Abstract
Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a ‘cohort of concern’ because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems. In this study, we employed human lymphoblastoid TK6 cells transduced with human cytochrome P450 (CYP) 2A6 to evaluate the genotoxicity of six nitrosamines that have been identified as impurities in drug products: N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), N-nitrosomethylphenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA), and N-nitrosodibutylamine (NDBA). Using flow cytometry-based assays, we found that 24-h treatment with NDEA, NEIPA, NMBA, and NMPA caused concentration-dependent increases in the phosphorylation of histone H2A.X (γH2A.X) in CYP2A6-expressing TK6 cells. Metabolism of these four nitrosamines by CYP2A6 also caused significant increases in micronucleus frequency as well as G2/M phase cell-cycle arrest. In addition, nuclear P53 activation was found in CYP2A6-expressing TK6 cells exposed to NDEA, NEIPA, and NMPA. Overall, the genotoxic potency of the six nitrosamine impurities in our test system was NMPA > NDEA ≈ NEIPA > NMBA > NDBA ≈ NDIPA. This study provides new information on the genotoxic potential of nitrosamines in human cells, complementing test results generated from traditional assays and partially addressing the issue of the relevance of nitrosamine genotoxicity for humans. The metabolically competent human cell system reported here may be a useful model for risk assessment of nitrosamine impurities found in drugs.
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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. YL was supported by an appointment to the Postgraduate Research Program at the National Center for Toxicological Research (NCTR) administered by the Oak Ridge Institute for Science Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration (FDA). We thank Drs. Robert Dorsam, Sruthi King, Naomi Kruhlak from CDER for their valuable comments regarding nitrosamine impurities and Dr. Tao Chen and Ms. Roberta Mittelstaedt for their critical review of this manuscript.
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This article reflects the views of the authors and does not necessarily reflect those of the U.S. Food and Drug Administration (FDA). Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Li, X., He, X., Le, Y. et al. Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s. Arch Toxicol 96, 3077–3089 (2022). https://doi.org/10.1007/s00204-022-03347-6
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DOI: https://doi.org/10.1007/s00204-022-03347-6