Advancing genotoxicity assessment with transcriptomic biomarkers: from 2D to 3D HepaRG spheroid models
Abstract
The development of New Approach Methodologies (NAMs) for genotoxicity testing is needed to support the transition toward animal-free human-relevant chemical genotoxicity assessment. In this context, we investigated the predictive power of two transcriptomic biomarkers developed in 2D cell systems (GENOMARK and TGx-DDI) in metabolically active 3D HepaRG spheroids to classify chemicals as genotoxic (GTx) or non-genotoxic (NGTx). Human-hepatic HepaRG spheroids were treated for 72 h with three different groups of chemicals: (i) five genotoxic chemicals: Lasiocarpine, cyclophosphamide, ethyl methanesulfonate, aflatoxin B1, benzo[a]pyrene; (ii) five non-genotoxic non-carcinogens: Aflatoxin B2, 2-deoxy-D-glucose, D-mannitol, caffeine, ampicillin trihydrate; and (iii) five non-genotoxic carcinogens: Perfluorooctanoic acid, diethanolamine, thioacetamide, phorbol-12-myristate-13-acetate, di(2-ethylhexyl) phthalate. Gene expression data from the treated HepaRG spheroids were collected using the high-throughput TempO-Seq technology and analyzed with the transcriptomic biomarkers GENOMARK and TGx-DDI. Results with both biomarkers generally aligned with existing knowledge for all three groups of tested chemicals, effectively distinguishing genotoxic from non-genotoxic chemicals. The results of this study indicate that combining the 3D HepaRG spheroid model with transcriptomic biomarkers offers a promising, human-relevant approach to complement traditional in vitro genotoxicity tests.
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BibTeX
@article{saleh2026advancing,
title = {{Advancing genotoxicity assessment with transcriptomic biomarkers: from 2D to 3D HepaRG spheroid models}},
author = {Mariam Saleh and Kevin Hogeveen and Anouck Thienpont and Emmanuel Demuynck and Valérie Fessard and Birgit Mertens and Ludovic Le Hégarat},
journal = {Toxicological Sciences},
year = {2026},
month = oct,
doi = {10.1093/toxsci/kfag095},
url = {https://anses.hal.science/anses-05799783v1},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Advancing genotoxicity assessment with transcriptomic biomarkers: from 2D to 3D HepaRG spheroid models
AU - Mariam Saleh
AU - Kevin Hogeveen
AU - Anouck Thienpont
AU - Emmanuel Demuynck
AU - Valérie Fessard
AU - Birgit Mertens
AU - Ludovic Le Hégarat
PY - 2026
DA - 2026/10/01
JO - Toxicological Sciences
DO - 10.1093/toxsci/kfag095
UR - https://anses.hal.science/anses-05799783v1
AB - The development of New Approach Methodologies (NAMs) for genotoxicity testing is needed to support the transition toward animal-free human-relevant chemical genotoxicity assessment. In this context, we investigated the predictive power of two transcriptomic biomarkers developed in 2D cell systems (GENOMARK and TGx-DDI) in metabolically active 3D HepaRG spheroids to classify chemicals as genotoxic (GTx) or non-genotoxic (NGTx). Human-hepatic HepaRG spheroids were treated for 72 h with three different groups of chemicals: (i) five genotoxic chemicals: Lasiocarpine, cyclophosphamide, ethyl methanesulfonate, aflatoxin B1, benzo[a]pyrene; (ii) five non-genotoxic non-carcinogens: Aflatoxin B2, 2-deoxy-D-glucose, D-mannitol, caffeine, ampicillin trihydrate; and (iii) five non-genotoxic carcinogens: Perfluorooctanoic acid, diethanolamine, thioacetamide, phorbol-12-myristate-13-acetate, di(2-ethylhexyl) phthalate. Gene expression data from the treated HepaRG spheroids were collected using the high-throughput TempO-Seq technology and analyzed with the transcriptomic biomarkers GENOMARK and TGx-DDI. Results with both biomarkers generally aligned with existing knowledge for all three groups of tested chemicals, effectively distinguishing genotoxic from non-genotoxic chemicals. The results of this study indicate that combining the 3D HepaRG spheroid model with transcriptomic biomarkers offers a promising, human-relevant approach to complement traditional in vitro genotoxicity tests.
SN - 1096-6080
SN - 1096-6099
ER -