目的 從腸道菌群的角度探討結(jié)構(gòu)相似的N-亞硝基乙基異丙胺（NEIPA）和N-亞硝基二異丙胺（NDIPA）的肝毒性強(qiáng)弱與其代謝途徑差異的關(guān)聯(lián)。方法 70只C57BL/6J小鼠隨機(jī)分為7組：對(duì)照組（0.5% CMC-Na），NEIPA低、中、高劑量（25、50、100 mg·kg^-1）組，NDIPA低、中、高劑量（25、50、100 mg·kg^-1）組，各組連續(xù)ig給藥7 d，隨后設(shè)置3周恢復(fù)期，并且分別在末次給藥和恢復(fù)期結(jié)束解剖動(dòng)物和觀察指標(biāo)。實(shí)驗(yàn)期間，每天觀察動(dòng)物一般狀態(tài)，每周測定體質(zhì)量。末次給藥后稱肝臟質(zhì)量、進(jìn)行血清生化檢查和肝組織病理學(xué)檢查。試驗(yàn)期間收集糞便，并進(jìn)行16S rRNA基因測序，比較其在腸道菌群結(jié)構(gòu)組成方面的差異。結(jié)果 與對(duì)照組相比，NEIPA組和NDIPA組在首次給藥后28 d內(nèi)均未觀察到異常癥狀或明顯體質(zhì)量下降，且NEIPA組和NDIPA組與對(duì)照組在臟器指數(shù)上無顯著差異；首次給藥后7、28 d時(shí)，NEIPA高劑量組在丙氨酸氨基轉(zhuǎn)移酶（ALT）水平上顯著升高（P＜0.01、0.001）；給藥7 d時(shí)，NDIPA高劑量組肌酸激酶（CK）水平顯著升高（P＜0.05），但在首次給藥后28 d時(shí)無顯著差異。NEIPA中、高劑量組可見肝細(xì)胞肥大/核巨大，病變發(fā)生率及病變程度隨劑量增加而增加；腸道菌群分析結(jié)果提示，與對(duì)照組相比，在給予NEIPA的小鼠中，Turicibacter、Lactobacillus、Akkermansia和Faecelbaculum的豐度顯著增加，Liilactobacilus、f_Lachnospiraceae、Lachnospiraceae_NK4A136_group的豐度降低；在給予NDIPA的小鼠中，f_Muribaculaceae、g_unclassifed_o-Clostridia_UCG-014、Lactobacillus和Akkermansia的豐度增加，Lachnospiraceae_NK4A136和liilactobacilus的豐度降低。結(jié)論 NEIPA僅比NDIPA多一個(gè)α-氫原子，但肝毒性存在一定差異，NEIPA增加了與膽汁酸代謝相關(guān)的Turicibacter的豐度，NDIPA增加了有促炎作用的o-Clostridia_UCG-014的豐度。NEIPA和NDIPA肝毒性風(fēng)險(xiǎn)的差異可能歸因于它們不同的代謝途徑，相關(guān)代謝途徑及機(jī)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

藥品監(jiān)管科學(xué)全國重點(diǎn)實(shí)驗(yàn)室課題“藥品雜質(zhì)遺傳毒性評(píng)價(jià)新技術(shù)和生物標(biāo)志物研究”（2023SKLDRS0128）