Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates

Abstract

Genotoxicity testing is critical for predicting adverse effects of pharmaceutical, industrial, and environmental chemicals. The alkaline comet assayis an established method for detecting DNA strandbreaks, however, the assay does not detect potentially carcinogenic bulky adducts that can arise whenmetabolic enzymes convert pro-carcinogens into ahighly DNA reactive products. To overcome this, weuse DNA synthesis inhibitors (hydroxyurea and 1-β-d-arabinofuranosyl cytosine) to trap single strandbreaks that are formed during nucleotide excision repair, which primarily removes bulky lesions. In thisway, comet-undetectable bulky lesions are convertedinto comet-detectable single strand breaks. Moreover, we use HepaRG™cells to recapitulatein vivometabolic capacity, and leverage the CometChip platform (a higher throughput more sensitive comet as-say) to create the ‘HepaCometChip’, enabling the detection of bulky genotoxic lesions that are missed bycurrent genotoxicity screens. The HepaCometChip thus provides a broadly effective approach for detection of bulky DNA adducts.