基于非靶向代谢组学的吡虫啉暴露致人肝细胞HepG2毒性效应研究  

The toxic effects of imidacloprid exposure on HepG2 cell based on non-targeted metabolomics

作　　者：周兴藩 孙谊然 朱晓俊 林梦文 白文琳[2] 张英英 张文平[2] ZHOU Xingfan;SUN Yiran;ZHU Xiaojun;LIN Mengwen;BAI Wenlin;ZHANG Yingying;ZHANG Wenping(Beijing Key Laboratory of Occupational Safety and Health,Institute of Urban Safety and Environmental Science,Beijing Academy of Science and Technology,Beijing 100054,China;School of Public Health,Shanxi Medical University,Taiyuan,Shanxi 030001,China;National Center for Occupational Safety and Health,NHC,Beijing 102308,China)

机构地区：[1]北京市科学技术研究院城市安全与环境科学研究所,职业安全健康北京市重点实验室,北京100054 [2]山西医科大学公共卫生学院,山西太原030001 [3]国家卫生健康委职业安全卫生研究中心,北京102308

出　　处：《环境与职业医学》2023年第2期216-223,229,共9页Journal of Environmental and Occupational Medicine

基　　金：北京市科学技术研究院改革与发展项目(BJAST-RD_202103&202109-10);职业安全健康北京市重点实验室开放课题项目(2F022020027)。

摘　　要：[背景]吡虫啉是一种被广泛使用于农业生产中的新烟碱类杀虫剂,在人群生物样本中检出率高。已有研究显示吡虫啉暴露与肝损伤相关性较高,但具体机制不明。[目的]观察吡虫啉暴露对人肝细胞HepG2毒性效应及其非靶向代谢谱的扰动影响,分析其主要参与的生物学过程和信号通路,探讨吡虫啉肝毒性可能的分子机制。[方法]制备人肝细胞HepG2细胞悬液接种于96孔板中,分为空白对照组、二甲基亚砜(DMSO)溶剂对照组和多种浓度的吡虫啉染毒组,每组设5个平行样。MTT法测定不同浓度吡虫啉(1、2.5、5、7.5、10 mmol·L^(-1))染毒8 h后的HepG2细胞存活率,分析其剂量-效应关系。选用细胞存活率为80%对应的吡虫啉暴露浓度(3 mmol·L^(-1))对HepG2细胞进行染毒,应用UHPLC-Q-TOF/MS技术进行非靶向代谢组学分析,并与空白对照组和DMSO溶剂对照组进行比较分析,筛选各组间差异代谢物,利用京都基因与基因组百科全书(KEGG)数据库进行注释,分析其富集的生物过程和相关信号通路。[结果]与空白对照组和DMSO溶剂对照组比较,吡虫啉暴露组HepG2细胞存活率降低,差异有统计学意义(P<0.001)。暴露于2.5mmol·L^(-1)吡虫啉8 h后,HepG2细胞存活率约为86%。非靶向代谢组学研究发现,暴露3 mmol·L^(-1)吡虫啉可引起HepG2细胞包括肌酸(变量权重值VIP=1.11,P<0.001)、精氨酸(VIP=1.47,P=0.048)、牛磺酸(VIP=4.28,P=0.001)、α-D-葡萄糖(VIP=1.90,P=0.006)等在内的61种差异代谢产物水平紊乱,主要涉及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路(P<0.001)、精氨酸和脯氨酸代谢(P=0.002)及半乳糖代谢(P=0.015)等。[结论]吡虫啉暴露能够显著抑制HepG2细胞存活率,干扰mTOR信号通路、精氨酸和脯氨酸代谢、半乳糖代谢等通路。[Background] Imidacloprid is a neonicotinoid insecticide that is widely used in agricultural production, with a high detection rate in human biological samples. Previous studies have shown a high correlation between imidacloprid exposure and liver injury, but the specific mechanism is still unknown.[Objective] To observe potential toxic effects of HepG2 cells and its perturbation of non-targeted metabolic profile after imidacloprid exposure, and to explore possible molecular mechanisms of hepatotoxicity of imidacloprid by analyzing invovlved biological processes and signaling pathways.[Methods] HepG2 cell suspension was prepared and seeded in a 96-well plate, which was divided into blank control group, dimethyl sulfoxide(DMSO) solvent control group and imidacloprid exposure groups with multiple concentrations. Each group was set with 5 parallel samples. The viability of HepG2 cells viability were determined after 8 h of exposure to different concentrations of imidacloprid(1, 2.5, 5, 7.5, 10 mmol·L^(-1)), and the dose-effect relationship was analyzed. A proper concentration(3 mmol·L^(-1)with 80%viability) was chosen for imidacloprid exposure, non-targeted metabolomic analysis was applied to the cultivated HepG2 cells using UHPLC-Q-TOF/MS technology, the differential metabolites between groups were screened, and the bioprocess and related signaling pathways of their enrichment were annotated using the Kyoto Encyclopedia of Genes and Genomes(KEGG) database.[Results] Compared to the other two groups, the survival rates of HepG2 cells in the imidacloprid exposure groups decreased. A survival rate of about 86% of HepG2 cells was found in HepG2 cells exposed to 2.5 mmol·L^(-1)imidacloprid exposure. The non-targeted metabolomics studies showed that 61 metabolites were significantly affected in HepG2 cells after 3 mmol·L^(-1)imidacloprid exposure, including creatine(variable importance in projection VIP=1.11, P < 0.001), arginine(VIP=1.47, P=0.048), taurine(VIP=4.28, P=0.001), and α-D-glucose(VIP=1.90, P=0.006). The

关 键 词：吡虫啉 HepG2细胞 非靶向代谢组学 MTOR信号通路 半乳糖代谢 

分 类 号：R114[医药卫生—卫生毒理学]