Analytical choices drive toxicogenomic potency estimates: a systematic evaluation of transcriptomic points of departure

Toxicological Sciences, vol. 209

Abstract

Omics technologies are increasingly integrated into next-generation risk assessment, yet quantitative toxicogenomics outcomes remain highly dependent on analytical choices, motivating a systematic evaluation of how bioinformatics workflows influence hazard characterization and transcriptomic Points of Departure (tPOD). Here, we applied 5 independent transcriptomics pipelines to a shared dataset of RPTEC/TERT1 kidney cells exposed to cisplatin across multiple concentrations and time points, comparing effects of pre-processing, benchmark concentration modeling, and pathway-based interpretation strategies. Across workflows, substantial variability was observed in gene-level benchmark concentrations (BMCs). This variability was associated with differences in normalization, filtering, and modeling software, although the present design does not isolate the contribution of individual workflow choices. Despite this variability, convergence increased at later time points as transcriptional responses strengthened, with 24 h consistently identified as the most sensitive time point at the gene level. Aggregation of gene-level BMCs into pathway-based metrics reduced variability but did not eliminate it, with pathway definition emerging as a major determinant of tPOD estimates. Notably, distinct pathway resources showed minimal gene overlap, and smaller, biologically coherent gene sets (e.g. co-expression modules and biomarker panels) produced lower and less dispersed BMCs compared with broader pathway annotations. Furthermore, direct modeling of pathway activity scores yielded systematically different tPODs relative to median-based aggregation, with method-dependent conservativeness influenced by pathway coverage and response strength. Overall, our findings demonstrate that both analytical workflow design and pathway selection critically shape toxicogenomic-derived potency estimates, highlighting the need for harmonized, transparent methodologies to enable robust application of transcriptomics in chemical safety assessment and regulatory decision-making.

Authors 18

1. Imke B. Bruns Aachen Division of Drug Discovery and Safety Leiden Academic Centre for Drug Research

   Centre for Human Drug Research

   Affiliation as printed

   Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University , Leiden, 2333 CC,

2. Dayna R. Schultz

   Aristotle University of Thessaloniki

   Affiliation as printed

   Environmental Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki , Thessaloniki, 54124,

   HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation , Balkan Center, Bldg. B, Thessaloniki, 57001,

3. Emmanuel Demuynck

   Vrije Universiteit Brussel

   Affiliation as printed

   Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel , Brussels, 1090,

   Scientific Direction of Chemical and Physical Health Risks, Sciensano , Brussels, 1050,

4. Friedel Dewulf
   Affiliation as printed

   Blue Growth Research Lab, Ghent University , Oostende, 8400,

5. Ioannis Theologidis

   Benaki Phytopathological Institute

   Affiliation as printed

   Laboratory of Toxicological Control of Pesticides, Scientific Directorate of Pesticides’ Control & Phytopharmacy, Benaki Phytopathological Institute , Athens, 14561,

6. Steven J. Kunnen Aachen Division of Drug Discovery and Safety Leiden Academic Centre for Drug Research

   Centre for Human Drug Research

   Affiliation as printed

   Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University , Leiden, 2333 CC,

7. Lukas S. Wijaya Aachen Division of Drug Discovery and Safety Leiden Academic Centre for Drug Research

   Centre for Human Drug Research

   Affiliation as printed

   Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University , Leiden, 2333 CC,

8. Ilias Frydas

   Aristotle University of Thessaloniki

   Affiliation as printed

   Environmental Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki , Thessaloniki, 54124,

   HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation , Balkan Center, Bldg. B, Thessaloniki, 57001,

9. Nafsika Papaioannou

   Aristotle University of Thessaloniki · National Hellenic Research Foundation

   Affiliation as printed

   Environmental Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki , Thessaloniki, 54124,

   HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation , Balkan Center, Bldg. B, Thessaloniki, 57001,

   National Hellenic Research Foundation , Athens, 11635,

10. Elisavet Renieri

    Aristotle University of Thessaloniki

    Affiliation as printed

    Environmental Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki , Thessaloniki, 54124,

    HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation , Balkan Center, Bldg. B, Thessaloniki, 57001,

11. Thanasis Papageorgiou

    Aristotle University of Thessaloniki

    Affiliation as printed

    Environmental Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki , Thessaloniki, 54124,

    HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation , Balkan Center, Bldg. B, Thessaloniki, 57001,

12. Dimosthenis Andreas Sarigiannis

    Aristotle University of Thessaloniki · National Hellenic Research Foundation

    Affiliation as printed

    Environmental Engineering Laboratory, Department of Chemical Engineering, Aristotle University of Thessaloniki , Thessaloniki, 54124,

    HERACLES Research Center on the Exposome and Health, Center for Interdisciplinary Research and Innovation , Balkan Center, Bldg. B, Thessaloniki, 57001,

    National Hellenic Research Foundation , Athens, 11635,

13. Kyriaki A. Machera

    Benaki Phytopathological Institute

    Affiliation as printed

    Laboratory of Toxicological Control of Pesticides, Scientific Directorate of Pesticides’ Control & Phytopharmacy, Benaki Phytopathological Institute , Athens, 14561,

14. Birgit Mertens
    Affiliation as printed

    Scientific Direction of Chemical and Physical Health Risks, Sciensano , Brussels, 1050,

15. Jana Asselman
    Affiliation as printed

    Blue Growth Research Lab, Ghent University , Oostende, 8400,

16. Carsten Weiß

    Karlsruhe Institute of Technology

    Affiliation as printed

    Institute of Biological and Chemical Systems—Biological Information Processing, Karlsruhe Institute of Technology , Eggenstein-Leopoldshafen, 76344,

17. Bob van de Water Aachen Division of Drug Discovery and Safety Leiden Academic Centre for Drug Research

    Centre for Human Drug Research

    Affiliation as printed

    Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University , Leiden, 2333 CC,

18. Giulia Callegaro Aachen Division of Drug Discovery and Safety Leiden Academic Centre for Drug Research

    Centre for Human Drug Research

    Affiliation as printed

    Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research, Leiden University , Leiden, 2333 CC,