Application of HepG2/C3A liver spheroids as a model system for genotoxicity studies

• Published in: Toxicology letters Vol. 345; pp. 34 - 45
• Main Authors: Coltman, Nicholas J., Coke, Brandon A., Chatzi, Kyriaki, Shepherd, Emma L., Lalor, Patricia F., Schulz-Utermoehl, Timothy, Hodges, Nikolas J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2021
• Subjects: 3D culture; 4-Nitroquinoline-1-oxide - metabolism; 4-Nitroquinoline-1-oxide - toxicity; Activation, Metabolic; Animal Testing Alternatives; Anthracenes - metabolism; Anthracenes - toxicity; Benzo(a)pyrene - metabolism; Benzo(a)pyrene - toxicity; Comet Assay; DNA Damage; Gene Expression Regulation, Enzymologic; Genotoxicity; Hep G2 Cells; Hepatocytes - drug effects; Hepatocytes - enzymology; Hepatocytes - pathology; HepG2; Histones - metabolism; Humans; Imidazoles - metabolism; Imidazoles - toxicity; In vitro; Liver - drug effects; Liver - enzymology; Liver - pathology; Phosphorylation; Spheroids, Cellular; Time Factors; HepG2; 3D culture; In vitro; Genotoxicity
• ISSN: 0378-4274; 1879-3169
• DOI: 10.1016/j.toxlet.2021.04.004

[Display omitted] •HepG2 liver spheroids as a liver like in vitro model for genotoxicity assessment.•Potential applications in early stage drug development.•Spheroid models will help to contribute to number of animals used in scientific research in the future. HepG2 cells continue to be a valuable tool in early drug discovery and pharmaceutical development. In the current study we develop a 3D in vitro liver model, using HepG2/C3A cells that is predictive of human genotoxic exposure. HepG2/C3A cells cultured for 7-days in agarose-coated microplates formed spheroids which were uniform in shape and had well defined outer perimeters and no evidence of a hypoxic core. Quantitative real-time-PCR analysis showed statistically significant transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, UG1A1, UGT1A3, UGT1A6, EPHX, NAT2) and genes linked to liver function (ALB, CAR) in 3D cultures. In response to three model pro-genotoxicants: benzo[a]pyrene, amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-aminoanthracene (2-AA), we observed further transcriptional upregulation of xenobiotic metabolising genes (CYP1A1, CYP1A2, NAT1/2, SULT1A2, UGT1A1, UGT1A3) compared to untreated spheroids. Consistent with this, spheroids were more sensitive than 2D monolayers to compound induced single- and double- stranded DNA-damage as assessed by the comet assay and γH2AX phosphorylation respectively. In contrast, levels of DNA-damage induced by the direct acting mutagen 4-nitroquinoline N-oxide (4NQO) was the same in spheroids and monolayers. In support of the enhanced genotoxic response in spheroids we also observed transcriptional upregulation of genes relating to DNA-damage and cellular stress response (e.g. GADD45A and CDKN1A) in spheroids. In conclusion, HepG2/C3A 3D spheroids are a sensitive model for in vitro genotoxicity assessment with potential applications in early stage drug development.