Kagan V E, Kuzmenko A I, Shvedova A A, Kisin E R, Li R, Martin I, Quinn P J, Tyurin V A, Tyurina Y Y, Yalowich J C
Department of Environmental and Occupational Health, University of Pittsburgh, 3343 Forbes Ave, PA 15260, USA.
Biochim Biophys Acta. 2003 Mar 17;1620(1-3):72-84. doi: 10.1016/s0304-4165(02)00509-3.
Myeloperoxidase (MPO)-catalyzed one-electron oxidation of endogenous phenolic constituents (e.g., antioxidants, hydroxylated metabolites) and exogenous compounds (e.g., drugs, environmental chemicals) generates free radical intermediates: phenoxyl radicals. Reduction of these intermediates by endogenous reductants, i.e. recycling, may enhance their antioxidant potential and/or prevent their potential cytotoxic and genotoxic effects. The goal of this work was to determine whether generation and recycling of MPO-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxychromane (PMC), by physiologically relevant intracellular reductants such as ascorbate/lipoate could be demonstrated in intact MPO-rich human leukemia HL-60 cells. A model system was developed to show that MPO/H(2)O(2)-catalyzed PMC phenoxyl radicals (PMC*) could be recycled by ascorbate or ascorbate/dihydrolipoic acid (DHLA) to regenerate the parent compound. Absorbance measurements demonstrated that ascorbate prevents net oxidation of PMC by recycling the phenoxyl radical back to the parent compound. The presence of DHLA in the reaction mixture containing ascorbate extended the recycling reaction through regeneration of ascorbate. DHLA alone was unable to prevent PMC oxidation. These conclusions were confirmed by direct detection of PMC* and ascorbate radicals formed during the time course of the reactions by EPR spectroscopy. Based on results in the model system, PMC* and ascorbate radicals were identified by EPR spectroscopy in ascorbate-loaded HL-60 cells after addition of H(2)O(2) and the inhibitor of catalase, 3-aminotriazole (3-AT). The time course of PMC* and ascorbate radicals was found to follow the same reaction sequence as during their recycling in the model system. Recycling of PMC by ascorbate was also confirmed by HPLC assays in HL-60 cells. Pre-loading of HL-60 cells with lipoic acid regenerated ascorbate and thus increased the efficiency of ascorbate in recycling PMC*. Lipoic acid had no effect on PMC oxidation in the absence of ascorbate. Thus PMC phenoxyl radical does not directly oxidize thiols but can be recycled by dihydrolipoate in the presence of ascorbate. The role of phenoxyl radical recycling in maintaining antioxidant defense and protecting against cytotoxic and genotoxic phenolics is discussed.
髓过氧化物酶（MPO）催化内源性酚类成分（如抗氧化剂、羟基化代谢产物）和外源性化合物（如药物、环境化学物质）的单电子氧化会产生自由基中间体：苯氧自由基。内源性还原剂对这些中间体的还原作用，即循环利用，可能会增强它们的抗氧化潜力和/或防止其潜在的细胞毒性和基因毒性作用。这项工作的目的是确定在富含MPO的完整人类白血病HL-60细胞中，是否能证明生理相关的细胞内还原剂（如抗坏血酸/硫辛酸）对维生素E同系物2,2,5,7,8-五甲基-6-羟基色满（PMC）的MPO催化苯氧自由基的生成和循环利用。开发了一个模型系统来表明MPO/H₂O₂催化的PMC苯氧自由基（PMC*）可以被抗坏血酸或抗坏血酸/二氢硫辛酸（DHLA）循环利用以再生母体化合物。吸光度测量表明，抗坏血酸通过将苯氧自由基循环回母体化合物来防止PMC的净氧化。在含有抗坏血酸的反应混合物中加入DHLA，通过抗坏血酸的再生延长了循环反应。单独的DHLA无法防止PMC氧化。通过电子顺磁共振波谱（EPR）直接检测反应过程中形成的PMC和抗坏血酸自由基，证实了这些结论。基于模型系统的结果，在加入H₂O₂和过氧化氢酶抑制剂3-氨基三唑（3-AT）后，通过EPR光谱在加载了抗坏血酸的HL-60细胞中鉴定出了PMC和抗坏血酸自由基。发现PMC和抗坏血酸自由基的时间进程与它们在模型系统中循环利用时遵循相同的反应顺序。在HL-60细胞中通过高效液相色谱（HPLC）分析也证实了抗坏血酸对PMC的循环利用。用硫辛酸预加载HL-60细胞可再生抗坏血酸，从而提高抗坏血酸循环利用PMC的效率。在没有抗坏血酸的情况下，硫辛酸对PMC氧化没有影响。因此，PMC苯氧自由基不会直接氧化硫醇，但在抗坏血酸存在的情况下可以被二氢硫辛酸循环利用。讨论了苯氧自由基循环利用在维持抗氧化防御以及保护细胞免受细胞毒性和基因毒性酚类物质侵害方面的作用。
