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五种农药及其二元、三元组合对人肝癌HepG2细胞的联合毒性

王天彩 陈晨 马朦朦 杨茜 李耘 钱永忠

王天彩, 陈晨, 马朦朦, 杨茜, 李耘, 钱永忠. 五种农药及其二元、三元组合对人肝癌HepG2细胞的联合毒性[J]. 农药学学报. doi: 10.16801/j.issn.1008-7303.2021.0021
引用本文: 王天彩, 陈晨, 马朦朦, 杨茜, 李耘, 钱永忠. 五种农药及其二元、三元组合对人肝癌HepG2细胞的联合毒性[J]. 农药学学报. doi: 10.16801/j.issn.1008-7303.2021.0021
Tiancai WANG, Chen CHEN, Mengmeng MA, Xi YANG, Yun LI, Yongzhong QIAN. Combined toxicity of five pesticides and their binary and ternary mixtures on HepG2 cells[J]. Chinese Journal of Pesticide Science. doi: 10.16801/j.issn.1008-7303.2021.0021
Citation: Tiancai WANG, Chen CHEN, Mengmeng MA, Xi YANG, Yun LI, Yongzhong QIAN. Combined toxicity of five pesticides and their binary and ternary mixtures on HepG2 cells[J]. Chinese Journal of Pesticide Science. doi: 10.16801/j.issn.1008-7303.2021.0021

五种农药及其二元、三元组合对人肝癌HepG2细胞的联合毒性

doi: 10.16801/j.issn.1008-7303.2021.0021
基金项目: 国家重点研发计划 (2018YFC1603000);国家自然科学基金项目 (31771894)
详细信息
    作者简介:

    王天彩,女,硕士研究生,E­mail:18810583803@163.com

    通讯作者:

    陈晨,通信作者 (Author for correspondence),男,博士,主要从事食品安全风险评估,E­mail:chenchen02@caas.cn

  • 中图分类号: S481.1;TQ450.2

Combined toxicity of five pesticides and their binary and ternary mixtures on HepG2 cells

  • 摘要: 为研究农产品中农药混合污染的联合毒性,选取生菜中应用广泛、广谱性强的5种农药(苯醚甲环唑、氯氰菊酯、烯酰吗啉、氯氟氰菊酯和啶虫脒)及其高频检出的二元、三元农药组合为研究对象,采用CCK-8 (Cell Counting Assay Kit-8) 法探究农药单剂及混合物对人肝癌HepG2细胞的增殖抑制毒性,基于单个农药剂量效应曲线按照等效应的联合毒性测定方法,应用浓度相加、独立作用和联合指数3个预测模型推导联合效应,并通过高内涵筛选系统分析农药混合物对人肝癌HepG2细胞凋亡的诱导作用。结果表明:不同农药对HepG2细胞增殖抑制的影响存在较大差异,但均具有明显的剂量-效应关系,其中苯醚甲环唑具有最强的细胞增殖抑制毒性,细胞活力的半数抑制浓度 (median effective concentration, EC50) 为24.72 μmol/L;其余农药毒性顺序为烯酰吗啉 > 氯氟氰菊酯 > 啶虫脒 > 氯氰菊酯。通过联合指数判定农药混合物的联合毒性效应的结果表明:所有二元组合均随农药浓度增大表现出细胞增殖抑制效应增强,联合效应由拮抗转为协同;三元组合烯酰吗啉 + 苯醚甲环唑 + 氯氰菊酯、烯酰吗啉 + 氯氰菊酯 + 啶虫脒的细胞增殖抑制效应分别为60%和18%时,联合效应由协同转为拮抗。同时,利用剂量减少指数量化农药混合物之间的协同效应研究表明,在上述两个三元组合的协同效应中,氯氰菊酯发挥较大作用。在凋亡毒性测定中,各农药单独处理组均会造成HepG2细胞凋亡率的显著提高,且呈剂量依赖性;三元组合烯酰吗啉 + 氯氰菊酯 + 啶虫脒在20.02~80.10 μmol/L浓度范围内可诱导细胞凋亡水平显著提升。本研究明确了农药单剂和混合物对HepG2细胞增殖和凋亡毒性具有明显的剂量依赖效应,可为混合农药毒性评价以及风险监测提供科学依据。联合指数模型可以定量地描述农药混合物在不同组分和浓度下的相互作用,对比浓度相加和独立作用模型,可较准确地预测混合物的联合毒性,是一种有效的生态毒理学风险评估工具。
  • 图  1  5种农药单剂 (A) 及二元 (B)、三元 (C) 农药组合暴露24 h对HepG2细胞增殖的抑制作用

    Figure  1.  Inhibitory effects of five individual pesticides (A), binary (B) and ternary (C) pesticides on HepG2 cell growth after 24 h treatment

    图  2  基于联合指数、浓度相加和独立作用模型的农药混合物细胞增殖毒性试验的实际和预测所得剂量-效应曲线

    Figure  2.  Actual and predicted concentration-response curves of cell growth inhibitory toxicity tests of pesticide mixtures based on combination index (CI), concentration addition (CA) and independent action (IA) models

    图  3  二元、三元混合农药的联合指数图

    Figure  3.  The combination index plot for binary and ternary pesticide mixtures

    图  4  基于CI指数模型分析得出的联合毒性强度及性质

    Figure  4.  The combined toxicity intensity and properties based on the analysis of CI model

    图  5  农药单剂及混合物对HepG2细胞凋亡的影响

    注:DIM、DIF、CYP和ACE分别表示烯酰吗啉、苯醚甲环唑、氯氰菊酯和啶虫脒,*** 分别表示与农药单剂处理组相比,差异显著 (P < 0.05) 和极显著 (P < 0.01)。

    Figure  5.  Effect of individual pesticides and mixtures on HepG2 cell apoptosis

    Note: DIM, DIF, CYP and ACE represent dimethomorph, difenoconazole, cypermethrin and acetamiprid, respectively. * and ** mean significant (P < 0.05) and extremely significant (P < 0.01) difference compared with the individual pesticide treatment group.

    表  1  三元农药组合对HepG2细胞增殖抑制毒性的联合作用

    Table  1.   The combined effects of ternary mixtures on HepG2 cell growth inhibitory toxicity

    农药组合
    Pesticide combination
    浓度比
    Concentration ratio
    10% 毒性效应水平
    10% Toxicity effect level
    20% 毒性效应水平
    20% Toxicity effect level
    CIDRICIDRI
    烯酰吗啉 + 苯醚甲环唑 + 氯氰菊酯
    dimethomorph + difenoconazole + cypermethrin
    1.54 : 1.00: 5.17 0.53 3.36 (烯酰吗啉 dimethomorph) 0.63 3.25 (烯酰吗啉 dimethomorph)
    8.42 (苯醚甲环唑 difenoconazole) 5.65 (苯醚甲环唑 difenoconazole)
    8.50 (氯氰菊酯 cypermethrin) 6.99 (氯氰菊酯 cypermethrin)
    烯酰吗啉 + 氯氰菊酯 + 啶虫脒
    dimethomorph + cypermethrin + acetamiprid
    1.00 : 3.35 : 1.95 0.18 14.14 (烯酰吗啉 dimethomorph) 1.43 1.88 (烯酰吗啉 dimethomorph)
    35.82 (氯氰菊酯 cypermethrin) 4.04 (氯氰菊酯 cypermethrin)
    12.50 (啶虫脒 acetamiprid) 1.54 (啶虫脒 acetamiprid)
    注:CI值是根据混合物的毒性效应曲线计算出的混合物在x%毒性效应水平下的联合指标值。CI < 1,CI = 1和CI > 1分别表示协同作用,相加作用和拮抗作用。DRI值是指在给定的效应水平上,与单独使用每种农药的剂量相比,混合物中每种农药的剂量可减少的倍数。Note: CI value is the combination index value of the mixture at the x% toxicity effect level calculated according to the toxicity effect curve of the mixture. CI < 1, CI = 1 and CI > 1 represent synergistic effect, additive effect and antagonistic effect, respectively.The DRI value is the number of times that the dose of each pesticide in the mixture can be reduced compared with the dose of each pesticide used alone at a given level of effect.
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  • 收稿日期:  2020-07-16
  • 录用日期:  2020-11-02
  • 网络出版日期:  2021-02-05

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