NO-MEDIATED NITROSATION OF MEIQX POTENTIATED BY HEMIN AND MPO

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。由杂环胺亚硝化形成的n -亚硝胺可能在食用熟肉和结肠炎症的人群中引发结肠癌。本研究研究了一氧化氮（NO）介导的2-氨基-3,8-二甲基咪唑[4,5-f]喹诺啉（MeIQx）的亚硝化作用，以及饮食（hemin）和炎症[NO、髓过氧化物酶（MPO）和H2O2]成分对亚硝化作用的影响。使用NO供体精胺NONOate （1.2 M NO/min）在pH 7.4和0.005 mM MeIQx条件下，NO自氧化产物达到检测限（HPLC回收总放射性的1%）。在生成H2O2（葡萄糖氧化酶/葡萄糖）的原位系统中，在10 M hemin或85 nM MPO的存在下，产物的形成分别增加了13倍或16倍。采用电喷雾质谱法分析亚硝化产物及其氯衍生物，确定产物为2-亚硝基氨基-3,8-二甲基咪唑[4,5-f]喹啉（N-NO-MeIQx）。仅在1.2 M NO/min时检测到NO自氧化亚硝化作用，H2O2不影响亚硝化作用。hemin的研究确定了增强所需的最小有效成分：1 M hemin， 1 M H2O2/min和0.012 M NO/min。hemin和MPO产生的活性氧（RNOS）对MeIQx的亲和力分别比NO自氧化产生的RNOS高4倍和3倍。用试验剂对亚硝化进行了表征。过氧化氢酶、超氧化物歧化酶、叠氮化物和NADH的结果与多重RNOS一致，亚硝化过程中缺乏过氧亚硝酸盐参与，氧化亚硝基化而不是亚硝化作用增强了过氧化物。用苯酚酯刺激人中性粒细胞，N-NO-MeIQx的形成及其被试验剂修饰与MPO一致，而与过氧亚硝酸盐不一致。因此，meqx的亚硝化以及hemin和MPO对其的增强提供了一种机制，通过这种机制，熟透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.