毒死蜱和苯醚甲环唑联合暴露诱导小鼠精原细胞凋亡
Apoptosis of GC-1 Cells Induced by Combined Exposure of Chlorpyrifos and Difenoconazole
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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 μmol·L-1和89.57 μmol·L-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。Abstract: 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 difenoconazole are widely-used pesticides. Existing studies have proved that these two pesticides have toxic effects on the reproductive system of mice, but there are few studies on the mechanism of their combined toxicities. In order to study the combined toxic effect of chlorpyrifos and difenoconazole on mouse spermatogonia and its related mechanism, 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 viability. The levels of reactive oxygen species (ROS), glutathione (GSH), and malondialdehyde (MDA), as well as the activity of superoxide dismutase (SOD) were measured after the cells were exposed to these two pesticides at their IC50 concentrations, alone or in combination. The mitochondrial membrane potential and the apoptosis of the cells were detected. The expression levels of apoptosis-related proteins, Cleaved-Caspase3, Cleaved-PARP and Bax were detected by Western blot. Results showed that the altered cellular morphology and reduced survival rate were significantly induced in the combined exposure. The IC50 values of chlorpyrifos and difenoconazole were approximately 158.5 μmol·L-1 and 89.57 μmol·L-1. Compared with that in the control group, the intracellular levels of ROS and MDA in the combined group increased, and the activity of SOD and the level of GSH decreased, indicating the oxidative stress. The promoted expressions of Cleaved-Caspase3, Cleaved-PARP and Bax were also detected. The combined exposure to chlorpyrifos and difenoconazole induced higher cytotoxicity than their exposure alone, revealing in the altered cellular morphology, the decreased mitochondrial membrane potentials, and the increased cellular oxidative stress level and expression levels of apoptosis-related proteins, which finally lead to the increased apoptosis of the cells.
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Key words:
- chlorpyrifos /
- difenoconazole /
- GC-1 cells /
- combined exposure /
- apoptosis
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