Published ahead of print on June 1, 2006, doi:10.1164/rccm.200510-1580OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200510-1580OC
Guanine Nitration in Idiopathic Pulmonary Fibrosis and Its Implication for CarcinogenesisDepartments of Cell Pathology, Microbiology, and Thoracic Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto; and Sumika Technoservice Corporation, Osaka, Japan Correspondence and requests for reprints should be addressed to Takaaki Akaike, M.D., Ph.D., Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. E-mail: takakaik{at}gpo.kumamoto-u.ac.jp
Rationale: Nitric oxide (NO)–induced nitrative stress of nucleic acids, as evidenced by guanine nitration, appears to be involved in inflammation-induced carcinogenesis. A high incidence of lung cancer in idiopathic pulmonary fibrosis (IPF) is the major reason for poor prognosis in patients with IPF. Objectives and Methods: We immunohistochemically analyzed the formation and localization of 8-nitroguanine in lung tissues from control subjects, patients with IPF, and patients with lung cancer. Main Results: Immunohistochemical analysis of control smoker and nonsmoker lungs showed weak immunoreactivity for 8-nitroguanine, mainly in cytoplasm of bronchial epithelial cells. In addition to the bronchial epithelial cells, metaplastic regenerated epithelial cells overlying dense fibrotic lesions in IPF showed strong 8-nitroguanine staining in the cytoplasm. The staining in these metaplastic cells colocalized with staining of inducible and endothelial NO synthases and 8-oxodeoxyguanosine, as evidenced by double-immunostaining analysis. Confocal and immunoelectron microscopy revealed localization of 8-nitroguanine in metaplastic epithelial cytoplasm, mostly in mitochondria. Appreciable 8-nitroguanine immunostaining was also observed in both nuclei and cytoplasm of malignant epithelial cells in squamous cell carcinoma. No significant difference was found in the epithelial 8-nitroguanine formation between control smokers and nonsmokers, but much higher guanine nitration was observed in patients with IPF than in control subjects and patients with lung cancer, via a quantitative immunofluorescence image analysis. Conclusions: The present study indicates that not only oxidative stress but also nitrative stress induced by NO may participate in the pathogenesis of epithelial cell damage and aberrant regeneration occurring in IPF. Thus, guanine nitration may be a major risk factor for lung cancer development in IPF.
Key Words: idiopathic pulmonary fibrosis/usual interstitial pneumonia • lung cancer • nitrative stress • 8-nitroguanine • tumorigenesis A high incidence of lung cancer in patients with idiopathic pulmonary fibrosis/usual interstitial pneumonia (IPF/UIP) is the major reason for the poor prognosis of these diseases (1–4). During remodeling of alveolar septa in lungs from patients with IPF/UIP, proliferation and metaplasia of epithelial cells are observed. These histopathologic changes are believed to be key etiologic findings for IPF/UIP, as part of the precancerous process leading to lung cancer (1–4). For example, previous studies showed that P53 and P21 are expressed in hyperplastic bronchial and alveolar epithelial cells from lung tissues in IPF/UIP (5). Tumorigenesis occurring in IPF/UIP may also be associated with p53 mutation caused by chronic DNA damage and repair, which would lead to p53 up-regulation (5). Lung epithelial cells are often exposed to various stresses because of direct access to outside air and thus are a primary target for reactive oxygen and nitrogen oxide species (ROS and RNS) generated during various pathologic events. For example, formation of ROS/RNS in the lung increases during infections, inflammation, and exposure to air pollutants and cigarette smoke (6, 7). Oxidative and nitrative stresses caused by ROS/RNS in lung epithelial cells are reportedly involved in pathophysiology of lung diseases, including pulmonary fibrosis and lung cancer. Increased oxidative DNA damage, as evidenced by enhanced 8-hydroxydeoxyguanosine (8-oxodeoxyguanosine) formation, was documented in dysplastic epithelial cells from patients with IPF/UIP (8). 8-Oxodeoxyguanosine formation also was increased in smokers' lung and lung cancer tissues compared with nonsmokers' lungs (9) and noncancerous control lungs (10). Intriguingly, the degree and profile of protein nitration are found to be altered in lung cancer tissues analyzed by proteomics (11). In this context, we reported that nitration of purine nucleosides, most typically by 8-nitroguanosine formation, occurs in vivo in airway epithelial cells of mice infected with pneumotropic viruses and in cells in culture, with the process depending on production of nitric oxide (NO) from inducible NO synthase (iNOS) (12, 13). Nitration of nucleotides in DNA and RNA by various RNS (e.g., peroxynitrite and nitrogen dioxide) was first suggested by Yermilov and colleagues and Masuda and colleagues, mainly on the basis of data obtained from a cell-free chemical reaction in vitro (14–16). This suggestion prompted us to propose that a nitrated nucleotide may be formed in vivo and in cultured cells and may be a biomarker for DNA and RNA damage induced by NO. We therefore produced antibodies that specifically recognize 8-nitroguanine so that guanine nitration could be readily identified in biological samples. Immunohistochemical studies with these antibodies did indicate formation of 8-nitroguanine in vivo and in cultured cells, as just mentioned. We recently discovered, however, that our antibodies could bind not only to 8-nitroguanine (or 8-nitroguanosine) but also to 8-nitroxanthine (see Figure 1) (17). Notwithstanding this cross-reactivity, our antibodies should be useful for evaluation of a nitration reaction or nitrative stress of nucleic acids in biological systems.
After our first report, Pinlaor and coworkers, by using a similar antibody, suggested that 8-nitroguanine, or related nitrated derivatives, may be formed in hamster livers infected with Opisthorchis viverrini (18) and in human gastric mucosa infected with Helicobacter pylori (19). Because these infections are risk factors for hepatic and gastric cancers (20, 21), nitrative stress of nucleic acids, as shown by immunostaining for nitrated guanine (purine) derivatives, appears to be involved in infection- or inflammation-induced carcinogenesis. More important, we recently found that appreciable amounts of 8-nitroguanine and 8-nitroxanthine can be recovered from human urine (17). However, whether nitrative stress is also induced in IPF/UIP and lung cancer remains unclear. It is important to explore nitration of purine bases, nucleosides, and nucleotides as a potential biomarker for nitrative nucleic acid damage and modification induced by NO in lung tissues from patients with IPF/UIP, because such stress may contribute to carcinogenesis in IPF/UIP. Therefore, we performed immunohistochemical studies of the lung tissue specimens from patients with IPF/UIP or lung cancer and from control subjects, by means of monoclonal antibodies for 8-nitroguanine (8-nitroguanosine). Also, we correlated nitrative stress with oxidative stress, as assessed by 8-oxodeoxyguanosine formation and its related repair system P53 expression in lung epithelium from patients with IPF/UIP or squamous cell carcinoma of the lung and from control subjects.
Characterization of Anti–8-Nitroguanine Antibody An enzyme-linked immunosorbent assay (ELISA) was performed as described previously (12). Briefly, each well of a 96-well microtiter plate was coated with 100 µl of the conjugate of 8-nitroguanine and bovine serum albumin (5 µg/ml) in phosphate-buffered saline (PBS), blocked with 0.5% gelatin, and washed three times with PBS containing 0.05% Tween 20 (washing buffer). Samples in the wells were incubated for 1 h with 0.1 ml of mouse monoclonal anti–8-nitroguanine or anti– 8-nitroguanosine (1G6) antibody (0.1 µg/ml each) in the presence or absence of various concentrations of 8-nitroguanine, 8-nitroguanosine, or 8-nitroxanthine dissolved in washing buffer. These monoclonal antibodies were produced according to the method reported in our earlier study (13). Wells were then washed with washing buffer three times and reacted with horseradish peroxidase (HRP)–conjugated anti-mouse IgG antibody, followed by reaction with 1,2-phenylenediamine dihydrochloride. The reaction was terminated by addition of 0.1 ml of 1.0 mol/L sulfuric acid, and absorbance at 490 nm was read by means of a micro-ELISA plate reader.
Patients
Light Microscopic Immunohistochemistry Frozen (4% paraformaldehyde, OCT-embedded) and paraffin-embedded tissues were used for immunohistochemistry. To prepare tissue sections obtained via the lung biopsy and thoracic surgery, a portion of each lung specimen was placed immediately in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH, 7.4) and fixed for 8 h, after which samples were embedded in paraffin, or they were sequentially washed: for 4 h in 10% sucrose in 0.01 M PBS (pH, 7.4; 4°C), 4 h in 20% sucrose in PBS, and overnight in 30% sucrose in PBS. The washed tissues were then snap-frozen in OCT embedding medium and stored at –80°C for filing at our department. The frozen tissues were cut into 6-µm sections and were used mainly for immunofluorescence and immunoelectron analyses. Formaldehyde-fixed, paraffin-embedded tissues were also prepared according to standard procedures from the frozen samples collected via the biopsy and surgical operation as described above, and were cut into 3-µm sections. These specimens were then deparaffinized and treated with proteinase K (20 µg/ml) for 15 min at room temperature before 8-nitroguanine immunostaining. The paraffin-embedded sections were incubated for 30 min with a biotin blocking system (Dako, Carpinteria, CA), and endogenous peroxidase activity was eliminated by the method of Isobe and colleagues (23). After treatment with normal goat serum, tissues were incubated overnight at 4°C with 10 µg/ml mouse anti–8-nitroguanine monoclonal antibody, and were then incubated with 1 µg/ml biotinylated goat anti-mouse IgG (Zymed, San Francisco, CA) and streptavidin-biotin-HRP (Dako, Glostrup, Denmark) for 1.5 h at room temperature. Bound antibody was visualized after incubation for 10 min in a Coplin jar with 100 ml of Tris-HCl buffer (pH, 7.6) containing 20 mg of diaminobenzidine (DAB) and 17 ml of 30% H2O2. Counterstaining was performed with Mayer's hematoxylin. Nonspecific labeling of primary antibody was evaluated with normal mouse serum. In some studies, frozen (4% paraformaldehyde, OCT-embedded) sections were also used for immunohistochemistry in the same manner as for paraffin-embedded sections. A differently labeled, second antibody, alkaline phosphatase-labeled goat anti-mouse IgG or fluorescein isothiocyanate (FITC)–labeled goat anti-mouse IgG, was used to confirm 8-nitroguanine immunoreactivity. To visualize the reaction for alkaline phosphatase, a section was stained red with a substrate consisting of 0.2 mM naphthol AS-MX phosphate, 1 mM Fast Red TR salt, and 1 mM levamisole (an inhibitor for endogenous alkaline phosphatase) in 0.1 M Tris-HCl buffer (pH, 8.2) for 20 min (Dako APAAP kit; Dako). To visualize fluorescence, specimens were examined with a filter set at 450 to 490 nm for FITC, based on an upright microscope (DMRB; Leica Lasertechnik, Heidelberg, Germany). Images were obtained by use of an Olympus DP70 digital camera and software (Olympus, Tokyo, Japan), with constant camera exposure settings throughout. The specificity of the anti–8-nitroguanine antibody used in this study was confirmed by an enzyme immunoassay (Figure 1), which indicated that the monoclonal anti–8-nitroguanine antibody bound exclusively to 8-nitroguanine and 8-nitroguanosine as well as to 8-nitroxanthine (solid lines in Figure 1). Therefore, to more specifically confirm nitration of guanine rather than xanthine, we used a new clone (1G6) of mouse monoclonal antibody specific for 8-nitroguanosine as illustrated in Figure 1 (broken lines). The 1G6 antibody was applied to the standard indirect immunohistochemistry via the method of Isobe and collleagues, as previously described, with normal goat serum and HRP-labeled goat anti-mouse IgG as a second antibody (Nichirei Biosciences, Inc., Tokyo, Japan) and with DAB for visualization. Each tissue sample was also stained with a series of antibodies for iNOS (NOS2; rabbit polyclonal; NeoMarkers, Fremont, CA), endothelial NOS (eNOS; NOS3; rabbit polyclonal; Sigma Chemical, St. Louis, MO), 8-oxodeoxyguanosine (mouse monoclonal; Japan Institute for the Control of Aging, Fukuroi, Japan), 3-nitrotyrosine (rabbit polyclonal; Sigma), P53 (mouse monoclonal; DO-7; Dako), proliferating cell nuclear antigen (PCNA) (mouse monoclonal; PC10; Dako), and cytokeratin (mouse monoclonal; AE1 + AE3; Dako). Serial sections stained with hematoxylin–eosin were used for the histopathologic analysis.
Immunofluorescence Image Analysis
Double-labeled Immunohistochemical Studies via Confocal Microscopy
Immunoelectron Microscopy
Statistical Analysis
Formation and Localization of 8-Nitroguanine in Lung Tissues from Control Subjects and Patients with IPF/UIP Lung tissues from control subjects of smokers had intact alveolar architecture with infiltration of a few inflammatory cells (Figure 2A). Immunohistochemical analysis of control lungs manifested weak immunoreactivity for 8-nitroguanine in the cytoplasm of bronchial epithelial cells and with alveolar macrophages and lymphocytes (Figure 2B). Cytoplasm of bronchial epithelial cells also demonstrated immunostaining for iNOS (Figure 2D), eNOS (Figure 2E), and 8-oxodeoxyguanosine (Figure 2F), but no immunoreactivity for P53 (Figure 2G), 3-nitrotyrosine (Figure 2H), and PCNA (data not shown) in serial sections. As a negative control, normal mouse serum used as a first antibody showed no positive staining (Figure 2C). Immunohistochemical analysis of control lungs from nonsmokers also showed weak immunoreactivity for 8-nitroguanine, iNOS, and 8-oxodeoxyguanosine in the cytoplasm of bronchial epithelial cells (Figures 3B–3D). However, there was no significant difference between nonsmokers and smokers among normal control subjects in immunoreactivity of 8-nitroguanine as detected by the immunofluorescence staining (Figures 2I, 2J, 3E, and 3F), and as determined by the quantitative immunofluorescence image analysis (Table 2).
In lungs of smokers with IPF/UIP, fibrotic zones showed a temporal heterogeneity, with dense acellular collagen, scattered fibroblastic foci, and intervening, nearly normal alveoli. Most fibrotic zones had honeycombing with complete destruction of the normal architecture (Figures 4A and 4B). In addition to immunostaining in bronchial epithelial cells, strong 8-nitroguanine staining occurred in the cytoplasm of metaplastic regenerated epithelial cells overlying the dense fibrotic lesions (Figure 4C), which was further confirmed by using alkaline phosphatase- and FITC-labeled second antibodies (Figures 4D and 5; Table 2). 8-Nitroguanine immunostaining was also detected in some inflammatory cells, such as macrophages and lymphocytes, and in endothelial cells around the lesions (Figures 4C, 4D, and 4J). Strong immunoreactivity reactions for iNOS (Figure 4E), eNOS (Figure 4F), and 8-oxodeoxyguanosine (Figure 4G) were found in the cytoplasm of these metaplastic epithelial cells and in alveolar macrophages and lymphocytes. Weak staining for 3-nitrotyrosine (Figure 4H), P53 (Figure 4I), and PCNA (data not shown) were seen in these metaplastic cells. Similar to the 8-nitroguanine immunostaining (Figures 4C and 4D), strong 8-nitroguanosine staining with 1G6 antibody (Figure 4J) was also found in the cytoplasm of metaplastic regenerated epithelial cells. It is also important that, as demonstrated in Figure 5 and Table 2, greater guanine nitration was evident in the epithelial lesion of IPF/UIP lungs than in control lungs.
Furthermore, in two cases of nonsmoking IPF/UIP, remarkable 8-nitroguanine staining was observed in the cytoplasm of metaplastic regenerated epithelial cells overlying the dense fibrotic lesions (data not shown), to the same extent as that of IPF/UIP with smoking, as evidenced by the quantitative immunofluorescence analysis (Table 2).
Tissue and Intracellular Localization of 8-Nitroguanine in IPF/UIP by Means of Double-labeled Immunohistochemical Analyses
It is intriguing that the fluorescence for 8-nitroguanine (Figure 7E, green) overlapped with that of mitochondria (Figure 7F, red), with the colocalization in metaplastic epithelial cells being apparent in Figure 7G (yellow). Figure 7H shows the same image as Figures 7E–7G but was obtained with Nomarski optics.
Immunoelectron Microscopic Analysis of Intracellular Formation of 8-Nitroguanine in the Lung in IPF/UIP
Formation of 8-Nitroguanine in Squamous Cell Carcinoma In lungs of patients with squamous cell carcinoma, malignant squamoid epithelial cells proliferated throughout the lung, which showed remodeled tissue with fibrotic and cystic changes (Figures 9A and 9B). Marked 8-nitroguanine immunostaining was observed in both nuclei and cytoplasm of these malignant cells (Figures 9C and 10A), although its level was lower than that of epithelial lesions in IPF/UIP, as indicated by the quantitative immunofluorescence analysis (Table 2). This intense immunostaining was also seen with alkaline phosphatase-labeled second antibody (Figure 9D). However, both nuclei and cytoplasm of these malignant cells manifested strong immunoreactivity for iNOS and 8-oxodeoxyguanosine (Figures 10B and 10D). Strong eNOS immunoreactivity was noted in the cytoplasm (Figure 10C), and marked P53 and PCNA immunoreactivity was observed in nuclei of the malignant cells (Figures 10E and 10F).
In this study, we verified for the first time that guanine nitration occurs in metaplastic epithelium of fibrotic lesions in IPF/UIP and in malignant epithelium in squamous cell carcinoma of the lung. We also clearly demonstrated much more extensive immunostaining for 8-nitroguanine in lung tissues from patients with IPF/UIP and squamous cell carcinoma than those from control smokers and nonsmokers. Saleh and colleagues previously documented, in lungs of patients with IPF/UIP, strong expression of iNOS, eNOS, and 3-nitrotyrosine, a biomarker of RNS, in macrophages, neutrophils, and alveolar epithelial cells (25). Also, Kuwano and coworkers demonstrated increased 8-oxodeoxyguanosine formation in lung epithelial cells from patients with IFP/UIP (8). We obtained similar results in this immunohistochemical analysis for IPF/UIP. In addition, we found evidence of marked 8-nitroguanine immunoreactivity in the cytoplasm of metaplastic epithelial cells in IPF/UIP. 8-Nitroguanine is formed in DNA and RNA as the result of nitration by RNS, such as peroxynitrite (12–17), which is produced by sustained generation of ROS and NO. Therefore, although the exact mechanism of ROS and RNS generation during IPF/UIP and lung cancer is not fully understood, increased NOS expression could be a major contributor to 8-nitroguanine formation in bronchial epithelial cells in these pulmonary diseases. We recently identified elevated levels of urinary excretion of 8-nitroguanine in smokers, compared with those in nonsmoking control subjects (17). Our present analysis, however, showed no significant enhancing effect of smoking on 8-nitroguanine formation in airway epithelial cells among control nonsmokers and smokers, except that there is some trend of increased immunofluorescence staining in smokers with IPF/UIP compared with that in nonsmokers with IPF/UIP (p > 0.05; Table 2). This inconsistent result may be due to distinct biological materials we examined (urine vs. lung tissues), or possibly because of completely different analytic approaches used in these studies (immunochemistry vs. high-performance liquid chromatography–electrochemical detection coupled with immunoaffinity purification). We nevertheless interpreted that smoking may not be a major inducer for 8-nitroguanosine formation in airway epithelial cells. More important, much extensive guanine nitration was observed in IPF/UIP than in squamous cell carcinoma of the lung (Table 2). This may suggest higher levels of nitrative stress occurring in the metaplastic epithelial cells during IPF/UIP than in lung cancer cells. Because 8-oxodeoxyguanosine in oxidatively modified DNA is believed to cause G:C to T:A transversion, which may promote carcinogenesis (26, 27), excessive production of ROS and resulting oxidative stress may be mutagenic and may contribute to carcinogenesis in during IPF/UIP. 8-Nitroguanine undergoes spontaneous depurination, which leads to the presence of apurinic sites in DNA (28) and thereby to G to T transversions (29). A recent study by Suzuki and colleagues indicated that 8-nitroguanine formed in DNA caused G to T transversion without depurination of the nitrated nucleotide (30). It is important to note that a weak but appreciable level of P53 immunoreactivity was detected in nuclei of metaplastic epithelial cells in IPF/UIP. Intense immunostaining of 8-nitroguanine together with P53 induction in metaplastic cells suggests that nitrative nucleic acid damage occurring in IPF tissues may contribute to the accumulation of P53. It has been reported that elevated expression of P53 in IPF tissues is associated with increased mutations of the p53 gene (31–33). The p53 tumor suppressor gene blocks replication of damaged DNA by arresting the cell cycle in the G1 phase and preventing cells from entering the S phase (34). Thus, impairment of P53 functions, possibly by its gene mutation, is an important step for multistage carcinogenesis, and in fact, the mutation of p53 gene was observed at the stage of mild dysplasia of bronchial epithelium (35). Taken together, guanine nitration, in addition to 8-oxodeoxyguanosine, may contribute to carcinogenesis during IPF/UIP by means of mutation of a gene such as p53 in metaplastic cells. We found via evaluation of the immunoelectron microscopic and double-labeled confocal fluorescence images that intracellular localization of 8-nitroguanine is near mitochondria at the circumference of the nucleus in the metaplastic epithelial cells. Mitochondria consume up to 90% of molecular oxygen used by the body, and 1 to 2% of the molecular oxygen metabolized by mitochondria is converted to ROS. NO and its derivatives (e.g., RNS) inhibit mitochondrial respiration and either stimulate or inhibit cell death depending on surrounding conditions (36). Therefore, mitochondria appear to be susceptible to oxidative and nitrative damage. Thus, our finding of mitochondria-related localization of 8-nitroguanine suggests that ROS and RNS may affect mitochondrial functions via DNA damage (37, 38), which would lead to impaired electron transport and additional damage to epithelial cells. Increased levels of exhaled NO associated with up-regulation of iNOS have been demonstrated in patients with primary lung cancer (39). Inoue and coworkers also reported that patients with lung cancer have increased 8-oxodeoxyguanosine levels in DNA from normal noncancerous areas in the lung periphery (10). Consistent with these earlier reports, our results showed appreciable immunoreactivity for iNOS, eNOS, 3-nitrotyrosine, and 8-oxodeoxyguanosine in malignant epithelial cells in squamous cell carcinoma of the lung. Our immunochemical assays also revealed 8-nitroguanine formation not only in the cytoplasm but also in the nuclei, together with intense immunostaining of P53 and PCNA, in malignant epithelial cells in squamous cell carcinoma. Because strong nuclear localization of 8-nitroguanine in cells was not observed in lungs from patients with IPF/UIP and in control lungs, this positive nuclear immunostaining seems to be a characteristic of squamous cell carcinoma. In addition, carcinoma cells may lack the appropriate repair systems for nitrative DNA damage in nuclei. Together, these results support our proposal that excessive generation of ROS and RNS may be involved in progression of lung cancer via induction of nitrative and oxidative DNA injury. In conclusion, our current work clearly indicates participation of not only oxidative stress but also of nitrative stress caused by 8-nitroguanine formation in epithelial damage in lungs of smokers and those of patients with IPF/UIP, which may increase the risk of lung cancer development in IPF/UIP.
The authors thank Ms. Judith B. Gandy for her excellent editing of the manuscript.
Supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), and the Ministry of Health, Labor, and Welfare of Japan. Originally Published in Press as DOI: 10.1164/rccm.200510-1580OC on June 1, 2006 Conflict Of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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