Systematic assessment of transcriptomic and phenotypic biological profiling for mechanism-based hazard assessment using target-annotated reference chemicals in renal proximal tubular epithelial cells
bioRxiv (Cold Spring Harbor Laboratory)
Abstract
Abstract Integrating high-throughput in vitro data into next-generation risk assessment (NGRA) workflows requires screening strategies that yield quantitative potency estimates and mechanistically interpretable biological signals. Transcriptomic and morphological profiling are increasingly adopted for early-stage hazard identification by enabling triage of substances for resource-intensive follow-up and prioritizing candidates most likely to present meaningful risk. In this study, we aimed to characterize biological concordance and uncertainty by quantifying how well high-throughput transcriptomics (HTTr) and Cell Painting PLUS (CPP) bioactivity profiles recover target-relevant biological signals in immortalized human renal proximal tubule epithelial RPTEC/TERT1 cells using 313 reference chemicals with high-confidence target annotations. Through quality control procedures and biological activity filters we yielded 142 reference chemicals spanning 66 different targets, which were systematically evaluated for biological concentration-responses by HTTr and CPP. HTTr was evaluated using TXG-MAPr-based qualitative and quantitative gene network activity analysis. HTTr showed the most prominent activity for targets that were highest expressed in RPTEC/TERT1 cells. Active chemical-pairs showed strong gene network activity correlation albeit with different potencies. Similarly, the highest transcriptomic concordance was observed for reference chemicals acting in the same pathway, such as EGFR/MEK or PI3K/AKT/mTOR. CPP often showed high sensitivity primarily at the organelle level providing limited statistical power for chemical grouping. Collectively, the results support HTTr and CPP as complementary early-tier assays within an in vitro weight-of-evidence safety testing framework. Although CPP is suitable as a cost-effective screening modality, HTTr offers higher mechanistic resolution for mode-of-action inference in high-throughput bioactivity screening and therefore remains necessary for high-confidence mechanistic interpretation.
Authors 11
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Hugo W van Kessel Aachen Division of Cell Systems and Drug Safety Leiden Academic Centre for Drug Research
Affiliation as printed
Division of Cell Systems and Drug Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands
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Federal Institute for Risk Assessment · Freie Universität Berlin
Affiliation as printed
German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR), Berlin, Germany
Institute of Biology, Free University of Berlin, Berlin, Germany
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Affiliation as printed
Division of Pharmaceutical Chemistry, Pharmacoinformatics Research Group, University of Vienna, Vienna, Austria
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Latvijas Organiskās Sintēzes Institūts
Affiliation as printed
Latvian Institute of Organic Synthesis, Riga, Latvia
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National Institutes of Health · National Institute of Environmental Health Sciences
Affiliation as printed
Division of Translational Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
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Affiliation as printed
Corteva Agriscience, Indianapolis, Indiana, USA
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Affiliation as printed
Division of Pharmaceutical Chemistry, Pharmacoinformatics Research Group, University of Vienna, Vienna, Austria
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Federal Institute for Risk Assessment
Affiliation as printed
German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR), Berlin, Germany
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Federal Institute for Risk Assessment
Affiliation as printed
German Centre for the Protection of Laboratory Animals (Bf3R), German Federal Institute for Risk Assessment (BfR), Berlin, Germany
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Giulia Callegaro Aachen Division of Cell Systems and Drug Safety Leiden Academic Centre for Drug Research
Affiliation as printed
Division of Cell Systems and Drug Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands
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Bob van de Water corresponding Aachen Division of Cell Systems and Drug Safety Leiden Academic Centre for Drug Research
Affiliation as printed
Division of Cell Systems and Drug Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands
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