SummaryNitroxides 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. 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. Gene expression profile studies of selected tissues were conducted to obtain early gene expression changes by Tempol in mice prior to changes in weight (14 days) and later when animal weights were reduced by Tempol (60 days). The majority of gene changes (both up- and down-regulated) resulting from the Tempol diet occurred in the liver (248 genes), followed by the muscle (150 genes), and brain (9 genes). The Tempol diet up-regulated genes related to mitochondrial function and fatty acid and/or lipid synthesis. Drug metabolizing genes were elevated at both 14 and 60 days of Tempol treatment. Glutathione metabolism genes and Nrf2 genes (which provide protection against free radicals) were up-regulated. We are currently evaluating the effect of Tempol on gene expression profiles in adipose tissue, in particular if proteins modulation can be observed from genes either up- or down-regulated. In on-going studies metabolic products in the urine from mice consuming Tempol either in the diet or by direct administration is being determined. Hundreds of metabolic products were altered by Tempol administration. Preliminary data indicates that 2,8-dihydroxylquinoline and its glucuonide were significantly elevated compared to control. Likewise, butyric acid, panthothenic acid, and isobutrylcarnitine are decreases as is fatty acid metabolism. Data evaluation is currently ongoing. Lastly, in a related project dealing with oxidative stress, we evaluated a different antioxidant (resveratrol) and its metabolic product piceatannol as potential protectors against oxidative stress (hydrogen peroxide, radiation). Piceatannol was found to provide concentration-dependent protection against hydrogen peroxide induced cytotoxcity; whereas, resveratrol markedly enhanced the cytotoxicity. Neither agent afforded protection against ionizing radiation in vitro or in vivo, suggesting that these agents should not be evaluated clinically for radiation effects.