Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR, 72079, USA.
Wiess School of Natural Sciences, Rice University, Houston, TX, 77005, USA.
Arch Toxicol. 2023 Oct;97(10):2785-2798. doi: 10.1007/s00204-023-03560-x. Epub 2023 Jul 24.
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.
N-亚硝胺杂质在人类药物中越来越多地被检测到。这是一个安全隐患，因为许多亚硝胺在细菌中具有致突变性，在啮齿动物模型中具有致癌性。通常，亚硝胺的致突变性和致癌活性需要细胞色素 P450 酶（CYPs）的代谢激活，在许多体外模型中，这些酶是使用鼠肝匀浆外源性提供的。关于亚硝胺在人类细胞系统中的遗传毒性仅有有限的数据。在这项研究中，我们使用代谢能力强的人 HepaRG 细胞，其代谢能力与原代人肝细胞相当，来评估八种亚硝胺[N-环戊基-4-亚硝基哌嗪（CPNP）、N-亚硝二丁胺（NDBA）、N-亚硝二乙胺（NDEA）、N-亚硝二甲胺（NDMA）、N-亚硝二异丙胺（NDIPA）、N-亚硝基乙基异丙胺（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。在我们用于培养 HepaRG 细胞的条件下，三维（3D）球体比 2D 单层细胞具有更高水平的 CYP 活性；因此，除了更传统的 2D 培养外，我们还使用 3D HepaRG 球体研究了这八种亚硝胺的遗传毒性。使用高通量彗星芯片测定法评估遗传毒性作为 DNA 损伤，并用基于流式细胞术的微核（MN）测定法评估有丝分裂后期加倍/断裂剂的遗传毒性。经过 24 小时处理，所有亚硝胺均在 3D 球体中诱导 DNA 损伤，而只有三种亚硝胺（NDBA、NDEA 和 NDMA）在 2D HepaRG 细胞中产生阳性反应。此外，这三种亚硝胺还导致 2D 和 3D HepaRG 模型中 MN 频率显著增加，而 NMBA 和 NMPA 仅在 3D HepaRG MN 测定中呈阳性。总体而言，我们的结果表明 HepaRG 球体可能为评估亚硝胺的遗传毒性提供一种敏感的基于人类的细胞系统。
