High-Throughput Transcriptomics Screen of ToxCast Chemicals in U-2 OS Cells

New approach methodologies (NAMs) aim to accelerate the pace of chemical risk assessment while simultaneously reducing cost and dependency on animal studies. High Throughput Transcriptomics (HTTr) is an emerging NAM in the field of chemical hazard evaluation for establishing in vitro points-of-departure and providing mechanistic insight. In the current study, 1,201 test chemicals were screened for bioactivity at eight concentrations using a 24-hour exposure duration in the human- derived U-2 OS osteosarcoma cell line with HTTr. Assay reproducibility was assessed using three reference chemicals that were screened on every assay plate. The resulting transcriptomics data were analyzed by aggregating signal from genes into signature scores using gene set enrichment analysis, followed by concentration-response modeling of signatures scores. Signature scores were used to predict putative mechanisms of action, and to identify biological pathway altering concentrations (BPACs). BPACs were consistent across replicates for each reference chemical, with replicate BPAC standard deviations as low as 5.6×10-3 µM, demonstrating the internal reproducibility of HTTr-derived potency estimates. BPACs of test chemicals showed modest agreement (R2 = 0.55) with existing phenotype altering concentrations from high throughput phenotypic profiling using Cell Painting of the same chemicals in the same cell line. Altogether, this HTTr based chemical screen contributes to an accumulating pool of publicly available transcriptomic data relevant for chemical hazard evaluation and reinforces the utility of cell based molecular profiling methods in estimating chemical potency and predicting mechanism of action across a diverse set of chemicals. In a previous study, we established experimental and computational workflows for HTTr screening in a human cell model (Harrill et al., 2021). The TempO-Seq human whole transcriptome assay (Yeakley et al., 2017) was used to identify biological pathway altering concentrations (BPACs) and characterize the biological activity of test chemicals. The present study applies those methods to a much larger chemical space. Here, we screened 1201 unique chemicals from the ToxCast chemical collection in U-2 OS cells using HTTr and the similar experimental conditions as described previously (Harrill et al., 2021).