黄钰婷¹,
李敬尧¹,
张琪¹,
王丹¹,
周群芳²,
卢晓霞¹
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

作者简介: 黄钰婷(1998-),女,硕士,研究方向为生态毒理学,E-mail:18012141642@pku.edu.cn.

基金项目: 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）


中图分类号: X171.5

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

Huang Yuting¹,
Li Jingyao¹,
Zhang Qi¹,
Wang Dan¹,
Zhou Qunfang²,
Lu Xiaoxia¹
1. Key Laboratory for Earth Surface Processes, Ministry of Education, College of Urban and Environmental Sciences, Peking University, Beijing 100871, China;
2. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China

CLC number: X171.5

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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（IC₁₀）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，nAChRs α7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IC₁₀值分别约为1.5 mmol·L^-1和2.0 mmol·L^-1。1mmol ·L^-1吡虫啉对nAChR α7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nAChR α7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。
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

Abstract: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 (IC₁₀) 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 nAChR α7 was determined by RT-qPCR and the protein expression of nAChR α7 was determined by Western Blot. The results showed that the IC₁₀ 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 nAChR α7, 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 nAChR α7 (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 significantly affected by the exposure to imidacloprid. This study demonstrated that imidacloprid and acetamiprid could produce a series of significant effects on cells at nonlethal concentrations, and the effect of imidacloprid was greater than that of acetamiprid.
Key words:imidacloprid/
acetamiprid/
SK-N-SH cells/
acute toxicity/
neurotoxicity/
nAChR α7 subunit.

Tomizawa M, Casida J E. Neonicotinoid insecticide toxicology:Mechanisms of selective action[J]. Annual Review of Pharmacology and Toxicology, 2005, 45:247-268

益军. 新烟碱类杀虫剂货紧价扬市场景气度提升仍将持续[J]. 农药市场信息, 2017, 5(30):29-31

Goulson D. An overview of the environmental risks posed by neonicotinoid insecticides[J]. Journal of Applied Ecology, 2013, 50(4):977-987

Seccia S, Fidente P, Barbini D A, et al. Multiresidue determination of nicotinoid insecticide residues in drinking water by liquid chromatography with electrospray ionization mass spectrometry[J]. Analytica Chimica Acta, 2005, 553(1-2):21-26

Xie W, Han C, Qian Y, et al. Determination of neonicotinoid pesticides residues in agricultural samples by solidphase extraction combined with liquid chromatographytandem mass spectrometry[J]. Journal of Chromatography A, 2011, 1218(28):4426-4433

Seccia S, Fidente P, Montesano D, et al. Determination of neonicotinoid insecticides residues in bovine milk samples by solid-phase extraction clean-up and liquid chromatography with diode-array detection[J]. Journal of Chromatography A, 2008, 1214(1-2):115-120

吕正标. 新烟碱类杀虫剂在水环境中的残留特征及潜在风险[D]. 杭州:浙江工业大学, 2020:22-25 Lv Z B. Residual characteristics and potential risks of neonicotinoids in waters[D]. Hangzhou:Zhejiang University of Technology, 2020:22-25(in Chinese)

谭颖, 张琪, 赵成, 等. 蔬菜水果中的新烟碱类农药残留量与人群摄食暴露健康风险评价[J]. 生态毒理学报, 2016, 11(6):67-81Tan Y, Zhang Q, Zhao C, et al. Residues of neonicotinoid pesticides in vegetables and fruit and health risk assessment of human exposure via food intake[J]. Asian Journal of Ecotoxicology, 2016, 11(6):67-81(in Chinese)

Wang L, Liu T Z, Liu F, et al. Occurrence and profile characteristics of the pesticide imidacloprid, preservative parabens, and their metabolites in human urine from rural and urban China[J]. Environmental Science & Technology, 2015, 49(24):14633-14640

Chen Q Y, Zhang Y Y, Li J H, et al. Serum concentrations of neonicotinoids, and their associations with lipid molecules of the general residents in Wuxi City, Eastern China[J]. Journal of Hazardous Materials, 2021, 413:125235

张琪, 赵成, 卢晓霞, 等. 新烟碱类杀虫剂对非靶标生物毒性效应的研究进展[J]. 生态毒理学报, 2020, 15(1):56-71Zhang Q, Zhao C, Lu X X, et al. Advances in research on toxic effects of neonicotinoid insecticides on non-target organisms[J]. Asian Journal of Ecotoxicology, 2020, 15(1):56-71(in Chinese)

Weber M, Motin L, Gaul S, et al. Intravenous anaesthetics inhibit nicotinic acetylcholine receptor-mediated currents and Ca2+ transients in rat intracardiac ganglion neurons[J]. British Journal of Pharmacology, 2005, 144(1):98-107

Yang W, Carmichael S L, Roberts E M, et al. Residential agricultural pesticide exposures and risk of neural tube defects and orofacial clefts among offspring in the San Joaquin Valley of California[J]. American Journal of Epidemiology, 2014, 179(6):740-748

Keil A P, Daniels J L, Hertz-Picciotto I. Autism spectrum disorder, flea and tick medication, and adjustments for exposure misclassification:The CHARGE (CHildhood Autism Risks from Genetics and Environment) case-control study[J]. Environmental Health, 2014, 13(1):3

Bal R, Erdogan S, Theophilidis G, et al. Assessing the effects of the neonicotinoid insecticide imidacloprid in the cholinergic synapses of the stellate cells of the mouse cochlear nucleus using whole-cell patch-clamp recording[J]. NeuroToxicology, 2010, 31(1):113-120

Kimura-Kuroda J, Komuta Y, Kuroda Y, et al. Nicotinelike effects of the neonicotinoid insecticides acetamiprid and imidacloprid on cerebellar neurons from neonatal rats[J]. PLoS One, 2012, 7(2):e32432

Zhou Y, Lu X X, Fu X F, et al. Development of a fast and sensitive method for measuring multiple neonicotinoid insecticide residues in soil and the application in parks and residential areas[J]. Analytica Chimica Acta, 2018, 1016:19-28

Hirano T, Minagawa S, Furusawa Y, et al. Growth and neurite stimulating effects of the neonicotinoid pesticide clothianidin on human neuroblastoma SH-SY5Y cells[J]. Toxicology and Applied Pharmacology, 2019, 383:114777

Cheng L, Lu Y Y, Zhao Z H, et al. Assessing the combined toxicity effects of three neonicotinoid pesticide mixtures on human neuroblastoma SK-N-SH and lepidopteran Sf-9 cells[J]. Food and Chemical Toxicology, 2020, 145:111632

邱敏, 李晓红, 黄兹锐, 等. 尼古丁对多种干细胞中烟碱型乙酰胆碱受体表达的影响[J]. 中国病理生理杂志, 2020, 36(7):1287-1293Qiu M, Li X H, Huang Z R, et al. Effects of nicotine on expression of nicotinic acetylcholine receptors in several kinds of stem cells[J]. Chinese Journal of Pathophysiology, 2020, 36(7):1287-1293(in Chinese)

Convertini P, Zhang J Y, de la Grange P, et al. Genome wide array analysis indicates that an amyotrophic lateral sclerosis mutation of FUS causes an early increase of CAMK2N2 in vitro[J]. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2013, 1832(8):1129-1135

Schroeder M E, Flattum R F. The mode of action and neurotoxic properties of the nitromethylene heterocycle insecticides[J]. Pesticide Biochemistry and Physiology, 1984, 22(2):148-160

潘承谕, 余晋霞, 田英, 等. 新烟碱类杀虫剂人群暴露水平及生殖毒性的研究进展[J]. 环境与职业医学, 2020, 37(11):1064-1069Pan C Y, Yu J X, Tian Y, et al. Research progress on human exposure levels and reproductive toxicity of neonicotinoids[J]. Journal of Environmental and Occupational Medicine, 2020, 37(11):1064-1069(in Chinese)

Le Novère N, Corringer P J, Changeux J P. The diversity of subunit composition in nAChRs:Evolutionary origins, physiologic and pharmacologic consequences[J]. Journal of Neurobiology, 2002, 53(4):447-456

Millar N S. Assembly and subunit diversity of nicotinic acetylcholine receptors[J]. Biochemical Society Transactions, 2003, 31(4):869-874

田华, 贾继民, 张建江, 等. 7种有机磷农药对不同来源乙酰胆碱酯酶活性的抑制作用比较[J]. 解放军预防医学杂志, 2016, 34(6):846-848Tian H, Jia J M, Zhang J J, et al. Comparative analysis of inhibition of seven orgnaophosphorus pesticides to activity of acetylcholinesterase (AChE) from different sources[J]. Journal of Preventive Medicine of Chinese, 2016, 34(6):846-848(in Chinese)

郭志英, 任启伟, 林琳. 谷胱甘肽GSH/GSSG氧化还原态各指标的测定[J]. 济宁医学院学报, 2007, 30(1):36-38Guo Z Y, Ren Q W, Lin L. Research of measuring glutathion GSH/GSSG redox state[J]. Journal of Jining Medical College, 2007, 30(1):36-38(in Chinese)