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Involvement of nitric oxide in acute lung inflammation induced by cigarette smoke in the mouse
Nitric Oxide 20 (2009) 175–181
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Involvement of nitric oxide in acute lung inflammation induced by cigarette smoke in the mouse Samuel Santos Valença a, Wagner Alves Pimenta a, Carlos Romualdo Rueff-Barroso a, Thiago Santos Ferreira a, Ângela Castro Resende b, Roberto Soares de Moura b, Luís Cristóvão Porto a,* a b
Laboratory of Tissue Repair, Department of Histology and Embryology, Rio de Janeiro State University, Brazil Department of Pharmacology, Institute of Biology Roberto Alcantara Gomes, Rio de Janeiro State University, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 14 August 2008 Revised 10 November 2008 Available online 6 December 2008 Keywords: Nitric oxide Cigarette smoke Inflammation Oxidative stress Mouse
a b s t r a c t Short-term exposure to cigarette smoke (CS) leads to acute lung inflammation (ALI) by disturbing oxidant/ antioxidant balance. Both CS exposure and lung inflammation are important risk factors in the pathogenesis of chronic obstructive pulmonary disease. Nitric oxide (NO) is an oxidant both present in CS and produced in the inflammatory response, but its role in the effects of CS exposure is unclear. Our aim was to study involvement of NO in a model of CS exposure. Groups of mice (male C57BL/6) exposed to CS (six cigarettes per day over five days) were simultaneously subjected to treatment with vehicle (CS), 60 mg/kg/day x-nitro-L-arginine methyl ester (CS + L-NAME), 20 mg/kg/day nitroglycerine (CS + NTG), or 120 mg/kg/day L-arginine (CS + L-arg). Bronchoalveolar lavage fluid was then aspirated to perform cell counts, and malondialdehyde (MDA), nitrite, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels were measured in lung homogenates. Macrophage and neutrophil counts were increased in the CS (p < 0.001) and CS + L-NAME groups (p < 0.05 and p < 0.01, respectively); the CS + NTG and CS + L-arg groups showed no differences from the control group. MDA was increased in the CS (p < 0.05) and CS + L-NAME (p < 0.01) groups when compared to the control group. Nitrite levels were decreased in the CS and CS + L-NAME groups (p < 0.001) and increased in the CS + NTG (p < 0.001) and CS + L-arg (p < 0.01) groups when compared to the control. CAT, SOD and GPx activities in the CS and CS + L-NAME groups were all significantly increased compared to the control group. Our results suggest that administration of NO donors or substrates may be a useful therapy in the treatment of ALI caused by CS. Ó 2008 Elsevier Inc. All rights reserved.
Chronic obstructive pulmonary disease (COPD) is a rapidly increasing global health problem, predicted to be the third leading cause of death in developed countries by 2020 [1]. COPD is characterized by slowly progressive and largely irreversible airflow limitation due to chronic bronchitis and/or emphysema, associated with an abnormal inflammatory response of the lungs [2]. Cigarette smoke (CS) has been identified as the most important risk factor for development of COPD [3]. Although the prevalence of smoking has fallen in developed countries, it continues to increase in many low- and middle-income countries, especially among young people and women [4]. An estimated 4.9 million (8.8% of the global total) deaths in 2000 were attributable to use of tobacco, which is 45% higher than the figure 1990. The increase was greatest in developing countries, which now account for 50% of the global mortality and 56% of the disease burden attributable to tobacco [5]. Men were almost four times more likely than women to be
* Corresponding author. Address: Departamento de Histologia e Embriologia, IBRAG, UERJ, Avenida Professor Manuel de Abreu, 444, 3° andar, Maracanã, 20.550170 Rio de Janeiro, RJ, Brazil. Fax: +55 21 2587 6511. E-mail address: [email protected] (L.C. Porto). 1089-8603/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2008.11.003
smokers. The prevalence of smoking among men was highest in the Western Pacific region. The differential in gender-specific smoking prevalence was narrowest in the Americas and Europe; it was widest in South-East Asia and the Western Pacific region. China’s growing cigarette consumption since 1975 has had a major bearing on the rise in global per capita consumption [6]. Although ALI caused by CS exposure does not model all aspects of COPD pathogenesis, the two processes do share certain hallmarks, including accumulation of inflammatory cells such as alveolar macrophages and neutrophils, and lung oxidative stress [7]. Oxidants and free radicals in the gas phase of CS (alkyl, peroxyl, nitric oxide and superoxide anion) and the tar phase (semiquinone) [8] can stimulate alveolar macrophages to produce reactive oxygen species (ROS) and release cytokines involved in migration of inflammatory cells into the lungs. Alveolar macrophages and neutrophils can generate ROS [9], resulting in an imbalance between oxidants and antioxidants such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) [10]. CS exposure is also associated with increased levels of malondialdehyde (MDA), a marker of oxidative stress [11]. This suggests the existence of an oxidative stress mechanism induced by CS in animals and humans.
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Nitric oxide (NO) is an oxidant both present in CS and synthesized de novo in cells from L-arginine by various isoforms of NO synthase (NOS), including constitutive neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3) [12]. NO can readily pass through the plasma membrane to exert its action on cells. It is involved in vessel dilatation, inhibition of platelet aggregation, and host defense [13]. Organic nitrates (RONO2) such as nitroglycerine and isosorbide dinitrate have been used for over a century in the treatment of various cardiovascular diseases [14–16]. It has been generally accepted that nitroglycerine undergoes biotransformation to NO, which then binds to and activates guanylate cyclase, catalyzing production of cyclic GMP [17]. In contrast, x-nitro-Larginine 4 methyl ester (L-NAME) is a NOS inhibitor which blocks the production of NO by both iNOS and constitutive NOS [18]. NO is produced by a variety of cells within the respiratory tract, including not only inflammatory but also epithelial cells [19]. It is generally believed that NO produced by iNOS is associated with pro-inflammatory and damaging effects [20]. However, L-arginine is an obligatory substrate for all NOS isoforms. Indeed, inhaled or orally administered L-arginine increases the production of NO in human airways, suggesting that availability of the cationic amino acid is rate limiting for NO production [21]. The purpose of the present study was to analyze the involvement of NO on inflammatory cell migration, nitrite values, lipid peroxidation and antioxidant enzyme activities (CAT, SOD and GPx) in mouse lung exposed to CS. For this, we used nitroglycerine (a donor of NO), L-arginine (a substrate for NO formation), and L-NAME (an inhibitor of NO formation) in combination with CS exposure in mice.
Experimental procedures Reagents Thiobarbituric acid, adrenaline, NADPH, trichloroacetic acid, nitroglycerine, x-nitro-L-arginine methyl ester, L-arginine, sulfanilamide, phosphoric acid, naphthylenediamide dihydrochloride, sodium nitrite and perchloric acid were purchased from Sigma Chemical (St. Louis, MO, USA). Pallflex filters were purchased from Imprint (São Paulo, Brazil). Diff-Quik was purchased from Baxter Dade AG (Dudingen, Switzerland). Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). Hydrogen peroxide was purchased from Vetec (Duque de Caxias, Brazil). Animals C57BL/6 male mice (8 weeks old—20–24 g) were purchased from the Veterinary Institute—Fluminense Federal University (Niterói, Brazil). Mice were housed (5 per cage) in a controlled environment room with a 12-h light/12-h dark cycle (lights on at 6 pm) and ambient temperature of 25 ± 2 °C (humidity 80%). The animals had free access to water and food. Acclimatization was performed during the two weeks before the experimental procedures. CS exposure C57BL/6 male mice (n = 40) were exposed to six commercial full-flavor filtered Virginia cigarettes (10 mg of tar, 0.9 mg of nicotine and 10 mg of carbon monoxide) per day for 5 days by using a smoking chamber described previously [11,22]. Briefly, each group of mice was placed in the inhalation chamber (40 cm long, 30 cm wide and 25 cm high), inside an exhaustion chapel. A cigarette was coupled to a plastic 60 mL syringe so
that puffs could be drawn in and subsequently expelled into the exposure chamber. We aspirated 1 L of smoke from one cigarette with this syringe (20 puffs of 50 mL) and immediately injected the puff into the chamber. The 10 animals of each group were maintained in this smoke-air condition for 6 min (3%), and the inhalation chamber was opened, by removing its cover, and the smoke evacuated for 1 min by exhaustion of the chapel. This cigarette exposure was repeated two times (2 6 min) with intervals of 1 min (exhaustion). We repeated this procedure three times per day (morning, noon and afternoon) resulting in 36 min of CS exposure to six cigarettes. Each cigarette smoked produced 300 mg/m3 of total particulate matter in the chamber (measured by weighing material collected on Pallflex filters) [22]. Carboxyhemoglobin (COHb) concentration was previously monitored in mice using the same experimental protocol and COHb was not toxic [23]. Groups (n = 10 each) were defined as mice exposed to CS and treated with vehicle (CS), mice exposed to CS and treated with 20 mg/kg/day of nitroglycerine (CS + NTG), mice exposed to CS and treated with 120 mg/kg/ day of L-arginine (CS + L-arg) and mice exposed to CS and treated with 60 mg/kg/day of L-NAME (CS + L-NAME). All treatments were performed by oral gavages once per day (simultaneously with CS exposure) and drugs were mixed with saline (vehicle). Mice exposed to ambient air were used as the control group (n = 10) and were subjected to oral gavages with vehicle. The doses of nitroglycerine [24], L-arginine [25] and L-NAME [26] were based on previous data from the literature with modification of the administration via. A separate group of C57BL/6 male mice (n = 5 for each group) were exposed to ambient air during five days by using the same protocol described above and simultaneously treated with vehicle (control group), 20 mg/kg/day of nitroglycerine (NTG), 120 mg/kg/day of L-arginine (L-arg) and 60 mg/kg/day of L-NAME (L-NAME). All procedures were carried out in accordance with conventional guidelines for experimentation with animals, and the local animal care and use committee approved the experimental protocol. Bronchoalveolar lavage (BAL) Twenty-four hours after the last exposure to ambient air or CS the animals were killed by cervical displacement. Lung airspaces were washed with buffered saline solution (500 lL) three times (final volume 1.2–1.5 mL). The BAL fluid was withdrawn and stored on ice. Total mononuclear and polymorphonuclear cell numbers were determined in a Zi Coulter counter (Beckman Coulter, Carlsbad, CA, USA). Differential cell counts were performed on cytospin preparations (Shandon, Waltham, MA, USA) stained with Diff-Quik. At least 200 cells per BAL sample were counted using standard morphologic criteria [11,22,23]. After BAL, lungs were immediately removed and homogenized on ice with 10% (w/v) PBS (pH 7.3) using an Ultra-TurraxÒ T 8 homogenizer (Toronto, Canada) and then centrifuged at 3000g for 5 min. Supernatants were kept at 20 °C for analysis of nitrite values, lipid peroxidation and antioxidant enzyme activities (CAT, SOD and GPx). Malondialdehyde assay As an index of lipid peroxidation we used the thiobarbituric acid reactive substances (TBARS) method for analyzing malondialdehyde products during an acid-heating reaction as previously described by Draper and co-workers [27]. Briefly, samples from lung homogenates were mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid; the samples were then heated in a boiling water bath for 30 min. TBARS levels were determined by absorbance at 532 nm and expressed as malondialdehyde equivalents (nm/mg protein).
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Nitrite assay Nitrite levels in lung homogenates were determined by a method based on the Griess reaction [28]. A total of 100 lL of sample was mixed with 100 lL of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylenediamide dihydrochloride in water) and incubated at room temperature for 10 min followed by measuring the absorbance in a plate reader at 550 nm (Bio-Rad Microplate Reader model 680, CA, USA). Nitrite concentrations in the samples were determined from a standard curve generated by different concentrations of sodium nitrite. Catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities CAT, SOD and GPx activities were determined in lung homogenates. CAT activity was measured by the rate of decrease in hydrogen peroxide concentration at 240 nm [29]. SOD activity was assayed by measuring inhibition of adrenaline auto-oxidation as absorbance at 480 nm [30]. GPx activity was measured by monitoring the oxidation of NADPH at 340 nm in the presence of H2O2 [31]. The total protein content in the samples from lung homogenates was determined by the method of Bradford [32]. Statistical analysis Data are expressed as means ± SEM. For comparison between control, CS, CS + NTG, CS + L-arg and CS + L-NAME, data concerning BAL cells, MDA, nitrite, CAT, SOD and GPx were analyzed with one-way ANOVA followed by the Student–Newman–Keuls post hoc test (p < 0.05). GraphPad InStat software was used to perform the statistical analyses (GraphPad InStat version 3.00 for Windows 95, GraphPad Software Inc.; San Diego, CA, USA).
Results
Fig. 1. (a) Effect of L-NAME, L-arginine and nitroglycerine on the migration of alveolar macrophages and neutrophils into the lungs of mice exposed to CS. Data are shown as means ± SEM from 10 mice for each group. *Compared to control group. #Compared to CS group. *p < 0.05, **p < 0.01, ***p < 0.001. #p < 0.05, ##p < 0.01, ### p < 0.001. (b) Effect of L-NAME, L-arginine and nitroglycerine on the migration of alveolar macrophages and neutrophils into the lungs of mice exposed to ambient air. Data are shown as means ± SEM from five mice for each group. *Compared to control group. #Compared to L-NAME group. #p < 0.05. ##p < 0.01, ###p < 0.001.
Inflammatory cell migration into the lungs C57BL/6 mice were exposed to six cigarettes a day for five consecutive days. BAL cell counts revealed a marked migration of inflammatory cells into the airway lumen for some of the experimental groups (Fig. 1a). The numbers of alveolar macrophages and neutrophils were increased in the CS group in comparison to the control group (p < 0.001). They were also significantly higher in the CS + L-NAME group compared to the control group (p < 0.05 and p < 0.01, respectively); however, the numbers of neutrophils remained significantly lower compared to the CS group (p < 0.01). Both the CS + NTG and CS + L-arg groups showed a marked reduction in the numbers of alveolar macrophages (p < 0.01 and p < 0.05, respectively) and neutrophils (p < 0.001) compared to the CS group. No statistical differences were observed among the CS + NTG, CS + L-arg and control groups. A separate group of C57BL/6 mice was exposed to ambient air for five consecutive days and treated with vehicle or with nitroglycerine (NTG), L-arginine and L-NAME (L-NAME) (Fig. 1b). The number of alveolar macrophages was reduced in the NTG group in comparison to the control group (p < 0.05) and the L-NAME group (p < 0.001). The number of alveolar macrophages was also reduced in the L-arg group in comparison to the L-NAME group (p < 0.05). The number of neutrophils was reduced in the NTG group (p < 0.01) and in the L-arg group (p < 0.05) in comparison to the L-NAME group.
Malondialdehyde content Fig. 2a shows the MDA equivalents in mouse lungs of the different experimental groups. We observed an increase in the levels of MDA in the CS and CS + L-NAME groups compared to the control group (p < 0.05 and p < 0.01, respectively). On the other hand, a decrease in MDA in the CS + NTG group was noted compared to the CS group (p < 0.05). MDA levels in the CS + NTG and CS + L-arg groups were similar to that of the control group. MDA levels in the CS and CS + L-NAME groups were similar. A separate group of C57BL/6 mice was exposed to ambient air for five consecutive days and treated with vehicle or with nitroglycerine (NTG), L-arginine or L-NAME (L-NAME) (Fig. 2b). We observed an increase in the level of MDA in the L-NAME group compared to the control group (p < 0.05). MDA levels in the NTG and L-arg groups were reduced in comparison with the L-NAME group (p < 0.05). MDA levels in the NTG and L-arg groups were similar to that of the control group. Nitrite content The results of evaluation of nitrite levels in mouse lungs are shown in Fig. 3. Mice in the CS group showed a lower level of nitrite compared to the control group (p < 0.001). Mice in the
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Fig. 3. Effect of L-NAME, L-arginine and nitroglycerine on the nitrite content of lung homogenates of mice exposed to CS. Data are shown as means ± SEM from eight mice for each group. *Compared to control group. #Compared to CS group. **p < 0.01, *** p < 0.001, ###p < 0.001.
Fig. 2. (a) Effect of L-NAME, L-arginine and nitroglycerine on the malondialdehyde (MDA) content of lung homogenates of mice exposed to CS. Data are shown as means ± SEM from eight mice for each group. *Compared to control group. # Compared to CS group. *p < 0.05, **p < 0.01, #p < 0.05. (b) Effect of L-NAME, Larginine and nitroglycerine on the malondialdehyde (MDA) content of lung homogenates of mice exposed to ambient air. Data are shown as means ± SEM from five mice for each group. *Compared to control group. #Compared to L-NAME group. *p < 0.05, #p < 0.05.
CS + L-NAME group showed the lowest level of nitrite formation in comparison to the CS and control groups (p < 0.001). In contrast, the levels of nitrite were increased in the CS + NTG and CS + L-arg groups compared to the CS (p < 0.001 and p < 0.001) and control groups (p < 0.001 and p < 0.01, respectively). CAT, SOD and GPx activities Fig. 4 shows CAT activity in mouse lungs. CAT activity was increased in the CS and CS + L-NAME groups compared to the control group (p < 0.01 and p < 0.001, respectively). CAT activity in the CS + L-NAME group was elevated compared to that of the CS group (p < 0.01). CAT activity in the CS + L-arg group was reduced in relation to that of the CS group (p < 0.01). Both the CS + NTG and CS + L-arg groups had CAT activity similar to that of the control group. Fig. 5 shows the SOD activity in each group based on measurement of inhibition of adrenaline auto-oxidation. An increase in SOD activity was observed in the CS and CS + L-NAME groups compared to the control group (p < 0.05 and p < 0.001). On the other hand, a reduction in SOD activity was noted in the CS + NTG and CS + L-arg groups in comparison to the CS (p < 0.001) and control groups (p < 0.05). Fig. 6 shows the GPx activity in mouse lungs based on measurement of the oxidation of NADPH. The CS and CS + L-NAME groups showed elevated levels of GPx activity
Fig. 4. Effect of L-NAME, L-arginine and nitroglycerine on the catalase (CAT) activity in lung homogenates of mice exposed to CS. Data are shown as means ± SEM from nine mice for each group. *Compared to control group. #Compared to CS group. ** p < 0.01, ***p < 0.001, ##p < 0.01.
compared to the control group (p < 0.01). The GPx activity of the CS + L-arg group was significantly lower than that of the CS group (p < 0.05). The GPx activities of both the CS + NTG and CS + L-arg groups were similar to that of the control group. Discussion Nitric oxide is a signaling molecule responsible for several diverse physiological and pathophysiological processes, and until now the prevailing hypothesis about NO has been that it contributes to toxicant-induced lung inflammation and injury [19,20,33,34]. The present study suggests a different role for NO in ALI induced by CS. We showed here that administration of an NO donor (by nitroglycerine) or NO substrate (by L-arginine) resulted in a reduction in the migration of inflammatory cells, lower oxidative stress and normalized antioxidant enzyme activities. The
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Fig. 5. Effect of L-NAME, L-arginine and nitroglycerine on the superoxide dismutase (SOD) activity in lung homogenates of mice exposed to CS. Data are shown as means ± SEM from nine mice for each group. *Compared to control group. # Compared to CS group. *p < 0.05, ***p < 0.001. ###p < 0.001.
Fig. 6. Effect of L-NAME, L-arginine and nitroglycerine on the glutathione peroxidase (GPx) activity in lung homogenates of mice exposed to CS. Data are shown as means ± SEM from nine mice for each group. *Compared to control group. # Compared to CS group. **p < 0.01, #p < 0.05.
action of NO here was different from its actions elicited by other lung inflammatory stimuli such as lipopolysaccharide or bleomycin [35,36]. Although NO has been described in several studies to be a mediator of the inflammatory response, for example by stimulating production of inflammatory cytokines and peroxynitrite (ONOO ) [37–39], beneficial effects of NO in the lungs have also been reported [40–44]. We found a reduction in both alveolar macrophages and neutrophils in the CS + NTG and CS + L-arg groups when compared to the control group. The specific action of NO on inflammatory cells is unknown, but we suggest that nitroglycerine or L-arginine may reduce the expression of endothelial cell adhesion molecules and consequently inflammatory cell migration to the alveolar space. Furthermore, NO was reported to be an important molecule for blocking the endothelium–leukocyte interaction [45–48]. Apoptosis of inflammatory cells prior to the fifth day of CS exposure through NO-activation of death domains is another hypothesis to
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explain reduced alveolar macrophages and neutrophils in the CS + NTG and CS + L-arg groups, due to involvement of NO in apoptosis [49–51]. The use of the NO-inhibitor L-NAME resulted in a reduction in the level of neutrophils compared with the CS group. Therefore, although CS itself decreased nitrite formation, further decreasing it with L-NAME actually reduced the inflammatory response to CS (in terms of neutrophil levels). Certainly, blocking endogenous NO production (with L-NAME) did not have as favorable a result as augmenting NO production (with nitroglycerine or L-Arg) in so far as reducing inflammation, but arguably both can work based on these results. Administration of just L-NAME to mice exposed to ambient air did not affect the levels of migration of alveolar macrophages and neutrophils into the lungs. However, treatment with nitroglycerine or L-arginine of mice exposed to ambient air reduced the numbers of alveolar macrophages and neutrophils when compared to the control group. These data help to explain the positive effects of NO donor or substrate in decreasing the inflammatory response by lowering cell recruitment into the lungs. Lipid peroxidation is an important marker of oxidative stress in the lung, and we showed in a previous study the association between ALI caused by CS and lipid peroxidation as analyzed by MDA [11,22]. We found MDA levels to be increased in CS-exposed mice in comparison with control animals. Treatment with nitroglycerine or L-arginine reduced the amount of MDA. This result is similar to that of Machova and co-workers [52] who showed that NO treatment inhibited lipid peroxidation in vitro and protected against cellular damage and cytotoxicity. NO has also been described as a scavenger of other far more toxic radicals, and therefore, an enhancer of defense mechanisms [53]. Consequently, lipid peroxidation was observed only in the CS + L-NAME and CS groups, not in the CS + NTG and CS + L-arg groups. In addition, administration of L-NAME to mice exposed to ambient air exposed led to increased MDA levels in lung homogenates, a result that suggests the occurrence of oxidative stress in this group when compared to the control group. Nitroglycerine and L-arginine increased nitrite (NO2 ) production in lung tissue, whereas administration of CS only or L-NAME did not, indicating reduced production of NO in the two latter groups. However, nitrite formation during CS exposure is unclear. A reduction in nitrite levels was reported by Hoyt and co-workers [54] together with a reduction in NO synthase (iNOS) mRNA and protein expression. In contrast, Wright and co-workers reported an increase in iNOS with CS exposure [55]. However, L-NAME, being an analogue of L-arginine, significantly lowers NO concentrations as it interacts with NOS, but this interaction does not result in NO production [53]. The lower nitrite levels that we observed in the CS + NTG and CS + L-arg groups confirm the efficacy of treatment with NO donor or substrate and led us to hypothesize the involvement of NO in blunting the inflammatory response. The increase in CAT, SOD and GPx activities in the lungs of CS mice reflects indirectly the generation of free radicals. The compensatory mechanism of lung cells in an acute model of CS exposure has been reported by us previously [23]. Interestingly, the lungs of mice exposed to long-term CS showed reduced levels of CAT, SOD and GPx activity (data not shown). We observed variations in CAT, SOD and GPx activities in the CS + NTG and CS + Larg groups, and in general these values were similar to those of the control group. Thus, the CS + NTG and CS + L-arg groups appeared to regulate the CAT, SOD and GPx activities to some degree. However, the highest CAT, SOD and GPx activities were found in the CS + L-NAME group. If the increase of CAT, SOD and GPx activities in CS-exposed animals reflects a disturbance of the balance between oxidants and antioxidants, then L-NAME enhanced this imbalance. We suggest that CS and L-NAME both disturb the balance between oxidants and antioxidants, favoring the oxidant side.
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In summary, we showed that NO supplementation by administration of nitroglycerine or L-arginine was able to reduce both CS-induced ALI and oxidative stress, and that it regulated CAT, SOD and GPx activities. The NO inhibitor L-NAME maintained the inflammatory status in the lungs of CS-exposed mice, contributed to the oxidative stress there, and induced a major imbalance between oxidants and antioxidants by maintaining high CAT, SOD and GPx activities, favoring the oxidant side. Lung inflammatory status, oxidative stress, and CAT, SOD and GPx activities from CS mice were to some degree similar to those of CS + L-NAME mice. Supplementation with NO may be a good alternative in the treatment of ALI caused by CS and in the prevention of COPD, diseases which involve oxidative stress in their pathogenesis. Acknowledgments This work was conducted with grants from National Council of Scientific and Technological Development (CNPq) and Rio de Janeiro State Research Agency (FAPERJ). References [1] D.A. Groneberg, K.F. Chung, Models of chronic obstructive pulmonary disease, Respir. Res. 5 (2004) 18. [2] D.E. O’Donnell, C.M. Parker, COPD exacerbations. 3. Pathophysiology, Thorax 61 (2006) 354–361. [3] S. Molet, C. Belleguic, H. Lena, N. Germain, C.P. Bertrand, S.D. Shapiro, J.M. Planquois, P. Delaval, V. Lagente, Increase in macrophage elastase (MMP-12) in lungs from patients with chronic obstructive pulmonary disease, Inflamm. Res. 54 (2005) 31–36. [4] M.A. Corrao, G.E. Guindon, V. Cokkinides, N. Sharma, Building the evidence base for global tobacco control, Bull. World Health Organ. 78 (2000) 884–890. [5] Y. Lacasse, F. Maltais, R.S. Goldstein, Smoking cessation in pulmonary rehabilitation: goal or prerequisite?, J Cardiopulm. Rehabil. 22 (2002) 148– 153. [6] K. Shibuya, C. Ciecierski, E. Guindon, D.W. Bettcher, D.B. Evans, C.J. Murray, WHO framework convention on tobacco control: development of an evidence based global public health treaty, BMJ 327 (2003) 154–157. [7] A. Churg, R.D. Wang, H. Tai, X. Wang, C. Xie, J. Dai, S.D. Shapiro, J.L. Wright, Macrophage metalloelastase mediates acute cigarette smoke-induced inflammation via tumor necrosis factor-alpha release, Am. J. Respir. Crit. Care Med. 167 (2003) 1083–1089. [8] W. MacNee, Oxidants/antioxidants and COPD, Chest 117 (2000) 303S–317S. [9] W. MacNee, I. Rahman, Is oxidative stress central to the pathogenesis of chronic obstructive pulmonary disease?, Trends Mol Med. 7 (2001) 55–62. [10] I. Rahman, I.M. Adcock, Oxidative stress and redox regulation of lung inflammation in COPD, Eur. Respir. J. 28 (2006) 219–242. [11] F. Silva Bezerra, S.S. Valenca, M. Lanzetti, W.A. Pimenta, P. Castro, V.L. Goncalves Koatz, L.C. Porto, Alpha-tocopherol and ascorbic acid supplementation reduced acute lung inflammatory response by cigarette smoke in mouse, Nutrition 22 (2006) 1192–1201. [12] C.M. Prado, E.A. Leick-Maldonado, L. Yano, A.S. Leme, V.L. Capelozzi, M.A. Martins, I.F. Tiberio, Effects of nitric oxide synthases in chronic allergic airway inflammation and remodeling, Am. J. Respir. Cell Mol. Biol. 35 (2006) 457–465. [13] L. Fakhrzadeh, J.D. Laskin, C.R. Gardner, D.L. Laskin, Superoxide dismutaseoverexpressing mice are resistant to ozone-induced tissue injury and increases in nitric oxide and tumor necrosis factor-alpha, Am. J. Respir. Cell Mol. Biol. 30 (2004) 280–287. [14] M.C. Gupta, M.M. Singh, A.K. Srivastava, R. Singh, V. Prakash, Sublingual isosorbide dinitrate, topical isosorbide dinitrate and topical nitroglycerine in salvaging ischaemic myocardium, J. Assoc. Physicians India 32 (1984) 1023– 1026. [15] K.C. Ferdinand, Isosorbide dinitrate and hydralazine hydrochloride: a review of efficacy and safety, Expert Rev. Cardiovasc. Ther. 3 (2005) 993–1001. [16] G. Yetik-Anacak, J.D. Catravas, Nitric oxide and the endothelium: history and impact on cardiovascular disease, Vascul. Pharmacol. 45 (2006) 268–276. [17] R.M. Gill, J.C. Braz, N. Jin, G.J. Etgen, W. Shen, Restoration of impaired endothelium-dependent coronary vasodilation in failing heart: role of eNOS phosphorylation and CGMP/cGK-I signaling, Am. J. Physiol. Heart Circ. Physiol. 292 (2007) H2782–H2790. [18] B. Jover, A. Mimran, Nitric oxide inhibition and renal alterations, J. Cardiovasc. Pharmacol. 38 (Suppl. 2) (2001) S65–S70. [19] A. van der Vliet, J.P. Eiserich, C.E. Cross, Nitric oxide: a pro-inflammatory mediator in lung disease?, Respir Res. 1 (2000) 67–72. [20] D.L. Laskin, L. Fakhrzadeh, J.D. Laskin, Nitric oxide and peroxynitrite in ozoneinduced lung injury, Adv. Exp. Med. Biol. 500 (2001) 183–190. [21] R.A. Dweik, The lung in the balance: arginine, methylated arginines, and nitric oxide, Am. J. Physiol. Lung Cell Mol. Physiol. 292 (2007) L15–L17.
[22] M. Lanzetti, F.S. Bezerra, B. Romana-Souza, A.C. Brando-Lima, V.L. Koatz, L.C. Porto, S.S. Valenca, Mate tea reduced acute lung inflammation in mice exposed to cigarette smoke, Nutrition 24 (2008) 375–381. [23] S.S. Valenca, F. Silva Bezerra, A.A. Lopes, B. Romana-Souza, M.C. Marinho Cavalcante, A.B. Lima, V.L. Goncalves Koatz, L.C. Porto, Oxidative stress in mouse plasma and lungs induced by cigarette smoke and lipopolysaccharide, Environ. Res. 108 (2008) 199–204. [24] P.S. Tsou, V. Addanki, H.L. Fung, Dissociation between superoxide accumulation and nitroglycerin-induced tolerance, J. Pharmacol. Exp. Ther. 327 (2008) 97–104. [25] S. Chatterjee, S. Premachandran, J. Shukla, T.B. Poduval, Synergistic therapeutic potential of dexamethasone and L-arginine in lipopolysaccharide-induced septic shock, J. Surg. Res. 140 (2007) 99–108. [26] J.Y. Kim, D. Kim, E.M. Lee, I. Choi, C.G. Park, K.S. Kim, J. Ha, S.J. Kim, J. Yang, Y.S. Kim, J.S. Han, S. Kim, J.S. Lee, C. Ahn, Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions, Transpl. Immunol. 12 (2003) 63–72. [27] H.H. Draper, E.J. Squires, H. Mahmoodi, J. Wu, S. Agarwal, M. Hadley, A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials, Free Radic. Biol. Med. 15 (1993) 353–363. [28] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannenbaum, Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids, Anal. Biochem. 126 (1982) 131–138. [29] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. [30] J.V. Bannister, L. Calabrese, Assays for superoxide dismutase, Methods Biochem. Anal. 32 (1987) 279–312. [31] L. Flohe, W.A. Gunzler, Assays of glutathione peroxidase, Methods Enzymol. 105 (1984) 114–121. [32] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [33] M. Eisenhut, Detection of adverse effects of endogenous nitric oxide in acute lung injury, Am. J. Respir. Crit. Care Med. 175 (2007) 1095–1096. [34] D. Ren, R. Sun, S. Wang, Role of inducible nitric oxide synthase expressed by alveolar macrophages in high mobility group box 1—induced acute lung injury, Inflamm. Res. 55 (2006) 207–215. [35] S.N. Giri, I. Biring, T. Nguyen, Q. Wang, D.M. Hyde, Abrogation of bleomycininduced lung fibrosis by nitric oxide synthase inhibitor, aminoguanidine in mice, Nitric Oxide 7 (2002) 109–118. [36] M.M. Rafi, P.N. Yadav, M. Reyes, Lycopene inhibits LPS-induced proinflammatory mediator inducible nitric oxide synthase in mouse macrophage cells, J. Food Sci. 72 (2007) S069–S074. [37] T.M. Wizemann, C.R. Gardner, J.D. Laskin, S. Quinones, S.K. Durham, N.L. Goller, S.T. Ohnishi, D.L. Laskin, Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia, J. Leukoc. Biol. 56 (1994) 759–768. [38] H. Yaren, H. Mollaoglu, B. Kurt, A. Korkmaz, S. Oter, T. Topal, T. Karayilanoglu, Lung toxicity of nitrogen mustard may be mediated by nitric oxide and peroxynitrite in rats, Res. Vet. Sci. 83 (2007) 116–122. [39] J.L. Zhou, G.H. Jin, Y.L. Yi, J.L. Zhang, X.L. Huang, Role of nitric oxide and peroxynitrite anion in lung injury induced by intestinal ischemia-reperfusion in rats, World J. Gastroenterol. 9 (2003) 1318–1322. [40] E. Esposito, S. Cuzzocrea, The role of nitric oxide synthases in lung inflammation, Curr. Opin. Investig. Drugs 8 (2007) 899–909. [41] J.L. Kang, W. Park, I.S. Pack, H.S. Lee, M.J. Kim, C.M. Lim, Y. Koh, Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation, J. Appl. Physiol. 92 (2002) 795–801. [42] H. Tavaf-Motamen, T.J. Miner, B.W. Starnes, T. Shea-Donohue, Nitric oxide mediates acute lung injury by modulation of inflammation, J. Surg. Res. 78 (1998) 137–142. [43] S.A. ter Horst, F.J. Walther, B.J. Poorthuis, P.S. Hiemstra, G.T. Wagenaar, Inhaled nitric oxide attenuates pulmonary inflammation and fibrin deposition and prolongs survival in neonatal hyperoxic lung injury, Am. J. Physiol. Lung Cell Mol. Physiol. 293 (2007) L35–L44. [44] T. Waldow, K. Alexiou, W. Witt, F.M. Wagner, V. Gulielmos, K. Matschke, M. Knaut, Attenuation of reperfusion-induced systemic inflammation by preconditioning with nitric oxide in an in situ porcine model of normothermic lung ischemia, Chest 125 (2004) 2253–2259. [45] X. Bessa, J.I. Elizalde, F. Mitjans, V. Pinol, R. Miquel, J. Panes, J. Piulats, J.M. Pique, A. Castells, Leukocyte recruitment in colon cancer: role of cell adhesion molecules, nitric oxide, and transforming growth factor beta1, Gastroenterology 122 (2002) 1122–1132. [46] C. Bouzin, A. Brouet, J. De Vriese, J. Dewever, O. Feron, Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy, J. Immunol. 178 (2007) 1505–1511. [47] M.Z. Jiang, H. Tsukahara, Y. Ohshima, Y. Todoroki, M. Hiraoka, M. Maeda, M. Mayumi, Effects of antioxidants and nitric oxide on TNF-alpha-induced adhesion molecule expression and NF-kappaB activation in human dermal microvascular endothelial cells, Life Sci. 75 (2004) 1159–1170. [48] T. Waldow, W. Witt, E. Weber, K. Matschke, Nitric oxide donor-induced persistent inhibition of cell adhesion protein expression and NFkappaB activation in endothelial cells, Nitric Oxide 15 (2006) 103–113. [49] M. Raveendran, J. Wang, D. Senthil, J. Wang, B. Utama, Y. Shen, D. Dudley, Y. Zhang, X.L. Wang, Endogenous nitric oxide activation protects against
S.S. Valença et al. / Nitric Oxide 20 (2009) 175–181 cigarette smoking induced apoptosis in endothelial cells, FEBS Lett. 579 (2005) 733–740. [50] P. Chanvorachote, U. Nimmannit, L. Wang, C. Stehlik, B. Lu, N. Azad, Y. Rojanasakul, Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin–proteasome-mediated degradation of FLICE inhibitory protein, J. Biol. Chem. 280 (2005) 42044–42050. [51] R.M. Levytskyy, Y.Z. Filyak, R.S. Stoika, Correlation between generation of nitric oxide and cell viability in human peripheral blood mononuclear cells and leukemic Jurkat T-cell line, Exp. Oncol. 26 (2004) 217–220. [52] J. Machova, M. Stefek, M. Kukan, M. Sinsky, K. Ondrias, L. Rackova, J. Navarova, V. Bauer, Involvement of L-arginine-nitric oxide system in the response of
181
isolated trachea to reactive oxygen species, Methods Find. Exp. Clin. Pharmacol. 25 (2003) 287–296. [53] G.A. Blaise, D. Gauvin, M. Gangal, S. Authier, Nitric oxide, cell signaling and cell death, Toxicology 208 (2005) 177–192. [54] J.C. Hoyt, R.A. Robbins, M. Habib, D.R. Springall, L.D. Buttery, J.M. Polak, P.J. Barnes, Cigarette smoke decreases inducible nitric oxide synthase in lung epithelial cells, Exp. Lung Res. 29 (2003) 17–28. [55] J.L. Wright, J. Dai, K. Zay, K. Price, C.B. Gilks, A. Churg, Effects of cigarette smoke on nitric oxide synthase expression in the rat lung, Lab. Invest. 79 (1999) 975–983.
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Nitric Oxide journal homepage: www.elsevier.com/locate/yniox
Involvement of nitric oxide in acute lung inflammation induced by cigarette smoke in the mouse Samuel Santos Valença a, Wagner Alves Pimenta a, Carlos Romualdo Rueff-Barroso a, Thiago Santos Ferreira a, Ângela Castro Resende b, Roberto Soares de Moura b, Luís Cristóvão Porto a,* a b
Laboratory of Tissue Repair, Department of Histology and Embryology, Rio de Janeiro State University, Brazil Department of Pharmacology, Institute of Biology Roberto Alcantara Gomes, Rio de Janeiro State University, Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 14 August 2008 Revised 10 November 2008 Available online 6 December 2008 Keywords: Nitric oxide Cigarette smoke Inflammation Oxidative stress Mouse
a b s t r a c t Short-term exposure to cigarette smoke (CS) leads to acute lung inflammation (ALI) by disturbing oxidant/ antioxidant balance. Both CS exposure and lung inflammation are important risk factors in the pathogenesis of chronic obstructive pulmonary disease. Nitric oxide (NO) is an oxidant both present in CS and produced in the inflammatory response, but its role in the effects of CS exposure is unclear. Our aim was to study involvement of NO in a model of CS exposure. Groups of mice (male C57BL/6) exposed to CS (six cigarettes per day over five days) were simultaneously subjected to treatment with vehicle (CS), 60 mg/kg/day x-nitro-L-arginine methyl ester (CS + L-NAME), 20 mg/kg/day nitroglycerine (CS + NTG), or 120 mg/kg/day L-arginine (CS + L-arg). Bronchoalveolar lavage fluid was then aspirated to perform cell counts, and malondialdehyde (MDA), nitrite, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels were measured in lung homogenates. Macrophage and neutrophil counts were increased in the CS (p < 0.001) and CS + L-NAME groups (p < 0.05 and p < 0.01, respectively); the CS + NTG and CS + L-arg groups showed no differences from the control group. MDA was increased in the CS (p < 0.05) and CS + L-NAME (p < 0.01) groups when compared to the control group. Nitrite levels were decreased in the CS and CS + L-NAME groups (p < 0.001) and increased in the CS + NTG (p < 0.001) and CS + L-arg (p < 0.01) groups when compared to the control. CAT, SOD and GPx activities in the CS and CS + L-NAME groups were all significantly increased compared to the control group. Our results suggest that administration of NO donors or substrates may be a useful therapy in the treatment of ALI caused by CS. Ó 2008 Elsevier Inc. All rights reserved.
Chronic obstructive pulmonary disease (COPD) is a rapidly increasing global health problem, predicted to be the third leading cause of death in developed countries by 2020 [1]. COPD is characterized by slowly progressive and largely irreversible airflow limitation due to chronic bronchitis and/or emphysema, associated with an abnormal inflammatory response of the lungs [2]. Cigarette smoke (CS) has been identified as the most important risk factor for development of COPD [3]. Although the prevalence of smoking has fallen in developed countries, it continues to increase in many low- and middle-income countries, especially among young people and women [4]. An estimated 4.9 million (8.8% of the global total) deaths in 2000 were attributable to use of tobacco, which is 45% higher than the figure 1990. The increase was greatest in developing countries, which now account for 50% of the global mortality and 56% of the disease burden attributable to tobacco [5]. Men were almost four times more likely than women to be
* Corresponding author. Address: Departamento de Histologia e Embriologia, IBRAG, UERJ, Avenida Professor Manuel de Abreu, 444, 3° andar, Maracanã, 20.550170 Rio de Janeiro, RJ, Brazil. Fax: +55 21 2587 6511. E-mail address: [email protected] (L.C. Porto). 1089-8603/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2008.11.003
smokers. The prevalence of smoking among men was highest in the Western Pacific region. The differential in gender-specific smoking prevalence was narrowest in the Americas and Europe; it was widest in South-East Asia and the Western Pacific region. China’s growing cigarette consumption since 1975 has had a major bearing on the rise in global per capita consumption [6]. Although ALI caused by CS exposure does not model all aspects of COPD pathogenesis, the two processes do share certain hallmarks, including accumulation of inflammatory cells such as alveolar macrophages and neutrophils, and lung oxidative stress [7]. Oxidants and free radicals in the gas phase of CS (alkyl, peroxyl, nitric oxide and superoxide anion) and the tar phase (semiquinone) [8] can stimulate alveolar macrophages to produce reactive oxygen species (ROS) and release cytokines involved in migration of inflammatory cells into the lungs. Alveolar macrophages and neutrophils can generate ROS [9], resulting in an imbalance between oxidants and antioxidants such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) [10]. CS exposure is also associated with increased levels of malondialdehyde (MDA), a marker of oxidative stress [11]. This suggests the existence of an oxidative stress mechanism induced by CS in animals and humans.
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Nitric oxide (NO) is an oxidant both present in CS and synthesized de novo in cells from L-arginine by various isoforms of NO synthase (NOS), including constitutive neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3) [12]. NO can readily pass through the plasma membrane to exert its action on cells. It is involved in vessel dilatation, inhibition of platelet aggregation, and host defense [13]. Organic nitrates (RONO2) such as nitroglycerine and isosorbide dinitrate have been used for over a century in the treatment of various cardiovascular diseases [14–16]. It has been generally accepted that nitroglycerine undergoes biotransformation to NO, which then binds to and activates guanylate cyclase, catalyzing production of cyclic GMP [17]. In contrast, x-nitro-Larginine 4 methyl ester (L-NAME) is a NOS inhibitor which blocks the production of NO by both iNOS and constitutive NOS [18]. NO is produced by a variety of cells within the respiratory tract, including not only inflammatory but also epithelial cells [19]. It is generally believed that NO produced by iNOS is associated with pro-inflammatory and damaging effects [20]. However, L-arginine is an obligatory substrate for all NOS isoforms. Indeed, inhaled or orally administered L-arginine increases the production of NO in human airways, suggesting that availability of the cationic amino acid is rate limiting for NO production [21]. The purpose of the present study was to analyze the involvement of NO on inflammatory cell migration, nitrite values, lipid peroxidation and antioxidant enzyme activities (CAT, SOD and GPx) in mouse lung exposed to CS. For this, we used nitroglycerine (a donor of NO), L-arginine (a substrate for NO formation), and L-NAME (an inhibitor of NO formation) in combination with CS exposure in mice.
Experimental procedures Reagents Thiobarbituric acid, adrenaline, NADPH, trichloroacetic acid, nitroglycerine, x-nitro-L-arginine methyl ester, L-arginine, sulfanilamide, phosphoric acid, naphthylenediamide dihydrochloride, sodium nitrite and perchloric acid were purchased from Sigma Chemical (St. Louis, MO, USA). Pallflex filters were purchased from Imprint (São Paulo, Brazil). Diff-Quik was purchased from Baxter Dade AG (Dudingen, Switzerland). Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA). Hydrogen peroxide was purchased from Vetec (Duque de Caxias, Brazil). Animals C57BL/6 male mice (8 weeks old—20–24 g) were purchased from the Veterinary Institute—Fluminense Federal University (Niterói, Brazil). Mice were housed (5 per cage) in a controlled environment room with a 12-h light/12-h dark cycle (lights on at 6 pm) and ambient temperature of 25 ± 2 °C (humidity 80%). The animals had free access to water and food. Acclimatization was performed during the two weeks before the experimental procedures. CS exposure C57BL/6 male mice (n = 40) were exposed to six commercial full-flavor filtered Virginia cigarettes (10 mg of tar, 0.9 mg of nicotine and 10 mg of carbon monoxide) per day for 5 days by using a smoking chamber described previously [11,22]. Briefly, each group of mice was placed in the inhalation chamber (40 cm long, 30 cm wide and 25 cm high), inside an exhaustion chapel. A cigarette was coupled to a plastic 60 mL syringe so
that puffs could be drawn in and subsequently expelled into the exposure chamber. We aspirated 1 L of smoke from one cigarette with this syringe (20 puffs of 50 mL) and immediately injected the puff into the chamber. The 10 animals of each group were maintained in this smoke-air condition for 6 min (3%), and the inhalation chamber was opened, by removing its cover, and the smoke evacuated for 1 min by exhaustion of the chapel. This cigarette exposure was repeated two times (2 6 min) with intervals of 1 min (exhaustion). We repeated this procedure three times per day (morning, noon and afternoon) resulting in 36 min of CS exposure to six cigarettes. Each cigarette smoked produced 300 mg/m3 of total particulate matter in the chamber (measured by weighing material collected on Pallflex filters) [22]. Carboxyhemoglobin (COHb) concentration was previously monitored in mice using the same experimental protocol and COHb was not toxic [23]. Groups (n = 10 each) were defined as mice exposed to CS and treated with vehicle (CS), mice exposed to CS and treated with 20 mg/kg/day of nitroglycerine (CS + NTG), mice exposed to CS and treated with 120 mg/kg/ day of L-arginine (CS + L-arg) and mice exposed to CS and treated with 60 mg/kg/day of L-NAME (CS + L-NAME). All treatments were performed by oral gavages once per day (simultaneously with CS exposure) and drugs were mixed with saline (vehicle). Mice exposed to ambient air were used as the control group (n = 10) and were subjected to oral gavages with vehicle. The doses of nitroglycerine [24], L-arginine [25] and L-NAME [26] were based on previous data from the literature with modification of the administration via. A separate group of C57BL/6 male mice (n = 5 for each group) were exposed to ambient air during five days by using the same protocol described above and simultaneously treated with vehicle (control group), 20 mg/kg/day of nitroglycerine (NTG), 120 mg/kg/day of L-arginine (L-arg) and 60 mg/kg/day of L-NAME (L-NAME). All procedures were carried out in accordance with conventional guidelines for experimentation with animals, and the local animal care and use committee approved the experimental protocol. Bronchoalveolar lavage (BAL) Twenty-four hours after the last exposure to ambient air or CS the animals were killed by cervical displacement. Lung airspaces were washed with buffered saline solution (500 lL) three times (final volume 1.2–1.5 mL). The BAL fluid was withdrawn and stored on ice. Total mononuclear and polymorphonuclear cell numbers were determined in a Zi Coulter counter (Beckman Coulter, Carlsbad, CA, USA). Differential cell counts were performed on cytospin preparations (Shandon, Waltham, MA, USA) stained with Diff-Quik. At least 200 cells per BAL sample were counted using standard morphologic criteria [11,22,23]. After BAL, lungs were immediately removed and homogenized on ice with 10% (w/v) PBS (pH 7.3) using an Ultra-TurraxÒ T 8 homogenizer (Toronto, Canada) and then centrifuged at 3000g for 5 min. Supernatants were kept at 20 °C for analysis of nitrite values, lipid peroxidation and antioxidant enzyme activities (CAT, SOD and GPx). Malondialdehyde assay As an index of lipid peroxidation we used the thiobarbituric acid reactive substances (TBARS) method for analyzing malondialdehyde products during an acid-heating reaction as previously described by Draper and co-workers [27]. Briefly, samples from lung homogenates were mixed with 1 mL of 10% trichloroacetic acid and 1 mL of 0.67% thiobarbituric acid; the samples were then heated in a boiling water bath for 30 min. TBARS levels were determined by absorbance at 532 nm and expressed as malondialdehyde equivalents (nm/mg protein).
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Nitrite assay Nitrite levels in lung homogenates were determined by a method based on the Griess reaction [28]. A total of 100 lL of sample was mixed with 100 lL of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthylenediamide dihydrochloride in water) and incubated at room temperature for 10 min followed by measuring the absorbance in a plate reader at 550 nm (Bio-Rad Microplate Reader model 680, CA, USA). Nitrite concentrations in the samples were determined from a standard curve generated by different concentrations of sodium nitrite. Catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities CAT, SOD and GPx activities were determined in lung homogenates. CAT activity was measured by the rate of decrease in hydrogen peroxide concentration at 240 nm [29]. SOD activity was assayed by measuring inhibition of adrenaline auto-oxidation as absorbance at 480 nm [30]. GPx activity was measured by monitoring the oxidation of NADPH at 340 nm in the presence of H2O2 [31]. The total protein content in the samples from lung homogenates was determined by the method of Bradford [32]. Statistical analysis Data are expressed as means ± SEM. For comparison between control, CS, CS + NTG, CS + L-arg and CS + L-NAME, data concerning BAL cells, MDA, nitrite, CAT, SOD and GPx were analyzed with one-way ANOVA followed by the Student–Newman–Keuls post hoc test (p < 0.05). GraphPad InStat software was used to perform the statistical analyses (GraphPad InStat version 3.00 for Windows 95, GraphPad Software Inc.; San Diego, CA, USA).
Results
Fig. 1. (a) Effect of L-NAME, L-arginine and nitroglycerine on the migration of alveolar macrophages and neutrophils into the lungs of mice exposed to CS. Data are shown as means ± SEM from 10 mice for each group. *Compared to control group. #Compared to CS group. *p < 0.05, **p < 0.01, ***p < 0.001. #p < 0.05, ##p < 0.01, ### p < 0.001. (b) Effect of L-NAME, L-arginine and nitroglycerine on the migration of alveolar macrophages and neutrophils into the lungs of mice exposed to ambient air. Data are shown as means ± SEM from five mice for each group. *Compared to control group. #Compared to L-NAME group. #p < 0.05. ##p < 0.01, ###p < 0.001.
Inflammatory cell migration into the lungs C57BL/6 mice were exposed to six cigarettes a day for five consecutive days. BAL cell counts revealed a marked migration of inflammatory cells into the airway lumen for some of the experimental groups (Fig. 1a). The numbers of alveolar macrophages and neutrophils were increased in the CS group in comparison to the control group (p < 0.001). They were also significantly higher in the CS + L-NAME group compared to the control group (p < 0.05 and p < 0.01, respectively); however, the numbers of neutrophils remained significantly lower compared to the CS group (p < 0.01). Both the CS + NTG and CS + L-arg groups showed a marked reduction in the numbers of alveolar macrophages (p < 0.01 and p < 0.05, respectively) and neutrophils (p < 0.001) compared to the CS group. No statistical differences were observed among the CS + NTG, CS + L-arg and control groups. A separate group of C57BL/6 mice was exposed to ambient air for five consecutive days and treated with vehicle or with nitroglycerine (NTG), L-arginine and L-NAME (L-NAME) (Fig. 1b). The number of alveolar macrophages was reduced in the NTG group in comparison to the control group (p < 0.05) and the L-NAME group (p < 0.001). The number of alveolar macrophages was also reduced in the L-arg group in comparison to the L-NAME group (p < 0.05). The number of neutrophils was reduced in the NTG group (p < 0.01) and in the L-arg group (p < 0.05) in comparison to the L-NAME group.
Malondialdehyde content Fig. 2a shows the MDA equivalents in mouse lungs of the different experimental groups. We observed an increase in the levels of MDA in the CS and CS + L-NAME groups compared to the control group (p < 0.05 and p < 0.01, respectively). On the other hand, a decrease in MDA in the CS + NTG group was noted compared to the CS group (p < 0.05). MDA levels in the CS + NTG and CS + L-arg groups were similar to that of the control group. MDA levels in the CS and CS + L-NAME groups were similar. A separate group of C57BL/6 mice was exposed to ambient air for five consecutive days and treated with vehicle or with nitroglycerine (NTG), L-arginine or L-NAME (L-NAME) (Fig. 2b). We observed an increase in the level of MDA in the L-NAME group compared to the control group (p < 0.05). MDA levels in the NTG and L-arg groups were reduced in comparison with the L-NAME group (p < 0.05). MDA levels in the NTG and L-arg groups were similar to that of the control group. Nitrite content The results of evaluation of nitrite levels in mouse lungs are shown in Fig. 3. Mice in the CS group showed a lower level of nitrite compared to the control group (p < 0.001). Mice in the
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Fig. 3. Effect of L-NAME, L-arginine and nitroglycerine on the nitrite content of lung homogenates of mice exposed to CS. Data are shown as means ± SEM from eight mice for each group. *Compared to control group. #Compared to CS group. **p < 0.01, *** p < 0.001, ###p < 0.001.
Fig. 2. (a) Effect of L-NAME, L-arginine and nitroglycerine on the malondialdehyde (MDA) content of lung homogenates of mice exposed to CS. Data are shown as means ± SEM from eight mice for each group. *Compared to control group. # Compared to CS group. *p < 0.05, **p < 0.01, #p < 0.05. (b) Effect of L-NAME, Larginine and nitroglycerine on the malondialdehyde (MDA) content of lung homogenates of mice exposed to ambient air. Data are shown as means ± SEM from five mice for each group. *Compared to control group. #Compared to L-NAME group. *p < 0.05, #p < 0.05.
CS + L-NAME group showed the lowest level of nitrite formation in comparison to the CS and control groups (p < 0.001). In contrast, the levels of nitrite were increased in the CS + NTG and CS + L-arg groups compared to the CS (p < 0.001 and p < 0.001) and control groups (p < 0.001 and p < 0.01, respectively). CAT, SOD and GPx activities Fig. 4 shows CAT activity in mouse lungs. CAT activity was increased in the CS and CS + L-NAME groups compared to the control group (p < 0.01 and p < 0.001, respectively). CAT activity in the CS + L-NAME group was elevated compared to that of the CS group (p < 0.01). CAT activity in the CS + L-arg group was reduced in relation to that of the CS group (p < 0.01). Both the CS + NTG and CS + L-arg groups had CAT activity similar to that of the control group. Fig. 5 shows the SOD activity in each group based on measurement of inhibition of adrenaline auto-oxidation. An increase in SOD activity was observed in the CS and CS + L-NAME groups compared to the control group (p < 0.05 and p < 0.001). On the other hand, a reduction in SOD activity was noted in the CS + NTG and CS + L-arg groups in comparison to the CS (p < 0.001) and control groups (p < 0.05). Fig. 6 shows the GPx activity in mouse lungs based on measurement of the oxidation of NADPH. The CS and CS + L-NAME groups showed elevated levels of GPx activity
Fig. 4. Effect of L-NAME, L-arginine and nitroglycerine on the catalase (CAT) activity in lung homogenates of mice exposed to CS. Data are shown as means ± SEM from nine mice for each group. *Compared to control group. #Compared to CS group. ** p < 0.01, ***p < 0.001, ##p < 0.01.
compared to the control group (p < 0.01). The GPx activity of the CS + L-arg group was significantly lower than that of the CS group (p < 0.05). The GPx activities of both the CS + NTG and CS + L-arg groups were similar to that of the control group. Discussion Nitric oxide is a signaling molecule responsible for several diverse physiological and pathophysiological processes, and until now the prevailing hypothesis about NO has been that it contributes to toxicant-induced lung inflammation and injury [19,20,33,34]. The present study suggests a different role for NO in ALI induced by CS. We showed here that administration of an NO donor (by nitroglycerine) or NO substrate (by L-arginine) resulted in a reduction in the migration of inflammatory cells, lower oxidative stress and normalized antioxidant enzyme activities. The
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Fig. 5. Effect of L-NAME, L-arginine and nitroglycerine on the superoxide dismutase (SOD) activity in lung homogenates of mice exposed to CS. Data are shown as means ± SEM from nine mice for each group. *Compared to control group. # Compared to CS group. *p < 0.05, ***p < 0.001. ###p < 0.001.
Fig. 6. Effect of L-NAME, L-arginine and nitroglycerine on the glutathione peroxidase (GPx) activity in lung homogenates of mice exposed to CS. Data are shown as means ± SEM from nine mice for each group. *Compared to control group. # Compared to CS group. **p < 0.01, #p < 0.05.
action of NO here was different from its actions elicited by other lung inflammatory stimuli such as lipopolysaccharide or bleomycin [35,36]. Although NO has been described in several studies to be a mediator of the inflammatory response, for example by stimulating production of inflammatory cytokines and peroxynitrite (ONOO ) [37–39], beneficial effects of NO in the lungs have also been reported [40–44]. We found a reduction in both alveolar macrophages and neutrophils in the CS + NTG and CS + L-arg groups when compared to the control group. The specific action of NO on inflammatory cells is unknown, but we suggest that nitroglycerine or L-arginine may reduce the expression of endothelial cell adhesion molecules and consequently inflammatory cell migration to the alveolar space. Furthermore, NO was reported to be an important molecule for blocking the endothelium–leukocyte interaction [45–48]. Apoptosis of inflammatory cells prior to the fifth day of CS exposure through NO-activation of death domains is another hypothesis to
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explain reduced alveolar macrophages and neutrophils in the CS + NTG and CS + L-arg groups, due to involvement of NO in apoptosis [49–51]. The use of the NO-inhibitor L-NAME resulted in a reduction in the level of neutrophils compared with the CS group. Therefore, although CS itself decreased nitrite formation, further decreasing it with L-NAME actually reduced the inflammatory response to CS (in terms of neutrophil levels). Certainly, blocking endogenous NO production (with L-NAME) did not have as favorable a result as augmenting NO production (with nitroglycerine or L-Arg) in so far as reducing inflammation, but arguably both can work based on these results. Administration of just L-NAME to mice exposed to ambient air did not affect the levels of migration of alveolar macrophages and neutrophils into the lungs. However, treatment with nitroglycerine or L-arginine of mice exposed to ambient air reduced the numbers of alveolar macrophages and neutrophils when compared to the control group. These data help to explain the positive effects of NO donor or substrate in decreasing the inflammatory response by lowering cell recruitment into the lungs. Lipid peroxidation is an important marker of oxidative stress in the lung, and we showed in a previous study the association between ALI caused by CS and lipid peroxidation as analyzed by MDA [11,22]. We found MDA levels to be increased in CS-exposed mice in comparison with control animals. Treatment with nitroglycerine or L-arginine reduced the amount of MDA. This result is similar to that of Machova and co-workers [52] who showed that NO treatment inhibited lipid peroxidation in vitro and protected against cellular damage and cytotoxicity. NO has also been described as a scavenger of other far more toxic radicals, and therefore, an enhancer of defense mechanisms [53]. Consequently, lipid peroxidation was observed only in the CS + L-NAME and CS groups, not in the CS + NTG and CS + L-arg groups. In addition, administration of L-NAME to mice exposed to ambient air exposed led to increased MDA levels in lung homogenates, a result that suggests the occurrence of oxidative stress in this group when compared to the control group. Nitroglycerine and L-arginine increased nitrite (NO2 ) production in lung tissue, whereas administration of CS only or L-NAME did not, indicating reduced production of NO in the two latter groups. However, nitrite formation during CS exposure is unclear. A reduction in nitrite levels was reported by Hoyt and co-workers [54] together with a reduction in NO synthase (iNOS) mRNA and protein expression. In contrast, Wright and co-workers reported an increase in iNOS with CS exposure [55]. However, L-NAME, being an analogue of L-arginine, significantly lowers NO concentrations as it interacts with NOS, but this interaction does not result in NO production [53]. The lower nitrite levels that we observed in the CS + NTG and CS + L-arg groups confirm the efficacy of treatment with NO donor or substrate and led us to hypothesize the involvement of NO in blunting the inflammatory response. The increase in CAT, SOD and GPx activities in the lungs of CS mice reflects indirectly the generation of free radicals. The compensatory mechanism of lung cells in an acute model of CS exposure has been reported by us previously [23]. Interestingly, the lungs of mice exposed to long-term CS showed reduced levels of CAT, SOD and GPx activity (data not shown). We observed variations in CAT, SOD and GPx activities in the CS + NTG and CS + Larg groups, and in general these values were similar to those of the control group. Thus, the CS + NTG and CS + L-arg groups appeared to regulate the CAT, SOD and GPx activities to some degree. However, the highest CAT, SOD and GPx activities were found in the CS + L-NAME group. If the increase of CAT, SOD and GPx activities in CS-exposed animals reflects a disturbance of the balance between oxidants and antioxidants, then L-NAME enhanced this imbalance. We suggest that CS and L-NAME both disturb the balance between oxidants and antioxidants, favoring the oxidant side.
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In summary, we showed that NO supplementation by administration of nitroglycerine or L-arginine was able to reduce both CS-induced ALI and oxidative stress, and that it regulated CAT, SOD and GPx activities. The NO inhibitor L-NAME maintained the inflammatory status in the lungs of CS-exposed mice, contributed to the oxidative stress there, and induced a major imbalance between oxidants and antioxidants by maintaining high CAT, SOD and GPx activities, favoring the oxidant side. Lung inflammatory status, oxidative stress, and CAT, SOD and GPx activities from CS mice were to some degree similar to those of CS + L-NAME mice. Supplementation with NO may be a good alternative in the treatment of ALI caused by CS and in the prevention of COPD, diseases which involve oxidative stress in their pathogenesis. Acknowledgments This work was conducted with grants from National Council of Scientific and Technological Development (CNPq) and Rio de Janeiro State Research Agency (FAPERJ). References [1] D.A. Groneberg, K.F. Chung, Models of chronic obstructive pulmonary disease, Respir. Res. 5 (2004) 18. [2] D.E. O’Donnell, C.M. Parker, COPD exacerbations. 3. Pathophysiology, Thorax 61 (2006) 354–361. [3] S. Molet, C. Belleguic, H. Lena, N. Germain, C.P. Bertrand, S.D. Shapiro, J.M. Planquois, P. Delaval, V. Lagente, Increase in macrophage elastase (MMP-12) in lungs from patients with chronic obstructive pulmonary disease, Inflamm. Res. 54 (2005) 31–36. [4] M.A. Corrao, G.E. Guindon, V. Cokkinides, N. Sharma, Building the evidence base for global tobacco control, Bull. World Health Organ. 78 (2000) 884–890. [5] Y. Lacasse, F. Maltais, R.S. Goldstein, Smoking cessation in pulmonary rehabilitation: goal or prerequisite?, J Cardiopulm. Rehabil. 22 (2002) 148– 153. [6] K. Shibuya, C. Ciecierski, E. Guindon, D.W. Bettcher, D.B. Evans, C.J. Murray, WHO framework convention on tobacco control: development of an evidence based global public health treaty, BMJ 327 (2003) 154–157. [7] A. Churg, R.D. Wang, H. Tai, X. Wang, C. Xie, J. Dai, S.D. Shapiro, J.L. Wright, Macrophage metalloelastase mediates acute cigarette smoke-induced inflammation via tumor necrosis factor-alpha release, Am. J. Respir. Crit. Care Med. 167 (2003) 1083–1089. [8] W. MacNee, Oxidants/antioxidants and COPD, Chest 117 (2000) 303S–317S. [9] W. MacNee, I. Rahman, Is oxidative stress central to the pathogenesis of chronic obstructive pulmonary disease?, Trends Mol Med. 7 (2001) 55–62. [10] I. Rahman, I.M. Adcock, Oxidative stress and redox regulation of lung inflammation in COPD, Eur. Respir. J. 28 (2006) 219–242. [11] F. Silva Bezerra, S.S. Valenca, M. Lanzetti, W.A. Pimenta, P. Castro, V.L. Goncalves Koatz, L.C. Porto, Alpha-tocopherol and ascorbic acid supplementation reduced acute lung inflammatory response by cigarette smoke in mouse, Nutrition 22 (2006) 1192–1201. [12] C.M. Prado, E.A. Leick-Maldonado, L. Yano, A.S. Leme, V.L. Capelozzi, M.A. Martins, I.F. Tiberio, Effects of nitric oxide synthases in chronic allergic airway inflammation and remodeling, Am. J. Respir. Cell Mol. Biol. 35 (2006) 457–465. [13] L. Fakhrzadeh, J.D. Laskin, C.R. Gardner, D.L. Laskin, Superoxide dismutaseoverexpressing mice are resistant to ozone-induced tissue injury and increases in nitric oxide and tumor necrosis factor-alpha, Am. J. Respir. Cell Mol. Biol. 30 (2004) 280–287. [14] M.C. Gupta, M.M. Singh, A.K. Srivastava, R. Singh, V. Prakash, Sublingual isosorbide dinitrate, topical isosorbide dinitrate and topical nitroglycerine in salvaging ischaemic myocardium, J. Assoc. Physicians India 32 (1984) 1023– 1026. [15] K.C. Ferdinand, Isosorbide dinitrate and hydralazine hydrochloride: a review of efficacy and safety, Expert Rev. Cardiovasc. Ther. 3 (2005) 993–1001. [16] G. Yetik-Anacak, J.D. Catravas, Nitric oxide and the endothelium: history and impact on cardiovascular disease, Vascul. Pharmacol. 45 (2006) 268–276. [17] R.M. Gill, J.C. Braz, N. Jin, G.J. Etgen, W. Shen, Restoration of impaired endothelium-dependent coronary vasodilation in failing heart: role of eNOS phosphorylation and CGMP/cGK-I signaling, Am. J. Physiol. Heart Circ. Physiol. 292 (2007) H2782–H2790. [18] B. Jover, A. Mimran, Nitric oxide inhibition and renal alterations, J. Cardiovasc. Pharmacol. 38 (Suppl. 2) (2001) S65–S70. [19] A. van der Vliet, J.P. Eiserich, C.E. Cross, Nitric oxide: a pro-inflammatory mediator in lung disease?, Respir Res. 1 (2000) 67–72. [20] D.L. Laskin, L. Fakhrzadeh, J.D. Laskin, Nitric oxide and peroxynitrite in ozoneinduced lung injury, Adv. Exp. Med. Biol. 500 (2001) 183–190. [21] R.A. Dweik, The lung in the balance: arginine, methylated arginines, and nitric oxide, Am. J. Physiol. Lung Cell Mol. Physiol. 292 (2007) L15–L17.
[22] M. Lanzetti, F.S. Bezerra, B. Romana-Souza, A.C. Brando-Lima, V.L. Koatz, L.C. Porto, S.S. Valenca, Mate tea reduced acute lung inflammation in mice exposed to cigarette smoke, Nutrition 24 (2008) 375–381. [23] S.S. Valenca, F. Silva Bezerra, A.A. Lopes, B. Romana-Souza, M.C. Marinho Cavalcante, A.B. Lima, V.L. Goncalves Koatz, L.C. Porto, Oxidative stress in mouse plasma and lungs induced by cigarette smoke and lipopolysaccharide, Environ. Res. 108 (2008) 199–204. [24] P.S. Tsou, V. Addanki, H.L. Fung, Dissociation between superoxide accumulation and nitroglycerin-induced tolerance, J. Pharmacol. Exp. Ther. 327 (2008) 97–104. [25] S. Chatterjee, S. Premachandran, J. Shukla, T.B. Poduval, Synergistic therapeutic potential of dexamethasone and L-arginine in lipopolysaccharide-induced septic shock, J. Surg. Res. 140 (2007) 99–108. [26] J.Y. Kim, D. Kim, E.M. Lee, I. Choi, C.G. Park, K.S. Kim, J. Ha, S.J. Kim, J. Yang, Y.S. Kim, J.S. Han, S. Kim, J.S. Lee, C. Ahn, Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions, Transpl. Immunol. 12 (2003) 63–72. [27] H.H. Draper, E.J. Squires, H. Mahmoodi, J. Wu, S. Agarwal, M. Hadley, A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials, Free Radic. Biol. Med. 15 (1993) 353–363. [28] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannenbaum, Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids, Anal. Biochem. 126 (1982) 131–138. [29] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. [30] J.V. Bannister, L. Calabrese, Assays for superoxide dismutase, Methods Biochem. Anal. 32 (1987) 279–312. [31] L. Flohe, W.A. Gunzler, Assays of glutathione peroxidase, Methods Enzymol. 105 (1984) 114–121. [32] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [33] M. Eisenhut, Detection of adverse effects of endogenous nitric oxide in acute lung injury, Am. J. Respir. Crit. Care Med. 175 (2007) 1095–1096. [34] D. Ren, R. Sun, S. Wang, Role of inducible nitric oxide synthase expressed by alveolar macrophages in high mobility group box 1—induced acute lung injury, Inflamm. Res. 55 (2006) 207–215. [35] S.N. Giri, I. Biring, T. Nguyen, Q. Wang, D.M. Hyde, Abrogation of bleomycininduced lung fibrosis by nitric oxide synthase inhibitor, aminoguanidine in mice, Nitric Oxide 7 (2002) 109–118. [36] M.M. Rafi, P.N. Yadav, M. Reyes, Lycopene inhibits LPS-induced proinflammatory mediator inducible nitric oxide synthase in mouse macrophage cells, J. Food Sci. 72 (2007) S069–S074. [37] T.M. Wizemann, C.R. Gardner, J.D. Laskin, S. Quinones, S.K. Durham, N.L. Goller, S.T. Ohnishi, D.L. Laskin, Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia, J. Leukoc. Biol. 56 (1994) 759–768. [38] H. Yaren, H. Mollaoglu, B. Kurt, A. Korkmaz, S. Oter, T. Topal, T. Karayilanoglu, Lung toxicity of nitrogen mustard may be mediated by nitric oxide and peroxynitrite in rats, Res. Vet. Sci. 83 (2007) 116–122. [39] J.L. Zhou, G.H. Jin, Y.L. Yi, J.L. Zhang, X.L. Huang, Role of nitric oxide and peroxynitrite anion in lung injury induced by intestinal ischemia-reperfusion in rats, World J. Gastroenterol. 9 (2003) 1318–1322. [40] E. Esposito, S. Cuzzocrea, The role of nitric oxide synthases in lung inflammation, Curr. Opin. Investig. Drugs 8 (2007) 899–909. [41] J.L. Kang, W. Park, I.S. Pack, H.S. Lee, M.J. Kim, C.M. Lim, Y. Koh, Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation, J. Appl. Physiol. 92 (2002) 795–801. [42] H. Tavaf-Motamen, T.J. Miner, B.W. Starnes, T. Shea-Donohue, Nitric oxide mediates acute lung injury by modulation of inflammation, J. Surg. Res. 78 (1998) 137–142. [43] S.A. ter Horst, F.J. Walther, B.J. Poorthuis, P.S. Hiemstra, G.T. Wagenaar, Inhaled nitric oxide attenuates pulmonary inflammation and fibrin deposition and prolongs survival in neonatal hyperoxic lung injury, Am. J. Physiol. Lung Cell Mol. Physiol. 293 (2007) L35–L44. [44] T. Waldow, K. Alexiou, W. Witt, F.M. Wagner, V. Gulielmos, K. Matschke, M. Knaut, Attenuation of reperfusion-induced systemic inflammation by preconditioning with nitric oxide in an in situ porcine model of normothermic lung ischemia, Chest 125 (2004) 2253–2259. [45] X. Bessa, J.I. Elizalde, F. Mitjans, V. Pinol, R. Miquel, J. Panes, J. Piulats, J.M. Pique, A. Castells, Leukocyte recruitment in colon cancer: role of cell adhesion molecules, nitric oxide, and transforming growth factor beta1, Gastroenterology 122 (2002) 1122–1132. [46] C. Bouzin, A. Brouet, J. De Vriese, J. Dewever, O. Feron, Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy, J. Immunol. 178 (2007) 1505–1511. [47] M.Z. Jiang, H. Tsukahara, Y. Ohshima, Y. Todoroki, M. Hiraoka, M. Maeda, M. Mayumi, Effects of antioxidants and nitric oxide on TNF-alpha-induced adhesion molecule expression and NF-kappaB activation in human dermal microvascular endothelial cells, Life Sci. 75 (2004) 1159–1170. [48] T. Waldow, W. Witt, E. Weber, K. Matschke, Nitric oxide donor-induced persistent inhibition of cell adhesion protein expression and NFkappaB activation in endothelial cells, Nitric Oxide 15 (2006) 103–113. [49] M. Raveendran, J. Wang, D. Senthil, J. Wang, B. Utama, Y. Shen, D. Dudley, Y. Zhang, X.L. Wang, Endogenous nitric oxide activation protects against
S.S. Valença et al. / Nitric Oxide 20 (2009) 175–181 cigarette smoking induced apoptosis in endothelial cells, FEBS Lett. 579 (2005) 733–740. [50] P. Chanvorachote, U. Nimmannit, L. Wang, C. Stehlik, B. Lu, N. Azad, Y. Rojanasakul, Nitric oxide negatively regulates Fas CD95-induced apoptosis through inhibition of ubiquitin–proteasome-mediated degradation of FLICE inhibitory protein, J. Biol. Chem. 280 (2005) 42044–42050. [51] R.M. Levytskyy, Y.Z. Filyak, R.S. Stoika, Correlation between generation of nitric oxide and cell viability in human peripheral blood mononuclear cells and leukemic Jurkat T-cell line, Exp. Oncol. 26 (2004) 217–220. [52] J. Machova, M. Stefek, M. Kukan, M. Sinsky, K. Ondrias, L. Rackova, J. Navarova, V. Bauer, Involvement of L-arginine-nitric oxide system in the response of
181
isolated trachea to reactive oxygen species, Methods Find. Exp. Clin. Pharmacol. 25 (2003) 287–296. [53] G.A. Blaise, D. Gauvin, M. Gangal, S. Authier, Nitric oxide, cell signaling and cell death, Toxicology 208 (2005) 177–192. [54] J.C. Hoyt, R.A. Robbins, M. Habib, D.R. Springall, L.D. Buttery, J.M. Polak, P.J. Barnes, Cigarette smoke decreases inducible nitric oxide synthase in lung epithelial cells, Exp. Lung Res. 29 (2003) 17–28. [55] J.L. Wright, J. Dai, K. Zay, K. Price, C.B. Gilks, A. Churg, Effects of cigarette smoke on nitric oxide synthase expression in the rat lung, Lab. Invest. 79 (1999) 975–983.