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Nucleic Acids Res. 2020 Feb 20;48(3):e13. doi: 10.1093/nar/gkz1077.

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

Author information

1
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
2
Toxicology Program, Integrated Laboratory Systems, Inc., Research Triangle Park, NC 27560, USA.
3
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
4
BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology, 138602 Singapore.
5
Department of Electrical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Abstract

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

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