A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment

Abstract

Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.

Three-dimensional HepaRG spheroids as a liver model to study human genotoxicity in vitro with the single cell gel electrophoresis assay

“…However, etoposide ETO was shown to be genotoxic using the highly predictive qPCR array (microarray prediction of 0.97) on 2D HepaRG cells. This result was obtained on a selection of 84 genes after 72 hrs of treatment with 30 µM ETO every day 10 . The discrepancy of the results between the two studies could be explained by the higher concentration and the longer time of exposure used in the later, as treatment for more than 24 hrs could affect the cell cycle of HepaRG cells.…”

“…This phenotype is stable in culture for several weeks and responds to various inducers 7–9 . Moreover, this model was shown to be suitable to detect genotoxic compounds with various tests: comet assay, micronucleus, γH2AX, as well as genotoxicity-targeted qPCR array 2,10–14 . However, some promutagen compounds have remained difficult to detect in 2D HepaRG cells, such as styrene and the aromatic amines IQ, MeIQX and 2,4-DAT, possibly due to low CYP2E1 and CYP1A2 activities and N-acetyl-, or sulfotransferase (NAT, SULT) activities 2,13,15 .…”

Assessment of the performance of the TGx‐DDI biomarker to detect DNA damage‐inducing agents using quantitative RT‐PCR in TK6 cells

“…Recently, Ates et al developed a 96‐well qPCR array for analyzing a proprietary gene expression signature of genotoxicity in HepaRG cells (Ates et al, ). While the experimental approach is similar to our study, this gene expression signature is analyzed after a 72 h exposure based on a reference set of known in vivo genotoxicants that contains aneugens (e.g., vinblastine) that are included in the non‐DDI category of the TGx‐DDI reference set.…”

Considerations for strategic use of high-throughput transcriptomics chemical screening data in regulatory decisions

“…High-throughput transcriptomics (HTTr) is a type of HTS that uses gene expression profiling as a highly-multiplexed endpoint for rapidly evaluating the biological effects of large numbers of chemicals. A variety of high-throughput transcriptomic technologies can be categorized by the breadth of genes measured, ranging from dozens of genes using qRT-PCR arrays [6, 7], hundreds or thousands of genes using targeted transcriptomic panels (i.e. L1000, S1500+) [8, 9], or the whole transcriptome using microarrays, RNA-Seq or targeted RNA-Seq [10, 11].…”