The Power of Resolution: Contextualized Understanding of Chemical-biological Interactions

Prediction of human response to chemical exposures is a major challenge in both pharmaceutical and toxicological research. Transcriptomics has been a powerful tool to explore chemical-biological interactions. However, limited throughput, high-costs and complexity of transcriptomic interpretations have yielded numerous studies lacking sufficient experimental context for predictive application. We utilized a novel high-throughput transcriptomics platform to explore a broad range of exposures to 24 reference compounds in both differentiated and undifferentiated human HepaRG cultures. Our goals were to 1) explore transcriptomic characteristics distinguishing liver injury compounds, 2) assess impacts of differentiation state on baseline and compound-induced responses (e.g., metabolically-activated), and 3) identify and resolve reference biological-response pathways and their quantitative translation to human exposures. Study data revealed the predictive utility of transcriptomic concentration-response modeling to quantitatively identify human liver injury compounds by their respective benchmark concentrations (BMCs), and model hepatic responses to classical reference compounds yielding plausibly-relevant estimations of human potency. BioProject accession (PRJNA497448) HepaRG cells were seeded at a density of ~2000 cells per well onto collagen(I)-coated 384-well plates and kept in culture only 3 days prior to compound exposures for maintain proliferation and the undifferentiated state. Cells were seeded using Williams E medium (ThermoFisher) supplemented with plating additive (Lonza, Catalog), and 24 hours later exchanged with William’s E medium supplemented with maintenance additive (Lonza) for the duration of cultures. After the initial 3/10 days, 50 μL of compound mixtures was transferred to the plates containing cells. Media was exchanged after the initial 48h of treatment. At the end of the 96-hour compound exposure period, exposure media was removed, and cells were washed once with 50 μL Phosphate-buffered saline (ThermoFisher) followed by addition of 20 μL of 1X TempO-Seq lysis buffer (Biospyder) to culture plates. Plates were then incubated for 15 minutes at room temperature and frozen at -80° C. TempO-Seq analysis was performed as described previously, at a targeted sequencing depth of 500 mapped read counts per transcript (Yeakley, et al., 2017).