NO-MEDIATED NITROSATION OF MEIQX POTENTIATED BY HEMIN AND MPO

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。由杂环胺亚硝化形成的N-亚硝胺可能会在食用优质红肉饮食和结肠发炎的人中引发结肠癌。本研究考察了一氧化氮(NO)介导的2-氨基-3，8-二甲基咪唑并[4，5-f]喹恶啉(MeIQx)亚硝化反应以及膳食(氯化血红素)和炎症成分[NO、髓过氧化物酶(MPO)和过氧化氢]对亚硝化反应的影响。使用NO供体精胺NONOate(1.2MNO/min)，在pH 7.4和0.005 mM MeIQx条件下，由于NO自氧化而产生的产物处于检测限(用高效液相色谱法回收总放射性的1%)。在原位生成过氧化氢(葡萄糖氧化酶/葡萄糖)的体系中，在10M氯化血红素或85 NM MPO存在下，产物形成分别增加了13倍或16倍。用电喷雾电离质谱仪对亚硝化产物及其氯代衍生物进行了分析，确定其为2-nitrosoamino-3，8-dimethylimidazo[4，5-f]quinoxaline(N-NO-MeQx)。NO自氧化反应仅在1.2M NO/min时被检测到，且不受H_2O_2的影响。用氯化血红素研究确定了增强作用所需的最低有效成分：1M氯化血红素、1M过氧化氢/分钟和0.012 M一氧化氮/分钟。氯化高铁血红素和MPO产生的活性氮氧物种(RNO)对MeIQx的亲和力分别是未自氧化的4倍和3倍。试验试剂用于表征亚硝化作用。结果过氧化氢酶、SOD、叠氮化物和NADH与多种RNO、过氧亚硝酸盐参与亚硝化、氧化亚硝化而不是亚硝化的过氧化增强作用相一致。用佛波酯刺激的人中性粒细胞，N-NO-MeIQx的形成和被测试剂修饰与MPO一致，而不是过氧亚硝酸根。因此，MeIQx的亚硝化及其被氯化血红素和MPO的增强提供了一种机制，通过这种机制，良好的红肉消费和炎症可以产生N-亚硝基化合物，并在炎症条件下引发结肠癌，如结肠炎。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. N-Nitrosamines formed by nitrosation of heterocyclic amines might initiate colon cancer in individuals consuming well-done red meat diets and with inflammatory conditions in their colon. This study investigates nitric oxide (NO)-mediated nitrosation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and the influence of dietary (hemin) and inflammatory [NO, myeloperoxidase (MPO), and H2O2] components on nitrosation. Using the NO donor spermine NONOate (1.2 M NO/min) at pH 7.4 with 0.005 mM MeIQx, a product due to NO autoxidation was at the limit of detection (1% of total radioactivity recovered by HPLC). Product formation increased 13- or 16-fold in the presence of 10 M hemin or 85 nM MPO, respectively, with an in situ system for generating H2O2 (glucose oxidase/glucose). The nitrosation product and its chloro derivative were analyzed by electrospray ionization mass spectrometry, and the product was determined to be 2-nitrosoamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-NO-MeIQx). Nitrosation by NO autoxidation was only detected at 1.2 M NO/min and was not affected by H2O2. Investigations with hemin determined minimum effective components necessary for potentiation: 1 M hemin, 1 M H2O2/min, and 0.012 M NO/min. The reactive nitrogen oxygen species (RNOS) produced by hemin and MPO had a 4- and 3-fold, respectively, greater affinity for MeIQx than those produced by NO autoxidation. Test agents were used to characterize nitrosation. Results with catalase, SOD, azide, and NADH are consistent with multiple RNOS, the lack of peroxynitrite involvement in nitrosation, and peroxidatic potentiation by oxidative nitrosylation rather than nitrosation. Using phorbol ester stimulated human neutrophils, the formation of N-NO-MeIQx and its modification by test agents was consistent with MPO and not peroxynitrite. Thus, nitrosation of MeIQx and its potentiation by hemin and MPO provide a mechanism by which well-done red meat consumption and inflammation can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.