3D Skin comet assay

Abstract

The comet assay is a widely used methodology, whose increased recognition for regulatory purposes led to the recent implementation of the “In Vivo Mammalian Alkaline Comet Assay OECD Testing Guideline” (OECD TG 489). Since it does not generally rely on proliferating cells, this methodology allows the investigation of DNA damage in any cell type or tissue which can be subjected to single cell isolation. Therefore, it is well suited to investigate effects of the first site of contact as well as organ-specific downstream effects, which are documented by the comet assay as primary DNA damage that may lead to clastogenic lesions or gene mutations. Increased DNA damage is signaled by increased migration of negatively charged DNA strands in the electric field which is recorded as increased fluorescence intensity, after appropriate staining of DNA. For the in vitro comet assay, so far, no regulatory guidelines are available. Considering the skin as the first site of contact of household products, pharmaceuticals, or cosmetics, the comet assay has been customized for the use with 3D reconstructed skin tissues, namely, the EpiDerm™ Full Thickness (EpiDerm™ FT; MatTek, MA) and the Phenion® Full-Thickness Skin Models. They consider both barrier function of the skin and organ- and species-specific metabolism, enabling exposure of chemicals relevant for the situation of use. A joint research project of five European and US-American laboratories has transferred and optimized the respective protocol to establish the 3D Skin Comet assay. First results of the ongoing validation, using Phenion® Full-Thickness Skin Models, have provided evidence for a good reproducibility and predictive capacity of the assay. In consequence, the 3D Skin Comet assay is considered a valuable new in vitro method for the assessment of genotoxicity of dermally exposed chemicals and drugs. It is intended to be used for following up on positive or equivocal results generated with dermally exposed substances in the standard in vitro genotoxicity tests, thereby filling a critical gap in the test battery.