Nitroxides as Protectors against Oxidative Stress Summary Nitroxides are proving to have broad utility in a number of disease processes and/or conditions that represent excessive oxidative stress. The fact that nitroxides exert activity over such a range of conditions speaks to the importance of free radical reactions in tissue. Likewise, it is becoming apparent that free radicals are important in normal molecular signaling pathways and related gene expression. In collaborative studies, the effects of chronic administration of Tempol (supplemented in food) of two mouse models that exhibit neurodegeneration and/or neurological damage have been evaluated. Iron regulatory protein 2 knockout mice (IRP2-/-) exhibit age-related neurodegeneration (similar to Parkinsons disease patients). Tempol treatment attenuated the progression of neurodegeneration in IRP2-/- mice. Tempol was also shown to be highly protective in an experimental autoimmune encephalomyelitis (EAE) mouse model. The EAE mouse model is an acute or chronic demyelinating autoimmune disease whose clinical manifestations of paralysis and quadriparesis that closely resemble those observed in Multiple Sclerosis patients. These studies are presently ongoing and may represent a new approach toward treating these diseases. We continue to search for the mechanism(s) of how 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. First, we have initiated studies using an in vitro lipogenesis system (3T3L1 cells) and have found that Tempol inhibits lipogenesis in this model. Further, we have found that in this system that induction of lipogenesis results in a reduction in HIF-1 alpha and an increase in prolyl hydroxylase;whereas, Tempol prevents the reduction of HIF-1 alpha and induction of prolyl hydroxylase. Studies are focused on the mechanism of this interaction, which may involve Tempol effects on the ferryl state of prolyl hydroxylase. Using this same model we have also found that Tempol prevents differiation-induced induction of PPAR-gamma and alpha along with FIAF, all of which are involved in fat storage and lipid metabolism. Further mechanistic studies are ongoing. Studies with the 3T3L1 cells have agreed with gene expression array studies evaluating tissue taken from age-matched control mice and mice on Tempol food supplementation for 1 month or 1 year. A number of genes have been identified in Tempol supplemented mice that are differentially up- or down-regulated in liver and brain tissue, including genes associated with glutathione metabolism (up-regulated). Genes associated with fat synthesis and storage were found to be modulated by Tempol. The gene array study suggested that hypoxic related genes were also modulated by Tempol. Finally, Tempol administration also significantly delayed the onset of tumors in Atm and p53 deficient mice and more recently in Fanconis Anemia knockout mice. We have recently similar to that observed in caloric restricted animals. Additionally in preliminary studies, we have shown that Tempol administration (in the food) decreases pristane-induced plasmocytomas in mice, further suggesting that Tempol may interfere/delay the onset of cancer induction. These studies will hopefully enable us to better understand the complex cellular/molecular mechanisms of nitroxides that trigger responses important in the antioxidant properties of nitroxides as well as those related to weight and the chemopreventive findings.