Harris W, Sachana M, Flaskos J, Hargreaves A J
School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.
Toxicol Appl Pharmacol. 2009 Oct 15;240(2):159-65. doi: 10.1016/j.taap.2009.07.028. Epub 2009 Jul 30.
In previous work we showed that sub-lethal levels of diazinon inhibited neurite outgrowth in differentiating N2a neuroblastoma cells. Western blotting analysis targeted at proteins involved in axon growth and stress responses, revealed that such exposure led to a reduction in the levels of neurofilament heavy chain, microtubule associated protein 1 B (MAP 1B) and HSP-70. The aim of this study was to apply the approach of 2 dimensional polyacrylamide gel electrophoresis and mass spectrometry to identify novel biomarkers of effect. A number of proteins were found to be up-regulated compared to the control on silver-stained gels. These were classified in to 3 main groups of proteins: cytosolic factors, chaperones and the actin-binding protein cofilin, all of which are involved in cell differentiation, survival or metabolism. The changes observed for cofilin were further confirmed by quantitative Western blotting analysis with anti-actin and anti-cofilin antibodies. Indirect immunofluorescence staining with the same antibodies indicated that the microfilament network was disrupted in diazinon-treated cells. Our data suggest that microfilament organisation is disrupted by diazinon exposure, which may be related to increased cofilin expression.
在之前的研究中，我们发现，二嗪农的亚致死剂量会抑制分化中的N2a神经母细胞瘤细胞的神经突生长。针对轴突生长和应激反应相关蛋白的蛋白质印迹分析表明，这种暴露会导致神经丝重链、微管相关蛋白1B（MAP 1B）和热休克蛋白70（HSP-70）水平降低。本研究的目的是应用二维聚丙烯酰胺凝胶电泳和质谱方法来鉴定新的效应生物标志物。在银染凝胶上，与对照组相比，发现有多种蛋白质上调。这些蛋白质分为3大类：胞质因子、伴侣蛋白和肌动蛋白结合蛋白cofilin，它们均参与细胞分化、存活或代谢。用抗肌动蛋白和抗cofilin抗体进行的蛋白质印迹定量分析进一步证实了观察到的cofilin的变化。用相同抗体进行的间接免疫荧光染色表明，在二嗪农处理的细胞中微丝网络遭到破坏。我们的数据表明，二嗪农暴露会破坏微丝组织，这可能与cofilin表达增加有关。
