Dequenching after photobleaching cytotoxicity test

DAP assay is a quantitative live cell cytotoxicity assay based on measurements on DNA alterations, which are assessed by the ability of DNA fluorophobes to establish different toxicity-dependent quenching levels in cells. The technology used is based on fluorescence Dequenching After Photobleaching. Therefore, DAP is a mechanistic-based assay that addresses DNA alterations in association (or not) with cell death. The protocol used is straightforward (single-step, no washings) and has been set up in HTS cell culture formats (96/384 well microplates). Mammalian or fish cell models (suspension or adherent) have been used e.g. HepG2, Caco-2 and primary cells. The results are expressed as IC50 values calculated from the concentration response curves using the following sigmoidal dose-response regression model: Y=Bottom + (Top-Bottom)/[1+10^(LogIC50-X)]. The following prediction model was established: Log(human blood LC50) = 0.967 x Log(DAP IC50) - 0.824.

The DAP technology is presented as potentially applicable to several toxicological areas such as acute toxicity, genotoxicity, chronic toxicity and carcinogenicity. The immediate application could be for human acute toxicity, although this has not been explicitly shown. For the other areas of application experimental data are lacking.