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基于荧光探针技术检测30种农兽药及其二元、三元组合对CYP3A4酶的联合毒性

朱新月 陈立森 何深贵 赵鑫 程林丽 崔京南

朱新月, 陈立森, 何深贵, 赵鑫, 程林丽, 崔京南. 基于荧光探针技术检测30种农兽药及其二元、三元组合对CYP3A4酶的联合毒性[J]. 农药学学报. doi: 10.16801/j.issn.1008-7303.2022.0013
引用本文: 朱新月, 陈立森, 何深贵, 赵鑫, 程林丽, 崔京南. 基于荧光探针技术检测30种农兽药及其二元、三元组合对CYP3A4酶的联合毒性[J]. 农药学学报. doi: 10.16801/j.issn.1008-7303.2022.0013
ZHU Xinyue, CHEN Lisen, HE Shengui, ZHAO Xin, CHENG Linli, CUI Jingnan. Detecting the combined toxicity of 30 pesticides and veterinary drugs and their binary and ternary combinations toward CYP3A4 based on fluorescence probe technology[J]. Chinese Journal of Pesticide Science. doi: 10.16801/j.issn.1008-7303.2022.0013
Citation: ZHU Xinyue, CHEN Lisen, HE Shengui, ZHAO Xin, CHENG Linli, CUI Jingnan. Detecting the combined toxicity of 30 pesticides and veterinary drugs and their binary and ternary combinations toward CYP3A4 based on fluorescence probe technology[J]. Chinese Journal of Pesticide Science. doi: 10.16801/j.issn.1008-7303.2022.0013

基于荧光探针技术检测30种农兽药及其二元、三元组合对CYP3A4酶的联合毒性

doi: 10.16801/j.issn.1008-7303.2022.0013
基金项目: 国家科技部重点研发计划 (2018YFC1603001).
详细信息
    作者简介:

    朱新月,zxy0812@mail.dlut.edu.cn

    通讯作者:

    崔京南,jncui@dlut.edu.cn

  • 中图分类号: S481.1;TQ450.2

Detecting the combined toxicity of 30 pesticides and veterinary drugs and their binary and ternary combinations toward CYP3A4 based on fluorescence probe technology

Funds: National Key R&D Program of China (2018YFC1603001)
  • 摘要: 在农业生产中混合使用多种农药或兽药越来越普遍,但因药剂联合毒性效应的不确定性而对人体健康产生严重威胁。本文基于酶抑制法原理,利用荧光探针 NEN (N-乙基-1,8-萘二甲酰亚胺) 直接检测CYP3A4酶的活性,建立了广谱筛查混合农兽药联合毒性效应的方法,并以常用的30种农兽药及其典型的23种二元和26种三元组合为研究对象,检测了农兽药混合物对CYP3A4酶的联合毒性效应,其中标准质量浓度根据食品国家安全标准规定的农兽药最大残留限量确定。结果表明:3种质量浓度梯度下对CYP3A4酶均具有协同作用的混合农兽药组合有克百威 + 多菌灵、克百威 + 吡虫啉、啶虫脒 + 烯酰吗啉、吡虫啉 + 多菌灵、氰氯菊酯 + 啶虫脒 + 烯酰吗啉、克百威 + 啶虫脒 + 多菌灵、吡虫啉 + 啶虫脒 + 多菌灵、吡虫啉 + 啶虫脒 + 烯酰吗啉、毒死蜱 + 啶虫脒 + 多菌灵和联苯菊酯 + 啶虫脒 + 多菌灵。当单一农兽药对CYP3A4酶活性的抑制率较高时,与其他农兽药混合后联合毒性效应呈拮抗作用,而当单一农兽药对酶活性的抑制率低于2%时,则与其他农兽药混合后联合毒性效应呈现不确定性。农兽药组合在低浓度下对CYP3A4酶的联合毒性往往存在较强的协同作用,但随着浓度的升高,联合毒性效应从协同变为拮抗作用。分析农兽药与CYP3A4酶之间的构效关系可知,含有芳氯基团的数量与对酶活性的抑制程度成正相关,含有3个芳氯及以上基团的农兽药对CYP3A4酶活性的抑制作用最为显著,抑制率在30%以上,如百菌清、毒死蜱、甲基毒死蜱、咪鲜胺等;含有2个芳氯或“强吸电子基团 + 1个芳氯”基团的农兽药,对CYP3A4酶活性的抑制作用较强,抑制率在18%以上,如苯醚甲环唑、哒螨灵和异菌脲等。具有氨基甲酸酯基团的农兽药单独作用于CYP3A4酶毒性较小或几乎没有毒性时,与其他农兽药混合后显示较强的协同作用。本研究建立的检测方法为广谱筛查混合农兽药联合毒性提供了新思路,检测结果可为进一步在细胞和动物水平制订农兽药混剂的风险评估方案设计提供依据。
  • 图  1  NEN对CYP3A4酶的荧光响应机制

    Figure  1.  The fluorescence response mechanism of the probe NEN toward CYP3A4

    图  2  30种农兽药在三种质量浓度梯度下对CYP3A4酶活性的抑制率 ( (a) 表1中农兽药编号1~15;(b) 表1中农兽药编号16~30)

    Figure  2.  The inhibition rate of 30 pesticides and veterinary drugs on CYP3A4 activity under three mass concentration gradients. (a) Pesticide and veterinary drug No. 1~15 in table 1. (b) Pesticide and veterinary drug No. 16~30 in table 1.

    图  3  23种典型的农兽药二元组合分别在3倍标准质量浓度 (a) 、标准质量浓度 (b) 、0.5倍标准质量浓度梯度 (c) 下对CYP3A4酶活性的抑制率实测值、加和计算值及差值柱状图 (注:图中编号参照表1)

    Figure  3.  The measured value, sum calculated value and difference histogram of inhibition rate of 23 typical pesticide and veterinary drug binary combinations on CYP3A4 activity under 3 times standard mass concentration (a), standard mass concentration (b) and 0.5 times standard mass concentration (c) respectively (Note: The number of each pesticide and veterinary drug was from Table 1)

    图  4  26种典型的农兽药三元组合分别在3倍标准质量浓度 (a) 、标准质量浓度 (b) 、0.5倍标准质量浓度梯度 (c) 下对CYP3A4酶活性的抑制率实测值、加和计算值及差值柱状图 (注:图中编号参照表1)

    Figure  4.  The measured value, sum calculated value and difference histogram of inhibition rate of 26 typical pesticide and veterinary drug ternary combinations on CYP3A4 activity under 3 times standard mass concentration (a), standard mass concentration (b) and 0.5 times standard mass concentration (c) respectively (Note: The number of each pesticide and veterinary drug was from Table 1)

    表  1  30种农兽药的编号、结构式、标准质量浓度及抑制率

    Table  1.   The number, structural formula, standard mass concentration and inhibition rate of 30 pesticides and veterinary drugs

    编号
    No.
    农兽药
    pesticide and
    veterinary drug
    结构式
    Structural
    formula
    标准质量浓度
    Standard mass
    concentration/(mg/L)
    抑制率
    Inhibition
    rate/%
    编号
    No.
    农兽药
    pesticide and
    veterinary drug
    结构式
    Structural
    formula
    标准质量浓度
    Standard mass
    concentration/(mg/L)
    抑制率
    Inhibition
    rate/%
    1 甲基毒死蜱
    chlorpyrifos-methyl
    1.00 23.5c±1.5 16 啶虫脒
    acetamiprid
    1.00 −10.8b±0.6
    2 毒死蜱
    chlorpyrifos
    1.00 50.1c±2.2 17 吡虫啉
    imidacloprid
    0.50 −1.9b±0.4
    3 氧化乐果
    omethoate
    0.02 −2.7a±0.3 18 多菌灵
    carbendazim
    0.50 −1.9c±0.2
    4 乐果
    dimethoate
    0.50 5.8b±0.7 19 咪鲜胺
    prochloraz
    0.017 73.2a±3.1
    5 甲胺磷
    methamidophos
    0.05 −6.5a±1.3 20 烯酰吗啉
    dimethomorph
    1.00 9.9c±1.1
    6 克百威
    carbofuran
    0.02 −1.2a±0.4 21 苯醚甲环唑
    difenoconazole
    0.008 61.2a±2.4
    7 速灭威
    MTMC
    0.05 0.0a±0.2 22 氟苯尼考
    florfenicol
    0.10 −1.6a±0.4
    8 联苯菊酯
    bifenthrin
    0.50 16.1b±1.3 23 呋喃妥因
    furantoin
    1.00 16.3c±1.6
    9 氰戊菊酯
    fenvalerate
    0.20 −4.4a±0.5 24 呋喃它酮
    furaltadone
    0.20 −3.9a±0.9
    10 氯氰菊酯
    cypermethrin
    1.00 17.3c±1.4 25 乙酰甲喹
    mequindox
    2.00 3.9c±0.7
    11 氯氟氰菊酯
    lambda-
    cyhalothrin
    0.50 6.4b±1.0 26 环丙沙星
    ciprofloxacin
    0.10 −2.9a±0.3
    12 哒螨灵
    pyridaben
    2.00 32.6c±2.6 27 恩诺沙星
    enrofloxacin
    0.10 −1.6a±0.5
    13 虫螨腈
    chlorfenapyr
    1.00 6.2c±1.5 28 奥硝唑
    ornidazole
    0.20 6.5a±0.8
    14 百菌清
    chlorothalonil
    0.167 63.7±2.8 29 替硝唑
    tinidazole
    0.20 −8.1a±0.4
    15 异菌脲
    iprodione
    5.00 8.6c±1.1 30 土霉素
    oxytetracycline
    0.20 −2.4a±0.3
    注:a、b、c分别表示农药在3倍标准浓度下、标准浓度及0.5倍标准浓度下的抑制率。Note: a, b and c represent the inhibition rates of pesticides and veterinary drugs at 3 times of standard concentration, standard concentration and 0.5 times of standard concentration respectively.
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  • [1] 董梦晓, 瞿玮, 谢书宇, 等. 兽用抗菌药复方注射剂研制进展[J]. 中国抗生素杂志, 2017, 42(10): 880-890. doi: 10.3969/j.issn.1001-8689.2017.10.011

    DONG M X, QU W, XIE S Y, et al. The development of compound injections for veterinary antibacterial agents[J]. Chin J Antibiot, 2017, 42(10): 880-890. doi: 10.3969/j.issn.1001-8689.2017.10.011
    [2] MINGLE C L, DARKO G, BORQUAYE L S, et al. Veterinary drug residues in beef, chicken, and egg from Ghana[J]. Chem Afr, 2021, 4(2): 339-348. doi: 10.1007/s42250-020-00225-5
    [3] LI Y, WANG W, GUO A H, et al. Screening and confirmation of pesticide residues in substitutional tea such as orange peel, lotus leaf, pueraria lobata and Pangdahai[J]. J hyg res, 2020, 49(5): 815-822.
    [4] YAO Q H, LI J, LIN Q, et al. Determination of seventeen insecticide residues in tremella fuciformis berk by QuEChERS-gas chromatography-tandem mass spectrometry[J]. J Anal Sci, 2021, 37(2): 211-216.
    [5] ZHAO G P, YANG F W, LI J W, et al. Toxicities of neonicotinoid-containing pesticide mixtures on nontarget organisms[J]. Environ Toxicol Chem, 2020, 39(10): 1884-1893. doi: 10.1002/etc.4842
    [6] LI Z, CHEN H, XU J, et al. Toxicological effects of three veterinary drugs and feed additives on fish[J]. J Ecol Rural Environ, 2006, 22(1): 84-86.
    [7] KANG H-S, LEE S-B, SHIN D, et al. Occurrence of veterinary drug residues in farmed fishery products in South Korea[J]. Food Control, 2018, 85: 57-65. doi: 10.1016/j.foodcont.2017.09.019
    [8] 马晨, 张群, 刘春华, 等. 海南芒果中农药多残留分析[J]. 农药学学报, 2021, 23(3): 552-560.

    MA C, ZHANG Q, LIU C H, et al. Analysis of multiple pesticide residues in mango of Hainan Province[J]. Chin J Pestic Sci, 2021, 23(3): 552-560.
    [9] LORENZO F, DAVID K. Alternatives to neonicotinoid insecticides for pest control: case studies in agriculture and forestry[J]. Environ sci pollut res int, 2015, 22(1): 135-147. doi: 10.1007/s11356-014-3628-7
    [10] LUAN Y, ZHAO J, HAN H, et al. Toxicologic effect and transcriptome analysis for short-term orally dosed enrofloxacin combined with two veterinary antimicrobials on rat liver[J]. Ecotox Environ Safe, 2021: 220.
    [11] CHENG L, LU Y, ZHAO Z, 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 Chem Toxicol, 2020: 145.
    [12] RANJAN R, RASTOGI N K, THAKUR M S. Development of immobilized biophotonic beads consisting of photobacterium leiognathi for the detection of heavy metals and pesticide[J]. J Hazard Mater, 2012, 225: 114-123.
    [13] SHI Y, DING W, ZHANG Z, et al. Determination of the acute toxicity of eight kinds of veterinary drug on V. qinghaiensis[J]. Acat Agr Boreali Sin, 2012, 21(6): 17-21.
    [14] JIN C, MAO L, SI X. Effects of single and combined pollution of three common sulfonamide veterinary drugs on root apical cell micronuclei of different crops[J]. J Agro Environ Sci, 2015, 34(4): 666-671.
    [15] 杨桂玲, 陈晨, 王彦华, 等. 几种农药多元组合对HepG2人肝癌细胞的联合毒性效应[J]. 农药学学报, 2019, 21(4): 453-460.

    YANG G L, CHEN C, WANG Y H, et al. Combined toxic effect of pesticides mixtures in HepG2 cells[J]. Chin J Pestic Sci, 2019, 21(4): 453-460.
    [16] 刘玉琼, 顾佳斌, 叶焕锋, 等. 毒死蜱对子代雄鼠性激素相关基因mRNA表达的影响[J]. 农药学学报, 2020, 22(5): 808-814.

    LIU Y Q, GU J B, YE H F, et al. Effects of chlorpyrifos on mRNA expression of genes related to testosterone synthesis in male offspring of mice[J]. Chin J Pestic Sci, 2020, 22(5): 808-814.
    [17] 刁晓平, 孙英健, 孙振钧, 等. 三种兽药在不同暴露系统对蚯蚓的急性毒性[J]. 农业环境科学学报, 2004, 23(4): 823-826. doi: 10.3321/j.issn:1672-2043.2004.04.046

    DIAO X P, SUN Y J, SUN Z J, et al. Acute toxicity of three kinds of veterinary drugs on earthworm (Eisenia fetida) under different exposure systems[J]. J Agro Environ Sci, 2004, 23(4): 823-826. doi: 10.3321/j.issn:1672-2043.2004.04.046
    [18] 王春琼, 曾彦波, 李苓, 等. 酶抑制法在农药残留快速检测中的研究进展[J]. 农业与技术, 2020, 40(16): 29-31.

    WANG C Q, ZENG Y B, LI L, et al. Research progress of enzyme inhibition method in rapid determination of pesticide residues[J]. Agric Technol, 2020, 40(16): 29-31.
    [19] NIWA T, YABUSAKI Y, HONMA K, et al. Contribution of human hepatic cytochrome P450 isoforms to regioselective hydroxylation of steroid hormones[J]. Can Metall Quart, 1998, 28(6): 539-547.
    [20] MURAYAMA N, NAKAMURA T, SAEKI M, et al. CYP3A4 gene polymorphisms influence testosterone 6beta-hydroxylation[J]. Drug metab and pharmacok, 2002, 17(2): 150-156. doi: 10.2133/dmpk.17.150
    [21] KESHAVA C, MCCANLIES E C, WESTON A. CYP3A4 polymorphisms - potential risk factors for breast and prostate cancer: A huge review[J]. Am J Epidemiol, 2004, 160(9): 825-841. doi: 10.1093/aje/kwh294
    [22] MA J S, WANG S H, ZHANG M L, et al. Simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma by UPLC-MS/MS and its application to cytochrome P450 activity study in rats[J]. Biomed Chromatogr, 2015, 29(8): 1203-1212. doi: 10.1002/bmc.3409
    [23] HANSEN J, PALMFELDT J, PEDERSEN K W, et al. Postmortem protein stability investigations of the human hepatic drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP3A4 using mass spectrometry[J]. J Proteomics, 2019, 194: 125-131. doi: 10.1016/j.jprot.2018.11.024
    [24] XIAO K, GAO J, WENG S J, et al. CYP3A4/5 activity probed with testosterone and midazolam: Correlation between two substrates at the microsomal and enzyme levels[J]. Mol Pharmaceut, 2019, 16(1): 382-392. doi: 10.1021/acs.molpharmaceut.8b01043
    [25] 戴子茹, 葛广波, 冯磊, 等. 细胞色素P4501A1亚型特异性双光子荧光探针及其生物学应用[C]//中国化学会第30届学术年会摘要集-第四分会: 生物分析和生物传感, 2016, 275-276.

    DAI Z R, GE G B, FENG L, et al. Characterization of an isoform specific two-photon fluorescent probe for human cytochrome P450 1A1 and its biological applications[C]//Abstracts of the 30th annual meeting of the Chinese Chemical Society - Chapter 4: bioanalysis and biosensors, 2016, 275-276.
    [26] NING J, WANG W, GE G B, et al. Target enzyme-activated two-photon fluorescent probes: A case study of CYP3A4 using a two-dimensional design strategy[J]. Angew Chem Int Edit, 2019, 58(29): 9959-9963. doi: 10.1002/anie.201903683
    [27] 中华人民共和国国家卫生健康委员会, 农业农村部, 国家市场监督管理总局. 食品安全国家标准 食品中农药最大残留限量: GB 2763-2021 [S]. 北京: 中国标准出版社, 2021.

    The State Health Commission of the people's Republic of China, the Ministry of agriculture and rural areas, the State Administration of market supervision and administration. National food safety standard Maximum residue limit of pesticides in food: GB 2763-2021 [S]. Beijing: China Standards Press, 2021.
    [28] 中华人民共和国国家卫生健康委员会, 农业农村部, 国家市场监督管理总局. 食品安全国家标准 食品中兽药最大残留限量: GB 31650-2019 [S]. 北京: 中国标准出版社, 2019.

    The State Health Commission of the people's Republic of China, the Ministry of agriculture and rural areas, the State Administration of market supervision and administration. National food safety standard Maximum residue limit of veterinary drugs in food: GB 31650-2019 [S]. Beijing: China Standards Press, 2019.
    [29] BERENBAUM M C. The expected effect of a combination of agents - the general-solution[J]. J Theor Biol, 1985, 114(3): 413-431. doi: 10.1016/S0022-5193(85)80176-4
    [30] 李静, 杨克礼, 孟丽, 等. 果蔬中四种农药残留二元混合物对人肝细胞L-O2的毒性探索[J]. 农村经济与科技, 2017, 28(S1): 62-64,131.

    LI J, YANG K L, MENG L, et al. Toxicity of binary mixtures of four pesticide residues in fruits and vegetables to human hepatocyte L-O2[J]. Rural Econ Sci Technol, 2017, 28(S1): 62-64,131.
    [31] 苟练, 廉楠, 王巧旭, 等. 有机磷及氨基甲酸酯类农药单独和联合作用对PC12细胞DNA损伤的影响[J]. 生态毒理学报, 2017, 12(4): 202-209.

    GOU L, LIAN N, WANG Q X, et al. Effects of organophosphorus and carbamate pesticides on DNA damage in PC12 cells alone and in combination[J]. Asian J Ecotoxicol, 2017, 12(4): 202-209.
    [32] 王天彩, 陈晨, 马朦朦, 等. 果蔬中四种农药残留二元混合物对人肝细胞L-O2的毒性探索五种农药及其二元、三元组合对人肝癌HepG2细胞的联合毒性[J]. 农药学学报, 2021, 23(3): 499-508.

    WANG T C, CHEN C, MA M M, et al. Combined toxicity of five pesticides and their binary and ternary mixtures on HepG2 cells[J]. Chin J Pestic Sci, 2021, 23(3): 499-508.
    [33] 王昕璐. 豇豆中残留农药对Neuro-2a细胞的联合毒性效应研究[D]. 北京: 中国农业科学院, 2021.

    WANG X L. Combined toxic effects of pesticide residues in cowpeas on neuro-2a cells[D]. Beijing: Chinese Academy of Agricultural Sciences, 2021.
    [34] AIKUAN W, AIQIN S, XIUQING Y, et al. Effects of chloromethane on wheat chlorophyll and soil respiration[J]. J Soil Water Conserv, 2007, 21(6): 162-164,186.
    [35] TRIPATHI A M, AGARWAL R A. Synergism in tertiary mixtures of pesticides[J]. Chemosphere, 1997, 35(10): 2365-2374. doi: 10.1016/S0045-6535(97)00221-X
    [36] LAETZ C A, BALDWIN D H, COLLIER T K, et al. The synergistic toxicity of pesticide mixtures: Implications for risk assessment and the conservation of endangered Pacific salmon[J]. Environ Heal Perspect, 2009, 117(3): 348-353. doi: 10.1289/ehp.0800096
    [37] NONG Q, QIN L, MO L, et al. The toxic interactions of long-term effects involving antibiotics and triazole fungicides on selenastrum capricornutum[J]. Asian J Ecotoxicol, 2019, 14(4): 140-149.
    [38] GROTEN J P, FERON V J, SÜHNEL J. Toxicology of simple and complex mixtures[J]. Trends Pharmacol Sci, 2001, 22(6): 316-322. doi: 10.1016/S0165-6147(00)01720-X
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出版历程
  • 收稿日期:  2021-10-11
  • 录用日期:  2022-01-12
  • 网络出版日期:  2022-03-08

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