Saleh A M, Vijayasarathy C, Masoud L, Kumar L, Shahin A, Kambal A
Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17 666, Al Ain, United Arab Emirates.
Toxicol Appl Pharmacol. 2003 Jul 1;190(1):47-57. doi: 10.1016/s0041-008x(03)00126-1.
The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.
有机磷化合物如对氧磷（POX）的毒性归因于其抗胆碱酯酶作用。最近，我们发现，在非胆碱能剂量（1至10 nM）下，POX（对硫磷的生物活性代谢产物）通过激活caspase-3，导致小鼠EL4 T淋巴细胞白血病细胞系发生凋亡性细胞死亡。在本研究中，我们使用caspase特异性抑制剂，扩展了我们的观察结果，以阐明POX刺激的凋亡所涉及的事件序列。用caspase-9特异性抑制剂zLEHD-fmk预处理EL4细胞，以剂量依赖性方式减弱了POX诱导的凋亡，而caspase-8抑制剂zIETD-fmk则没有效果。此外，在zLEHD-fmk存在的情况下，POX处理后caspase-9、-8和-3的激活被完全抑制，这表明caspase 9依赖性线粒体途径参与了POX刺激的凋亡。事实上，在体外和体内条件下，通过蛋白质印迹分析评估，POX引发了细胞色素c从线粒体到细胞质的剂量和时间依赖性转位。对细胞色素c释放机制的研究表明，POX破坏了线粒体跨膜电位。这种作用和细胞色素c释放均不依赖于caspase激活，因为caspase家族的通用抑制剂zVAD-fmk对这两个过程均无影响。最后，POX处理还导致促凋亡分子Bax的时间依赖性上调和向线粒体的转位。zVAD-fmk对这一事件的抑制表明，Bax向线粒体的激活和转位是在caspase级联反应激活之后。结果表明，POX通过破坏线粒体跨膜电位，直接作用于线粒体，导致细胞色素c释放到细胞质中，随后激活caspase-9，从而诱导EL4细胞凋亡。抑制这一特定途径可能是一种有用的策略，以尽量减少有机磷中毒。
