Nitroxides as Protectors Against Oxidative Stress

用作EPR自旋标记的氮氧化物（如tempol）已被证明具有超氧化物歧化酶（SOD）活性，并且在保护细胞免受各种氧化应激（包括过氧化氢、超氧化物、有机氢过氧化物、氧化还原循环化疗药物和电离辐射）方面非常有效。我们已经证明，Tempol可以保护体外细胞和小鼠免受电离辐射。因此，氮氧化物代表了一类新的辐射保护剂，可以广泛用于保护人类免受辐射。重要的是，我们已经证明tempol不能保护啮齿动物的肿瘤组织；我们认为其机制涉及正常组织与肿瘤组织的差异代谢减少特性。携带肿瘤的动物模型的体内电子顺磁共振成像研究表明，与正常组织相比，肿瘤中氮氧化物的减少速度更快。最近的研究表明，缺乏葡萄糖6磷酸脱氢酶（G6PD）的细胞将氮氧化物还原为羟胺的速度比对照细胞慢得多，这表明这一重要的生化途径在氮氧化物还原中的作用。我们目前正在研究G6PD在肿瘤和正常组织中的状态。使用氮氧化物自旋探针，功能性EPR成像系统还将使我们能够绘制组织中的氧水平以及研究组织的各种氧化还原参数。我们将继续研究氮氧化物介导的辐射防护机制。最近的研究表明，只有氧化形式的氮氧化物（而不是还原形式）提供辐射防护。有趣的是，当氨基在氮氧化物环上的不同位置被取代时，辐射防护增强，这表明与细胞内目标（如DNA）结合的重要性可能是辐射防护的必要组成部分。研究还在继续，以评估局部应用于大鼠直肠的tempol和其他氮氧化物的辐射防护性能。由于直肠是前列腺癌和/或宫颈癌患者放疗期间受损的主要正常组织，如果我们的临床前研究证明是阳性的，我们将考虑在临床上使用tempol来保护直肠。我们目前的研究是针对氮氧化物输送到直肠组织的方法，以优化氮氧化物浓度。我们也在体内模型中研究氮氧化物单独或附加在大分子（如白蛋白）上的活性。由于这些药物很容易穿透细胞膜，它们可能用于其他医学研究领域，如缺血/再灌注损伤研究、预防白内障、炎症过程和衰老。我们最近的研究表明，大鼠脑缺血后给予tempol可显著减少与缺血/再灌注相关的梗死体积。初步研究表明，对p53基因敲除小鼠长期给予tempol（在食物或饮用水中）可延长其寿命。P53基因敲除小鼠在出生几个月后因肿瘤快速诱导死亡。给药后寿命延长35 ~ 70%。这种作用的机制是未知的，目前是一个主要的焦点。同样，我们已经证明，长期给小鼠服用tempol会导致体重减轻，我们认为这会影响瘦素和解偶联蛋白的水平。由于氮氧化物很容易穿透细胞膜，是有效的抗氧化剂，它们可能用于其他医学研究领域，如缺血/再灌注损伤研究，预防白内障，炎症过程和衰老。最后，为了更好地了解tempol处理在分子水平上的影响，我们已经开始使用cDNA微阵列进行基因表达研究。

Nitroxides (such as tempol) which have been used as EPR spin labels have been shown to exhibit superoxide dismutase (SOD) activity and are quite effective agents in protecting cells against a wide variety of oxidative stresses including hydrogen peroxide, superoxide, organic hydroperoxides, redox-cycling chemotherapy drugs, and ionizing radiation. We have demonstrated that Tempol protects both cells in vitro and mice against ionizing radiation. Thus, the nitroxides represent a new class of radiation protectors that may have widespread use in protecting humans against radiation. Importantly, we have shown that tempol does not protect rodent tumor tissue; the mechanism of which we believe involves differential metabolic reduction properties of normal versus tumor tissue. In vivo electron paramagnetic resonance imaging studies in a tumor-bearing animal model has shown more rapid reduction of nitroxides in tumor compared to normal tissue. Recent studies have shown that cells deficient in glucose 6 phosphate dehydrogenase (G6PD) reduce the nitroxide to the hydroxylamine much slower than control cells suggesting a role for this important biochemical pathway in nitroxide reduction. We are presently studying G6PD status in tumor versus normal tissue. Using nitroxide spin probes, the functional EPR imaging system will also enable us to map out oxygen levels in tissue as well as study various redox parameters of tissue.We continue to study the mechanism(s) of nitroxide-mediated radioprotection. Recent studies have shown that only the oxidized form of the nitroxide (as opposed to the reduced form) provides radioprotection. Interestingly, when amino groups are substituted at various positions on the nitroxide ring, radioprotection increases, suggesting the importance perhaps of binding to intracellular targets such as DNA as a necessary component of radioprotection. Studies continue toward evaluating the radioprotective properties of tempol and other nitroxides applied topically to the rectum of rats. Since the rectum is a major normal tissue damaged during radiotherapy for patients with prostate and/or cervix cancer, we will consider using tempol clinically to protect the rectum should our pre-clinical studies prove positive. Our present studies are directed on nitroxide delivery methods to rectal tissue to optimize nitroxide concentration. We are also investigating in in vivo models, the activity of nitroxides alone or appended to macromolecules such as albumin. Since these agents readily penetrate cell membranes, they may be of use in other areas of medical research such as ischemia/reperfusion injury studies, prevention of cataracts, inflammatory processes and aging. We have recently shown that tempol administration after induced ischemia of rat brain markedly reduced the infarct volume associated with ischemia/reperfusion. Preliminary studies have indicated that long term administration of tempol (in the food or drinking water) to p53 knockout mice extends their life span. p53 knockout mice die several months after birth due to rapid tumor induction. Tempol administration extended the life span of these animals ~35-70%. The mechanism of this effect is unknown and is presently a major focus. Likewise, we have shown that long-term administration of tempol to mice results in weight reduction, which we believe impacts leptin and perhaps uncoupling proteins levels. 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. Lastly, to better understand the effects of tempol treatment at the molecular level we have initiated gene expression studies using cDNA microarrays.