中国药物警戒 ›› 2026, Vol. 23 ›› Issue (5): 494-499.
DOI: 10.19803/j.1672-8629.20260220

• 亚硝胺类药物相关杂质安全性研究专栏 • 上一篇    下一篇

亚硝胺杂质体外PIG-A基因突变试验研究

寇小旋1,2, 田冶1,2△, 邵子轩1,2, 牛茜怡1,2, 姜华1,2, 文海若1,2,*   

  1. 1中国食品药品检定研究院安全评价研究所,北京 100176;
    2药品监管科学全国重点实验室,北京 102629
  • 收稿日期:2026-03-17 发布日期:2026-05-20
  • 通讯作者: *文海若,女,博士,研究员,遗传毒理学。E-mail: wenhairuo@nifdc.org.cn
  • 作者简介:寇小旋,女,在读硕士,遗传毒理学。△为并列第一作者。
  • 基金资助:
    国家自然科学基金资助项目（82473889）; 药品监管科学全国重点实验室课题“药品杂质遗传毒性评价新技术和生物标志物研究”（2023SKLDRS0128）

In vitro PIG-A Gene Mutation Assay for Evaluation of N-nitrosamine Impurities

KOU Xiaoxuan1,2, TIAN Ye1,2△, SHAO Zixuan1,2, NIU Qianyi1,2, JIANG Hua1,2, WEN Hairuo1,2,*   

  1. 1Institute of Safety Evaluation, National Institutes for Food and Drug Control, Beijing 100176, China;
    2State Key Laboratory of Drug Regulatory Science, Beijing 102629, China
  • Received:2026-03-17 Published:2026-05-20

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摘要： 目的 探索基于TK6细胞的体外PIG-A基因突变试验方法的可靠性和灵敏性,建立适宜评价亚硝胺杂质的试验体系。方法 TK6细胞经免疫磁珠分离法实施自发突变细胞清除,基于亚硝胺杂质致突变风险评价的体外代谢条件需求,在仓鼠S9代谢活化条件下分别以二甲基亚砜（DMSO）为阴性对照、N-亚硝基二乙胺（NDEA）作为阳性对照（31.25~2 000 μg·mL-1）,应用高通量流式检测法检测对TK6细胞CD55和CD59表型的影响。基于已建立的试验条件,分别检测2种与药物结构相关的亚硝胺杂质,即1-环戊基-4-硝基哌嗪（CPNP, 50~300 μg·mL-1）和N-亚硝基布美他尼（250~2 000 μg·mL-1）对TK6细胞CD55和CD59表型的影响。结果 最后1次清除后TK6细胞的自发突变率降至0.007 2%。非代谢活化条件下,NDEA所有剂量组与阴性对照组相比无显著性变化;仓鼠S9代谢活化条件下,NDEA为500 μg·mL-1及以上浓度时TK6细胞的PIG-A基因突变率与阴性对照组相比显著性增加。仓鼠S9代谢活化条件下,150、300 μg·mL-1 CPNP浓度的PIG-A基因突变率与阴性对照组相比显著性增加,N-亚硝基布美他尼所有剂量组无显著性差异;非代谢活化条件下,CPNP、N-亚硝基布美他尼的所有剂量组与阴性对照组相比均无显著性差异。结论 基于TK6细胞的体外PIG-A基因突变试验方法在仓鼠S9代谢活化条件下可有效评价亚硝胺杂质的致突变性风险,未来可与二代测序技术相结合进一步进行亚硝基类化合物的遗传毒性评价。

关键词: 亚硝胺杂质, PIG-A基因突变试验, TK6细胞, 代谢活化

Abstract: Objective To explore the reliability and sensitivity of an in vitro PIG-A gene mutation assay based on TK6 cells, and establish an experimental system for evaluating N-nitrosamine impurities. Methods Immunomagnetic bead separation was used to eliminate the spontaneous mutation rate of the PIG-A gene in TK6 cells. As required by the in vitro metabolic activation for mutagenicity assessment of nitrosamine impurities, the assay was performed with hamster liver S9 metabolic activation. DMSO served as the negative control, and NDEA (31.25-2 000 μg·mL-1) as the positive control. High-throughput flow cytometry was used to detect changes in the CD55 and CD59 phenotypes of TK6 cells. Under the established conditions, two nitrosamine impurities related to pharmaceutical structures were tested: 1-cyclopentyl-4-nitrosopiperazine (CPNP, 50-300 μg·mL-1) and N-nitrosobumetanide (250-2 000 μg·mL-1). Results The spontaneous mutation frequency decreased to 0.007 2% after the final elimination. In the absence of metabolic activation, no significant differences were observed in any of the NDEA groups compared with the negative control. In the presence of hamster S9, the PIG-A gene mutation frequency was significantly increased at NDEA concentrations of 500 μg·mL-1 and above. Under S9 activation, the mutation frequency was significantly elevated in the 150 and 300 μg·mL-1 CPNP groups, but no significant differences were found in any of the N-nitrosobumetanide groups. Without metabolic activation, neither CPNP nor N-nitrosobumetanide showed significant differences at any dose compared with the negative control. Conclusion The in vitro PIG-A gene mutation assay based on TK6 cells can effectively detect the mutagenic risk of N-nitrosamine impurities. In the future, it can be combined with next-generation sequencing technology to conduct genotoxicity evaluation of nitrosamine compounds.

Key words: N-nitrosamine Impurities, PIG-A Gene Mutation Assay, TK6 Cells, Metabolic Activation

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