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Dose- and Time-Dependence of Radiation-Induced Nitric Oxide Formation in Mice as Quantified with Electron Paramagnetic Resonance
NITRIC OXIDE: Biology and Chemistry Vol. 5, No. 1, pp. 47–52 (2001) doi:10.1006/niox.2000.0321, available online at http://www.idealibrary.com on
Dose- and Time-Dependence of Radiation-Induced Nitric Oxide Formation in Mice as Quantified with Electron Paramagnetic Resonance Hidehiko Nakagawa, Nobuo Ikota, Toshihiko Ozawa, and Yashige Kotake* ,1 Department of Bioregulation Research, National Institute for Radiological Sciences, Inage, Chiba 263 Japan; and *Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104
Received June 7, 2000, and in revised form September 11, 2000; published online January 16, 2001
In vivo nitric oxide (NO) formation was quantified in mice after exposure to high-dose whole-body X-ray irradiation. NO produced and accumulated in the livers of irradiated mice was determined using NO trapping method with iron-dithiocarbamate complex combined with electron paramagnetic resonance (EPR) spectroscopy. When mice were irradiated with 50 Gy X-ray, NO formation peaked in approximately 3 h after the irradiation was terminated. Dose-dependence study indicated that NO formation measured 5 h after irradiation was leveled off at the dose higher than 50 Gy. Administration of NO synthase inhibitor, N G-monomethyl L-arginine (L-NMMA) shortly after irradiation completely abolished the NO signal, indicating that radiation-induced NO is produced through L-arginine-dependent NO synthase pathways. These results suggest that irradiation of X-ray initiates inflammation processes, resulting in delayed NO synthase expression and NO formation. © 2001 Academic Press
Key Words: radiation; ionizing radiation; X-ray; nitric oxide; NO trapping; EPR; electron paramagnetic resonance; mouse.
1
To whom correspondence and reprint requests should be addressed. Fax: (405) 271-1437. E-mail: yashige-kotake@omrf. ouhsc.edu. 1089-8603/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Nitric oxide (NO) has been shown to be an important messenger molecule in various physiological activities in animals and humans. A larger amount of NO is produced in some pathological states such as in sepsis, arthritis, and diabetes. There have been studies that indicate that the irradiation of ionizing radiation to living animals causes NO formation in various organs. For example, the increase in NO level in the kidney in a rat radiation nephritis model has been shown indirectly by the up-regulation of cyclic GMP (1). Radiation neumatitis in rats has been shown to be mediated by radiation-induced NO formation (2). In cellular systems, ionizing radiation induced NO synthase in the presence of exogenous interferon-␥ (3, 4). In contrast, exogenous NO has been shown to be radio-protective in vivo (5). The initial response of living systems to radiation exposure is the damage in cellular components including DNA. Repair enzymes are activated to fix minor damage to DNA; however, if this damage is lethal, necrotic or apoptotic cell death will follow. 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 47
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act as signaling molecules to initiate inflammation (6). The initial step for this inflammation is likely to be the activation of inflammatory transcription factors such as nuclear factor B (NF-B) (6 – 8), followed by the expression of inflammatory cytokines and enzymes. In fact, Brach et al. demonstrated that ionizing radiation induced expression and biding activity of NF-B in human leukemia cells (9, 10). NF-B inhibition decreased the induced production of cytokines and NO in cells and in vivo (11–14). NO produced in inflammation has been shown to predominantly originate from inducible isoform of NO synthase (iNOS). Although quantitative determination of radiationinduced NO in living systems is important in the evaluation of radiation trauma, it 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 EPR spectroscopy is unique and may be the only available method so far (15–17). This method utilizes the in vivo reaction of NO with an administered iron–sulfur complex 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 (18). But the sensitivity of the wholebody EPR spectroscopy is limited. Therefore, when the concentration of NO in the tissue is low, a section of organ is subjected to ex vivo EPR analyses (19, 20). By using this method, Voevodskaya and Vanin were the first to show that NO is produced in multiple organs in mice after whole-body ␥-ray irradiation (21). A lipophilic iron complex, iron-diethyl dithiocarbamate (Fe-DETC) 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 with different routes, thus the in vivo concentration of the iron-DETC complex was difficult to estimate. In the present study, we used a water soluble iron complex of D-N-methyl glucamine dithiocarbamate (Fe-MGD). EPR signals from the trapped NO (NOiron-MGD complex) were recorded in mouse liver tissues 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. The dose range studied was from 0 to 100 Gy.
EXPERIMENTAL PROCEDURES
Animals and Materials Female ICR mice, 5 weeks old, were obtained from SLC (Hamamatsu Japan). The sodium salt of MGD was synthesized and purified in our laboratory. NO gas for the synthesis of the authentic NO complex of iron-MGD was purchased from Sumitomo Seika (Osaka, Japan). Tris base was obtained from Sigma Chemical Co. (St. Louis, MO). NO synthase inhibitor L-N-monomethyl arginine (L-NMMA) and other chemicals, such as starting materials for the MGD synthesis, were purchased from Wako Pure Chemical Ind. (Osaka Japan).
Irradiation and NO Trapping Experiments X-ray irradiation was carried out by placing animals in compartments of a home-made restraint made of 5-mm-thick acrylic resin. Five animals were placed in the restraint separately and irradiated at once using a PANTAK-320S X-ray generator (Shimadzu Corp., Kyoto Japan) with a dose rate of 3.69 Gy/min. Irradiation time was adjusted so that the animal received the specified total doses. Subsequently, animals were returned to a cage and left at ambient conditions for the specified period. Control animals were placed in the same restraint and stayed there for the same period of time as the irradiated animals. The detection of NO produced in the livers of mice was conducted using the NO trapping method with the iron-MGD complex. A Tris solution of iron sulfate (200 mM) and MGD (600 mM) was administered intraperitoneally to mice, 30 min later the mice were subjected to euthanasia with carbon dioxide asphyxiation, and the liver specimens were obtained. A piece of liver tissue was mounted on a quartz tissue sample cell (Radical Research, Hino Japan) for EPR measurement. EPR spectra were recorded at room temperature in a JEOL RE1X EPR spectrometer. The concentration of NO in the liver
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
RADIATION-INDUCED NO FORMATION
FIG. 1. X-band EPR spectrum from NO complex of iron-MGD in the liver tissue, 3 h after mouse whole-body X-ray irradiation (50 Gy). Mice were irradiated with 50 Gy X-ray with the dose rate of 3.7 Gy/min, and after 3 h MGD-iron solution was administered intraperitoneally. Thirty minutes later, the liver specimen was placed on the EPR tissue-sample cell to record EPR spectrum. The amplitude of the lowest field line in the EPR spectrum was used to determine the concentration of NO complex.
tissue was determined by comparing the signal intensity of the authentic NO complex of MGD, which was synthesized from NO gas and MGD-iron complex. Six animals were used for each condition (dose and time lapsed) to determine NO formation. Statistical analysis was performed using ANOVA followed by Students’ t test as a posttest.
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FIG. 2. The dependence of NO produced in the livers of mice on the dose of X-ray irradiation. Mice were irradiated with various doses of X-ray, and 5 h later an EPR signal from the liver tissue was recorded (n ⫽ 6 for each treatment). The height of the EPR signal was calibrated into the concentration of NO-complex (NOadduct), and plotted as a function of X-ray dose.
was terminated. Usually, six animals were tested at one dose and their EPR signal intensities were averaged. In Fig. 3 the dependence of the averaged EPR signal intensities on the time lapsed after X-ray irradiation was terminated. The radiation dose was fixed at 50 Gy.
RESULTS
The levels of NO produced in the livers of mice after the whole-body X-ray irradiation were dependent on the total dose as well as the time lapsed after irradiation. NO produced in mice with no treatment (no restraint and no irradiation) yielded negligible levels of EPR signals. However, NO production in nonirradiated control animals, which experienced the same restraints as the irradiated animals were higher than no restraint controls, indicating that a stress caused by the confinement resulted NO formation. Radiation dose dependence of NO formation in irradiated mice were determined over the dose range from 0 to 100 Gy and the result is illustrated in Figs. 1 and 2. In this experiment, the NO level in the liver was determined 5 h after the irradiation
FIG. 3. The time course of NO formation in the livers of mice after whole-body X-ray irradiation. Mice were irradiated with X-ray (50 Gy), and at various times after irradiation (n ⫽ 6 for each waiting period), the heights of EPR signals of NO complex (NO-adduct) in the liver tissue was recorded. The height of the EPR signal was calibrated into the concentration of NO-complex and plotted as a function of time lapsed after irradiation.
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
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FIG. 4. The effect of the NO synthase inhibitor L-NMMA on the radiation-induced NO formation in vivo. Mice were irradiated with 50 Gy X-ray and administered with L-NMMA (100 mg/kg) three times (1, 3, and 5 h after irradiation). 5.5 h later liver NO was measured (n ⫽ 3 for each treatment). NO formation in L-NMMA-treated mice was completely abolished.
In order to confirm that NO formation after irradiation was L-arginine-dependent, i.e., caused by NO synthase activity, the effect of NOS inhibitor L-NMMA was tested. After X-ray irradiation (50 Gy), mice were injected (ip) with L-NMMA (100 mg/kg) three times 1, 3, and 5 h after irradiation and 5.5 h later liver NO was measured (n ⫽ 3). The result illustrated in Fig. 4 shows a complete inhibition of NO formation in mice treated with the inhibitor. DISCUSSION
In these experiments, we have shown that NO formation after the whole-body X-ray irradiation of mice can be quantified by means of an EPR NO trapping method with an iron-dithiocarbamate complex. It is clear that NO was not produced by the direct radiolysis of nitrogen-containing molecules because NO was not detected immediately after irradiation. The administration of the NOS inhibitor L-NMMA completely blocked the NO formation (Fig. 4), indicating that NO was formed via L-argininedependent NOS pathways. The presence of lag time after the termination of irradiation until the appearance of the EPR signal
suggests that NO was produced as a result of gene induction processes that were initiated by irradiation. NO formation was also shown to be dependent on the radiation dose (Fig. 2). At 10 Gy dose, NO formation was only slightly less than the amount produced by the 100 Gy irradiation, suggesting that a major part of induction has occurred at less than 10 Gy. At the high dose, it is possible that the cytokine-producing cells may have been damaged by irradiation, which ultimately results in the reduced NOS and NO. NO production was dependent on the time lapsed after irradiation. There was a rapid rise in 1 to 3 h after irradiation and reached at the maximum level in 3 to 5 h (Fig. 2). The NO level in 3 h after irradiation indicated a large error range (⫾ 73%), suggesting that there was a variation of the time at which the maximum NO level was obtained. Such variation may have been caused by animal individuality. In cellular systems, ionizing radiation has been shown to mediate the increase of expression and binding activity of NF-B (9, 10). In human KG-1 myeloid leukemia cells, at a dose of 20 Gy, the increase in NF-B binding activity was maximal at 2 to 4 h after irradiation (9). Because NF-B activation is considered to be a prerequisite for iNOS induction, the delay period in vivo before maximum NO formation (3 to 5 h) in the present experiment is consistent with cellular NF-B results. Although NO formation in LPS infused animals has been shown to be cytokine-mediated (22), one study demonstrated that radiation-induced NO formation is also cytokine-mediated, i.e., ␥-ray irradiation to the brain of living rats induced multiple cytokines genes in brain (23, 24). We speculate that necrosis which occurs at the initial stage of irradiation causes the cell rupture, from which calcium and endotoxin-like factors could be released. These factors could act as inflammatory stimuli to cause induced production of cytokines and iNOS. However, this assumption has not been tested. Also, it is tempting to speculate that delayed NO formation may play a role in cancer radiation therapy because NO has been shown to induce apoptosis in cancer cells (25, 26). In summary, the present study showed that the amount of L-arginine-dependent NO produced after
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
RADIATION-INDUCED NO FORMATION
mice were exposed to whole-body X-ray irradiation is strongly dependent on radiation dose and a function of the time lapsed after irradiation was terminated.
ACKNOWLEDGMENT We thank Science and Technology Agency, Japan, for the bilateral collaboration fellowship award.
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sion of interleukin 6 by human fibroblasts involving activation of nuclear factor-kappa B. J. Biol. Chem. 268, 8466 – 8472. 11. Kleinert, H., Euchenhofer, C., Ihrig-Biedert, I., and Forstermann, U. (1996). Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-kappa B. Mol. Pharmacol. 49, 15–21. 12. Liu, S. F., Ye, X., and Malik, A. B. (1997). In vivo inhibition of nuclear factor-kappa B activation prevents inducible nitric oxide synthase expression and systemic hypotension in a rat model of septic shock. J. Immunol. 159, 3976 –3983.
1. Cohen, E. P., Fish, B. L., and Moulder, J. E. (1996). The role of nitric oxide in radiation nephropathy. Arch. Physiol. Biochem. 104, 200 –206.
13. Sherman, M., Aeberhard, E., Wong, V., Griscavage, J., and Ignarro, L. (1993). Pyrrolidine dithiocarbamate inhibits induction of nitric oxide synthase activity in rat alveolar macrophages. Biochem. Biophys. Res. Commun. 191, 1301– 1308.
2. Nozaki, Y., Hasegawa, Y., Takeuchi, A., Fan, Z. H., Isobe, K. I., Nakashima, I., and Shimokata, K. (1997). Nitric oxide as an inflammatory mediator of radiation pneumonitis in rats. Am. J. Physiol. 272, L651–L658.
14. Schreck, R., Meier, B., Mannel, D., Droge, W., and Baeuerle, P. (1992). Dithiocarbamates as potent inhibitors of nuclear factor kappa B activation in intact cells. J. Exp. Med. 175, 1181–1194.
3. Ibuki, Y., and Goto, R. (1997). Enhancement of NO production from resident peritoneal macrophages by in vitro gamma-irradiation and its relationship to reactive oxygen intermediates. Free Rad. Biol. Med. 22, 1029 – 1035.
15. Mordvintcev, P., Mulsch, A., Busse, R., and Vanin, A. (1991). On-line detection of nitric oxide formation in liquid aqueous phase by electron paramagnetic resonance spectroscopy. Anal. Biochem. 199, 142–146.
REFERENCES
4. McKinney, L. C., Aquilla, E. M., Coffin, D., Wink, D. A., and Vodovotz, Y. (1998). Ionizing radiation potentiates the induction of nitric oxide synthase by IFN-gamma and/or LPS in murine macrophage cell lines: Role of TNF-alpha. J. Leuk. Biol. 64, 459 – 466.
16. Komarov, A., Mattson, D., Jones, M., Singh, P., and Lai, C. (1993). In vivo spin trapping of nitric oxide in mice. Biochem. Biophys. Res. Commun. 195, 1191–1198. 17. Kotake, Y., Moore, D., Sang, H., and Reinke, L. (1999). Continuous monitoring of in vivo nitric oxide formation using EPR analysis in bile flow. Nitric Oxide 3, 114 –122.
5. Liebmann, J., DeLuca, A. M., Coffin, D., Keefer, L. K., Venzon, D., Wink, D. A., and Mitchell, J. B. (1994). In vivo radiation protection by nitric oxide modulation. Cancer Res. 54, 3365–3368.
18. Yoshimura, T., Yokoyama, H., Fujii, S., Takayama, F., Oikawa, K., and Kamada, H. (1996). In vivo EPR detection and imaging of endogenous nitric oxide in lipopolysaccharidetreated mice. Nat. Biotechnol. 14, 992–994.
6. Schreck, R., Albermann, K., and Baeuerle, P. (1992). Nuclear factor kappaB: An oxidative stress-responsive transcription factor of eukaryotic cells. Free Rad. Res. 17, 231– 237.
19. Komarov, A., and Lai, C. (1995). Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy. Biochim. Biophys. Acta. 1272, 29 –36.
7. Christman, J., Lancaster, L., and Blackwell, T. (1998). Nuclear Factor kappaB: A pivotal role in the systemic inflammatory response syndrome and new target for the therapy. Intensive Care Med. 24, 1131–1138.
20. Miyajima, T., and Kotake, Y. (1995). Spin trapping agent, phenyl N-tert-butyl nitrone, inhibits induction of nitric oxide synthase in endotoxin-induced shock in mice. Biochem. Biophys. Res. Commun. 215, 114 –121.
8. Sen, R., and Baltimore, D. (1986). Multiple nuclear factors interact with the immunoglobulin enhancer sequences. Cell 46, 705–716.
21. Voevodskaya, N., and Vanin, A. (1992). Gamma-irradiation potentiates L-arginine-dependent nitric oxide formation in mice. Biochem. Biophys. Res. Commun. 186, 1423– 1428.
9. Brach, M. A., Hass, R., Sherman, M. L., Gunji, H., Weichselbaum, R., and Kufe, D. (1991). Ionizing radiation induces expression and binding activity of the nuclear factor kappa B. J. Clin. Invest. 88, 691– 695. 10. Brach, M. A., Gruss, H. J., Kaisho, T., Asano, Y., Hirano, T., and Herrmann, F. (1993). Ionizing radiation induces expres-
22. Sang, H., Wallis, G. L., Stewart, C. A., and Kotake, Y. (1999). Expression of cytokines and activation of transcription factors in lipoplysaccharide administered rats and their inhibition by phenyl N-tert-butylnitrone (PBN). Arch. Biochem. Biophys. 363, 341–348.
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23. Hong, J. H., Chiang, C. S., Campbell, I. L., Sun, J. R., Withers, H. R., and McBride, W. H. (1995). Induction of acute phase gene expression by brain irradiation. Int. J. Radiat. Oncol. Biol. Phys. 33, 619 – 626.
macher, V., and Umansky, V. (1999). Nitric-oxide-induced apoptosis in human leukemic lines requires mitochondrial lipid degradation and cytochrome C release. Blood 93, 2342–2352.
24. Chiang, C. S., Hong, J. H., Stalder, A., Sun, J. R., Withers, H. R., and McBride, W. H. (1997). Delayed molecular responses to brain irradiation. Int. J. Radiat. Biol. 72, 45–53.
26. Cui, S., Reichner, J. S., Mateo, R. B., and Albina, J. E. (1994). Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or -independent mechanisms. Cancer Res. 54, 2462– 2467.
25. Ushmorov, A., Ratter, F., Lehmann, V., Dro¨ge, W., Schirr-
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
Dose- and Time-Dependence of Radiation-Induced Nitric Oxide Formation in Mice as Quantified with Electron Paramagnetic Resonance Hidehiko Nakagawa, Nobuo Ikota, Toshihiko Ozawa, and Yashige Kotake* ,1 Department of Bioregulation Research, National Institute for Radiological Sciences, Inage, Chiba 263 Japan; and *Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104
Received June 7, 2000, and in revised form September 11, 2000; published online January 16, 2001
In vivo nitric oxide (NO) formation was quantified in mice after exposure to high-dose whole-body X-ray irradiation. NO produced and accumulated in the livers of irradiated mice was determined using NO trapping method with iron-dithiocarbamate complex combined with electron paramagnetic resonance (EPR) spectroscopy. When mice were irradiated with 50 Gy X-ray, NO formation peaked in approximately 3 h after the irradiation was terminated. Dose-dependence study indicated that NO formation measured 5 h after irradiation was leveled off at the dose higher than 50 Gy. Administration of NO synthase inhibitor, N G-monomethyl L-arginine (L-NMMA) shortly after irradiation completely abolished the NO signal, indicating that radiation-induced NO is produced through L-arginine-dependent NO synthase pathways. These results suggest that irradiation of X-ray initiates inflammation processes, resulting in delayed NO synthase expression and NO formation. © 2001 Academic Press
Key Words: radiation; ionizing radiation; X-ray; nitric oxide; NO trapping; EPR; electron paramagnetic resonance; mouse.
1
To whom correspondence and reprint requests should be addressed. Fax: (405) 271-1437. E-mail: yashige-kotake@omrf. ouhsc.edu. 1089-8603/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Nitric oxide (NO) has been shown to be an important messenger molecule in various physiological activities in animals and humans. A larger amount of NO is produced in some pathological states such as in sepsis, arthritis, and diabetes. There have been studies that indicate that the irradiation of ionizing radiation to living animals causes NO formation in various organs. For example, the increase in NO level in the kidney in a rat radiation nephritis model has been shown indirectly by the up-regulation of cyclic GMP (1). Radiation neumatitis in rats has been shown to be mediated by radiation-induced NO formation (2). In cellular systems, ionizing radiation induced NO synthase in the presence of exogenous interferon-␥ (3, 4). In contrast, exogenous NO has been shown to be radio-protective in vivo (5). The initial response of living systems to radiation exposure is the damage in cellular components including DNA. Repair enzymes are activated to fix minor damage to DNA; however, if this damage is lethal, necrotic or apoptotic cell death will follow. 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 47
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act as signaling molecules to initiate inflammation (6). The initial step for this inflammation is likely to be the activation of inflammatory transcription factors such as nuclear factor B (NF-B) (6 – 8), followed by the expression of inflammatory cytokines and enzymes. In fact, Brach et al. demonstrated that ionizing radiation induced expression and biding activity of NF-B in human leukemia cells (9, 10). NF-B inhibition decreased the induced production of cytokines and NO in cells and in vivo (11–14). NO produced in inflammation has been shown to predominantly originate from inducible isoform of NO synthase (iNOS). Although quantitative determination of radiationinduced NO in living systems is important in the evaluation of radiation trauma, it 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 EPR spectroscopy is unique and may be the only available method so far (15–17). This method utilizes the in vivo reaction of NO with an administered iron–sulfur complex 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 (18). But the sensitivity of the wholebody EPR spectroscopy is limited. Therefore, when the concentration of NO in the tissue is low, a section of organ is subjected to ex vivo EPR analyses (19, 20). By using this method, Voevodskaya and Vanin were the first to show that NO is produced in multiple organs in mice after whole-body ␥-ray irradiation (21). A lipophilic iron complex, iron-diethyl dithiocarbamate (Fe-DETC) 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 with different routes, thus the in vivo concentration of the iron-DETC complex was difficult to estimate. In the present study, we used a water soluble iron complex of D-N-methyl glucamine dithiocarbamate (Fe-MGD). EPR signals from the trapped NO (NOiron-MGD complex) were recorded in mouse liver tissues 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. The dose range studied was from 0 to 100 Gy.
EXPERIMENTAL PROCEDURES
Animals and Materials Female ICR mice, 5 weeks old, were obtained from SLC (Hamamatsu Japan). The sodium salt of MGD was synthesized and purified in our laboratory. NO gas for the synthesis of the authentic NO complex of iron-MGD was purchased from Sumitomo Seika (Osaka, Japan). Tris base was obtained from Sigma Chemical Co. (St. Louis, MO). NO synthase inhibitor L-N-monomethyl arginine (L-NMMA) and other chemicals, such as starting materials for the MGD synthesis, were purchased from Wako Pure Chemical Ind. (Osaka Japan).
Irradiation and NO Trapping Experiments X-ray irradiation was carried out by placing animals in compartments of a home-made restraint made of 5-mm-thick acrylic resin. Five animals were placed in the restraint separately and irradiated at once using a PANTAK-320S X-ray generator (Shimadzu Corp., Kyoto Japan) with a dose rate of 3.69 Gy/min. Irradiation time was adjusted so that the animal received the specified total doses. Subsequently, animals were returned to a cage and left at ambient conditions for the specified period. Control animals were placed in the same restraint and stayed there for the same period of time as the irradiated animals. The detection of NO produced in the livers of mice was conducted using the NO trapping method with the iron-MGD complex. A Tris solution of iron sulfate (200 mM) and MGD (600 mM) was administered intraperitoneally to mice, 30 min later the mice were subjected to euthanasia with carbon dioxide asphyxiation, and the liver specimens were obtained. A piece of liver tissue was mounted on a quartz tissue sample cell (Radical Research, Hino Japan) for EPR measurement. EPR spectra were recorded at room temperature in a JEOL RE1X EPR spectrometer. The concentration of NO in the liver
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
RADIATION-INDUCED NO FORMATION
FIG. 1. X-band EPR spectrum from NO complex of iron-MGD in the liver tissue, 3 h after mouse whole-body X-ray irradiation (50 Gy). Mice were irradiated with 50 Gy X-ray with the dose rate of 3.7 Gy/min, and after 3 h MGD-iron solution was administered intraperitoneally. Thirty minutes later, the liver specimen was placed on the EPR tissue-sample cell to record EPR spectrum. The amplitude of the lowest field line in the EPR spectrum was used to determine the concentration of NO complex.
tissue was determined by comparing the signal intensity of the authentic NO complex of MGD, which was synthesized from NO gas and MGD-iron complex. Six animals were used for each condition (dose and time lapsed) to determine NO formation. Statistical analysis was performed using ANOVA followed by Students’ t test as a posttest.
49
FIG. 2. The dependence of NO produced in the livers of mice on the dose of X-ray irradiation. Mice were irradiated with various doses of X-ray, and 5 h later an EPR signal from the liver tissue was recorded (n ⫽ 6 for each treatment). The height of the EPR signal was calibrated into the concentration of NO-complex (NOadduct), and plotted as a function of X-ray dose.
was terminated. Usually, six animals were tested at one dose and their EPR signal intensities were averaged. In Fig. 3 the dependence of the averaged EPR signal intensities on the time lapsed after X-ray irradiation was terminated. The radiation dose was fixed at 50 Gy.
RESULTS
The levels of NO produced in the livers of mice after the whole-body X-ray irradiation were dependent on the total dose as well as the time lapsed after irradiation. NO produced in mice with no treatment (no restraint and no irradiation) yielded negligible levels of EPR signals. However, NO production in nonirradiated control animals, which experienced the same restraints as the irradiated animals were higher than no restraint controls, indicating that a stress caused by the confinement resulted NO formation. Radiation dose dependence of NO formation in irradiated mice were determined over the dose range from 0 to 100 Gy and the result is illustrated in Figs. 1 and 2. In this experiment, the NO level in the liver was determined 5 h after the irradiation
FIG. 3. The time course of NO formation in the livers of mice after whole-body X-ray irradiation. Mice were irradiated with X-ray (50 Gy), and at various times after irradiation (n ⫽ 6 for each waiting period), the heights of EPR signals of NO complex (NO-adduct) in the liver tissue was recorded. The height of the EPR signal was calibrated into the concentration of NO-complex and plotted as a function of time lapsed after irradiation.
Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.
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FIG. 4. The effect of the NO synthase inhibitor L-NMMA on the radiation-induced NO formation in vivo. Mice were irradiated with 50 Gy X-ray and administered with L-NMMA (100 mg/kg) three times (1, 3, and 5 h after irradiation). 5.5 h later liver NO was measured (n ⫽ 3 for each treatment). NO formation in L-NMMA-treated mice was completely abolished.
In order to confirm that NO formation after irradiation was L-arginine-dependent, i.e., caused by NO synthase activity, the effect of NOS inhibitor L-NMMA was tested. After X-ray irradiation (50 Gy), mice were injected (ip) with L-NMMA (100 mg/kg) three times 1, 3, and 5 h after irradiation and 5.5 h later liver NO was measured (n ⫽ 3). The result illustrated in Fig. 4 shows a complete inhibition of NO formation in mice treated with the inhibitor. DISCUSSION
In these experiments, we have shown that NO formation after the whole-body X-ray irradiation of mice can be quantified by means of an EPR NO trapping method with an iron-dithiocarbamate complex. It is clear that NO was not produced by the direct radiolysis of nitrogen-containing molecules because NO was not detected immediately after irradiation. The administration of the NOS inhibitor L-NMMA completely blocked the NO formation (Fig. 4), indicating that NO was formed via L-argininedependent NOS pathways. The presence of lag time after the termination of irradiation until the appearance of the EPR signal
suggests that NO was produced as a result of gene induction processes that were initiated by irradiation. NO formation was also shown to be dependent on the radiation dose (Fig. 2). At 10 Gy dose, NO formation was only slightly less than the amount produced by the 100 Gy irradiation, suggesting that a major part of induction has occurred at less than 10 Gy. At the high dose, it is possible that the cytokine-producing cells may have been damaged by irradiation, which ultimately results in the reduced NOS and NO. NO production was dependent on the time lapsed after irradiation. There was a rapid rise in 1 to 3 h after irradiation and reached at the maximum level in 3 to 5 h (Fig. 2). The NO level in 3 h after irradiation indicated a large error range (⫾ 73%), suggesting that there was a variation of the time at which the maximum NO level was obtained. Such variation may have been caused by animal individuality. In cellular systems, ionizing radiation has been shown to mediate the increase of expression and binding activity of NF-B (9, 10). In human KG-1 myeloid leukemia cells, at a dose of 20 Gy, the increase in NF-B binding activity was maximal at 2 to 4 h after irradiation (9). Because NF-B activation is considered to be a prerequisite for iNOS induction, the delay period in vivo before maximum NO formation (3 to 5 h) in the present experiment is consistent with cellular NF-B results. Although NO formation in LPS infused animals has been shown to be cytokine-mediated (22), one study demonstrated that radiation-induced NO formation is also cytokine-mediated, i.e., ␥-ray irradiation to the brain of living rats induced multiple cytokines genes in brain (23, 24). We speculate that necrosis which occurs at the initial stage of irradiation causes the cell rupture, from which calcium and endotoxin-like factors could be released. These factors could act as inflammatory stimuli to cause induced production of cytokines and iNOS. However, this assumption has not been tested. Also, it is tempting to speculate that delayed NO formation may play a role in cancer radiation therapy because NO has been shown to induce apoptosis in cancer cells (25, 26). In summary, the present study showed that the amount of L-arginine-dependent NO produced after
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RADIATION-INDUCED NO FORMATION
mice were exposed to whole-body X-ray irradiation is strongly dependent on radiation dose and a function of the time lapsed after irradiation was terminated.
ACKNOWLEDGMENT We thank Science and Technology Agency, Japan, for the bilateral collaboration fellowship award.
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