Using the HepaCometChip Assay for Broad‐Spectrum DNA Damage Analysis

Abstract

Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the “HepaCometChip” (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single‐strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay

Genotoxicity assessment in HepaRG™ cells as a new approach methodology follow up to a positive response in the human TK6 cell micronucleus assay: Naphthalene case study

“…Naphthalene was tested for DNA damage in the HepaRG™ CometChip® assay using a 3‐day repeat dosing protocol as detailed in Owiti et al, 2022. After the conduct of a dose range finder study using eight concentrations of naphthalene, 6 exposure concentrations of naphthalene were tested based on cytotoxicity for DNA damage ranging from 39 μM to 1.25 mM (Figure 2).…”

“…Naphthalene was tested for the induction of MN in HepaRG™ cells using a 3‐day repeat dosing protocol as detailed in Buick et al (2020) and Owiti et al (2022). After the conduct of a dose range finder study, 12 exposure concentrations of naphthalene were tested for MN induction ranging from 340 μM to 2.5 mM (Figure 3).…”

“…We suggest that S9 mediated positives from the in vitro MN assay can be followed in human relevant NAMs employing metabolic competent HepaRG™ cells with CometChip for DNA damage and the MN assay for chromosomal aberrations (Buick et al, 2020, 2021; Owiti et al, 2022), rather than the conduct of in vivo Comet and MN assays in rodents using up to 90 animals when conducted independently. We have also extended these traditional genetic toxicology endpoints in HepaRG™ cells with the transcriptomics TGx DDI biomarker to assess MOA (Buick et al, 2020, 2021).…”

“…A detailed protocol for the cell culture of NoSpin® HepaRG™ cells (Lonza, Durham NC), chemical exposures, and conduct of the CometChip® assay has been published (Buick et al, 2021; Owiti et al, 2022). The details regarding the conduct of HepaRG™ MN assay have been published in detail (Buick et al, 2020).…”

“…The details regarding the conduct of HepaRG™ MN assay have been published in detail (Buick et al, 2020). For the studies in this manuscript, we used the same 3‐day repeat exposure regimen as done previously and described in detail (Buick et al, 2020, 2021; Owiti et al, 2022). For the HepaRG™ CometChip® 6 concentrations of naphthalene were tested, for the HepaRG™ MN assay 12 concentrations of naphthalene were tested.…”