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. We have completed an in vitro study to identify the most efficient nitroxide for protection purposes. Over 110 nitroxides were evaluated in a structure activity relationship study. We have identified 6 nitroxides that afford significantly more radioprotection than tempol (the first nitroxide shown to have radioprotective properties) and have also identified 3 analogs that radiosensitize aerobic cells. These agents will be evaluated and compared with tempol in vivo. Large quantities of several of the six protective nitroxides are being synthesized for further study of these newly discovered protectors. We have recently shown that heme proteins exposed to oxidants form highly toxic ferryl moieties and that nitroxides detoxify these toxic species and confer enhanced catalase-like activity to heme species. Reasoning in an analogous fashion we are investigating the affects of nitroxides as modulators of nitric oxide synthase because intermediates within the enzyme which depend on heme redox chemistry may be altered in the presence of nitroxides. We are also investigating in in vivo models, the activity of nitroxides 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. Nitroxides (such as tempol) which have been used as electron paramagnetic resonance (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. Different nitroxides analogues that do not influence blood pressure when administered to animals have been positively identified as radioprotectors thus eliminating the hemodynamic concerns of tempol administration. Recent studies 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 EPR imaging studies in one tumor-bearing animal model has shown more rapid reduction of nitroxides in tumor compared to normal tissue. We are presently seeking to identify and define cellular and physiological factors responsible for this differential effect using our newly constructed functional EPR imaging instrumentation for small animals. 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.Studies are presently underway evaluating the radioprotective properties of tempol 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.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. Lastly, since these agents 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. It has recently been shown that tempol administration after induced ischemia of rat brain markedly reduced the infarct volume associated with ischemia/reperfusion.
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