Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Jan 21;100(2):685-90.
doi: 10.1073/pnas.0235623100. Epub 2003 Jan 8.

8-nitroguanosine formation in viral pneumonia and its implication for pathogenesis

Takaaki Akaike  1 , Shinichiro Okamoto, Tomohiro Sawa, Jun Yoshitake, Fumio Tamura, Koji Ichimori, Kiminori Miyazaki, Kazumi Sasamoto, Hiroshi Maeda
Affiliations

8-nitroguanosine formation in viral pneumonia and its implication for pathogenesis

Takaaki Akaike et al. Proc Natl Acad Sci U S A. .

Abstract

For many diseases, mediation of pathogenesis by nitric oxide (NO) has been suggested. In this study, we explored NO-induced viral pathogenesis with a focus on nucleic acid damage as evidenced by 8-nitroguanosine formation in vivo. Wild-type mice and littermate mice deficient in inducible NO synthase (iNOS) were infected with influenza or Sendai virus. Formation of 8-nitroguanosine in virus-infected lungs was assessed immunohistochemically with an antibody specific for 8-nitroguanosine. Extensive nitration of RNA either treated with peroxynitrite or obtained from cultured RAW 264 cells expressing iNOS was readily detected by this antibody. Strong 8-nitroguanosine immunostaining was evident primarily in the cytosol of bronchial and bronchiolar epithelial cells of virus-infected wild-type mice but not iNOS-deficient mice. This staining colocalized with iNOS immunostaining in the lung. 8- Nitroguanosine staining disappeared after addition of exogenous authentic 8-nitroguanosine during the antibody reaction and after pretreatment of tissues with sodium hydrosulfite, which reduces 8-nitroguanosine to 8-aminoguanosine. NO was generated in excess in lungs of wild-type mice but was eliminated in iNOS-deficient mice after virus infection; this result also correlated well with formation of 8-nitroguanosine and 3-nitrotyrosine. One consequence of the lack of iNOS expression was marked improvement in histopathological changes in the lung and the lethality of the infection without effects on cytokine responses and viral clearance. It is intriguing that 8-nitroguanosine markedly stimulated superoxide generation from cytochrome P450 reductase and iNOS in vitro. The present data constitute a demonstration of 8-nitroguanosine formation in vivo and suggest a potential role for NO-induced nitrative stress in viral pathogenesis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Efficacy of 8-nitroguanosine formation by peroxynitrite in nucleic acids and nucleosides and specificity of anti-8-nitroguanosine antibody. (A) Guanosine (Guo, 0.1 mg/ml), deoxyguanosine (dGuo, 0.1 mg/ml), DNA (calf thymus, 0.5 mg/ml), and RNA (yeast tRNA, 0.5 mg/ml) were reacted with peroxynitrite in 100 mM sodium phosphate buffer (pH 7.4) followed by 10 min of incubation at room temperature. 8-Nitroguanosine (8-NitroGuo) thus formed was quantified by HPLC/electrochemical-detection analysis as described by Yermilov et al. (29). (B) Competitive enzyme immunoassay for the reaction of anti-8-nitroguanosine antibody with 8-nitroguanosine–BSA conjugate. Gua, guanine; 3-NitroTyr, 3-nitrotyrosine; 8-OxoGua, 8-oxoguanine. (Inset) Binding of anti-8-nitroguanosine antibody to BSA or 8-nitroguanosine–BSA conjugate fixed on microtiter plates. (C) Endogenous formation of 8- nitroguanosine in cells. Total RNA from RAW 264 cells stimulated or unstimulated with IFN-γ and lipopolysaccharide were analyzed by slot blotting coupled with the immunoperoxidase method for visualization of binding of anti-8-nitroguanosine antibody. Peroxynitrite-treated total RNA from CV-1 cells served as a positive control. (Lower) Immunoreactivity of total RNA from RAW 264 cells and peroxynitrite-treated RNA that were treated with 0.5 M Na2S2O4 in 0.1 M Tris⋅HCl buffer (pH 9.0) for 5 min at room temperature before adsorption onto the membrane. RNA was stained with ethidium bromide and Sybr green II (Molecular Probes).
Figure 2
Figure 2
8-Nitroguanosine immunohistochemistry in virus-infected lungs. (A a–e) Immunostaining in lungs obtained 0 (control noninfection), 3, 6, 8, and 10 days after influenza virus infection, respectively. (Af) Immunostaining for Sendai virus-infected lung (8 dpi). (B Upper) Tissue section (8 dpi, influenza) stained with anti-8-nitroguanosine antibody. (B Lower) The same section viewed by a confocal laser scanning microscope (Fluoroview FV300, Olympus, Nagano, Japan), in which strong fluorescence due to emission of Vector red is evident in the cytosol.
Figure 3
Figure 3
Specificity of immunostaining with anti-8-nitroguanosine antibody and colocalization of this staining with iNOS immunostaining in virus-infected lung. 8-Nitroguanosine immunostaining in lungs infected with influenza virus was nullified by authentic 8-nitroguanosine (8-NitroGuo, 1 mM) (A) and by pretreatment of tissue sections with Na2S2O4 (B). (C) Localization of 8- nitroguanosine immunostaining compared with immunostaining of iNOS. Serial adjacent sections were used for each panel (A–C). (D) 8-Nitroguanosine immunostaining in the lung of an iNOS−/− mouse infected with influenza virus (8 dpi).
Figure 4
Figure 4
3-Nitrotyrosine and NO formation in influenza virus-infected lungs from wild-type and iNOS-deficient mice. (A) HPLC/electrochemical-detection analysis of protein in BAL fluid was used to determine tyrosine nitration. Concentrations of protein-bound 3-nitrotyrosine in BAL fluid (means ± SE, n = 3) are plotted versus time. (B) Immunohistochemistry for 3-nitrotyrosine formation in lung tissues. (C) Typical ESR signals of the NO-N-dithiocarboxy(sarcosine)-Fe2+ adduct for each corresponding group in B. The amounts of NO-N-dithiocarboxy(sarcosine)-Fe2+ as assessed by double integration of ESR spectra (15) are shown (means ± SE, n = 3) (P < 0.01 by t test for the value for iNOS+/+ vs. values for iNOS+/− and iNOS−/−).
Figure 5
Figure 5
Survival and pathological pulmonary changes in wild-type and iNOS-deficient mice after influenza (A) and Sendai (B) virus infections. The time profile of survival of virus-infected mice (Left) and histopathological changes at 8 dpi (hematoxylin and eosin staining) (Right) are shown. The statistical difference in survival rates (wild-type vs. iNOS-deficient mice) was analyzed by Fisher's exact probability test.
Figure 6
Figure 6
8-Nitroguanosine-stimulated Oformula image generation from P450 reductase (A) and iNOS (B). (A) The complete reaction system contained 8-nitroguanosine (8-NitroGuo) (10 μM), P450 reductase (0.2 μM), NADPH (0.1 mM), diethylenetriamine pentaacetic acid (0.1 mM), and DMPO (45 mM) in sodium phosphate buffer (25 mM, pH 7.4) and was incubated for 1 min at room temperature. SOD, superoxide dismutase (310 units/ml). The ESR signal of DMPO-OOH adduct (aN = 1.43 mT, aformula image = 1.15 mT, and aformula image = 0.13 mT) was observed in the complete reaction system (28). (B) iNOS (0.2 μM) was incubated with NADPH (0.1 mM) in the absence or presence of 10 μM 8-nitroguanosine, and ESR analysis was performed in the same manner as described for A. L-Arg, L-arginine (1 mM). (C) Increase in DMPO-OOH formation with the iNOS/NADPH system after addition of various concentrations of 8-nitroguanosine (means ± SD, n = 3).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akaike T, Maeda H. In: Nitric Oxide: Biology and Pathobiology. Ignarro L J, editor. San Diego: Academic; 2000. pp. 733–745.
    1. Nathan C. J Clin Invest. 1997;100:2417–2423. - PMC - PubMed
    1. Estévez A G, Crow J P, Sampson J B, Reiter C, Zhuang Y, Richardson G J, Tarpey M M, Barbeito L, Beckman J S. Science. 1999;286:2498–2500. - PubMed
    1. Granger D L, Hibbs J B, Jr, Perfect J R, Durack D T. J Clin Invest. 1988;81:1129–1136. - PMC - PubMed
    1. James S L. Microbiol Rev. 1995;59:533–547. - PMC - PubMed

Publication types

LinkOut - more resources