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目的研究毒死蜱(chlorpyrifos,CPF)与克百威(carbofuran,CF)单独及联合染毒对体外培养细胞株的毒性,并阐明联合作用模式。方法应用不同浓度的CPF(0、50、100、200和400μmol/L)和CF(0、25、50、100和200μmol/L)分别单独染毒大鼠嗜铬细胞瘤株PC12细胞12 h,联合效应研究分别在低剂量(CPF 50μmol/L,CF 25μmol/L)和高剂量(CPF 200μmol/L,CF 100μmol/L)水平下混合染毒。染毒结束后,采用丁酰硫代胆碱法检测乙酰胆碱酯酶(ACh E)活性,二氯荧光素二乙酸酯(DCFH-DA)荧光探针法测定细胞内活性氧(ROS)的生成,硫代巴比妥酸(TBA)法测定细胞中脂质过氧化产物丙二醛(MDA)含量,黄嘌呤氧化酶法测定细胞中超氧化物歧化酶(SOD)活性,5,5’-二硫代双硝基苯甲酸(DTNB)显色法测定细胞中谷胱甘肽过氧化物酶(GPx)活性。结果毒死蜱、克百威单独染毒时,对ACh E活性的抑制作用、PC12细胞内ROS的生成量、SOD及GPx的活性均显著高于对照组(P<0.01),MDA含量无明显变化。2×2析因设计分析联合效应结果表明,较低剂量水平下,CPF与CF之间无交互作用,较高剂量水平,CPF、CF之间存在交互作用(P<0.01),作用模式主要为协同作用。结论毒死蜱、克百威单独及联合染毒都具有细胞毒性,联合作用模式主要为协同作用,氧化应激可能是其产生细胞毒性的机制之一。
Objective To study the toxicity of chlorpyrifos (CPF) and carbofuran (CF) to cultured cell lines in vitro and in combination mode. Methods PC12 cells were exposed to different concentrations of CPF (0, 50, 100, 200 and 400 μmol / L) and CF (0, 25, 50, 100 and 200 μmol / The combined effect study was mixed with low dose (CPF 50μmol / L, CF 25μmol / L) and high dose (CPF 200μmol / L, CF 100μmol / L) After the exposure, acetylcholinesterase (AChE) activity was measured by butyrylthiocholine method, and the formation of intracellular reactive oxygen species (ROS) was measured by fluorescent probe method with dichlorofluorescein diacetate (DCFH-DA) The content of malondialdehyde (MDA) in lipid peroxidation cells was measured by thiobarbituric acid (TBA) method. The activity of superoxide dismutase (SOD) in cells was measured by xanthine oxidase method. Thiobis-nitrobenzoic acid (DTNB) colorimetric assay for determination of glutathione peroxidase (GPx) activity in cells. Results The inhibitory effect of chlorpyrifos and carbofuran alone on ACh E activity, the production of ROS and the activity of GPx in PC12 cells were significantly higher than those in the control group (P <0.01), while the contents of MDA did not change significantly. The results of 2 × 2 factorial design analysis showed that there was no interaction between CPF and CF at higher dose levels and interaction between CPF and CF at higher dose levels (P <0.01). The mode of action was mainly Synergy. Conclusions Both chlorpyrifos and carbofuran are cytotoxic in single and combined groups. The combined mode of action is mainly synergistic. Oxidative stress may be one of the mechanisms of cytotoxicity.