新烟碱类杀虫剂吡虫啉和啶虫脒对人神经母细胞瘤SK-N-SH细胞的毒性作用  

Toxic Effects of Neonicotinoid Insecticides Imidacloprid and Acetamiprid on SK-N-SH Cells

作　　者：黄钰婷 李敬尧 张琪[1] 王丹[1] 周群芳[2] 卢晓霞[1] Huang Yuting;Li Jingyao;Zhang Qi;Wang Dan;Zhou Qunfang;Lu Xiaoxia(Key Laboratory for Earth Surface Processes,Ministry of Education,College of Urban and Environmental Sciences,Peking University,Beijing 100871,China;State Key Laboratory of Environmental Chemistry and Ecotoxicology,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences,Beijing 100085,China)

机构地区：[1]地表过程分析与模拟教育部重点实验室,北京大学城市与环境学院,北京100871 [2]中国科学院生态环境研究中心环境化学与生态毒理学国家重点实验室,北京100085

出　　处：《生态毒理学报》2021年第5期59-69,共11页Asian Journal of Ecotoxicology

基　　金：国家自然科学基金资助项目(41771528,41991330);环境化学与生态毒理学国家重点实验室开放基金资助项目(KF2016-15)。

摘　　要：吡虫啉和啶虫脒是目前使用较广的2种新烟碱类杀虫剂,它们在环境、食品和人体样品中的普遍残留,对人体健康构成威胁,但目前关于其对人的神经毒性仍知之甚少。本文以人神经母细胞瘤SK-N-SH细胞为模型,采用体外细胞实验,研究不同浓度吡虫啉和啶虫脒暴露对细胞活力、细胞形态、烟碱型乙酰胆碱受体(nAChRs)α7亚基的mRNA和蛋白表达、乙酰胆碱酯酶活性以及氧化应激的影响,为这2种杀虫剂的健康风险评价提供依据。实验中,先将SK-N-SH细胞分别暴露于不同浓度的吡虫啉和啶虫脒24 h,通过测定细胞活力,确定2种杀虫剂的10%抑制浓度(IC_(10))值。在此基础上,设定3个梯度的低暴露浓度(0.01、0.1和1 mmol·L^(-1))和溶剂对照,研究吡虫啉和啶虫脒对SK-N-SH细胞其他指标的影响(暴露24 h后)。细胞活力、氧化损伤和乙酰胆碱酯酶活性分别用相应的试剂盒测定,细胞形态用倒置光学显微镜观察,nAChRsα7亚基的mRNA和蛋白表达分别用RT-qPCR和Western Blot测定。结果表明,吡虫啉和啶虫脒的IC_(10)值分别约为1.5 mmol·L^(-1)和2.0 mmol·L^(-1)。1 mmol·L^(-1)吡虫啉对nAChRα7的mRNA表达和蛋白表达均显著提高,对乙酰胆碱酯酶活性有显著抑制,并引起细胞显著的氧化应激(P<0.05)。0.1 mmol·L^(-1)吡虫啉对乙酰胆碱酯酶活性有显著抑制(P<0.05)。1 mmol·L^(-1)啶虫脒对nAChRα7的mRNA表达和蛋白表达均有显著提高(P<0.05)。为进一步揭示吡虫啉的影响,对0.1 mmol·L^(-1)吡虫啉暴露组和溶剂对照组细胞进行了转录组分析,发现吡虫啉暴露对一些神经退行性疾病相关基因及其他一些重要通路相关基因有显著影响。本研究证明,吡虫啉和啶虫脒在非致死浓度条件下会对细胞产生一系列显著影响,吡虫啉的影响大于啶虫脒。Imidacloprid and acetamiprid are two kinds of widely-used neonicotinoid insecticides,and their residues are commonly found in the environment,food and human bodies,which poses a serious threat to public health.Besides the available data on the toxicological effects of these two insecticides,their neurotoxicity still remains unclear.In this paper,using human neuroblastoma SK-N-SH cell model,in vitro experiment was performed to study the effects of imidacloprid and acetamiprid on cell vitality,nicotinic acetylcholine receptors(nAChRs)α7 mRNA and protein expression,acetyl cholinesterase(AChE)activity,and oxidative stress.SK-N-SH cells were exposed to imidacloprid and acetamiprid at different concentrations for 24 h,and the 10%inhibition concentration(IC_(10))values of the two insecticides were determined by measuring cell viability.On this basis,three lower exposure concentrations(0.01,0.1 and 1 mmol·L^(-1))were set to study the effects of imidacloprid and acetamiprid on other indexes of SK-N-SH cells(after exposure for 24 h).Cell viability,cell morphology,AChE activity,and oxidative stress were determined by the corresponding kits,the gene expression of nAChRα7 was determined by RT-qPCR and the protein expression of nAChRα7 was determined by Western Blot.The results showed that the IC_(10) values of imidacloprid and acetamiprid were about 1.5 mmol·L^(-1)and 2.0 mmol·L^(-1),respectively.Imidacloprid at 1 mmol·L^(-1)significantly promoted the mRNA and protein expressions of nAChRα7,inhibited AChE activity,and caused oxidative stress(P<0.05).Imidacloprid at 0.1 mmol·L^(-1)significantly inhibited AChE activity(P<0.05).Acetamiprid at 1 mmol·L^(-1)significantly promoted the mRNA and protein expressions of nAChRα7(P<0.05).To further reveal the effect of imidacloprid,transcriptome analysis of cells from 0.1 mmol·L^(-1)imidacloprid exposure group and solvent control group(sequenced with Illumina HiSeq 2500)was performed and it turned out that genes associated with some neurodegenerative diseases were significant

关 键 词：吡虫啉 啶虫脒 SK-N-SH细胞 急性毒性 神经毒性 nAChRα7亚基 

分 类 号：X171.5[环境科学与工程—环境科学]