目的 从肠道菌群的角度探讨结构相似的N-亚硝基乙基异丙胺（NEIPA）和N-亚硝基二异丙胺（NDIPA）的肝毒性强弱与其代谢途径差异的关联。方法 70只C57BL/6J小鼠随机分为7组：对照组（0.5% CMC-Na），NEIPA低、中、高剂量（25、50、100 mg·kg^-1）组，NDIPA低、中、高剂量（25、50、100 mg·kg^-1）组，各组连续ig给药7 d，随后设置3周恢复期，并且分别在末次给药和恢复期结束解剖动物和观察指标。实验期间，每天观察动物一般状态，每周测定体质量。末次给药后称肝脏质量、进行血清生化检查和肝组织病理学检查。试验期间收集粪便，并进行16S rRNA基因测序，比较其在肠道菌群结构组成方面的差异。结果 与对照组相比，NEIPA组和NDIPA组在首次给药后28 d内均未观察到异常症状或明显体质量下降，且NEIPA组和NDIPA组与对照组在脏器指数上无显著差异；首次给药后7、28 d时，NEIPA高剂量组在丙氨酸氨基转移酶（ALT）水平上显著升高（P＜0.01、0.001）；给药7 d时，NDIPA高剂量组肌酸激酶（CK）水平显著升高（P＜0.05），但在首次给药后28 d时无显著差异。NEIPA中、高剂量组可见肝细胞肥大/核巨大，病变发生率及病变程度随剂量增加而增加；肠道菌群分析结果提示，与对照组相比，在给予NEIPA的小鼠中，Turicibacter、Lactobacillus、Akkermansia和Faecelbaculum的丰度显著增加，Liilactobacilus、f_Lachnospiraceae、Lachnospiraceae_NK4A136_group的丰度降低；在给予NDIPA的小鼠中，f_Muribaculaceae、g_unclassifed_o-Clostridia_UCG-014、Lactobacillus和Akkermansia的丰度增加，Lachnospiraceae_NK4A136和liilactobacilus的丰度降低。结论 NEIPA仅比NDIPA多一个α-氢原子，但肝毒性存在一定差异，NEIPA增加了与胆汁酸代谢相关的Turicibacter的丰度，NDIPA增加了有促炎作用的o-Clostridia_UCG-014的丰度。NEIPA和NDIPA肝毒性风险的差异可能归因于它们不同的代谢途径，相关代谢途径及机制有待深入研究。

Objective To investigate the relationship between the hepatotoxicity of N-nitrosoethyl isopropylamine (NEIPA) and Nnitroso-diisopropylamine (NDIPA) and their differences in metabolic pathways from the perspective of intestinal flora. Methods Seventy C57BL/6J mice were randomly divided into seven groups: the control group (0.5% CMC-Na), the low, medium and high dose NEIPA groups (25, 50, and 100 mg·kg^-1), and the low, medium and high dose NDIPA groups (25, 50, and 100 mg·kg^-1). Each group was ig administered for seven consecutive days, followed by a three-week recovery period. Animals were dissected and indicators were observed at the end of the last administration and at the end of the recovery period, respectively. During the experiment, clinical symptoms were observed daily and weight was measured weekly. After the last dose, liver was weighed, serum biochemical examination and liver histopathology were performed. During the trial, stool was collected and 16S rRNA gene was sequenced to compare the differences in the structural composition of intestinal flora. Results Compared with the solvent group, no abnormal symptoms or significant weight loss were observed in NEIPA group and NDIPA group within 28 d after the first dose, and there was no significant difference in organ coefficient between NEIPA group and NDIPA group and the solvent control group. At D7 and D28 after administration, the ALT level in the high-dose group (100 mg·kg^-1) of NEIPA group was significantly increased (P < 0.01, 0.001); CK levels in the high-dose group (100 mg·kg^-1) of the NDIPA group were significantly increased at D7 after administration (P < 0.05), but there was no significant difference at D28 after administration. In NEIPA group (50 and 100 mg·kg^-1), hepatocyte hypertrophy and nuclear giant were observed, and the incidence and degree of lesion increased with the increase of dose. The results of intestinal microflora analysis suggested that the abundance of Turicibacter, Lactobacillus, Akkermansia and Faecelbaculum were significantly increased in mice given NEIPA compared with the solvent group. The abundance of Liilactobacilus, f_Lachnospiraceae, Lachnospiraceae_NK4A136_group decreased. In mice given NDIPA, the abundance of f_Muribaculaceae, g_unclassifed_oClostridia_UCG-014, Lactobacillus and Akkermansia increased, the abundance of Lachnospiraceae_NK4A136 and liilactobacilus decreased. Conclusion NEIPA has only one more α-hydrogen atom than NDIPA, but there is some difference in hepatotoxicity. NEIPA increases the abundance of Turicibacter, which is related to bile acid metabolism, and NDIPA increases the abundance of oClostridia_UCG-014, which has pro-inflammatory effects. The difference in hepatotoxicity risk between NEIPA and NDIPA may be attributed to their different metabolic pathways, and relevant metabolic pathways and mechanisms need to be further studied.

R965

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）