Bhattacharya R, Lakshmana Rao P V
Division of Pharmacology and Toxicology, Defence Research and Development Establishment, Jhansi Road, 474 002 (M.P.), Gwalior, India.
Toxicol Lett. 2001 Feb 3;119(1):59-70. doi: 10.1016/s0378-4274(00)00309-x.
Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was attenuated by Zn2+ (modulator of Ca2+ dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the 'apoptotic' type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5-3 h) of various pharmacological agents viz., Trolox (antioxidant), EGTA (Ca2+ modulator) and aurintricarboxylic acid (ATA; Ca2+/Mg2+ dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox also increased the LD(50) of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.
氰化物会抑制线粒体呼吸链酶细胞色素氧化酶，从而导致组织中毒性缺氧。它主要被视为一种神经毒素，但其其他毒性表现也有充分记录。最近也证实了氰化物可诱导神经元细胞凋亡。与此同时，我们还报道了氰化钾（KCN）会在大鼠胸腺细胞中产生广泛的细胞毒性和DNA片段化。DNA损伤对细胞外Ca2+水平升高敏感，并可被Zn2+（Ca2+依赖性核酸内切酶的调节剂）、N-乙酰半胱氨酸（自由基清除剂）和地尔硫䓬（Ca2+通道阻滞剂）减弱。在这项工作的延续中，在本研究中我们表明，5 mM KCN诱导的细胞毒性和DNA片段化之前，线粒体完整性（MTT法和罗丹明-123染色）和细胞核活力（碘化丙啶摄取）丧失，这是由活性氧的产生（DCHF-DA染色）介导的。DNA损伤还伴有核碎裂（Hoechst 33342染色），这是一种表征“凋亡”型细胞死亡的现象。通过各种药理试剂即托洛克斯（抗氧化剂）、乙二醇双四乙酸（EGTA；Ca2+调节剂）和金精三羧酸（ATA；Ca2+/Mg2+依赖性核酸内切酶抑制剂）的预处理（0.5小时）、同时处理或后处理（0.5 - 3小时），对KCN的体外毒性损伤进行了挑战。此外，槲皮素（抗氧化剂）仅作为同时处理进行了测试，发现无效。基于各种生化指标和DNA片段化（定量和定性），发现托洛克斯的同时处理在体外减轻氰化物毒性方面最有效。这种保护可归因于对氧化应激介导的细胞损伤的干预，这是DNA损伤之前的早期事件。EGTA和ATA都无法预防这种损伤。与EGTA和ATA分别为1.8倍和1.6倍相比，托洛克斯还使小鼠中KCN的半数致死剂量（LD(50)）增加了2.5倍。
