A Modern Genotoxicity Testing Paradigm: Integration of the High-Throughput CometChip® and the TGx-DDI Transcriptomic Biomarker in Human HepaRG™ Cell Cultures.

Julie K Buick,Stephen S Ferguson,Matthew J Meier,Carole L Yauk,Andrew Williams,Carol D Swartz,Rémi Gagné,Bevin P Engelward,Leslie Recio

doi:10.3389/fpubh.2021.694834

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.

Highlights

New tools and approaches are urgently needed to allow regulatory agencies worldwide to evaluate a backlog of chemicals for potential adverse human health effects [1,2,3,4,5,6,7]
TGx biomarkers are useful for this purpose as they enable rapid extraction of mechanistic data from high-throughput transcriptomic (HTTr) data [36], which is more compelling when paired with a measure of DNA damage
Here we have combined a measure of DNA damage with the TGx-DDI genomic biomarker in physiologically-relevant human HepaRGTM cell cultures for hazard identification and quantitative analysis of genotoxic potential of DDI and non-DDI chemicals

Summary

Introduction

New tools and approaches are urgently needed to allow regulatory agencies worldwide to evaluate a backlog of chemicals for potential adverse human health effects [1,2,3,4,5,6,7]. Twenty-first century toxicology requires more affordable tests that are higherthroughput, higher-content, human-relevant, and mechanistic in nature for effective chemical evaluation [8,9,10,11,12,13]. Transcriptomic biomarkers are defined gene sets that produce reproducible changes for altered key events in adverse outcome pathways. These biomarkers can be used to identify chemical mode of action (MoA) and to guide chemical prioritization and classification [14,15,16,17,18,19]. The use of in vitro genomic biomarkers to predict specific toxicological responses reduces the subjectivity of interpretation for complex genomic data sets and can facilitate the use of genomics for human health risk assessment

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