Departments of 1 Physiology and 2 Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa 259-11; and 3 Electro-Chemical and Cancer Institute, Chofu, Tokyo 182-0022, Japan
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ABSTRACT |
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The purposes of this study were 1) to identify the nitric oxide (NO) synthase (NOS) isoform responsible for NO-mediated radiation-induced lung injury, 2) to examine the formation of nitrotyrosine, and 3) to see whether nitrotyrosine formation and lung injury are reduced by an inducible NOS (iNOS) inhibitor, aminoguanidine. The left hemithorax of rats was irradiated (20 Gy), and the degree of lung injury, the expression of NOS isoforms, and the formation of nitrotyrosine and superoxide were examined after 2 wk. iNOS mRNA was induced, and endothelial NOS mRNA was markedly increased in the irradiated lung. Nitrotyrosine was detected biochemically and immunohistochemically. Aminoguanidine prevented acute lung injury as indicated by decreased protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid and improved NMR parameters and histology. Furthermore, the formation of nitrotyrosine was significantly reduced in the aminoguanidine group. We conclude that iNOS induction is a major factor in radiation-induced lung injury and that nitrotyrosine formation may participate in the NO-induced pathogenesis.
nitric oxide; nitric oxide synthase; aminoguanidine
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INTRODUCTION |
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IRRADIATION is one of the established therapies for chest malignancies, but the risk of radiation pneumonitis followed by fibrosis limits the dose of irradiation or may even contraindicate its application. Studies on the pathogenesis of radiation-induced lung injury indicate that alveolar macrophages (AMs) or epithelial cells play an important role (3, 9, 15). The currently accepted mechanism of injury is that irradiation stimulates these cells to produce various cytokines that lead to tissue damage and subsequent fibrosis. Recently, Nozaki et al. (13) showed that expression of inducible nitric oxide (NO) synthase (iNOS) increases in the irradiated lung and inhibition of NO production attenuated radiation-induced lung injury in a rat model. They suggested that NO is a significant mediator of radiation-induced acute lung injury. However, the responsible isoform of NOS and the mechanism of NO-induced toxicity remain to be clarified. NO has been shown to have a cytotoxic function in addition to its well-known vasodilating and signal transduction functions (27). The toxicity of NO is due to both NO itself and NO-derived reactive oxidants (1, 17). Because most of these reactive oxidants are not directly detectable in vivo, 3-nitrotyrosine is often used as a footprint of NO-derived injury because it is a nitration product of tyrosine residues in proteins or nonproteins (7, 24). Tyrosine nitration may alter protein structure and cellular function (10, 25, 30).
The purposes of this study were 1) to examine the expression of NOS isoforms in irradiated lungs, 2) to establish whether iNOS is responsible for radiation-induced lung injury, and 3) to measure nitrotyrosine formation in the injured lung and evaluate the effect of an iNOS inhibitor on both nitrotyrosine formation and lung injury.
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MATERIALS AND METHODS |
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Animals
Specific pathogen-free male Wistar rats (n = 44) aged 9-10 wk (SLC, Shizuoka, Japan) were used. All rats were kept under specific pathogen-free conditions until used. All experiments complied with the guidelines for animal experiments of the School of Medicine, Tokai University (Kanagawa, Japan).Experimental Protocol
The experiments were performed 2 wk after irradiation because acute lung damage peaked at week 2 in previous studies by Kawana et al. (9) and Shioya et al. (19). The early damage appears to play a key role in initiating the inflammatory cascade that leads to the development of radiation pneumonitis and fibrosis.The rats were anesthetized with 50 mg/kg of pentobarbital sodium
intraperitoneally, and the left hemithorax was irradiated with an
absolute dose of 20 Gy in one fraction, delivered by a 60Co teletherapy
unit (Theratron, Ottawa, Canada). The right hemithorax and other organs
were shielded during irradiation. The rats were killed with an
overdose of pentobarbitol sodium, and the left lungs were
isolated for NMR measurement, histological examination, and measurement
of NOS mRNA and tissue nitrotyrosine. For histological examination, the
lungs were fixed with Formalin-saline, and for measurement of NOS mRNA
and nitrotyrosine formation, the lungs were frozen with liquid nitrogen
and stored at 80°C until used. For bronchoalveolar lavage
(BAL), two different groups of rats were prepared. One group of rats
was prepared to obtain inflammatory cells in BAL fluid (BALF) and lung
tissue for the measurement of superoxide
(O
2) production. The other group was
prepared to obtain BALF for measurement of the concentrations of
nitrite and nitrate
(NO
2/NO
3) and protein and the activity of lactate dehydrogenase (LDH). BAL was
performed only in the irradiated left lung immediately after death through a plastic tube inserted in the left main
bronchus while the right main bronchus was ligated.
Experimental Groups
The rats were divided into the following four groups: 1) control group (C group), 2) irradiated group (R group), 3) radiation plus aminoguanidine (AG) group (R+AG group), and 4) aminoguanidine group (AG group). In the R+AG group, the rats were treated with aminoguanidine 2 h before irradiation followed by a daily subcutaneous injection of 50 mg/kg and oral administration of 2 g/l in the drinking water. In the AG group, AG was provided on the same schedule as in the R+AG group.Measurement of NOS mRNA
The frozen lung tissue was homogenized in ISOGEN (Nippon Gene, Tokyo, Japan), and RNA was extracted. The expression of mRNA for iNOS and endothelial NOS (eNOS) was measured with the RT-PCR method as previously described (14, 20). Briefly, equal amounts of RNA were reverse transcribed into cDNA. The RT products were amplified with primers for both iNOS and eNOS designed from rat gene sequences (14, 20), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a standard. After amplification, each PCR mixture was electrophoresed through 1% agarose gel, which was stained with ethidium bromide. Each gel was photographed under ultraviolet (UV) light with the same exposure and development time. The bands of the positive film were scanned, and the density of each PCR product was evaluated with National Institutes of Health (NIH) Image software. The ratio of NOS gene product to GAPDH gene product was used for semiquantification.Measurement of
NO2/NO
3
and Protein Concentrations and LDH Activity in BALF
The protein concentration and LDH activity in BALF were measured as indexes of lung damage with a modification of the Lowry method and an enzyme assay kit (Promega, Madison, MI), respectively.
Measurement of NMR Transverse Relaxation Time in Lung Tissue
NMR relaxation analysis provides information about the molecular damages in the tissue through interactions between the protons of water and the protons of macromolecules. The spin-spin relaxation time (T2) is a sensitive measure of detecting tissue damage even when there was no evidence of damage on histological examination in the early stage of radiation lung injury (19). Immediately after death, a sample 2 mm in diameter and 8 mm in length was excised from the passively collapsed left lung. Each sample was placed in a 90-MHz Fourier-transform NMR spectrometer (FX90A, JEOL, Tokyo, Japan), and NMR T2 was measured by the Carr-Purcell-Meiboom-Gill method. The spin-echo signals were Fourier transformed, and the T2 decay curves for water peak were determined. The T2 decay curves of peripheral lung tissue have been shown to be multiexponential and can be fitted with two components. Values of the two T2 components, fast (T2f) and slow (T2s), which reflect changes in the intracellular and extracellular water, respectively, were determined by an iterative least squares curve fitting (19).Biochemical Detection of Nitrotyrosine Formation in the Lung
The frozen lung was homogenized with Milli-Q water and hydrolyzed as previously reported (6). Briefly, homogenates were hydrolyzed in 0.1% phenol containing 6 N HCl at 110°C in a vessel rack (JASCO) for 24 h. Separation of tyrosine and nitrotyrosine was achieved by HPLC on a Nucleosil 5-µm C-18 reverse-phase column (15 cm × 4.6 mm) with a guard column (JASCO). The column was eluted with 50 mmol/l of KH2PO4-H3PO4 (pH 3.01) containing 10% methanol at a flow rate of 1 ml/min through an isocratic pump, and the peaks were measured with an UV detector set at 274 nm. The level of nitrotyrosine is expressed as the percentage of nitrotyrosine to total tyrosine.Immunohistochemical Detection of Nitrotyrosine
A paraffin-embedded sample was sliced and treated with 0.1% trypsin in Tris · HCl buffer. Endogenous peroxidase was blocked with hydroperoxide in methanol. Nitrotyrosine was stained with nitrotyrosine antibody (2). Antibody binding was then visualized by a diaminobenzidine-peroxidase reaction with a peroxidase-labeled second antibody (mouse and rabbit cocktail). A negative control was prepared in each group with the same protocol except for the primary antibody to exclude nonspecific staining. The specimens were counterstained with methyl green.Measurement of O2 Production
by BALF Cells and Tissue Cells
Because inflammatory cells that invade the lung tissue (lung cells) cannot be collected by the BAL procedure, tissue cells were obtained from the left lung after the BAL procedure as previously described (28). Briefly, the tissue was minced and incubated in Hanks' balanced salt solution with collagenase and DNase at 37°C for 90 min, then filtered through a cloth (32-µm pore diameter) into RPMI 1640 medium containing 10% fetal calf serum. The adherent cells, which include AMs, polymorphonuclear neutrophils (PMNs), and interstitial macrophages were resuspended in PBS until used.
The total number of BALF and lung cells was counted with a hemocytometer. Composition of the cells was performed on cytospun preparations stained with Diff-Quik hematoxylin and eosin (Cytospin, Shandon Instruments, Cheshire, UK).
O2 production was measured with the
chemiluminescence method with an
O
2-specific chemiluminescent probe,
2-methyl-6-( p-methoxyphenyl)-3,7-dihydroimidazo(1,2-a)pyrazin-3-one (Tokyo Kasei, Tokyo, Japan) (12) in the presence of 50 ng of phorbol
12-myristate 13-acetate as previously described (23). To terminate
O
2 production, 50 U of superoxide dismutase were added. The amount of O
2
production is expressed as counts per second per cell × total
cell count.
Statistical Analysis
Data are expressed as means ± SD. To compare more than three groups, Tukey's multiple comparison test was used (29). A probability value of 0.05 or less was considered significant. ![]() |
RESULTS |
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NOS Induction and
NO2/NO
3
Generation
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Figure 2 shows the concentration of
NO2/NO
3
in BALF. Although an increase in vascular permeability in the R group
may influence the results,
NO
2/NO
3 was significantly increased in the R group, but the increase was attenuated in the R+AG group. There was no significant difference in
values between the C and R+AG groups.
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Lung Injury
We measured the protein concentration and LDH activity in BALF as indexes of lung injury (Fig. 3, A and B, respectively). Protein concentration and LDH activity were increased in the R group, but these increases were completely prevented by AG treatment. Changes in the NMR relaxation times T2f and T2s of the lung are shown in Fig. 4. The T2f and T2s increased in the R group, and AG treatment attenuated the increase in T2f and T2s, although the T2s in the R+AG group increased compared with that in the C group, suggesting mild interstitial damage. In the AG control group, there was no significant difference from the control value in protein concentration, LDH activity, or NMR T2.
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Nitrotyrosine Formation
Nitrotyrosine was not detected in the C group, but it increased in the R group and the increase was attenuated by AG treatment (Fig. 5).
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Immunohistochemical Detection of Nitrotyrosine
Nitrotyrosine staining was not observed in the C group but was present in the R group (Fig. 6). Nitrotyrosine-positive regions tended to coincide with sites of lung tissue injury, being located in the vicinity of airways, alveolar epithelia, and AMs. In the R+AG group, nitrotyrosine staining was not as marked as that in the R group.
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Cellular Composition of Cells in BALF and in Lung Tissue
BALF cells. Figure 7 shows the total number and composition of BALF cells. The significant decrease in the number of BALF cells in the R and R+AG groups probably reflects the direct fatal effect of irradiation on the inflammatory cells that reside in lung tissue. Also, it may reflect a methodological limitation of the BAL procedure; i.e., firmly adhering cells cannot be lavaged. However, it is clear that the PMN fraction increases and monocytes emerge in the R and R+AG groups. O
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Lung cells. The number of lung cells obtained was comparable in
the C, R, and R+AG groups (10.39 ± 4.77, 15.08 ± 7.04, and 10.87 ±1.42, respectively; Fig. 8). AMs were
decreased and PMNs were increased in the R group. The pattern of cell
composition in the R+AG group was intermediate between those of the
control and R groups. O2 production by
total cells was significantly greater in the R and R+AG groups than in
the C group (Fig. 9). There was no
significant difference in O
2 production between the R and R+AG groups.
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DISCUSSION |
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In this study, we observed an increased expression of both eNOS and
iNOS mRNAs together with an increased concentration of NO2/NO
3
in BALF from irradiated lung tissue after 2 wk. Treatment with an iNOS
inhibitor attenuated the injury without affecting the expression of
eNOS and iNOS. Although the increase in eNOS is a novel finding, an
increase of iNOS in the irradiated lung was already reported by Nozaki et al. (13), who showed that the inhibition of NO formation by
N-nitro-L-arginine methyl ester, a nonspecific NOS
inhibitor, attenuated the injury. In this study, we were able to show
that a relatively iNOS-specific inhibitor, AG, attenuated all indexes of the injury, indicating that iNOS, not eNOS, is the major source of
NO production and is primarily responsible for inducing the injury.
Although the role of eNOS in radiation-induced lung injury remains to
be determined, it may represent regenerating epithelial cells because a
marked increase in eNOS occurs in denuded aorta or in the repair stage
of inflammation (16, 18).
The nitrotyrosine-to-total tyrosine ratio was increased in the
irradiated lung. Furthermore, nitrotyrosine staining was prominent in
the areas where the destruction was severe. The nitrotyrosine-positive cells were identified as AMs and epithelial cells of the alveolus and
airways. These cell types are known to express iNOS protein and mRNA
(13), indicating that they may be major sources of NO production.
Although nitrotyrosine formation was decreased by AG treatment in terms
of both biochemical and immunohistochemical criteria, the degree of
decrease was less compared with that in the injury because almost
complete protection against injury except the change in MNR
T2s was observed. This suggests that tyrosine nitration is
not the only mechanism causing injury. Another plausible mediator of
the injury is O2, which has been shown
to play an important role in ischemia-reperfusion injury and
hypoxic or hyperoxic lung injury (8, 21, 22). The generation of
O
2 was increased in lung cells in the
irradiated group compared with that in the C group. However, increased
generation was also observed in the AG-treated group in which the
injury was markedly attenuated, indicating a limited direct role of
O
2 in causing the injury.
Tyrosine nitration can influence cellular function in addition to
altering protein structure and activity (10, 25, 30). The demonstration
of nitrotyrosine in atherosclerotic lesions (2) has shed light on its
formation in various pathological conditions (26, 27). Originally,
nitrotyrosine was considered to be a footprint of peroxynitrite, which
is a potent oxidant formed by the reaction of NO and
O2 (4). However, myeloperoxidase,
which is abundant in granules of neutrophils, was also shown to play a
role in the nitration reaction (5). Because we did not perform
myeloperoxidase staining or measure myeloperoxidase activity in the
injured lung, the relative importance of these pathways in
radiation-induced lung injury remains to be examined.
In conclusion, the effect of iNOS inhibition on radiation-induced lung injury and nitrotyrosine formation may imply a therapeutic strategy for this clinically important side effect of radiation therapy.
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ACKNOWLEDGEMENTS |
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We thank M. Tsuda, M. Seo, Y. Shinozaki, Y. Takahari and K. Naitoh for technical assistance.
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FOOTNOTES |
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This work was supported by Grant 09671630 for Scientific Research from the Ministry of Education, Science, and Culture, Japan, and a grant from Tokai School of Medicine Research Aid (Kanagawa, Japan).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Tsuji, Dept. of Physiology, Tokai Univ. School of Medicine, Isehara, Kanagawa 259-11, Japan (E-mail: ctsuji{at}is.icc.u-tokai.ac.jp).
Received 30 August 1999; accepted in final form 29 November 1999.
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