8 NITRO 2 DEOXYGUANOSINE, SPECIFIC MARKER OF OXIDATION BY REACTIVE NITROGEN

8 硝基 2 脱氧鸟苷，活性氮氧化的特异性标记物

• 批准号: 6665803
• 负责人: J BYUN
• 金额: $ 15.75万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6665803
• 关键词: biological products; biomedical resource; cardiovascular system; female; human subject; intestines; lipids; male; spectrometry

Reactive intermediates generated by phagocytes damage DNA and may contribute to the link between chronic inflammation and cancer. Myeloperoxidase, a heme protein secreted by activated phagocytes, is a potential catalyst for such reactions. Recent studies demonstrate that this enzyme uses hydrogen peroxide (H2O2) and nitrite (NO2-) to generate reactive nitrogen species which convert tyrosine to 3-nitrotyrosine. We now report that activated human neutrophils use myeloperoxidase, H2O2 and NO2- to nitrate 2'-deoxyguanosine, one of the nucleosides of DNA. Through HPLC, UV/visible spectroscopy and mass spectrometry, the two major products of this reaction were identified as 8-nitroguanine and 8-nitro-2'-deoxyguanosine. Nitration required each component of the complete enzymatic system and was inhibited by catalase and heme poisons. However, it was independent of chloride ion and little affected by scavengers of hypochlorous acid, suggesting that the reactive agent is a ni trogen dio xide-like species that results from a one-electron oxidation of NO2- by myeloperoxidase. Alternatively, 2'-deoxyguanosine might be oxidized directly by the enzyme to yield a radical species which subsequently reacts with NO2- or NO2( to generate the observed products. Human neutrophils stimulated with phorbol ester also generated 8-nitroguanine and 8-nitro-2'-deoxyguanosine. The reaction required NO2- and was inhibited by catalase and heme poisons, implicating myeloperoxidase in the cell-mediated pathway. These results indicate that human neutrophils use the myeloperoxidase-H2O2-NO2- system to generate reactive species that can nitrate the C-8 position of 2'-deoxyguanosine. Our observations raise the possibility that reactive nitrogen species generated by myeloperoxidase and other peroxidases contribute to nucleobase oxidation and tissue injury at sites of inflammation.

吞噬细胞产生的反应中间体会损伤 DNA，并可能 有助于慢性炎症和癌症之间的联系。 髓过氧化物酶是一种由活化的吞噬细胞分泌的血红素蛋白， 此类反应的潜在催化剂。 最近的研究表明 该酶使用过氧化氢 (H2O2) 和亚硝酸盐 (NO2-) 产生活性氮，将酪氨酸转化为 3-硝基酪氨酸。 我们现在报道激活的人类中性粒细胞使用 髓过氧化物酶、H2O2 和 NO2- 转化为硝酸盐 2'-脱氧鸟苷，其中之一 DNA的核苷。 通过 HPLC、紫外/可见光谱和 通过质谱分析，该反应的两个主要产物是 鉴定为8-硝基鸟嘌呤和8-硝基-2'-脱氧鸟苷。 硝化 需要完整酶系统的每个组件，并且是 受过氧化氢酶和血红素毒物抑制。 然而，它是独立的 氯离子含量低，受次氯酸清除剂影响小 酸，表明反应剂是二氧化氮类 NO2- 的单电子氧化反应产生的物质 髓过氧化物酶。 或者，2'-脱氧鸟苷可能被氧化 直接通过酶产生自由基物质，随后 与 NO2- 或 NO2( 反应生成观察到的产物。人类 佛波酯刺激的中性粒细胞也产生 8-硝基鸟嘌呤和8-硝基-2'-脱氧鸟苷。 所需反应 NO2- 并被过氧化氢酶和血红素毒物抑制，暗示 细胞介导途径中的髓过氧化物酶。 这些结果表明 人类中性粒细胞利用髓过氧化物酶-H2O2-NO2-系统 产生可硝化 C-8 位的活性物质 2'-脱氧鸟苷。 我们的观察提出了这样的可能性： 髓过氧化物酶和其他物质产生的活性氮 过氧化物酶有助于核碱基氧化和组织损伤 炎症部位。