Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models

Evaluating the mutagenicity of N-nitrosodimethylamine in 2D and 3D HepaRG cell cultures using error-corrected next generation sequencing

“…As was the case with the previous results from 24-h treatments, the 72-h NDMA treatments of 3D cultures produced higher levels of cytotoxicity, DNA strand breaks, and MN formation in the spheroids than in 2D cultures (Fig. 2 ), likely due to a 2.7-fold increase in CYP2E1 expression in the spheroids (Seo et al 2023a , 2023b ). It has been noted that species with higher levels of CYP2E1 enzyme have higher sensitivity to NDMA compared to other species (Cross and Ponting 2021 ).…”

“…The present study employed a 72-h treatment with NDMA and observed concentration-dependent increases in MFs in both 2D and 3D HepaRG cell models (Tables 1 , 2 , 3 ). The rationale for conducting a 72-h treatment is that NDMA induced DNA damage and MN formation following a 24-h treatment, but an additional incubation was required for detecting the MN in both 2D and 3D models (Guo et al 2020b ; Seo et al 2023a , 2023b ). Previous studies have characterized the proliferative capacity of HepaRG cell models and demonstrated that a portion of cells in HepaRG spheroids express the proliferation marker Ki67 (Clayton et al 2018 ) and that bipotent HepaRG hepatocyte-like cells can divide without losing differentiation (Cerec et al 2007 ).…”

“…NDMA was chosen for the present study because it is a well characterized mutagen that requires metabolic activation to exert its genotoxic and carcinogenic effects (IARC 1978 ; Kirkland et al 2016 ). In addition, there currently is renewed interest in the genotoxicity of NDMA due to its recent identification as an impurity in several popular human drugs used for the treatment of common diseases such as hypertension, heartburn, and diabetes (FDA 2021 ; Seo et al 2023b ). Our previous studies confirmed that NDMA induced significant DNA strand breakage and MN formation following a 24-h treatment of both 2D and 3D HepaRG cell models without the addition of liver S9 fractions (Seo et al 2022 , 2023a , 2023b ).…”

“…In addition, there currently is renewed interest in the genotoxicity of NDMA due to its recent identification as an impurity in several popular human drugs used for the treatment of common diseases such as hypertension, heartburn, and diabetes (FDA 2021 ; Seo et al 2023b ). Our previous studies confirmed that NDMA induced significant DNA strand breakage and MN formation following a 24-h treatment of both 2D and 3D HepaRG cell models without the addition of liver S9 fractions (Seo et al 2022 , 2023a , 2023b ). The present study assessed NDMA-induced mutations following a 72-h treatment in both 2D and 3D HepaRG models with further optimized DS and HiFi Sequencing techniques.…”