Computerized predictive approaches of genotoxicity and mutagenesis in 3D Hepoid of normal and transformed human hepatocytes

The low specificity of standard in vitro genotoxicity tests in mammalian cells continues to necessitate animal experimentation, raising ethical concerns that conflict with the principles of the 3Rs (Replacement, Reduction, and Refinement). This underscores the urgent need for reliable alternative in vitro assays. Given the liver's critical role in xenobiotic detoxification and bioactivation, we developed an advanced 3D in vitro model of human hepatocytes that supports the proliferation and long-term differentiation of primary human hepatocytes and HepaRG cells. Using a range of methodologies, including the γH2AX assay, the comet assay, the micronucleus test, and transcriptomic analysis, we investigated DNA damage induced by well-known genotoxic carcinogens such as methylmethane sulfonate (MMS), mitomycin C (MMC), colchicine, vinblastine, 1,2-dimethylhydrazine (DMH), and aflatoxin B₁ (AFB₁), as well as non-genotoxic carcinogens, including di-2-ethylhexyl phthalate (DEHP) and methylcarbamate. Our data demonstrate the reliability of the 3D human hepatocyte Hepoid model in replicating in vivo results. We effectively discriminated between genotoxic and non-genotoxic carcinogenic molecules using complementary methods analyzed with advanced computational approaches. We further studied the in vitro genotoxic potential of four main Heterocyclic Aromatic Amines (HAAs) including 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2- amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which are food-borne and environmental contaminants suspected to contribute to hepatocellular carcinoma. Our results validate the Hepoid model as a reliable in vitro system for assessing the genotoxic and mutagenic risks of chemicals and confirm the ability of HAAs to induce DNA damage in a highly differentiated human liver model.