genes to humans, small size, high reproductive rate, easy gene editing and relatively low costs. Thus, a number of protocols have been developed that allow the use of this vertebrate model for toxic effect evaluation at various biological levels, including genotoxicity, using the comet assay technique.The comet assay or single-cell gel electrophoresis is a popular and sensitive method to study DNA damage in cells, which is described in this chapter. Briefly, cells suspended in agarose on a microscope slide are lysed, denatured, electrophoresed, neutralized, and stained to study the migration of DNA strand breaks. As a result, cells with increased DNA damage present a high fluorescence intensity and an increase of comet tail length. For the visual score, comets are classified according to the head integrity, tail intensity, and tail length into five classes, namely, class 0 until class 4 (comets with high damage and with almost all the DNA in the tail). These data are used to calculate the Genetic Damage Index (GDI) expressed as Arbitrary Units (AU).