Abstract
BackgroundModern 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.MethodsHepaRG™ 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.ResultsA 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.ConclusionsThis 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.
Highlights
Twenty-first century toxicology necessitates alternative test methods that are more efficient and effective to evaluate the backlog of chemicals requiring assessment [1,2,3,4,5,6]
The present study investigates the performance of the TGx-DDI biomarker in HepaRGTM cells using RNA-Seq
Human HepaRGTM cells were exposed to increasing concentrations of 10 chemicals, five of which are wellcharacterized for their ability to cause DNA damage
Summary
Twenty-first century toxicology necessitates alternative test methods that are more efficient and effective to evaluate the backlog of chemicals requiring assessment [1,2,3,4,5,6]. In vitro genotoxicity tests have been performed in rodent cell lines such as CHO, V79, CHL, and L5178Y, in addition to human TK6 lymphoblastoid cells and peripheral blood lymphocytes [19, 20] These cell types have various limitations, a significant one being their lack of xenobiotic metabolism required for both activation and detoxification [21]. A generation in vitro testing strategy would benefit from the use of human-relevant cell models with metabolic capabilities that more effectively mimic in vivo metabolism without the potential complications and limitations of exogenous S9 addition or the use of PHHs [30, 33,34,35]. 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
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