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
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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。1 mmol·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。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 non-lethal concentrations, and the effect of imidacloprid was greater than that of acetamiprid.
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Key words:
- imidacloprid /
- acetamiprid /
- SK-N-SH cells /
- acute toxicity /
- neurotoxicity /
- nAChR α7 subunit
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