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

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.

Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform

“…Although 3D spheroids expressed significantly higher mRNA levels and activities of CYP3A4 and 1A2, AFB1 induced greater cytotoxicity, but not significant DNA damage in 3D spheroids compared to 2D cultured cells. AFB1 at 2.5 and 5 μM induced statistically significant increases in % tail DNA in 2D HepaRG cells after a 3-h exposure, while extended exposures of 24-h and 52-h showed no significant increases in DNA damage (Buick et al, 2021;Le Hegarat et al, 2010). AFB1 is known to induce mainly bulky DNA damage (Weng et al, 2017), but the alkaline comet assay is not sensitive to detect potentially carcinogenic bulky adducts.…”

Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip® assay in TK6 cells

“…Initial development and validation of the TGx-DDI genomic biomarker was completed in human TK6 cells using Agilent microarray technology (Buick et al, 2015;Li et al, 2015;Williams et al, 2015;Yauk et al, 2016;Li et al, 2017). The biomarker has since been validated using other gene expression platforms, including qPCR (Cho et al, 2019), NanoString nCounter ® digital counting (Li et al, 2017) and RNA-Sequencing (Buick et al, 2020;Buick et al, 2021). Further validation demonstrated the effectiveness of the TGx-DDI biomarker using metabolically competent human HepaRG ™ cells (Buick et al, 2015;Buick et al, 2020;Buick et al, 2021).…”

“…Analysis was done at 4 h to align with the gene expression analysis experimental design. Control and exposed TK6 cells were loaded into the CometChip ® wells and were allowed to settle by gravitational forces into the microwells, as previously described (Wood et al, 2010;Buick et al, 2021). Briefly, the cells were encased in a 1% low melting point agarose overlay and then lysed in cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10 with 1% Triton X-100 and 10%…”

“…The TGx-DDI biomarker classifies agents as DDI or non-DDI based on the specific expression profiles of these biomarker genes. The biomarker has been validated in the presence and absence of metabolic activation (i.e., rat liver S9), in two different human cell lines (i.e., TK6 cells and HepaRG ™ cells), and using a number of different gene expression technologies, including DNA microarray, quantitative PCR arrays, HT digital detection (i.e., nCounter ® ) and RNA sequencing (i.e., AmpliSeq and TempO-Seq ® ) (Buick et al, 2015;Li et al, 2015;Li et al, 2017;Corton et al, 2018;Cho et al, 2019;Li et al, 2019;Buick et al, 2020;Buick et al, 2021). In addition, a HT screening approach has recently been reported using a direct-lysate nCounter ® approach (Chen et al, 2022).…”

A Collaborative Initiative to Establish Genomic Biomarkers for Assessing Tumorigenic Potential to Reduce Reliance on Conventional Rodent Carcinogenicity Studies

“…The Workgroup will capitalize on experience the group has with the TGx-DDI biomarker, a transcriptomic biomarker currently under regulatory review by the FDA through the Center for Drug Evaluation and Research Biomarker Qualification Program ( FDA, 2021 ). This 64-gene biomarker was developed to enable differentiating true positive DNA damage-inducing (DDI) agents from non-DDI irrelevant positive agents using human TK6 cells ( Li et al , 2015 , 2017 ) and human liver HepaRG cells ( Buick et al , 2020 , 2021 ; Corton et al , 2018 , 2019 ) and is composed mostly of genes that are under control of p53 ( Corton et al , 2019 ). Experience with the TGx-DDI biomarker by the HESI collaborative group has been invaluable in understanding the process required to build toward acceptance of any new biomarker for regulatory drug development applications, which first requires precisely defining and acquiring consensus on the need statement and regulatory context of use, followed by assessment of the benefits and risks of biomarker use ( Leptak et al , 2017 ).…”