Tomita M, Okuyama T, Ishikawa T, Hidaka K, Nohno T
Departments of Legal Medicine, Kawasaki Medical School, Kurashiki 701-0192, Japan.
Free Radic Res. 2001 Feb;34(2):193-202. doi: 10.1080/10715760100300181.
Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25-50 microM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.
百草枯（PQ）是一种著名的肺毒性物质，通过提高细胞内超氧化物水平发挥其毒性作用。此外，促炎细胞因子的产生可能通过活性氧（ROS）和一氧化氮（NO）与PQ诱导的炎症过程相关联。然而，NO在PQ诱导的细胞损伤中的作用一直存在争议。为了探讨这个问题，我们通过将A549细胞暴露于外源性NO供体NOC18或细胞因子（肿瘤坏死因子-α、白细胞介素-1β和干扰素-γ）以及PQ来研究NO对A549细胞的影响。尽管外源性NO供体本身对乳酸脱氢酶（LDH）的释放没有影响，但当细胞暴露于高浓度的NOC18和PQ时，观察到显著的释放。通过相差显微镜确定了由1 mM NOC18加0.2 mM PQ引起的这种细胞损伤。另一方面，添加到培养基中的25 - 50 microM NOC18产生的NO提高了线粒体的MTT还原活性，表明NO对细胞有有益作用。用细胞因子孵育A549细胞会增加诱导型一氧化氮合酶（iNOS）的表达和亚硝酸盐积累，导致LDH释放。PQ进一步增强了这种释放。NOS抑制剂可以完全阻止亚硝酸盐水平的升高，而用它们抑制NO产生并不能减弱LDH的泄漏。另一方面，ROS清除酶超氧化物歧化酶和过氧化氢酶抑制了LDH的泄漏，而它们对亚硝酸盐水平的升高没有影响。这些结果表明，超氧化物而非NO在PQ/细胞因子引起的细胞损伤中起关键作用。我们的体外模型表明，NO根据产生的浓度和使用的模型系统具有有益和有害的作用。
