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This study was designed to determine if radiation-mediated activation of the apoptotic pathways would be influenced by antioxidants and if a correlation would be found between radioprotection and changes in transduction pathways. Human lymphoblastoid TK6 cells, known to undergo apoptosis as a result of radiation, were irradiated (6 Gy) with and without antioxidants, and then whole-cell lysates were collected. Parallel studies were conducted to assess the survival (clonogenic assay) and apoptotic index. The impacts of two nitroxide antioxidants, tempol and CAT-1, differing in cell permeability, as well as the sulfhydryl antioxidant N-acetyl- l -cysteine (L-NAC), were estimated. Changes in apoptotic pathway proteins and p53 were assessed by Western blotting. Fraction of apoptotic cells was determined by flow cytometry. Tempol (10 mM), which readily enters cells, partially radioprotected TK6 cells against clonogenic killing, but had no effect on radiation-induced apoptotic parameters such as cleaved caspase 3 or cleaved PARP. Tempol alone did not induce cytotoxicity, yet did increase cleaved PARP levels. The radiation-induced increase in p53 protein was partly inhibited by tempol, but was unaffected by CAT-1 and L-NAC. Both CAT-1 (10 mM), which does not enter cells, and L-NAC (10 mM) had no radioprotective effect on cell survival. Although L-NAC did not protect against radiation-induced cytotoxicity, it completely inhibited radiation-induced increase in cleaved caspase 3 and cleaved PARP. Collectively, the results question the validity of using selected apoptosis pathway members as sole indicators of cytotoxicity.

Reactive oxygen-derived species and particularly OH radicals can degrade hyaluronic acid (HA), resulting in a loss of viscosity and a subsequent decrease in its effectiveness as a joint-lubricating agent. The production of OH in the vicinity of HA can be catalyzed by bound redox-active metals, which participate in the Haber-Weiss reaction. Damage to HA can also occur as a result of hypochlorite formed by myeloperoxidase (MPO). The protective reagents commonly used to inhibit oxidative stress-induced degradation of HA include antioxidative enzymes, such as SOD and catalase, chelators that coordinate metal ions rendering them redox-inactive, and scavengers of radicals, such as OH, as well as nonradical reactive species. In recent years, stable cyclic nitroxides have also been widely used as effective antioxidants. In many cases, nitroxide antioxidants operate catalytically and mediate their protective effect through an exchange between their oxidized and reduced forms. It was anticipated, therefore, that nitroxides would protect HA from oxidative degradation as well. On the other hand, nitroxides serve as catalysts in many oxidation reactions of alcohols, sugars and polysaccharides, including hyalouronan. Such opposite effects of nitroxides on oxidative degradation are particularly intriguing and the aim of the present study was to examine their effect on HA when subjected to diverse forms of oxidative stress. The results indicate that nitroxides protect HA from OH radicals generated enzymatically or radiolytically. The protective effect is attributable neither to the scavenging of OH nor to the oxidation of reduced metal, but to the reaction of nitroxides with secondary carbohydrate radicals-most likely peroxyl radicals.

Stable free radical nitroxides are potent antioxidants possessing superoxide dismutase- and catalase-mimetic activity that protect cells and animals against a variety of oxidative insults. Tempol, as a representative nitroxide, was evaluated for its influence on weight maintenance and spontaneous tumor incidence in C3H mice. Tempol administered in either the drinking water or food did not show any untoward effects and prevented animals from becoming obese. Tempol-treated animals' leptin levels were reduced. Long-term treatment with Tempol significantly decreased tumorigenesis when compared to controls (10 vs. 40%, respectively). Selected tissues from Tempol-treated animals exhibited elevated levels of mitochrondrial uncoupling protein-2 (UCP-2) and HSP70. The present data suggest that nitroxides upregulate UCP-2, obviate weight gain, and decrease age-related spontaneous tumor incidence. As a class, nitroxides may provide overall health benefits by contributing to decreased obesity and tumor incidence.

The means by which neutrophils within the body ward off infectious and neoplastic processes by the activation of molecular oxygen, as well as how such mechanisms dysfunction, is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminol-amplified chemiluminescence surveillance of O2-. and H2O2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by PMA-stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such findings. The kinetics of O2-. production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin trapping combined with electron spin resonance detection, and ferricytochrome c reduction. The effects of pH and O2 level on luminol-amplified CL were determined using hypoxanthine/xanthine oxidase to produce O2-. and H2O2 in cell-free systems. As we have found by electron spin resonance and ferricytochrome c reduction, stimulated neutrophils continued to generate O2-. for several hours, yet when luminol-amplified CL was used to continuously follow radical production, CL was shortly lost. Similar loss of CL was observed with continuous enzymatic formation of O2-. and H2O2. The failure of the CL assay to report O2-. and H2O2 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged O2-. fluxes does not terminate radical production, and even when pH, [O2], and reagents are optimized, the use of luminol-amplified CL is not a valid assay for continuous monitoring of O2-. and H2O2 generated by either stimulated neutrophils or in cell-free systems.