Seo Ji-Eun, He Xiaobo, Bryant Matthew, Atrakchi Aisar H, McGovern Timothy J, Davis Bruno Karen L, Heflich Robert H, Guo Xiaoqing
Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA; Office of Scientific Coordination, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA.
Office of Scientific Coordination, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA.
Chem Biol Interact. 2025 Aug 1;416:111538. doi: 10.1016/j.cbi.2025.111538. Epub 2025 May 2.
N-nitrosamines have been increasingly detected in human drugs, raising serious safety concerns due to their potential mutagenicity and carcinogenicity. In order to expand upon the human data available on these drug impurities, we previously used metabolically competent HepaRG human hepatoma cells to evaluate the genotoxicity of eight small-molecule 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)]. In this study, we used the comet assay to further investigate the DNA damage induced by the eight nitrosamines in primary human hepatocytes (PHHs) from three individual donors and primary macaque hepatocytes (PMHs) from freshly isolated livers of six rhesus macaques. In addition, expression of genes encoding Phase I and II metabolic enzymes and the activities of the enzymes were compared in PHHs and PMHs, and Western blot was used to analyze protein biomarkers of DNA damage and apoptosis in PMHs. All eight nitrosamines induced significant DNA damage in both PHHs and PMHs; with the exception of NDMA, higher fold increases in % tail DNA were detected in PMHs compared to PHHs. Greater interindividual variability in CYP gene expression, enzyme activities, and DNA damage responses was observed in PHHs compared to PMHs. Benchmark concentration (BMC) modeling analysis showed that PHHs had more conservative BMC values than PMHs for most nitrosamines tested. Nonetheless, correlation analysis demonstrated that DNA damage data generated by PMHs and 3D HepaRG spheroids were comparable to those of PHHs. Western blot analysis suggested a potential role for the ethyl group in regulating protein expression in the DNA damage and apoptosis pathways for nitrosamines. Overall, this study provides human-relevant DNA damage responses for the eight nitrosamines and indicates that differences in genotoxic potency between PHHs and PMHs are likely related to CYP enzyme activity.
N-亚硝胺类物质在人类药物中被越来越多地检测到，由于其潜在的致突变性和致癌性，引发了严重的安全担忧。为了扩充关于这些药物杂质的现有人类数据，我们之前使用具有代谢活性的HepaRG人肝癌细胞评估了8种小分子亚硝胺 [N-环戊基-4-亚硝基哌嗪（CPNP）、N-亚硝基二丁胺（NDBA）、N-亚硝基二乙胺（NDEA）、N-亚硝基二甲胺（NDMA）、N-亚硝基二异丙胺（NDIPA）、N-亚硝基乙异丙胺（NEIPA）、N-亚硝基-N-甲基-4-氨基丁酸（NMBA）和N-亚硝基甲基苯胺（NMPA）] 的遗传毒性。在本研究中，我们使用彗星试验进一步研究了这8种亚硝胺在来自3名个体供体的原代人肝细胞（PHH）和来自6只恒河猴新鲜分离肝脏的原代猕猴肝细胞（PMH）中诱导的DNA损伤。此外，比较了PHH和PMH中编码I相和II相代谢酶的基因表达以及这些酶的活性，并使用蛋白质印迹法分析PMH中DNA损伤和凋亡的蛋白质生物标志物。所有8种亚硝胺在PHH和PMH中均诱导了显著的DNA损伤；除NDMA外，与PHH相比，PMH中检测到的尾DNA百分比增加倍数更高。与PMH相比，在PHH中观察到CYP基因表达、酶活性和DNA损伤反应的个体间变异性更大。基准浓度（BMC）建模分析表明，对于大多数测试的亚硝胺，PHH的BMC值比PMH更保守。尽管如此，相关性分析表明，PMH和3D HepaRG球体产生的DNA损伤数据与PHH的数据具有可比性。蛋白质印迹分析表明，乙基在调节亚硝胺的DNA损伤和凋亡途径中的蛋白质表达方面可能发挥作用。总体而言，本研究提供了这8种亚硝胺与人类相关的DNA损伤反应，并表明PHH和PMH之间遗传毒性效力的差异可能与CYP酶活性有关。
