Performance of the comet assay in a high-throughput version

Abstract

The high-throughput comet assay was developed to reduce the processing time and to increase sample-throughput of the assay as described by Tice et al. (RR. Tice, E. Agurell, D. Anderson, B. Burlinson, A. Hartmann, H. Kobayashi, Y. Miyamae, E. Rojas, JC. Ryu, YF. Sasaki. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing, Environ. Mol. Mutagen.35 (2000) 206-221). This high-throughput version allows for the processing of up to 400 samples per day. The basis of the new assay is a 96-well plate (multichamber plate, MCP) suitable for electrophoresis. After exposure of the cells to genotoxic agents, the walls of the MCP are separated from the bottom plate. All 96 samples together then go through lysis, alkaline unwinding, electrophoresis, neutralization, and staining. In this study, the first concentration-dependent results obtained with the high-throughput version are shown and a comparison is made with the standard version of the comet assay using five representative chemicals with different genotoxic properties. These genotoxic chemicals are methyl methanesulfonate (MMS) and ethylnitrosourea, which form small alkylation adducts, 4-nitroquinoline-1-oxide for bulky adducts, cisplatin for DNA cross-links, and H 2O 2 for direct DNA breakage. For medium and high effective concentrations a standard deviation of 3–20% for three replicates (25 comets per sample) was determined. A comparison of the standard assay with the high-throughput version revealed similar results for MMS and H 2O 2. The integrated viability assay (FDA assay), which was performed after chemical treatment and before detachment of the bottom from the walls of the MCP, did not influence the outcome of the comet formation. In conclusion, the high-throughput version of the comet assay facilitates the determination of genotoxicity in cases where large numbers of samples have to be measured, such as during testing of industrial chemicals, biomonitoring of environmental samples, and early screening of drug candidates for genotoxicity/photogenotoxicity. For such applications the cost- and time-saving of the high-throughput method provides substantial advantages over the standard comet assay.