L-NAME and L-arginine differentially ameliorate cigarette smoke-induced emphysema in mice.

Nitric oxide (NO) represents one of the most important intra- and extracellular mediators and takes part in both biologic and pathologic processes. This study aimed to verify the treatment with an NO inhibitor and an NO substrate in pulmonary emphysema induced by cigarette smoke (CS) in a murine model. We compared N-acetylcysteine (NAC), a precursor of glutathione, to G-nitro-L-arginine-methyl ester or L-NAME (LN), which is an NO inhibitor, and to l-arginine (LA), which is a substrate for NO formation. Mice were divided into several groups: control, CS, CS + LN, CS + LA, and CS + NAC. Control and CS groups were treated daily with a vehicle, while CS + LN, CS + LA, and CS + NAC groups were treated daily with LN (60 mg/kg), LA (120 mg/kg) and NAC (200 mg/kg), respectively. The bronchoalveolar lavage was analyzed and the lungs were removed for histological and biochemical analysis. CS increases neutrophil number. Neutrophil number was lowest in CS + LN, followed by CS + LA. The lungs of CS + LN, CS + LA and CS + NAC mice were protected compared to the lungs of CS mice, but not equal to the quality of lungs in control mice. The CS group also exhibited increased oxidative stress, which was also present in the CS + LN group and to a lesser extent in the CS + LA group. Tissue inhibitor of metalloproteinase 1 and 2 increased in the CS + LN group and to a lesser extent in the CS + LA group relative to the control group. These results suggest that LN and LA treatment protected the mouse lung from CS. However, NAC treatment was more than LN and LA. We suggest that the protection conferred by LN treatment requires a balance between proteases and antiproteases, and that protection conferred by LA treatment involves the balance between oxidants and antioxidants.

Long-term exposure to cigarette smoke impairs lung function and increases HMGB-1 expression in mice.

Cigarette smoke (CS)-induced emphysema is caused by a continuous inflammatory response in the lower respiratory tract. The development of the condition is believed to be mediated by oxidant-antioxidant imbalance. This paper describes the effects of long-term CS exposure on alveolar cell recruitment, antioxidant defense systems, activity of extracellular matrix metalloelastases, expression of metalloelastase MMP-12, and high mobility group box-1 protein (HMGB-1). Ten C57Bl/6 mice were exposed to 12 cigarettes-a-day for 60 consecutive days, while 10 control animals were exposed to ambient air. After sacrifice, bronchoalveolar lavage fluid (BALF) was removed, and lung tissue underwent biochemical and histological analyses. In CS-exposed animals influx of alveolar macrophages and neutrophils into BALF, lung static elastance, and expression of MMP-12 and HMGB-1 were significantly increased while the activity of antioxidant enzyme was significantly reduced in comparison with control group. Thus, we demonstrated for the first time that long-term CS exposure decreased antioxidant defenses concomitantly with impaired lung function, which was associated with HMGB-1 expression.

Involvement of nitric oxide in acute lung inflammation induced by cigarette smoke in the mouse.

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), 60mg/kg/day omega-nitro-l-arginine methyl ester (CS+l-NAME), 20mg/kg/day nitroglycerine (CS+NTG), or 120mg/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.

Effects of oral nicotine on rat liver stereology

Nicotine is the more abundant component in cigarette smoke. Because nicotine is first metabolized in the liver, our aim was to investigate the effects of nicotine on this organ by biochemical and stereological methods. Male Wistar rats were treated with oral nicotine (ON) diluted in drinking water during 32 days. The control group was treated with drinking water in the same period. Rats were sacrificed 24 hours after last day, the blood was collected and the liver was removed. Lipidogram was performed by enzymatic method and collagen fibers, fat globules and hepatocytes were count in the liver by stereological methods. We observed in control group preserved hepatocytes, with no presence of inflammatory cells. However in the ON group was possible to observe varied size of hepatocytes with cloudy cellular limits and histoarchitecture loss. Capillaries were fully of red blood cells. We observed also an increase of fat globules with small size. We observed in leucogram a reduction of leukocytes number (lymphocytes, neutrophils and monocytes) in the ON group in comparison to control group. The lipidogram showed an increase of triglycerides and total cholesterol for ON group when compared to control group. Moreover, a reduction of HDL- cholesterol was observed in ON group when compared to control group. Numerical density of hepatocytes was lesser in ON group when compared to control group. We suggest harmful effects of oral nicotine in liver induced by toxic mechanism with reduction of hepatocytes number and disturbance in lipid metabolism.

La nicotina es el componente más abundante en el humo del cigarrillo. Primero porque la nicotina es metabolizada en el hígado, nuestro objetivo fue investigar los efectos de la nicotina sobre este órgano por métodos bioquímicos y estereológicos. Hombre rata Wistar fueron tratados con la nicotina oral (NO) diluida en el agua potable durante 32 días. El grupo control fue tratado con agua potable en el mismo período. Las ratas se sacrificaron 24 horas después del último día, se recogió la sangre y el hígado fue retirado. Lipidogram se realizó por el método enzimático y fibras de colágeno, grasa y hepatocitos se cuentan en el hígado mediante métodos estereológicos. Hemos observado en el grupo control hepatocitos conservados, sin presencia de células inflamatorias. Sin embargo en el grupo ON fue posible observar variado tamaño de hepatocitos con nubes y claros límites celulares y histoarchitecture pérdida. Capilares están plenamente de los glóbulos rojos. Se observó también un aumento de grasa con pequeño tamaño. Hemos observado en leucogram una reducción del número de glóbulos blancos (linfocitos, neutrófilos y monocitos) en el grupo ON, en comparación con el grupo control. El lipidogram mostró un aumento de los triglicéridos y de colesterol total ON grupo comparado con el grupo control. Por otra parte, una reducción del HDL-colesterol se observó en el grupo ON, en comparación con el grupo control. Densidad numérica de los hepatocitos fue menor en el grupo ON, en comparación con el grupo control. Sugerimos oral efectos nocivos de la nicotina en el hígado inducido por tóxicos con mecanismo de reducción de número de hepatocitos y alteraciones en el metabolismo lipídico.

Alpha-tocopherol and ascorbic acid supplementation reduced acute lung inflammatory response by cigarette smoke in mouse.

OBJECTIVE: Short-term cigarette smoke (CS) exposure leads to acute lung inflammation through its influence over oxidants/antioxidants imbalance. Antioxidant vitamins such as ascorbic acid and alpha-tocopherol interact with oxidizing radicals. It is not clear if antioxidant supplementation can reduce inflammatory lung responses. Thus our aim was to analyze the effects of vitamin supplementation on the lungs of mice exposed to six cigarettes per day with histologic, cytological, and biochemical methods. METHODS: C57BL/6 mice were exposed to ambient air (control) or CS from 3, 6, 9, 12, or 15 cigarettes daily for up to 5 d. Mice alveolar macrophages and polymorphonuclear cells were counted in the bronchoalveolar lavage. Groups of CS animals received 50 mg/kg of ascorbic acid daily and/or 50 mg/kg of alpha-tocopherol daily as an oral supplementation (CS+C, CS+E, CS+C+E, respectively) 12 h before CS exposure. Thiobarbituric acid-reactive substances were detected and western blot to nuclear factor-kappaB were performed in lung extracts; metalloprotease-12 and tumor necrosis factor-alpha positive alveolar macrophages were quantified in the lungs processed for immunohistochemistry of the animals exposed to the smoke from six cigarettes daily for 5 d. RESULTS: The number of alveolar macrophages and polymorphonuclear cells in bronchoalveolar lavage (cells x 10(3)/mL) in mice exposed to CS were increased and CS with vitamin supplementation groups presented bronchoalveolar lavage cells similar to those of control. Thiobarbituric acid-reactive substances values were reduced in vitamin supplementation groups when compared with CS and the lower value was found in the CS+C+E group. Metalloprotease-12 and tumor necrosis factor-alpha were more evident in CS as much as nuclear factor-kappaB activation when compared with control and vitamin supplementation groups. CONCLUSION: Our results showed that CS induced acute lung inflammation. The inflammatory process after cigarette exposures was reduced by ascorbic acid, alpha-tocopherol, or more efficiently by both vitamin supplementations.

Light cigarette smoke-induced emphysema and NFkappaB activation in mouse lung.

Light cigarette (LC) exposure is supposed to be less hazardous with a decreased incidence of cancer and tobacco-associated diseases. C57BL/6 mouse groups were subjected to smoke from 3, 6 or 12 LC for 60 days and compared with mice exposed to ambient air (EAA) in order to study lung injury by morphometrical and biochemical methods. Bronchoalveolar lavage (BAL) analysis and histology and stereology were performed. Tissue from the right lung was used for measuring thiobarbituric acid reactive substances (TBARS) and Western blot analysis. One way anova was performed followed by the Student-Newman Keuls post-test (P < 0.05). The cellular content of BAL was 95% alveolar macrophages in all groups except in mice exposed to 3 LC, where 23% neutrophils were observed. Emphysema was not observed in three and 6 LC, but it was found in 12 LC parallel to increased volume density (Vv) of airspaces from 61.0 +/- 0.6 (EAA) to 80.9 +/- 1.0 (12 LC) and decreased Vv of elastic fibres from 17.8 +/- 0.9 (EAA) to 11.8 +/- 0.6 (12 LC). All exposed groups to LC showed low TBARS levels compared with mice EAA. Lung tissue from animals exposed to 12 LC showed decreased tissue inhibitor of metalloprotease-2 and increased matrix metalloprotease-12 detection, which suggests an imbalance in extracellular matrix (ECM). Increased tumour necrosis factor-alpha and nuclear factor-kappaB detection were observed in exposed groups to LC when compared with mice EAA. The data suggest that LC is so dangerous to lungs as full-flavour cigarettes inducing ECM imbalance and emphysema.