Detection of Urethane-Induced Genotoxicity in Vitro Using Metabolically Competent Human 2D and 3D Spheroid Culture Models.

In vitro genotoxicity studies are a quick and high throughput approach to assess the genotoxic potential of chemicals; however, the reliability of these tests and their relevance to in vivo effects depends on the choice of representative cell line and optimisation of assay conditions. For chemicals like urethane that require specific metabolic activation to cause genotoxicity, it is important that in vitro tests are conducted using cell lines exhibiting the activity and induction of CYP450 enzymes, including CYP2E1 enzyme that is important in the metabolism of urethane, at a concentration representing actual or perceived chemical exposure. We compared 2D MCL-5 cells and HepG2 cells with 3D HepG2 hanging drop spheroids to determine the genotoxicity of urethane using the micronucleus assay. Our 2D studies with MCL-5 did not show any statistically significant genotoxicity [99% relative population doubling (RPD)] compared to controls for concentrations and time point tested in vitro. HepG2 cells grown as 2D indicated that exposure to urethane of up to 30 mM for 23 h did not cause any genotoxic effect (102% RPD) but, at higher concentrations, genotoxicity was produced with only 89-85% RPD. Furthermore, an exposure of 20-50 mM for 23 h using 3D hanging drop spheroid assays revealed a higher MN frequency, thus exhibiting in vitro genotoxicity of urethane in metabolically active cell models. In comparison with previous studies, this study indicated that urethane genotoxicity is dose, sensitivity of cell model (2D vs. 3D) and exposure dependent.