Environmental Biochemistry and Molecular Biology Laboratory, Department of Biochemistry, University College of Medical Sciences and G.T.B. Hospital (University of Delhi), Dilshad Garden, Delhi 110 095, India.
J Biochem Mol Toxicol. 2010 Sep-Oct;24(5):286-92. doi: 10.1002/jbt.20337.
The molecular mechanism for noncholinergic toxicity of phosphamidon, an extensively used organophosphate pesticide, is still not clear. The aim of the present study is to find the possible molecular mechanism of this pesticide to induce apoptosis and the role of different drugs for attenuation of such effects. Human peripheral blood mononuclear cells (PBMC) were incubated with increasing concentrations of phosphamidon (0-20 μM) for 6-24 h. The MTT assay reveals that phosphamidon induces cytotoxicity in a dose-dependent manner. Cellular glutathione (GSH) is depleted in a dose-dependent manner from 55% to 70% at concentrations between 10 and 20 μM. The percentage of cells that bind to Annexin-V, which is a representative of cells either undergoing apoptosis or necrosis during 24 h incubation, increases in a dose-dependent manner. Above 5 μM, significant necrosis of cells was observed. DNA fragmentation assay revealed that at low concentration of phosphamidon (1 μM), no appreciable change in DNA fragmentation was seen; however, distinct fragmentation was observed beyond 2.5 μM. Phosphamidon was found to cause significant depletion of GSH, which correlates well with the percentage of cells undergoing apoptosis. An increasing trend in levels of cytochrome c was observed with increasing concentration of phosphamidon, indicating that the apoptotic effect of phosphamidon is mediated through cytochrome c release. Coadministration of the antioxidants N-acetylcysteine and curcumin attenuated phosphamidon-induced apoptosis. This further supports our hypothesis that oxidative stress, as indicated by GSH depletion, results in the induction of apoptosis by release of cytochrome c.
敌百虫是一种广泛使用的有机磷农药，其非胆碱能毒性的分子机制尚不清楚。本研究旨在寻找这种农药诱导细胞凋亡的可能分子机制，以及不同药物减轻这种作用的作用。将人外周血单核细胞（PBMC）与递增浓度的敌百虫（0-20 μM）孵育 6-24 小时。MTT 测定法显示敌百虫以剂量依赖性方式诱导细胞毒性。细胞谷胱甘肽（GSH）在 10-20 μM 浓度范围内以剂量依赖性方式从 55%到 70%耗竭。在 24 小时孵育期间，与 Annexin-V 结合的细胞百分比以剂量依赖性方式增加，Annexin-V 是细胞处于凋亡或坏死状态的代表性指标。超过 5 μM 时，观察到细胞明显坏死。DNA 片段化分析显示，在低浓度敌百虫（1 μM）下，DNA 片段化无明显变化；然而，超过 2.5 μM 时，明显观察到片段化。敌百虫导致 GSH 显著耗竭，这与细胞凋亡的百分比很好地相关。随着敌百虫浓度的增加，细胞色素 c 的水平呈上升趋势，表明敌百虫的凋亡作用是通过细胞色素 c 释放介导的。抗氧化剂 N-乙酰半胱氨酸和姜黄素的共给药减轻了敌百虫诱导的凋亡。这进一步支持了我们的假设，即 GSH 耗竭所指示的氧化应激导致细胞色素 c 释放诱导凋亡。
