Abstract
N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.
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Acknowledgements
This work was partly supported by funding from the Center for Drug Evaluation and Research (CDER) Regulatory Science Research program. HX was supported by an appointment to the Postgraduate Research Program and JY was supported by an appointment to the Summer Student Research Program; both programs are administered for CDER by the Oak Ridge Institute for Science Education through an interagency agreement between the U.S. Department of Energy and the U.S. FDA. We thank Drs. David Keire, Robert Dorsam, Sruthi King, Naomi Kruhlak from CDER for their valuable comments regarding nitrosamine impurities and Drs. Tao Chen and Li-Rong Yu from NCTR for their critical review of this manuscript.
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Seo, JE., Yu, J.Z., Xu, H. et al. Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models. Arch Toxicol 97, 2785–2798 (2023). https://doi.org/10.1007/s00204-023-03560-x
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DOI: https://doi.org/10.1007/s00204-023-03560-x