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Targeted RNA-seq (TempO-Seq) of temporal concentration responses to drug exposures in PHH and HepaRG cells

ABSTRACT: Targeted RNA-seq (TempO-Seq) of temporal concentration responses to drug exposures in PHH and HepaRG cells

PROVIDER: PRJEB60510 | ENA |

REPOSITORIES: ENA

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			Action	DRS
		Other
	ERR10982100.fastq.gz	Fastqsanger.gz
	ERR10982200.fastq.gz	Fastqsanger.gz
	ERR10982400.fastq.gz	Fastqsanger.gz
	ERR10982500.fastq.gz	Fastqsanger.gz

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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 human hepatic HepaRG cell line is often considered as the closest surrogate to primary culture of normal human hepatocytes (PHH) for toxicity studies. However, differentiated HepaRGcells express very low levels of the cytochrome P450 2D6 (CYP2D6) protein, which is essential for the biotransformation of nearly 25% of drugs on the market. To overcome this limitation, infection of progenitor HepaRG cells were performed using lentiviral particles containing a transgene encoding a single mRNA translated into a polypeptide undergoing proteolytic cleavage via the T2A peptide to produce both human CYP2D6 and GFP proteins. Differentiated HepaRG cells transduced with this lentivirus stably expressed GFP and catalytically active CYP2D6 enzyme at levels close to those found in PHH with high CYP2D6 activities. Using the CYP2D6 transgenic HepaRG cells, we showed that tramadol was metabolized into both, N- and O-desmethyl tramadol as observed in human serum in contrast with the production of N-desmethyl tramadol only in parental HepaRG cells via the CYP3A4 activity. Similarly, after perhexiline (PHX) treatments, higher IC50 were found in CYP2D6 expressing HepaRG cells associated to lower mitochondrial damages compared to those found in parental cells for the same PHX concentrations. Gene profiling between parental and transgenic cells demonstrated that the CYP2D6 expressing HepaRG cells had kept their ability to proliferate and differentiate with low impact on the expression of the hepatocyte specific functions, however, we identified a limited set of genes including NXF3 and TRIM63 that were up-regulated in CYP2D6 expressing HepaRG cells. By knocking-down GFP and CYP2D6 expression using CRISP/Cas9 technology in the transgenic cell line, we demonstrated that NXF3 and TRIM63 induction was triggered by the mRNA encoding GFP and CYP2D6 but not by the lentiviral transgene inserted into the HepaRG cell genome. Together, these data confirmed that the CYP2D6 transgenic HepaRG cells represent a suitable optimized transgenic model of HepaRG cells to evaluate biotransformation and toxicity of specific compounds metabolized by CYP2D6.

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Targeted RNA-seq (TempO-Seq) of temporal concentration responses to drug exposures in PHH and HepaRG cells

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