High-throughput Transcriptomics Screen of 137 Per- and Polyfluoroalkyl Substances (PFAS) in U-2 OS Cells

High-throughput transcriptomics (HTTr) was undertaken to quantify and characterize the biological effects of chemicals observed in human-derived in vitro test systems. A set of 137 structurally diverse PFAS was tested using the HTTr assay in the human-derived cell type U-2 OS (osteosarcoma). The PFAS studies were carried out as a part of a larger screening study of a total of 1323 chemicals. Chemicals were randomized to treatment plates (cells plus media) in triplicate. Plates also contained reference treatments for quality control purposes. 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. For this analysis, a catalog of 11,037 gene expression signatures collated from multiple public sources were analyzed using concentration-response modeling of signature enrichment scores determined using single-sample gene set enrichment analysis (ssGSEA) (Barbie et al. 2009) with DESeq2-moderated fold change values as input (Love et al. 2014). A sample was considered active if 25 or more signatures were concentration-responsive and the BPAC was calculated as the 5th percentile of benchmark concentrations (BMCs) of active signatures. 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 larger chemical space focused on Per- and Polyfluoroalkyl Substances (PFAS). Here, we screened 144 PFAS chemical samples corresponding to 137 unique PFAS substances in U-2 OS (osteosarcoma) cells using HTTr and experimental conditions similar to those described previously (Harrill et al., 2021). Each chemical sample was tested in a eight-point dilution series with one technical replicate well per chemical sample and dose level combination per culture, repeated across three independent cultures. For most chemical samples, the tested concentration range was 0.03 to 100 µM with half-log spacing. The highest tested concentration for some chemicals was as low as 16.7 µM, but half-log spacing across eight dilutions was used for all chemicals. U-2 OS cells were treated with PFAS chemicals for 24 hours prior to sampling for TempO-Seq analysis.