Mutation accumulation following extended exposure of human HepaRG cells to a genotoxic carcinogen

Accurately evaluating chemical risk may benefit from the development of human-relevant models capable of capturing the effects of treatments that closely mimic real-world environmental and pharmaceutical exposures. Building on our previous work (Seo et al. Arch Toxicol 98:1919-1935),¹ where cytotoxicity compromised mutation detection following short-term treatments, this study investigated mutation accumulation in both 2D and 3D HepaRG cultures following 7- and 14-day exposures with relatively non-cytotoxic <i>N</i>-nitrosodimethylamine (NDMA) concentrations. A multi-endpoint approach was employed to assess NDMA-induced DNA damage, micronucleus formation, and mutagenesis. Seven-day NDMA treatments were not cytotoxic but produced concentration-dependent increases in DNA damage and mutations. After 14 days of exposure, the highest NDMA concentrations produced no more than 30% cytotoxicity, and induced greater mutation frequencies compared to the 7-day exposures. Overall, the mutation frequencies induced by NDMA exhibited concentration- and treatment-duration-dependent relationships in both 2D and 3D HepaRG cultures, with notably higher mutation frequencies in 3D spheroids than in 2D cultures. Quantitative analysis by benchmark concentration (BMC) modeling demonstrated lower BMC values in 3D spheroids compared to their 2D counterparts. The predominant mutation in NDMA-treated cultures was T→C transition. These findings indicate the value of extended exposure periods for conducting <i>in vitro</i> genotoxicity testing in HepaRG cells.