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A Transcriptomic Biomarker to Classify Chemical-Induced Histone Deacetylase Inhibition in Human HepaRG Cells

ABSTRACT: New approach methods (NAMs), such as human-derived in vitro models integrated with transcriptomic biomarkers, offer an efficient, animal-free means to predict toxic responses. Biomarkers including GENOMARK and TGx-DDI enable high-throughput transcriptomic analysis and accurate genotoxicant classification. Histone deacetylase inhibitors (HDACi), a class of epigenotoxicants, disrupt chromatin structure and gene expression, which may lead to impaired DNA repair, apoptosis, and cell cycle perturbation. A transcriptomic biomarker (TGx-HDACi) was previously developed in human TK6 cells to classify HDACi; however, its performance in other cell types is suboptimal. Herein, we conducted a biomarker discovery study to identify a transcriptomic signature capable of discriminating HDACi from non-HDACi compounds in human HepaRG cells, a metabolically competent hepatic cell line increasingly used in genotoxicity testing. HepaRG cells were exposed to 22 reference chemicals (8 HDACi, 14 non-HDACi) at five increasing concentrations in a 72-hour repeated exposure design, and whole transcriptome profiling was performed using TempO-Seq with maximum concentrations selected based on cytotoxicity and solubility thresholds. One concentration per compound was used for biomarker development. A nearest shrunken centroid (NSC) approach identified a 102-gene panel (TGx-HDACi-HepaRG) that achieved 100% predictive accuracy by cross-validation and correctly classified compounds across multiple independent analytical approaches. Concentration-response evaluation further demonstrated the biomarker’s ability to identify exposure levels associated with HDAC inhibition. These findings establish TGx-HDACi-HepaRG as a promising transcriptomic biomarker for epigenotoxicity screening in a metabolically competent human liver model. As this study represents the biomarker discovery phase, future work will focus on external validation using independent chemical sets and case studies to support its application in mechanism-informed chemical risk assessment.

ORGANISM(S): Homo sapiens

PROVIDER: GSE341318 | GEO | 2026/07/27

REPOSITORIES: GEO

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Project description:Background: Modern testing paradigms seek to apply human-relevant cell culture models and integrate data from multiple test systems to accurately inform potential hazards and modes of action for chemical toxicology. In genetic toxicology, the use of metabolically competent human hepatocyte cell culture models provides clear advantages over other more commonly used cell lines that require the use of external metabolic activation systems, such as rat liver S9. HepaRG™ cells are metabolically competent cells that express Phase I and II metabolic enzymes and differentiate into mature hepatocyte-like cells, making them ideal for toxicity testing. We assessed the performance of the flow cytometry in vitro micronucleus (MN) test and the TGx-DDI transcriptomic biomarker to detect DNA damage-inducing (DDI) chemicals in human HepaRG™ cells after a 3-day repeat exposure. The biomarker, developed for use in human TK6 cells, is a panel of 64 genes that accurately classifies chemicals as DDI or non-DDI. Herein, the TGx-DDI biomarker was analyzed by Ion AmpliSeq whole transcriptome sequencing to assess its classification accuracy using this more modern gene expression technology as a secondary objective. Methods: HepaRG™ cells were exposed to increasing concentrations of 10 test chemicals (six genotoxic chemicals, including one aneugen, and four non-genotoxic chemicals). Cytotoxicity and genotoxicity were measured using the In Vitro MicroFlow® kit, which was run in parallel with the TGx-DDI biomarker. Results: A concentration-related decrease in relative survival and a concomitant increase in MN frequency were observed for genotoxic chemicals in HepaRG™ cells. All five DDI and five non-DDI agents were correctly classified (as genotoxic/non-genotoxic and DDI/non-DDI) by pairing the test methods. The aneugenic agent (colchicine) yielded the expected positive result in the MN test and negative (non-DDI) result by TGx-DDI. Conclusions: This next generation genotoxicity testing strategy is aligned with the paradigm shift occurring in the field of genetic toxicology. It provides mechanistic insight in a human-relevant cell-model, paired with measurement of a conventional endpoint, to inform the potential for adverse health effects. This work provides support for combining these assays in an integrated test strategy for accurate, higher throughput genetic toxicology testing in this metabolically competent human progenitor cell line.

Project description:Introduction: 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 CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq® as an integrated genotoxicity testing approach. Objective: To determine if the high-throughput CometChip® assay and TGx-DDI biomarker analysis can be combined using human HepaRG™ cells to provide an efficient next-generation genotoxicity screening approach to identify DNA damage-inducing (DDI) chemicals. Methods: Metabolically competent human HepaRG™ cell cultures were exposed to increasing concentrations of 12 chemicals (9 DDI and 3 non-DDI) using a repeat exposure design (0 hr, 24 hr, 48 hr) in 96-well plates to measure and classify chemicals based on their ability to damage DNA. After a 4 hr recovery period, exposed cells were used to perform the Trevigen CometChip assay to assess DNA damage or used for TempO-Seq® S1500+ Targeted Transcriptome Sequencing for TGx-DDI classification purposes. Bioinformatics and statistical tools were used to classify chemicals as DDI or non-DDI using three separate analyses. Benchmark concentration analysis was also conducted for both assays for the purposes of potency ranking. Results: In combination, the CometChip® assay and the TGx-DDI 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 for all 12 chemicals were highly concordant (within four-fold) and resulted in identical potency rankings. Conclusions: These results suggest that these two assays can be integrated for efficient, high-throughput identification of DNA damage in HepaRG™ cells. This study provides evidence for the complementarity of the high-throughput CometChip® assay with TGx-DDI biomarker analysis. Integration of these two tests provides an effective and high-throughput approach to identify DDI agents. This work is a first step in accomplishing a more integrated genotoxicity testing strategy to derive mechanistic information to better inform human health risk assessment in a high-throughput manner.

Project description:The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion paper, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or non-genotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and non-genotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4h and collected 0h, 4h and 20h post-exposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24h. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as non-genotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24h, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the classifier is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells. This experiment examined the whole genome transcriptional response of TK6 cells exposed to phenobarbital for 4 hours followed by recovery times of 4h and 20h in fresh media (8h and 24h time points, respectively) at 3 different concentrations, including a low (1 mM), a medium (3.2 mM), and a high (10 mM) concentration, in addition to a vehicle control (+S9). Each concentration and time point had 3 biological replicates, which were pooled for gene expression analysis. There were a total of 6 samples (12 arrays) included in the final analysis using a two-colour dye swap design.

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A Transcriptomic Biomarker to Classify Chemical-Induced Histone Deacetylase Inhibition in Human HepaRG Cells

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