A Modern Genotoxicity Testing Paradigm: Integration of the High-Throughput CometChip® and the TGx-DDI Transcriptomic Biomarker in Human HepaRG™ Cell Cultures
- DOI
- Language of the publication
- English
- Date
- 2021-08-18
- Type
- Article
- Author(s)
- Buick, Julie K.
- Williams, Andrew
- Meier, Matthew J.
- Swartz, Carol D.
- Recio, Leslie
- Gagné, Rémi
- Ferguson, Stephen S.
- Engelward, Bevin P.
- Yauk, Carole L.
- Publisher
- Frontiers Media
Abstract
Higher-throughput, mode-of-action-based assays provide a valuable approach to expedite chemical evaluation for human health risk assessment. In this study, we combined the high-throughput alkaline DNA damage-sensing CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq®, as an integrated genotoxicity testing approach. We used metabolically competent differentiated human HepaRG™ cell cultures to enable the identification of chemicals that require bioactivation to cause genotoxicity. We studied 12 chemicals (nine DDI, three non-DDI) in increasing concentrations to measure and classify chemicals based on their ability to damage DNA. The CometChip® classified 10/12 test chemicals correctly, missing a positive DDI call for aflatoxin B1 and propyl gallate. The poor detection of aflatoxin B1 adducts is consistent with the insensitivity of the standard alkaline comet assay to bulky lesions (a shortcoming that can be overcome by trapping repair intermediates). The TGx-DDI biomarker accurately classified 10/12 agents. TGx-DDI correctly identified aflatoxin B1 as DDI, demonstrating efficacy for combined used of these complementary methodologies. Zidovudine, a known DDI chemical, was misclassified as it inhibits transcription, which prevents measurable changes in gene expression. Eugenol, a non-DDI chemical known to render misleading positive results at high concentrations, was classified as DDI at the highest concentration tested. When combined, the CometChip® assay and the TGx-DDI biomarker were 100% accurate in identifying chemicals that induce DNA damage. Quantitative benchmark concentration (BMC) modeling was applied to evaluate chemical potencies for both assays. The BMCs for the CometChip® assay and the TGx-DDI biomarker were highly concordant (within 4-fold) and resulted in identical potency rankings. These results demonstrate that these two assays can be integrated for efficient identification and potency ranking of DNA damaging agents in HepaRG™ cell cultures.
Plain language summary
Health Canada is responsible for the evaluation of health risks caused by chemical exposure. Genotoxic chemicals can damage DNA, which can result in inherited genetic diseases and cancer. Thus, genotoxicity testing is a critical component of chemical testing. Current genotoxicity testing approaches seek to apply cells in culture that are relevant to humans and to integrate data from multiple assays to accurately identify genotoxic chemicals. The present study evaluated the applicability of an integrated approach including a high-throughput comet assay that enables measurement of levels of DNA breaks and the TGx-DDI biomarker to classify DNA damage-inducing (DDI) chemicals in human HepaRG™ cell cultures. The biomarker, developed previously in a different human cell line (i.e., TK6 cells), measures changes in 64 specific genes indicative of DNA damage. Further objectives of this study included additional validation of the performance of the biomarker in HepaRG™ cells using RNA sequencing (a technique used to measure changes in gene expression). HepaRG™ cells were exposed to increasing concentrations of 12 test chemicals (9 DDI and 3 non-DDI). Cell survival, DNA damage (by comet assay) and DDI chemical classification using the TGx-DDI biomarker were measured in exposed and control cells. The comet assay correctly identified 10/12 test chemicals, missing a DDI call for aflatoxin B1 and propyl gallate; whereas, the TGx-DDI accurately classified 10/12 chemicals, missing a positive call for zidovudine, and misclassifying eugenol as DDI at the highest test concentration. All non-DDI chemicals were correctly classified. When both assays were used in combination, 100% accuracy was reached in identifying chemicals that induce DNA damage, supporting their integration for genotoxicity assessment. This testing strategy provides information on how a chemical is damaging DNA in human cells in culture and enables accurate and rapid identification of DNA damaging chemicals, thereby supporting the human health risk assessment of chemicals that are potentially genotoxic.
Subject
- Health,
- Health and safety