High-throughput Transcriptomics Screen of ToxCast Chemicals in HepaRG Cells

With thousands of chemicals in commerce and the environment, rapid identification of potential hazards is a critical need. Combining broad molecular profiling with targeted in vitro assays, such as high-throughput transcriptomics (HTTr) and receptor screening assays, could improve identification of chemicals that perturb key molecular targets associated with adverse outcomes. We aimed to link transcriptomic readouts to individual molecular targets and integrate transcriptomic predictions with orthogonal receptor-level assays in a proof-of-concept framework for chemical hazard prioritization. Transcriptomic profiles generated via TempO-Seq in U-2 OS and HepaRG cell lines were used to develop signatures comprised of genes uniquely responsive to reference chemicals for distinct molecular targets. These signatures were applied to 75 reference and 1,126 non-reference chemicals screened via HTTr in both cell lines. Selective bioactivity towards each signature was determined by comparing potency estimates against the bulk of transcriptomic bioactivity for each chemical. Chemicals predicted by transcriptomics were confirmed for target bioactivity and selectivity using available orthogonal assay data from US EPA’s ToxCast program. A subset of 37 selectively acting chemicals from HTTr that did not have sufficient orthogonal data were prospectively tested using one of five receptor-level assays. Of the 1,126 non-reference chemicals screened, 201 demonstrated selective bioactivity in at least one transcriptomic signature, and 57 were confirmed as selective nuclear receptor agonists. Chemicals bioactive for each signature were significantly associated with orthogonal assay bioactivity, and signature-based points-of-departure were equally or more sensitive than biological pathway altering concentrations in 81.2% of signature-prioritized chemicals. Prospective profiling found that 18 of 37 (49%) chemicals without prior orthogonal assay data were bioactive against the predicted receptor. Our work demonstrates that integrating transcriptomics with targeted orthogonal assays in a tiered framework can support Next Generation Risk Assessment by informing putative molecular targets and prioritizing chemicals for further testing. NOTE: this GEO entry only includes the HepaRG cell line data; the U-2 OS cell line data can be located via GEO accession number GSE274318. High-throughput transcriptomics (HTTr) screens of 1,201 chemicals in U-2 OS and HepaRG cell lines were used as source data for all transcriptomic analyses. Both screens utilized the same experimental design and bioinformatics pipeline for initial derivation of transcriptional potency values. In brief, DMSO-solubilized chemical stock solutions were provided frozen from the US EPA ToxCast chemical inventory management contractor (EvoTec, Princeton, NJ), and stored at -80°C prior to dose plate preparation. U-2 OS osteosarcoma and HepaRG hepatocyte cell lines were used for chemical exposure. Chemicals were tested at 8 nominal concentrations ranging from 0.03—100 µM using 0.5-log spacing with a final DMSO concentration of 0.05%. All exposures were conducted in triplicate using independent cell cultures. Transcriptomic profiles generated via TempO-Seq in U-2 OS and HepaRG cell lines were used to develop signatures comprised of genes uniquely responsive to reference chemicals for distinct molecular targets. These signatures were applied to 75 reference and 1,126 non-reference chemicals screened via HTTr in both cell lines. Selective bioactivity towards each signature was determined by comparing potency estimates against the bulk of transcriptomic bioactivity for each chemical. NOTE: this GEO entry only includes the HepaRG cell line data; the U-2 OS cell line data can be located via GEO accession number GSE274318.