毒死蜱和苯醚甲环唑联合暴露诱导小鼠精原细胞凋亡_潘志辉.pdf

生态毒理学报Asian Journal of Ecotoxicology第 17 卷 第 6 期 2022 年 12 月Vol.17,No.6 Dec.2022 基金项目:国家重点研发计划课题(2018FYC1603001)第一作者:潘志辉(1997),男,硕士研究生,研究方向为农药毒性效应,E-mail: *通信作者(Corresponding author),E-mail:#共同通信作者(Co-corresponding author),E-mail:DOI:10.7524/AJE.1673-5897.20220301002潘志辉,席令仪,孙景然,等.毒死蜱和苯醚甲环唑联合暴露诱导小鼠精原细胞凋亡J.生态毒理学报,2022,17(6):441-451Pan Z H,Xi L Y,Sun J R,et al.Apoptosis of GC-1 cells induced by combined exposure of chlorpyrifos and difenoconazole J.Asian Journal of Ecotoxi-cology,2022,17(6):441-451(in Chinese)毒死蜱和苯醚甲环唑联合暴露诱导小鼠精原细胞凋亡潘志辉,席令仪,孙景然,刘伟丽,吴瑾#,房彦军*军事科学院军事医学研究院 环境医学与作业医学研究所,天津 300050收稿日期:2022-03-01 录用日期:2022-04-17摘要:现代农业生产中为有效防治病虫害、保证农产品质量,多种农药混用现象突出。毒死蜱、苯醚甲环唑都是广泛使用的农药,现有研究证明这 2 种农药单独暴露对小鼠生殖系统具有毒性作用,但目前对二者联合暴露机制的研究较少。为了研究毒死蜱和苯醚甲环唑联合暴露对小鼠精原细胞的毒性作用及机制,先将 GC-1 细胞分别暴露于不同浓度的毒死蜱和苯醚甲环唑24 h,通过测定细胞活力,确定 2 种农药的半抑制浓度(IC50)值。以 2 种药物的 IC50浓度进行单独和联合暴露,测定其活性氧(ROS)、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、丙二醛(MDA)指标水平,分析暴露后细胞线粒体膜电位和细胞凋亡等变化;通过 Western blot 检测凋亡相关蛋白 Cleaved-Caspase3、Cleaved-PARP 和 Bax 的表达。结果表明,毒死蜱和苯醚甲环唑暴露导致 GC-1 细胞形态发生明显改变、存活率显著降低,毒死蜱和苯醚甲环唑的 IC50分别为 158.5 mol L-1和 89.57 molL-1。与对照组相比,联合暴露组细胞内 ROS 含量和 MDA 含量明显增加,而 SOD 活性和 GSH 含量明显降低,引起细胞显著的氧化应激。联合暴露组中 Cleaved-PARP、Cleaved-Caspase3 和 Bax 蛋白表达水平显著提高。毒死蜱、苯醚甲环唑联合暴露对细胞毒性的影响明显高于其单独暴露组,导致 GC-1 细胞存活率降低并改变细胞形态,引起细胞氧化应激,线粒体膜电位下降,凋亡相关基因和蛋白的表达水平升高,最终诱导细胞凋亡。关键词:毒死蜱;苯醚甲环唑;小鼠精原细胞;联合暴露;细胞凋亡文章编号:1673-5897(2022)6-441-11 中图分类号:X171.5 文献标识码:AApoptosis of GC-1 Cells Induced by Combined Exposure of Chlorpyrifosand DifenoconazolePan Zhihui,Xi Lingyi,Sun Jingran,Liu Weili,Wu Jin#,Fang Yanjun*Institute of Environmental and Operational Medicine,Academy of Military Medical Science,Academy of Military Science,Tianjin300050,ChinaReceived 1 March 2022 accepted 17 April 2022Abstract:In order to effectively control diseases and insect pests and ensure the quality of agricultural products,the mixed use of multiple pesticides is prominent in modern agricultural production.Chlorpyrifos and difenocon-azole are widely-used pesticides.Existing studies have proved that these two pesticides have toxic effects on the re-productive system of mice,but there are few studies on the mechanism of their combined toxicities.In order tostudy the combined toxic effect of chlorpyrifos and difenoconazole on mouse spermatogonia and its related mecha-442 生态毒理学报第 17 卷nism,GC-1 cells were exposed to chlorpyrifos and difenoconazole at different concentrations for 24 h,respectively.The half inhibition concentration(IC50)values of the two pesticides were determined by measuring the cell viabili-ty.The levels of reactive oxygen species(ROS),glutathione(GSH),and malondialdehyde(MDA),as well as theactivity of superoxide dismutase(SOD)were measured after the cells were exposed to these two pesticides at theirIC50concentrations,alone or in combination.The mitochondrial membrane potential and the apoptosis of the cellswere detected.The expression levels of apoptosis-related proteins,Cleaved-Caspase3,Cleaved-PARP and Bax weredetected by Western blot.Results showed that the altered cellular morphology and reduced survival rate were sig-nificantly induced in the combined exposure.The IC50values of chlorpyrifos and difenoconazole were approximate-ly 158.5 mol L-1and 89.57 mol L-1.Compared with that in the control group,the intracellular levels of ROSand MDA in the combined group increased,and the activity of SOD and the level of GSH decreased,indicating theoxidative stress.The promoted expressions of Cleaved-Caspase3,Cleaved-PARP and Bax were also detected.Thecombined exposure to chlorpyrifos and difenoconazole induced higher cytotoxicity than their exposure alone,re-vealing in the altered cellular morphology,the decreased mitochondrial membrane potentials,and the increased cel-lular oxidative stress level and expression levels of apoptosis-related proteins,which finally lead to the increasedapoptosis of the cells.Keywords:chlorpyrifos;difenoconazole;GC-1 cells;combined exposure;apoptosis 现代农业中,使用农药的种类和数量越来越多。农药是把“双刃剑”,它能解放农业生产力、减少农产品产量损失,但滥用农药会造成环境污染、人畜中毒等严重后果,人们通过皮肤接触、饮食摄入和呼吸暴露于农药1。农药对生物具有多种毒性,主要包括生殖毒性、内分泌干扰毒性和神经系统毒性等,对人类健康造成严重威胁。长期低剂量接触有机磷农药会降低男性精子存活率,影响受精能力及精液的染色质结构2。毒死蜱是一种广谱有机磷农药,在世界范围内广泛使用,其半衰期较长,能够长时间存在于土壤和水中3。毒死蜱可以损伤神经系统、生殖系统和免疫系统等,现有研究表明其可导致雄性大鼠精子数量减少、生存能力降低,睾酮等生殖激素水平明显降低4。长期暴露于毒死蜱会导致各种生殖系统缺陷,比如男性生殖系统功能退化和不育5。VahabiBarzi 等6的研究表明,毒死蜱损伤小鼠生殖系统,诱导胚胎发育中的细胞凋亡,进而导致流产。毒死蜱暴露后,诱导 GC-1 细胞活性氧(ROS)水平升高,并导致细胞凋亡7。三唑类杀菌剂是一类具有独特1,2,4-三唑环结构的杀菌剂,在农业生产中应用非常广泛8。苯醚甲环唑作为三唑类中应用广泛的农药之一,更易进入环境中造成污染,对人体健康产生威胁。长期暴露于苯醚甲环唑降低海洋青鳉鱼下一代的繁殖力和生存力9。Li 等10的研究结果表明,苯醚甲环唑诱导小鼠巨噬细胞核溶解,提高细胞凋亡率。苯醚甲环唑暴露后,显著降低大鼠睾丸内精子数量,诱导组织结构改变和 DNA 损伤11。此外,苯醚甲环唑诱导 SH-SY5Y 细胞 DNA 损伤,提高细胞凋亡率12。已有研究报道单一农药以及农药和金属物质混用对生物的毒性作用,鲜有报道研究多种农药联合暴露的生殖毒性作用。精子的产生是一个复杂而连续的过程,包括精原细胞增殖、精母细胞减数分裂和精子细胞向精子的分化,其中,精原细胞在精子产生中起着至关重要的作用,在维持正常精子数量方面具有重要的生理意义13-14。目前的研究表明,毒死蜱和苯醚甲环唑在单独暴露时具有明显的生殖毒性,但关于毒死蜱和苯醚甲环唑联合暴露生殖毒性效应与机制的研究较少。因此,以小鼠精原细胞 GC-1 为模型,通过对不同浓度农药进行细胞活力检测,测定毒死蜱和苯醚甲环唑单独和联合暴露后其 ROS、超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)和丙二醛(MDA)等氧化应激指标水平,并验证凋亡相关蛋白的表达情况,从而研究毒死蜱和苯醚甲环唑单独和联合暴露对 GC-1 细胞的毒性作用,为评估这 2 种农药对生殖系统的毒性作用提供依据。1 材料与方法(Materi