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

Seo, Ji-Eun; Le, Yuan; Revollo, Javier; Miranda-Colon, Jaime; Xu, Hannah; McKinzie, Page; Mei, Nan; Chen, Tao; Heflich, Robert H.; Zhou, Tong; Robison, Timothy; Bonzo, Jessica A.; Guo, Xiaoqing

doi:10.1007/s00204-024-03731-4

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

Journal Article · 07 April 2024 · Archives of Toxicology

DOI: https://doi.org/10.1007/s00204-024-03731-4 · OSTI ID:2471338

Seo, Ji-Eun ^[1]; Le, Yuan ^[1]; Revollo, Javier ^[1]; Miranda-Colon, Jaime ^[1]; Xu, Hannah ^[1]; McKinzie, Page ^[1]; Mei, Nan ^[1]; Chen, Tao ^[1]; Heflich, Robert H. ^[1]; Zhou, Tong ^[2]; Robison, Timothy ^[3]; Bonzo, Jessica A. ^[3];

^[1]

US Food and Drug Administration (USFDA), Jefferson, AR (United States). National Center for Toxicological Research
US Food and Drug Administration (USFDA), Rockville, MD (United States). Center for Veterinary Medicine
US Food and Drug Administration (USFDA), Silver Spring, MD (United States). Center for Drug Evaluation and Research

Human liver-derived metabolically competent HepaRG cells have been successfully employed in both two-dimensional (2D) and 3D spheroid formats for performing the comet assay and micronucleus (MN) assay. In the present study, we have investigated expanding the genotoxicity endpoints evaluated in HepaRG cells by detecting mutagenesis using two error-corrected next generation sequencing (ecNGS) technologies, Duplex Sequencing (DS) and High-Fidelity (HiFi) Sequencing. Both HepaRG 2D cells and 3D spheroids were exposed for 72 h to N-nitrosodimethylamine (NDMA), followed by an additional incubation for the fixation of induced mutations. NDMA-induced DNA damage, chromosomal damage, and mutagenesis were determined using the comet assay, MN assay, and ecNGS, respectively. The 72-h treatment with NDMA resulted in concentration-dependent increases in cytotoxicity, DNA damage, MN formation, and mutation frequency in both 2D and 3D cultures, with greater responses observed in the 3D spheroids compared to 2D cells. The mutational spectrum analysis showed that NDMA induced predominantly A:T → G:C transitions, along with a lower frequency of G:C → A:T transitions, and exhibited a different trinucleotide signature relative to the negative control. These results demonstrate that the HepaRG 2D cells and 3D spheroid models can be used for mutagenesis assessment using both DS and HiFi Sequencing, with the caveat that severe cytotoxic concentrations should be avoided when conducting DS. With further validation, the HepaRG 2D/3D system may become a powerful human-based metabolically competent platform for genotoxicity testing.

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Research Organization:: Oak Ridge Institute for Science and Education (ORISE), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC); USFDA

Grant/Contract Number:: SC0014664

OSTI ID:: 2471338

Journal Information:: Archives of Toxicology, Journal Name: Archives of Toxicology Journal Issue: 6 Vol. 98; ISSN 0340-5761

Publisher:: SpringerCopyright Statement

Country of Publication:: United States

Language:: English

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

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