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.

Keywords: CometChip; DNA damage; HepaCometChip; bulky lesions; comet assay; genotoxicity; metabolic activation; single-strand breaks.

Figure 1

Overview of the workflow for growing and treating cells for the CometChip assay. Schematic diagram depicting the major steps in HepaRG cell handling and chemical treatments described in Basic Protocol 1. Briefly, HepaRG cells are plated in 96‐well collagen‐coated plates and the medium is renewed. The cells are then chemically treated for 3 days, after which cells are harvested and the CometChip assay is performed. See text for details.

Figure 2

The CometChip Protocol. Schematic diagram depicting the major steps in the CometChip protocol following chemical treatment of HepaRG cells, as described in Basic Protocol 2. Cells are loaded onto the CometChip and excess cells washed away. The cells are then capped with low melting agarose and lysed overnight. Following cell lysis, the DNA is unwound and electrophoresed. Following electrophoresis, the DNA is stained and comet images are captured using a fluorescent microscope. See text for details.

Figure 3

Representative image of HepaRG cells. Image of HepaRG cells at D14 of differentiation (i.e., 28 days after seeding of undifferentiated cells). Two distinctive cell populations are observed: the well‐differentiated hepatocyte‐like cells (raised) that express a full array of human hepatocyte functions and the primitive biliary cells (flat). Scale bar: 100 μm.

Figure 4

HepaCometChip sample data showing HepaRG cells treated with EMS. (A) Representative comet images of HepaRG cells exposed to varying concentrations of ethyl methane sulfonate (EMS). Scale bar = 100 μm. (B) Dose response to EMS in HepaRG cells. % tail in DNA represented as the mean of four experiments ± SD.

Figure 5

HepaCometChip sample data showing HepaRG cells treated with B[a]P in the presence of HU and AraC. (A) Representative comet images of HepaRG cells exposed to no benzo[a]pyrene (B[a]P) and 8 μM B[a]P in the presence and absence of HU and AraC. Scale bar = 100 μm. (B) Dose response to B[a]P in HepaRG cells in the presence and absence of HU and AraC. % tail in DNA represented as the mean of four experiments ± SD.