B (NF-B), is followed by the expression of inflammatory cytokines and enzymes.
12. Dose- and Time- Dependence of Radiation-Induced Nitric Oxide Formation in Mice as Quantified with Electron Paramagnetic Resonance
Hidehiko Nakagawa, Nobuo Ikota and Toshihiko Ozawa
Keywords: nitric oxide, iNOS, X-ray irradiation, electron paramagnetic resonance, iron dithiocarbamate complex
Nitric oxide (NO) has been shown to be an important messenger molecule in various physiological activities in animals and humans. A large amount of NO is produced in some pathological states such as sepsis, arthritis, and diabetes. There have been studies that indicate that exposing living animals to ionizing radiation caused NO formation in various organs. In cellular systems, ionizing radiation induced NO synthase in the presence of exogenous interferon-gamma. In contrast, exogenous NO has been shown to be radio-protective in vivo. The initial response of living systems to radiation exposure is damage in cellular components. In addition to acute damage, ionizing radiation also results in the expression of early phase inflammatory genes, although the mechanism of this gene induction is not understood. The resulting production of inflammatory cytokines is believed to cause the induction of the inducible isoform of NO synthase (iNOS) and NO formation. Free radicals that are produced by ionizing radiation are believed to act as signaling molecules to initiate inflammation. The initial step involving a inflammatory transcription factor such as nuclear factor B (NF-B), is followed by the expression of inflammatory cytokines and enzymes.
Although quantitative determination of radiation-induced NO in living systems it important in the evaluation of radiation trauma, is has been hampered by the unstable and elusive nature of the molecule NO. For the measurement of NO level in organs of animal models, the NO trapping method combined with electron paramagnetic resonance (EPR) spectroscopy is unique. This method utilizes the in vivo reaction of NO with an administered iron-sulfur cmplex that results in the formation of a stable EPR-active NO-iron complex. This complex is relatively stable in living animals and can be detected by EPR using whole-body EPR spectroscopy. But the sensitivity of the whole-body EPR spectroscopy is limited. Therefore, when the concentration of NO in the tissue is low, an organ section is subjected to ex vivo EPR analyses. By using this method, Voevodskaya and Vanin were the first to show that NO is produced in multiple organs in mice after whole-body gamma-ray irradiation. A lipophilic iron complex, iron-diethyl dithiocarbamate (Fe-DETC) was used as a trapping compound and the EPR measurement was made at liquid nitrogen temperature. Because the iron-DETC complex is not water soluble, iron and DETC had to be separately administered to the animal by different routes, thus the in vivo concentration of the iron-DETC complex was difficult to estimate. In this study, we used a water soluble iron complex of D-N-methyl glucamine dithiocarbamate (Fe-MGD). EPR signals from the trapped NO (NO-iron-MGD complex) were recorded in mouse liver tissue at room temperature, which allowed us to determine the accurate time course of NO formation after irradiation and the dependence of NO levels on the radiation dose. In vivo NO formation was quantified in mice after exposure to high-dose whole-body X-ray irradiation, ranging from 0 to 100 Gy. NO produced and accumulated in the livers of irradiated mice was determined. When mice were irradiated with 50 Gy X-rays, NO formation peaked in approximately 3 to 5 h after the irradiation was terminated. A dose-dependence study indicated that NO formation measured 5 h after irradiation leveled off for doses higher than 50 Gy. Administration of NO synthase inhibitor, N(G)-monomethyl L-arginine (L-NMMA) shortly after irradiation completely eliminated the NO signal, indicating that radiation-induced NO is produced through L-arginine-dependent NO synthase pathways. These results suggest that X-ray irradiation initiates inflammation processes, resulting in delayed NO synthase expression and NO formation.
Publications:
Nakagawa, H., Ikota, N., Ozawa, T. and Kotake, Y.: Nitric Oxide, 5, 47-52, 2001.
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