Unknown,Transcriptomics,Genomics,Proteomics

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

ABSTRACT: For the purpose of mechanism-based risk assessment, we investigated temporal concentration-dependent responses in cultures of PHH and HepaRG cells exposed to three drugs, acetaminophen (APAP), cyclosporine A (CSA) and valproic acid (VPA), that have a high liability for drug-induced liver injury. To facilitate the identification of early KEs and visualisation of their development over time, samples were collected at 4 time points, namely 8 and 24 hours (single exposure), 48 and 72 hours (daily repeated exposure), after exposure to a broad concentration range. Concentrations were selected based on the reported total Cmax of each drug. As these are approved drugs currently on the market, they are not expected to induce overt adverse effects around Cmax. Therefore, the selected maximum concentration was set at approximately 30x Cmax, whilst the minimum tested concentration was approximately 0.1x Cmax. The large concentration range allowed to apply benchmark concentration (BMC) modelling on individual genes as well as gene co-expression networks to derive in vitro transcriptomics benchmark concentrations (BMCs) and assess their suitability to be used as PoD in chemical risk assessment.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): synthetic construct

SUBMITTER: Steven Kunnen

PROVIDER: E-MTAB-12668 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Efforts to develop alternatives which can at least partially replace some of the currently used in vivo tests are ongoing. The recently ended FP6 European project carcinoGENOMICS had the goal to use the combination of toxicogenomics and in vitro cell culture models for identification of genotoxic- and non-genotoxic carcinogen-specific gene signatures. In this study is presented a part of the outcome of the project and in particular the performance of the gene classifier derived after exposure of the HepaRG cell line to prototypical hepatocarcinogens. Upon analyzing the data at a gene and a pathway level by using diverse biostatistical approaches, a clear-cut separation of the genotoxic from the non-genotoxic hepatocarcinogens and non-carcinogens was achieved (up to 88% correct prediction). The most characteristic pathway for genotoxic exposure was DNA damage. Further to show the robustness of the HepaRG model, the interlaboratory reproducibility of 3 blindly tested compounds was assessed. The results showed between 20% and 35% reproducibility. The subsequent classification of the 3 blindly tested compounds resulted in correct prediction of the genotoxicant, whereas the other two compounds were misclassified. In conclusion, the combination of transcriptomics and HepaRG in vitro cell model provides a solid basis for the detection of the genotoxic potential of unknown chemicals. HepaRG cells were exposed to 15 selected compounds for 72 hours, i.e. 5 GTX (N-nitrosomorpholine, NMP; Hydroquinone, HQO; Hydrazine dihydrochloride, HHC; 2-acetylaminofluorene, TAF; 2-amino-3-methylimidazo(4,5-f)quinoline, AMQ), 5 NGTX (Fumonisin B1, FMB; Cyclosporine A, CsA; Acetamide, ACE; Diethylhexyl Phthalate, DHP; Ethanol, ETH), 5 NC (4-acetylaminofluorene, FAF; D,L-Menthol, DLM; Benzoin, BEN; Benzyl Alcohol, BEA; Triclosan, TRI). The IC10 concentrations (reducing cell viability by 10%) were determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT test) (number of replicates n=3). Further RNA samples were generated by exposure of the HepaRG cells to the MTT-determined IC10 for a period of 24h and 72h. Complementary RNA targets were prepared and hybridized according to the manufacturer's procedures on high-density oligonucleotide microarrays (i.e. Affymetrix U133 Plus 2.0 GeneChip). Finally,alaysis was performed at a gene and pathway level. In the analysis, control (DMSO) samples were re-normalized with several sample groups and new processed data were created for each analysis. Thus, some of the Samples in this Series represent a re-analysis of other Samples and CEL files are identical.

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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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