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.