Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Ngo, Le P. et al. “Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates.” Nucleic Acids Research 48 (2020): e13 © 2020 The Author(s) As Published 10.1093/nar/gkz1077 Publisher Oxford University Press (OUP) Version Final published version Citable link https://hdl.handle.net/1721.1/124825 Terms of Use Creative Commons Attribution 4.0 International license Detailed Terms https://creativecommons.org/licenses/by/4.0/ Published online 11 December 2019 Nucleic Acids Research, 2020, Vol. 48, No. 3 e13 doi: 10.1093/nar/gkz1077 Sensitive CometChip assay for screening potentially carcinogenic DNA adducts by trapping DNA repair intermediates Le P. Ngo 1, Norah A. Owiti1, Carol Swartz2, John Winters2, Yang Su3,JingGe1, Aoli Xiong4, Jongyoon Han1,4,5, Leslie Recio2, Leona D. Samson1,3 and Downloaded from https://academic.oup.com/nar/article-abstract/48/3/e13/5661085 by MIT Libraries user on 05 March 2020 Bevin P. Engelward1,* 1Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, 2Toxicology Program, Integrated Laboratory Systems, Inc., Research Triangle Park, NC 27560, USA, 3Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA, 4BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology, 138602 Singapore and 5Department of Electrical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received October 29, 2018; Revised October 08, 2019; Editorial Decision October 27, 2019; Accepted November 19, 2019 ABSTRACT thus provides a broadly effective approach for detec- tion of bulky DNA adducts. Genotoxicity testing is critical for predicting ad- verse effects of pharmaceutical, industrial, and en- vironmental chemicals. The alkaline comet assay INTRODUCTION is an established method for detecting DNA strand Injury to genetic material can lead to debilitating heritable breaks, however, the assay does not detect poten- diseases, cancer, neurodegeneration and accelerated aging tially carcinogenic bulky adducts that can arise when (1–4). Therefore, regulatory agencies worldwide require that metabolic enzymes convert pro-carcinogens into a all pharmaceuticals be tested for their genotoxic potential highly DNA reactive products. To overcome this, we (https://www.fda.gov/media/71980/download). In contrast, use DNA synthesis inhibitors (hydroxyurea and 1- despite the fact that >2000 new chemicals are being ␤-D-arabinofuranosyl cytosine) to trap single strand produced by industry every year (https://ntp.niehs.nih.gov/ breaks that are formed during nucleotide excision re- annualreport/2017/2017annualreportdownloadpdf.pdf), pair, which primarily removes bulky lesions. In this the vast majority of these industrial chemicals have not way, comet-undetectable bulky lesions are converted been tested for their genotoxic potential. A major barrier into comet-detectable single strand breaks. More- to such testing is the need for a high throughput (HT) sensitive assay for DNA damage in mammalian cells (5). over, we use HepaRG™ cells to recapitulate in vivo Although there have been recent advances in HT assays for metabolic capacity, and leverage the CometChip plat- genotoxicity (6), most of these technologies depend on in- form (a higher throughput more sensitive comet as- direct measures of DNA damage, such as phosphorylation say) to create the ‘HepaCometChip’, enabling the de- of histones [e.g. ␥H2AX formation (7)] or gene induction tection of bulky genotoxic lesions that are missed by [i.e. p53 activation (8,9)]. While there are several methods current genotoxicity screens. The HepaCometChip for direct detection of DNA damage (e.g. alkaline elution *To whom correspondence should be addressed. Tel: +1 617 258 0260; Fax: +1 617 258 0499; Email: [email protected] Present addresses: Le P. Ngo, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Norah A. Owiti, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Carol Swartz, Toxicology Program, Integrated Laboratory Systems, Inc., Research Triangle Park, NC 27560, USA. John Winters, Toxicology Program, Integrated Laboratory Systems, Inc., Research Triangle Park, NC 27560, USA. Yang Su, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Jing Ge, Navigant Consulting, Boston, MA 02110, USA. Aoli Xiong, BioSystems and Micromechanics, Singapore-MIT Alliance for Research and Technology, 138602 Singapore. Jongyoon Han, Department of Biological Engineering and Department of Electrical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Leslie Recio, Toxicology Program, Integrated Laboratory Systems, Inc., Research Triangle Park, NC 27560, USA. Leona D. Samson, Department of Biological Engineering and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Bevin P. Engelward, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. C The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. e13 Nucleic Acids Research, 2020, Vol. 48, No. 3 PAGE 2 OF 17 and mass spectrometry), these assays are laborious and enzymes in the BER pathway can act independently, such low throughput. The alkaline comet assay is a promising that the subsequent SSB resolution steps are rate-limiting platform as it detects single-strand breaks (SSBs; for a once the damaged base has been removed (28–30), leading list of abbreviations, see Supplemental Table S1), abasic to an accumulation of SSB intermediates that are detectable sites and other alkali sensitive sites. However, the assay using the comet assay (27). has a critical blind spot, due to its inability to detect bulky While many base lesions are repaired by BER, bulky DNA lesions, a class of lesions that are often carcinogenic lesions are repaired primarily by nucleotide excision re- (3,10,11). Here, we describe methods to overcome this pair (NER) (10,11,31,32)(Figure2A). The NER pathway limitation. is highly coordinated in both prokaryotes and eukaryotes (31,33). In eukaryotes, the NER pathway operates by as- Downloaded from https://academic.oup.com/nar/article-abstract/48/3/e13/5661085 by MIT Libraries user on 05 March 2020 sembling more than a dozen different proteins prior to com- The comet assay mencing repair (34). As such, once repair is initiated, the The comet assay is an established method for detecting process is extremely efficient, thus minimizing the presence DNA strand breaks, and is based upon the underlying prin- of SSB intermediates (35). Specifically, NER involves two ciple that fragmented DNA migrates more readily through major steps: endonucleolytic cleavage 5 to the adduct, re- an agarose matrix under electrophoresis compared to intact pair synthesis and endonucleolytic cleavage 3 to the adduct DNA. The comet assay works because nuclear DNA is nor- (11,35), which together result in removal of an oligonu- mally highly supercoiled and thus does not readily migrate, cleotide containing the offending lesion. The incision 5 to while loops and fragments migrate more readily through the damage site is made by the ERCC1-XPF endonuclease the agarose matrix (12,13). The result is a comet-like shape, and is followed by recruitment of DNA polymerases and where the percent DNA in the comet tail is proportional to initiation of DNA repair synthesis. Repair synthesis cre- the levels of DNA strand breaks. ates a flap 3 to the original lesion, which is then cleaved While the comet assay is relatively simple and sensi- by the structure-specific endonuclease XPG, prior to com- tive, it is low-throughput, it has poor reproducibility, and pletion of repair synthesis and ligation (36,37). NER SSB the imaging and analysis methods are laborious. To over- intermediates can be detected using the alkaline comet as- come these limitations, the CometChip was previously de- say (38–40), but the required preassembly of the repair com- veloped (14,15). The basis for the CometChip is an agarose plex means that the signal is very weak due to the ephemeral microwell array. Briefly, cells are loaded into microwells by nature of the NER SSB intermediates. gravity, and excess cells are removed by shear force (Figure 1). By creating a mammalian cell microarray, overlapping Detecting bulky lesions using CometChip comets are prevented, and the comets lie on a shared focal plane. As a result, it is possible to capture multiple comets Here, we exploit methods for inhibiting repair synthesis (>50) in a single image rather than imaging each comet indi- as a means for prolonging the presence of NER SSB in- vidually as is done for the traditional comet assay. With au- termediates. Specifically, our approach is to use hydrox- tomated image analysis and reduced experimental noise, the yurea (HU) and 1-␤-D-arabinofuranosyl cytosine (AraC),