Convenient, multi-well plate-based DNA damage response analysis using DT40 mutants is applicable to a high-throughput genotoxicity assay with characterization of modes of action.

Chemists continually synthesize myriad new chemicals (∼2,000/year), some of which make their way into the environment or otherwise pose possible threats to humans who potentially become exposed to the compounds. Regulators must determine whether these, along with the glut (∼80,000) of existing, chemicals are toxic and at what exposure levels. An important component of this determination is to ascertain the mode of action (MOA) of each compound as it relates to the pathway the compound uses to induce genotoxicity. Several assays have traditionally been used to reveal these effects to the genome: the Ames test, tests with yeast and mammalian cell lines, and animal studies. Previously, we described a new multi-well plate-based method which makes use of the DT40 isogenic cell line and its dozens of available mutants knocked out in DNA repair and cell cycle pathways and we now provide a detailed protocol of the further improvement of the assay. Although the DT40 line has existed for some time and has been used in numerous studies of DNA repair pathways, little use has been made of this valuable resource for toxicological investigations. Our method introduces the 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide dye scheme determination of cell survival in a manner that greatly increases throughput and reduces cost while maintaining reasonable sensitivity. Although this new genotoxicity assay requires validation with many more mutagens before becoming an established, regulatory decision-making analysis tool, we believe that this method will be very advantageous if eventually added to the repertoire of those investigating MOAs of potentially genotoxic substances.