Wink D A, Feelisch M, Fukuto J, Chistodoulou D, Jourd'heuil D, Grisham M B, Vodovotz Y, Cook J A, Krishna M, DeGraff W G, Kim S, Gamson J, Mitchell J B
Tumor Biology Section, National Cancer Institute, Bethesda, Maryland 20892, USA.
Arch Biochem Biophys. 1998 Mar 1;351(1):66-74. doi: 10.1006/abbi.1997.0565.
In addition to the broad repertoire of regulatory functions nitric oxide (NO) serves in mammalian physiology, the L-arginine:NO pathway is also involved in numerous pathophysiological mechanisms. While NO itself may actually protect cells from the toxicity of reactive oxygen radicals in some cases, it has been suggested that reactive nitrogen oxide species formed from nitric oxide synthase (NOS) can be cytotoxic. In addition to NO, the one electron reduction product NO- has been proposed to be formed from NOS. We investigated the potential cytotoxic role of nitroxyl (NO-), using the nitroxyl donor Angelis's salt, (AS; sodium trioxodinitrate, Na2N2O3) as the source of NO-. As was found to be cytotoxic to Chinese hamster V79 lung fibroblast cells over a concentration range of 2-4 mM. The presence of equimolar ferricyanide (Fe(III)-(CN6)3-), which converts NO- to NO, afforded dramatic protection against AS-mediated cytotoxicity. Treatment of V79 cells with L-buthionine sulfoximine to reduce intracellular glutathione markedly enhanced AS cytotoxicity, which suggests that GSH is critical for cellular protection against the toxicity of NO-. Further experiments showed that low molecular weight transition metal complexes associated with the formation of reactive oxygen species are not involved in AS-mediated cytotoxicity since metal chelators had no effect. However, under aerobic conditions, AS was more toxic than under hypoxic conditions, suggesting that oxygen dramatically enhanced AS-mediated cytotoxicity. At a molecular level, AS exposure resulted in DNA double strand breaks in whole cells, and this effect was completely prevented by coincubation of cells with ferricyanide or Tempol. The data in this study suggest that nitroxyl may contribute to the cytotoxicity associated with an enhanced expression of the L-arginine:NO pathway under different biological conditions.
除了一氧化氮（NO）在哺乳动物生理学中发挥的广泛调节功能外，L-精氨酸：NO途径还参与众多病理生理机制。虽然在某些情况下，NO本身实际上可能保护细胞免受活性氧自由基的毒性，但有人提出，由一氧化氮合酶（NOS）形成的活性氮氧化物具有细胞毒性。除了NO，有人提出由NOS形成单电子还原产物NO-。我们使用硝酰基供体安吉利斯盐（AS；三氧二硝酸钠，Na2N2O3）作为NO-的来源，研究了硝酰基（NO-）的潜在细胞毒性作用。结果发现，在2-4 mM的浓度范围内，AS对中国仓鼠V79肺成纤维细胞具有细胞毒性。等摩尔铁氰化物（Fe(III)-(CN6)3-）的存在可将NO-转化为NO，从而显著保护细胞免受AS介导的细胞毒性。用L-丁硫氨酸亚砜亚胺处理V79细胞以降低细胞内谷胱甘肽水平，可显著增强AS的细胞毒性，这表明谷胱甘肽对于细胞抵抗NO-毒性的保护作用至关重要。进一步的实验表明，与活性氧形成相关的低分子量过渡金属络合物不参与AS介导的细胞毒性，因为金属螯合剂没有作用。然而，在有氧条件下，AS比在缺氧条件下毒性更大，这表明氧气显著增强了AS介导的细胞毒性。在分子水平上，AS暴露导致全细胞DNA双链断裂，而细胞与铁氰化物或Tempol共同孵育可完全阻止这种效应。本研究中的数据表明，在不同生物学条件下，硝酰基可能导致与L-精氨酸：NO途径表达增强相关的细胞毒性。
