Ultraviolet B light-induced nitric oxide/peroxynitrite imbalance in keratinocytes--implications for apoptosis and necrosis.

Elevation of nitric oxide (NO*) can either promote or inhibit ultraviolet B light (UVB)-induced apoptosis. In this study, we determined real-time concentration of NO* and peroxynitrite (ONOO(-)) and their role in regulation of membrane integrity and apoptosis. Nanosensors (diameter 300-500 nm) were used for direct in situ simultaneous measurements of NO* and ONOO(-) generated by UVB in cultured keratinocytes and mice epidermis. An exposure of keratinocytes to UVB immediately generated ONOO(-) at maximal concentration of 190 nm followed by NO(*) release with a maximal concentration of 91 nm. The kinetics of UVB-induced NO*/ONOO(-) was in contrast to cNOS agonist stimulated NO*/ONOO(-) from keratinocytes. After stimulating cNOS by calcium ionophore (CaI), NO* release from keratinocytes was followed by ONOO(-) production. The [NO*] to [ONOO(-)] ratio generated by UVB decreased below 0.5 indicating a serious imbalance between cytoprotective NO* and cytotoxic ONOO(-)-a main component of nitroxidative stress. The NO*/ONOO(-) imbalance increased membrane damage and cell apoptosis was partially reversed in the presence of free radical scavenger. The results suggest that UVB-induced and cNOS-produced NO* is rapidly scavenged by photolytically and enzymatically generated superoxide (O(2) (-)) to produce high levels of ONOO(-), which enhances oxidative injury and apoptosis of the irradiated cells.

Peroxynitrite formation in radiation-induced salivary gland dysfunction in mice.

Xerostomia frequently arises in patients with head and neck malignancies that are treated by radiation. However, the mechanisms responsible for the destruction of the salivary gland remain unknown. We previously established a xerostomia model of mice and identified the pathway through which nitric oxide (NO) affects the pathogenesis of radiation-induced salivary gland dysfunction. Although the toxicity of NO alone is modest, NO with superoxide anion (O2(*-)) rapidly forms peroxynitrite (ONOO), a more powerful toxic oxidant. In this study, we used the experimental model to examine: 1) when NO and O2(*-) production is maximum in the salivary gland after irradiation;2) whether peroxynitrite, as assessed by nitrotyrosine production, is responsible for salivary gland dysfunction; and 3) the effect of the iNOS selective inhibitor, aminoguanidine (AG), on nitrotyrosine formation. The increases in production of NO and O2(*-) in the salivary gland peaked on day 7 after irradiation. Nitrotyrosine detected immunohistochemically was significantly reduced by AG in the salivary gland. On the basis of these results, we concluded that NO together with O2(*-) forms the more reactive ONOO, which might be an important pathogenic factor in radiation-induced salivary gland dysfunction.

Melatonin nitrosation promoted by NO*2; comparison with the peroxynitrite reaction.

N-nitroso species have recently been detected in animal tissues. Protein N-nitrosotryptophan is the best candidate for this N-nitroso pool. N-nitrosation of N-blocked trytophan derivatives like melatonin (MelH) by N2O3 or peroxynitrite (ONOOH/ONOO- ) has been observed under conditions of pH and reagent concentrations similar to in vivo conditions. We studied the reaction of NO*2 with MelH. When NO*2 was synthesized by gamma-irradiation of aqueous neutral solutions of nitrate under anaerobic conditions, detected oxidation and nitration of MelH were negligible. In the presence of additional nitrite, when NO* was also generated, formation of 1-nitrosomelatonin increased with nitrite concentration. Nitrosation is not due to N2O3 but could proceed via successive additions of NO*2 and NO*. For comparison, peroxynitrite was infused into a solution of MelH under air leading to the same products as those detected in irradiated solutions but in different proportions. In the presence of additional nitrite, the formation of nitroderivatives increased significantly while N-formylkynuramine and 1-nitrosomelatonin were maintained at similar levels. Mechanistic implications are discussed.

Changes in superoxide dismutase activity and peroxynitrite content in rat peritoneal macrophages exposed to He-Ne laser radiation.

The formation of reactive oxygen and nitrogen species by rat peritoneal macrophages induced by a low-intensity He-Ne laser radiation (LR) was studied in this work. It was found that the formation of reactive oxygen species, nitric oxide, and peroxynitrite as well as changes in the activity of superoxide dismutase (SOD) depended to a large extent on the LR dose. In particular, it was found that activation of SOD at low LR doses was accompanied by nitric oxide level increase, while the level of peroxynitrite showed no significant changes. On the other hand, an enhanced LR dose inhibited the enzyme, and this was accompanied by peroxynitrite accumulation. All the measurements were carried out the day after LR treatment. The revealed regularities consequently demonstrate the existence of a deferred LR action on macrophages associated with the production of reactive oxygen and nitrogen species.