High-throughput screening strategy for photogenotoxic potential of pharmaceutical substances using fluorescent intercalating dye

The aim of the present study was to provide an intercalator-based photogenotoxicity (IBP) assay as a high-throughput in vitro screening system for predicting the photogenotoxic potential of pharmaceutical substances. The conditions of the high-throughput IBP assay using thiazole orange (TO), a fluorescent intercalating dye, were optimized and validated by a fluorescence titration experiment and reproducibility/robustness test. The IBP assay was applied to 27 phototoxic and 5 weak/non-phototoxic commercially available compounds, and other phototoxicity screenings were also carried out for comparison; these included the reactive oxygen species (ROS) assay for overall phototoxic potential and the DNA-photocleavage assay for photogenotoxic risk. According to the results from the comparative experiments, a decreased level of intercalated TO in the IBP assay could theoretically be related to the DNA-photocleaving behaviors of phototoxic drugs, but not to their ROS-generating abilities. The IBP assay could predict the photodynamic DNA impairment caused by irradiated drugs with a prediction accuracy of 78%. These findings suggest that the IBP assay could be a fast and reliable tool for predicting the photogenotoxic potential of a large number of drug candidates at early stages of drug discovery.