Pacelli R, Wink D A, Cook J A, Krishna M C, DeGraff W, Friedman N, Tsokos M, Samuni A, Mitchell J B
Radiation Biology Branch, National Cancer Institute, Bethesda, Maryland 20892-1002, USA.
J Exp Med. 1995 Nov 1;182(5):1469-79. doi: 10.1084/jem.182.5.1469.
Previously, we reported that nitric oxide (NO) provides significant protection to mammalian cells from the cytotoxic effects of hydrogen peroxide (H2O2). Murine neutrophils and activated macrophages, however, produce NO, H2O2, and other reactive oxygen species to kill microorganisms, which suggests a paradox. In this study, we treated bacteria (Escherichia coli) with NO and H2O2 for 30 min and found that exposure to NO resulted in minimal toxicity, but greatly potentiated (up to 1,000-fold) H2O2-mediated killing, as evaluated by a clonogenic assay. The combination of NO/H2O2 induced DNA double strand breaks in the bacterial genome, as shown by field-inverted gel electrophoresis, and this increased DNA damage may correlate with cell killing. NO was also shown to alter cellular respiration and decrease the concentration of the antioxidant glutathione to a residual level of 15-20% in bacterial cells. The iron chelator desferrioxamine did not stop the action of NO on respiration and glutathione decrease, yet it prevented the NO/H2O2 synergistic cytotoxicity, implicating metal ions as critical participants in the NO/H2O2 cytocidal mechanism. Our results suggest a possible mechanism of modulation of H2O2-mediated toxicity, and we propose a new key role in the antimicrobial macrophagic response for NO.
此前，我们报道过一氧化氮（NO）能为哺乳动物细胞提供显著保护，使其免受过氧化氢（H2O2）的细胞毒性作用。然而，小鼠中性粒细胞和活化巨噬细胞会产生NO、H2O2及其他活性氧物质来杀灭微生物，这就产生了一个矛盾。在本研究中，我们用NO和H2O2处理细菌（大肠杆菌）30分钟，通过克隆形成试验评估发现，暴露于NO只会产生极小的毒性，但却极大地增强了（高达1000倍）H2O2介导的杀菌作用。如交变脉冲凝胶电泳所示，NO/H2O2组合会诱导细菌基因组中的DNA双链断裂，而这种增加的DNA损伤可能与细胞杀伤有关。NO还被证明会改变细胞呼吸，并使细菌细胞内抗氧化剂谷胱甘肽的浓度降至15 - 20%的残留水平。铁螯合剂去铁胺并不能阻止NO对呼吸和谷胱甘肽减少的作用，但它能防止NO/H2O2的协同细胞毒性，这表明金属离子是NO/H2O2杀细胞机制中的关键参与者。我们的结果提示了一种调节H2O2介导毒性的可能机制，并且我们提出NO在抗菌巨噬细胞反应中具有新的关键作用。
