Nitroxides as Protectors Against Oxidative Stress

我们实验室的广泛研究表明，氮氧化物(如坦普尔)是有效的抗氧化剂和抗电离辐射损伤的保护剂。我们先前已经证明，氮氧化物介导的氧化应激保护的潜在机制(S)包括超氧化物歧化酶和过氧化氢酶样活性以及自由基反应。然而，最近的研究主要集中在氮氧化物治疗是否会影响基因表达。用几种不同氮氧化物类似物的无毒浓度处理的细胞，包括不能进入细胞(和保护细胞)的氮氧化物，都表现出相似的基因表达模式。氮氧化物处理上调的显著基因包括热休克蛋白(HSP)家族、还原酶基因以及与Wnt/β-连环蛋白途径相关的基因。这些研究有望使我们更好地理解氮氧化物触发我们在动物身上观察到的反应的复杂的细胞/分子机制。例如，长期服用坦波尔(在食物或饮用水中)可显著减轻体重，并降低小鼠的自发肿瘤发病率。服用坦泊尔的动物骨骼肌线粒体解偶联蛋白2(UCP2)和热休克蛋白(HSP)的表达增加。在乳腺癌细胞中使用UCP2的初步瞬时转染研究表明，对于施加氧化应激的试剂，该蛋白具有保护作用。此外，我们还用持续给药的方式治疗ATM缺陷小鼠。ATM缺陷小鼠是人类癌症易感综合征共济失调-毛细血管扩张的模型，表现为氧化应激和损伤，以及DNA损伤诱导的细胞周期检查点。值得注意的是，坦波尔治疗通过延长胸腺淋巴瘤的潜伏期，显著延长了这些小鼠的寿命。坦普尔处理可降低细胞内活性氧水平、组织氧化损伤和应激反应，以及辐射诱导的DNA双链断裂。此外，在检查点缺陷的ATM缺陷小鼠中，坦普尔可剂量依赖性地延长S时相。这些实验提供了强有力的证据，证明氮氧化物抗氧化剂可以作为新的化学预防药物用于癌症易感综合征。最后，我们正在继续研究坦普尔对正常组织的差异性辐射防护，而不是肿瘤。最近的研究表明，在分割放射治疗前10分钟给予temol并不能保护SCC小鼠肿瘤的生长。目前正在进行评估肺、皮肤和肾脏等正常组织的研究。由于氮氧化物很容易穿透细胞膜，是有效的抗氧化剂，它们可能在医学研究的其他领域中使用，如缺血/再灌注损伤研究、预防白内障、炎症过程和衰老。

Extensive studies in our laboratory have demonstrated that nitroxides (such as tempol) are effective antioxidants and protectors against ionizing radiation damage. We have previously demonstrated that the potential mechanism(s) for nitroxide-mediated protection against oxidative stress include superoxide dismutase- and catalase-like activity and radical-radical reactions. However, more recent studies have focused on whether nitroxide treatment impacts gene expression. Cells treated with non-toxic concentrations of several different nitroxide analogues, including a nitroxide incapable of cellular entry (and protection) all exhibit similar patterns of gene expression. Prominent genes upregulated by nitroxide treatment include the heat shock protein (HSP) family, reductive enzyme genes, and genes associated with the Wnt/beta-catenin pathway. These studies will hopefully enable us to better understand the complex cellular/molecular mechanisms of nitroxides that trigger responses we have observed in animals. For example, long-term administration of tempol (in the food or drinking water) results in dramatic weight reduction and a decrease in spontaneous tumor incidence in mice. Animals maintained on tempol exhibit increased expression in mitochondrial uncoupling protein 2 (UCP2) and HSP in skeletal muscle. Preliminary transient transfection studies using UCP2 in breast cancer cells indicate a protective role for this protein with respect to agents imposing oxidative stress. Further we have treated ATM-deficient mice with continuous administration of tempol. The ATM-deficient mouse is a model for a human cancer prone syndrome ataxia-telangiectasia which displays oxidative stress and damage, as well as DNA damage induced cell cycle checkpoints. Remarkably, tempol treatment resulted in a dramatically increased lifespan of these mice by prolonging the latency to thymic lymphomas. Tempol treatment reduced the elevated level of reactive oxygen species, tissue oxidative damage and stress, and radiation induced DNA double strand breaks. In addition, tempol induced a dose-dependent prolongation of S-phase in the checkpoint defective ATM-deficient mice. These experiments provide strong evidence that nitroxide antioxidants can be used as novel chemopreventative agents in cancer prone syndromes. Lastly, we are continuing our studies on the differential radioprotection of tempol toward normal tissues as opposed to tumor. Recent studies indicate that tempol administered 10 min prior to fractionated radiation treatment does not protect SCC murine tumor growth. Studies are currently underway to evaluate normal tissues such as lung, skin, and kidney. Since nitroxides readily penetrate cell membranes and are potent antioxidants, they may be of use in other areas of medical research such as ischemia/reperfusion injury studies, prevention of cataracts, inflammatory processes, and aging.