NO2-exposed Rats Research Articles

Increased Levels of 8-Isoprostane in EBC of NO2-Exposed Rats

Several epidemiological studies have shown the impact on respiratory health of pollution of nitrogen dioxide (NO2), particulate matter (PM10), and ozone (O3) as an environmental mixture. However, the influence of individual components of airborne pollutants is less well known. Our study examined the cumulative effects of a single pollutant, NO2, on sensitized rats by measurement of isoprostane release in exhaled breath condensate (EBC). Three groups of six rats were used: (1) controls (only exposed to air), (2) sensitized and challenged by ovalbumin and exposed to air, and (3) sensitized, challenged by ovalbumin, and exposed to NO2. There was no marked change in 8-isoprostane levels in EBC of sensitized rats, whereas a significant increase of 8-isoprostane was found in rats sensitized and exposed to NO2. Data indicate effect of exposure to NO2 is evident as increased 8-isoprostane levels in EBC, a relevant marker for assessment of pulmonary inflammation or oxidant stress and conventionally found in EBC of asthmatic subjects.

Phenotypical and functional characterization of alveolar macrophage subpopulations in the lungs of NO2-exposed rats

BackgroundAlveolar macrophages (AM) are known to play an important role in the regulation of inflammatory reactions in the lung, e.g. during the development of chronic lung diseases. Exposure of rats to NO2 has recently been shown to induce a shift in the activation type of AM that is characterized by reduced TNF-α and increased IL-10 production. So far it is unclear, whether a functional shift in the already present AM population or the occurrence of a new, phenotypically different AM population is responsible for these observations.MethodsAM from rat and mice were analyzed by flow cytometry for surface marker expression and in vivo staining with PKH26 was applied to characterize newly recruited macrophages. Following magnetic bead separation, AM subpopulations were further analyzed for cytokine, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP) mRNA expression using quantitative RT-PCR. Following in vitro stimulation, cytokines were quantitated in the culture supernatants by ELISA.ResultsIn untreated rats the majority of AM showed a low expression of the surface antigen ED7 (CD11b) and a high ED9 (CD172) expression (ED7-/ED9high). In contrast, NO2 exposure induced the occurrence of a subpopulation characterized by the marker combination ED7+/ED9low. Comparable changes were observed in mice and by in vivo labeling of resident AM using the dye PKH26 we could demonstrate that CD11b positive cells mainly comprise newly recruited AM. Subsequent functional analyses of separated AM subpopulations of the rat revealed that ED7+ cells showed an increased expression and production of the antiinflammatory cytokine IL-10 whereas TNF-α production was lower compared to ED7- AM. However, iNOS and IL-12 expression were also increased in the ED7+ subpopulation. In addition, these cells showed a significantly higher mRNA expression for the matrix metalloproteinases MMP-7, -8, -9, and -12.ConclusionNO2 exposure induces the infiltration of an AM subpopulation that, on the one hand may exert antiinflammatory functions by the production of high amounts of IL-10 but on the other hand may contribute to the pathology of NO2-induced lung damage by selective expression of certain matrix metalloproteinases.

Impaired superoxide radical production by bronchoalveolar lavage cells from NO 2-exposed rats

Production of superoxide radicals is a central property of professional phagocytes used to combat invading microorganisms. Even though the number of macrophages and neutrophils is often increased in the lungs of patients with chronic lung diseases, these patients frequently suffer from bacterially induced exacerbations. To understand the underlying mechanisms, we investigated the production of superoxide radicals by bronchoalveolar lavage (BAL) cells in a rat NO 2 exposure model (10 ppm NO 2 for 1, 3, or 20 days). We showed that cells from NO 2-exposed animals display a significantly impaired superoxide radical release after zymosan stimulation. The use of specific inhibitors (antimycin or diphenyleneiodonium [DPI]) revealed that the major enzyme systems, NADPH oxidase and complex III of the respiratory chain, are affected. In addition, we investigated gene expression and enzyme activities of antioxidant enzymes. mRNA expression was significantly enhanced for glutathione peroxidase (GPx)-3 and CuZn-superoxide dismutase (SOD) in BAL cells from animals exposed 3 and 20 days, and GPx and SOD enzyme activities were increased in BAL cells from rats exposed 20 days. In conclusion, concomitant occurrence of reduced production and increased scavenging of superoxide radicals resulted in the drastically impaired release of these radicals from BAL cells of NO 2-exposed rats.

Effect of N-acetylcysteine treatment on NO2-impaired type II pneumocyte surfactant metabolism.

Inhalation of nitrogen dioxide (NO2) is known to alter the composition of the bronchoalveolar lavage (BAL) and to impair the surfactant metabolism of type II pneumocytes. However, information is sparse as to whether application of the widely used antioxidant N-acetylcysteine (NAC) is capable of preventing or reducing these alterations. The aim of the study was to investigate if in vivo administration of NAC to NO2-inhaling rats protected BAL parameters and physiology of type II pneumocytes from impairment. For this purpose, rats were exposed to 720 p.p.m. h-1 NO2, that was applied continuously, intermittently or repeatedly. During inhalation one group of rats received saline and the other group received NAC antioxidant (200 mg kg-1, intraperitoneally) once a day. The BAL protein and phospholipid content increased most in the continuously and repeatedly NO2-exposed rats when compared to the controls, while the intermittent exposure did not change these parameters. Application of NAC led to a marked decrease of the protein elevation for the continuously and intermittently exposed groups, but exhibited no influence on the BAL phospholipid. Surprisingly, all NO2 exposure modes elevated the glutathione content (reduced and oxidized) in the BAL. Application of NAC clearly decreased the content of both forms of glutathione in the continuously and the repeatedly NO2-exposed groups. Phospholipid synthesis, measured by choline uptake into type II cells, was increased most after continuous NO2 inhalation. The NAC reduced this increase moderately. Whereas choline uptake by type II cells was obviously stimulated by NO2, the stimulated secretion of phosphatidylcholine from these cells was decreased by this oxidant. Only continuous exposure reduced this activity markedly. The NAC clearly restored the impaired secretion activity in the cells from the continuously NO2-exposed animals. Since the efficacy of NAC in the prevention of NO2-induced impairments in the surfactant system is striking mainly in the continuously exposed group, we suggest that administration of NAC to NO2-induced lung injury partially restores altered BAL components and the impaired physiology of type II pneumocytes.

α-tocopherol improves impaired physiology of rat type II pneumocytes isolated from experimentally injured lungs

Oxidant stress delivered by nitrogen dioxide (NO2) inhalation impairs the function of extracellular surfactant as well as surfactant phospholipid metabolism in type II pneumocytes. Because protection against oxidant stress is important to normal lung function, the lung contains a variety of antioxidants, including vitamin E. Whether administration of this antioxidant during NO2 inhalation attenuates NO2-induced alterations in phospholipid metabolism in type II pneumocytes has not been studied. We exposed rats to identical NO2 body doses (720 p.p.m. x h) using continuous, intermittent, or repetitive protocols. During exposure periods, the animals received daily intramuscular injections of vitamin E (25 mg kg-1). We isolated type II pneumocytes from NO2-exposed rats and evaluated them for cell yield and viability, as well as for synthesis and secretion of phosphatidylcholine (PC) as measures of surfactant metabolism. The yield of type II pneumocytes was significantly elevated from animals that had been exposed continuously to NO2 whereas in intermittently and repeatedly exposed rats, cell yield was similar to yield from control animals. Viability of the isolated cells was similar in controls and all NO2 exposure protocols. Vitamin E treatment of the NO2-exposed rats neither changed cell yield nor cell viability. Phospholipid de novo synthesis, as estimated by choline incorporation into PC, was increased most after continuous NO2 inhalation whereas in the other conditions there was only a slight increase. Vitamin E administration further increased phospholipid synthesis; this difference reached statistical significance only in the case of intermittent NO2 exposure. Secretion of phosphatidylcholine from type II cells was only reduced after continuous NO2 inhalation and administration of the antioxidant reduced the impairment. Because vitamin E appears to preserve the ability of type II pneumocytes isolated from NO2-exposed rats to synthesize and secrete surfactant lipid, we conclude that administration of vitamin E may mitigate NO2-induced lung injury.

Biochemical and morphological changes in lung tissue and isolated lung cells of rats induced by short-term nitrogen dioxide exposure

To investigate the effects of repeated exposure to nitrogen dioxide (NO2) on antioxidant enzymes in lung tissue and isolated lung cells, rats were continuously exposed to 20 mg/m3 NO2 (10.6 ppm) for 4 days. The activities of glucose-6-phosphate dehydrogenase (G6PDH), glutathione reductase (GR), and glutathione peroxidase (GSHPx) were measured in the cytosolic fraction of lung tissue of both control and NO2-exposed rats as well as in isolated alveolar macrophages (AMs) and type II cells. Qualitative and quantitative changes in AM and type II cells were studied by electron microscopy and by morphometric analyses using enzyme and immunohistochemistry. NO2 exposure resulted in significantly increased pulmonary activities of G6PDH, GR, and GSHPx, both expressed per lung and per gram of lung weight. Morphometric data show that NO2 exposure significantly increased the number of type II cells, predominantly in the centriacinar region, indicating proliferation of epithelium following cellular injury. Type II cells in lungs of NO2-exposed rats had a squamous, less cuboidal appearance with more lamellar bodies compared to type II cells in lungs of control rats. Compared to control lungs, a higher number of macrophages could be isolated from NO2-exposed lungs, while numbers of type II cells isolated from lungs of control and NO2-exposed rats were the same. Isolated type II cells from control and NO2-exposed rats were polymorphic, with a small number of lamellar bodies and without polarity. Isolated macrophages were rounded and contained many filopodia. NO2 exposure caused increases in the activities of G6PDH and GSHPx in isolated type II cells and of GSHPx in isolated macrophages, when expressed per number of cells. Macrophages and type II cells isolated from control and NO2-exposed rats and re-exposed in vitro to NO2, showed no differences in phagocytosis and viability features. Our results indicate that NO2-induced increases in pulmonary antioxidant enzymes are also reflected in isolated AM and type II cells. Since these lung cells do not display a decreased sensitivities toward an in vitro NO2 exposure, overall increase in antioxidant enzyme activities do not seem to play the most pivotal role in controlling cellular NO2 sensitivity and oxidant defence. Combined data from biochemical, morphological, and morphometric analyses of lungs and lung cells suggest that lung cell and tissue oxidant sensitivity and defence largely depends on the cell and tissue organisation, i.e., cell numbers and morphology as well as the ratio of surface area to cytoplasmic volume.

Biochemical detection of type I cell damage after nitrogen dioxide-induced lung injury in rats

We have previously shown that injury to lung epithelial type I cells can be detected biochemically by measuring the airway fluid content of a type I cell-specific protein, rTI40, in a model of severe acute lung injury [M. C. McElroy, J.-F. Pittet, S. Hashimoto, L. Allen, J. P. Wiener-Kronish, and L. G. Dobbs. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L181-L186, 1995]. The first objective of the present study was to evaluate the utility of rTI40 in the assessment of alveolar injury in a model of milder acute lung injury. Rats were exposed to 18 parts/ million NO2 for 12 h; control rats received filtered air for 12 h. In NO2-exposed rats, the total amount of rTI40 in bronchoalveolar fluid was elevated 2-fold compared with control values (P < 0.001); protein concentration was 8.5-fold of control values (P < 0.001). The increase in rTI40 was associated with morphological evidence of injury to type I cells limited to the proximal alveolar regions of the lung. The second objective was to correlate the severity of alveolar type I cell injury with functional measurements of lung epithelial barrier integrity. NO2 inhalation stimulated distal air space fluid clearance despite a significant increase in lung endothelial and epithelial permeability to protein. These data demonstrate that rTI40 is a useful biochemical marker for mild focal injury and that exposure to NO2 alters lung barrier function. Taken together with our earlier studies, these results suggest that the quantity of recoverable rTI40 can be used as an index of the severity of damage to the alveolar epithelium.

Interaction of air pollutants and pulmonary allergic responses in experimental animals

Asthma, which is primarily an allergic type of respiratory disease, has increased in the U.S and Europe by 30% over the last decade. Air pollution may play a role in this rise, since during episodes of smog, hospital admissions due to asthma increase. Ambient air quality has generally improved since the Clean Air Act was implemented in 1971 however, and has led some investigators to suggest that the increased risk of asthma is associated with a deterioration of indoor air quality through the introduction of closed ventilation systems and constant climate control. Thus, although the direct health effects of acute and chronic air pollutant exposure are not in dispute, emphasis on the sources and location of exposure is changing from outdoors to the home environment and workplace. The few experimental studies which have investigated the interaction of air pollutants with allergic disease have shown that exposure to O 3 or NO 2 can increase levels of allergen-specific antibody and may augment allergic symptoms. These experiments are reviewed along with a study conducted in our laboratory which demonstrated the enhancing effect of NO 2 exposure on immune responses and pulmonary inflammation following sensitization and pulmonary challenge with house dust mite allergen (HDM). In this study, rats exposed to 5 ppm NO 2 for 3 h after each immunization had significantly higher levels of serum IgE and local IgA, IgG and IgE antibody than air controls. Lymphocyte activity in the spleen and local lymph nodes, and pulmonary inflammatory cells were also increased in NO 2-exposed rats. The results show that exposure to NO 2 enhances immune responsiveness and the severity of pulmonary inflammation following antigen challenge. Since allergic individuals and most asthmatics also have increased immunity to these proteins, the possibility that air pollutant exposure enhances immune responses to allergens and thus exacerbates immune-mediated lung disease exists.

Lung surfactant components in bronchoalveolar lavage after inhalation of NO2 as markers of altered surfactant metabolism.

To study the effects of nitrogen dioxide (NO2) inhalation on lung lavage surfactant components as markers of an altered surfactant metabolism in type II pneumocytes, rats were exposed to atmospheres with increasing NO2 concentrations (0.8, 5.0, and 10.0 ppm) over 1 and 3 days. After exposure lung lavage was performed and surfactant components as well as lavageable cells analyzed. An increased number of total lavage cells was found with increasing concentration and duration of NO2 exposure. Cell distribution showed an elevation in the number of granulocytes and lymphocytes whereas the number of macrophages was diminished. The amount of total lavage protein revealed an increase related to NO2 concentration and duration. Also the content of lavage phospholipid was increased, with a decreased portion of phosphatidylcholine (PC). Further analyses of PC showed a diminished composition of saturated fatty acids but an elevated content of the unsaturated portion. Functional studies on surfactant phospholipid extracts exhibited comparable values for the surface tension at equilibrium, as well as for the maximal and minimal surface tension of animals exposed to 0.8 ppm NO2 and controls. Higher NO2 concentrations (5 and 10 ppm) resulted in increased values for surface tension compared to controls. This was also observed with purified surfactant that was obtained from controls and from NO2-exposed rats. These experiments show that in vitro exposure of purified surfactant to NO2 atmospheres was more effective than exposure in vivo. When the structure of the surfactant proteins A was studied it was found not to be altered by the NO2. The data clearly demonstrate that NO2 inhalation impaired function of surfactant components that may be used as markers of altered surfactant metabolism.

Changes in the Response of Lung Mast Cells Isolated from Rats and Guinea Pigs Exposed to Nitrogen Dioxide

To clarify the relationship between exposure to nitrogen dioxide (NO2) and mast-cell responses, rats and guinea pigs were exposed to 0, 1.0, 2.0, and 4.0 ppm NO2 for 12 wk. Although lung wet weights were not changed in both rats and guinea pigs, the number of lung cells from 2.0 and 4.0 ppm NO2-exposed rats were significantly decreased compared to that of control. No difference was observed in the number of lung mast cells from rats and guinea pigs exposed to NO2. in lung mast cells from rats, immunoglobulin E (IgE) mediated histamine release was slightly decreased, but A23187-induced histamine release was not changed. On the contrary, in lung mast cells from guinea pigs, IgE-mediated histamine release was increased in a dose-dependent fashion, though no changes in A23187-induced histamine release were observed. These results suggest that different sensitivity for NO2 exposure exists in lung mast cells from rats and guinea pigs.

Neutrophil recruitment and degranulation during induction of emphysema in the rat by nitrogen dioxide.

Rats exposed to 30 ppm NO2 continuously for 3 to 6 wk developed mild centriacinar air-space enlargement and mild interstitial fibrosis. The emphysematous changes did not become more severe after 6 wk with continued NO2 exposure for as long as 14 additional weeks. Elastase inhibitory capacity (EIC) and the total protein concentration increased acutely in lung lavage fluid, consistent with the histologic evidence of pulmonary edema, then subsided but remained above control levels for the entire exposure period. The ratio of EIC to total protein remained unchanged relative to that in control animals. Neutrophils accumulated in the lung and were recoverable by lavage with the same relative concentration profile as observed for total protein. The average elastase activity per neutrophil in cells recovered from the air space by lung lavage was 60% less than in cells recovered from peripheral blood. This was true for control or NO2-exposed rats. Thus, emphysematous changes in the NO2-exposed rat were accompanied by a marked neutrophil recruitment concomitant with an increased neutrophil elastase burden in the lung, which may have directly contributed to lesion development.

Decreased phagocytosis and superoxide anion production in alveolar macrophages of rats exposed to nitrogen dioxide.

Effects of nitrogen dioxide (NO2) on pulmonary defense systems were investigated by determining phagocytosis and Superoxide anion (O2−) production by alveolar macrophages (AM) of rats. Rats were exposed to 8 ppm NO2 for 1 to 7 days and 4 ppm NO2 for 1 to 10 days, respectively. The phagocytic activity of AM was determined by taking yeast particles into the cells. The O2− production by AM was determined at rest during phagocytosis of zymosan particles and stimulation with phorbol myristate acetate (PMA). The suppression of phagocytosis of AM was observed in cells from rats exposed to 8 ppm NO2 for 5 and 7 days. The suppression was also shown in AM from rats exposed to 4 ppm NO2 for 7 days. There were remarkable decreases in the O2− production by AM from 8 ppm NO2-exposed rats at rest during zymosan phagocytosis and PMA stimulation on and after day 3. The O2− production by AM from 4 ppm NO2-exposed rats at rest and during zymosan phagocytosis decreased on days 3, 5, and 10, but remained unchanged on day 7. The O2− production by PMA-stimulated AM from 4 ppm NO2-exposed rats decreased on day 3. The results suggest that such diminution in phagocytosis and O2− production by AM from NO2-exposed rats demonstrated the adverse effects on the pulmonary early defense systems and potentially causes bacterial infections.

Nitrogen Dioxide Exposure???Influence on Rat Testes

Prolonged intermittent exposure to subanesthetic concentrations of nitrous oxide (N2O) can impair spermatogenesis in the LEW/f mai rat. USP purity standards state that N2O used for medical purposes may contain other oxides of nitrogen such as NO and NO2 as impurities at concentrations up to one part per million (ppm) each. The question thus arises as to whether prolonged exposure to N2O may be associated with adverse health effects from oxides of nitrogen other than N2O, particularly NO2. NO2 has demonstrated biological toxicity at concentrations as low as 0.3 ppm. In this study, rats were exposed to filtered air with and without 1 ppm NO2 for 7 hr/day, 5 days/week, for 21 days. No gross or light microscopy abnormality was found in the testes of NO2-exposed rats. Serum vitamin B12 levels were within normal limits. The findings indicate that NO2 exposure under the test conditions does not in itself impair spermatogenesis or alter B12 levels.

Reflex cardiopulmonary responses by stimulation to type J receptors in rats exposed to NO2.

To examine the role of the vagal pathway on the cardiopulmonary functions in NO2-exposed rats, phenyl diguanide, which stimulates type J receptors in the lungs, was injected to control and exposed rats at a constant dose. Based on a statistical test, a decrease in the heart rate (HR) after the injection was observed in the groups exposed to 20 ppm NO2 for 1.5 h and 3h, and 10 ppm for 24 h. On the other hand, an increase in respiratory rate (RR) was observed in the groups exposed to 10 ppm for 3 h and 4 ppm for 1 wk. No change in HR and RR was found in the group exposed to 0.4 ppm for 4 wk. These results suggest that the augmentation of the reflex cardiopulmonary responses due to stimulation to the type J receptors was produced by exposures with a higher concentration of NO2.

Changes in phospholipid biosynthetic enzymes in Type II cells and alveolar macrophages isolated from rat lungs after NO 2 exposure

The activities of phospholipid biosynthesizing enzymes and subcellular marker enzymes were measured in type II cells and alveolar macrophages isolated from rat lungs 48 hr after exposure to air or 40 ppm NO 2. DNA and protein increased in cell fractions from NO 2-exposed lungs, but NO 2 exposure had no effect on the percentage of type II cells in isolated cell fractions. A general increase in cell content of biosynthetic enzyme activities (units/mg DNA) was observed in type II cells from NO 2-exposed rats, but no change was detected in the activity of the microsomal marker enzyme, NADPH cytochrome c reductase. Glycerolphosphate acyltransferase and choline phosphotransferase increased 171 and 168%, respectively, and phosphatidate phosphohydrolase increased 69%. Glycerolphosphate phosphatidyltranferase increased 143% and succinate cytochrome c reductase, the mitochondrial marker enzyme, increased 111%. Type II cells from control lungs contained a greater than threefold higher activity of the phosphatidylglycerol synthesizing enzyme, glycerolphosphate phosphatidyltransferase, compared to alveolar macrophages, and comparable activities of the enzymes of phosphatidylcholine synthesis and of the microsomal and mitochondrial reductases. Exposure to NO 2 resulted in a significant increase in NADPH cytochrome c reductase activity (46%) in macrophages, but no change in any biosynthetic enzymes. The increases in protein content and activity of phospholipid biosynthetic enzymes in type II cells are consistent with a general hypertrophy of type II cells which includes stimulation of surfactant phospholipid biosynthesis 2 days after exposure to NO 2, when type II cell proliferation is occurring.

The effects of intermittent nitrogen dioxide exposure on vitamin E-deficient and -sufficient rats

In two separate experiments rats fed vitamin E-deficient, normal or high vitamin E-supplemented diets were intermittently exposed to 15 ppm ± 1.0 ppm nitrogen dioxide (NO 2) over a 5-week (4 days/week, total of 31.5 h exposure) or an 18-week (5 days/week, total of 93.5 h exposure) period. In the 5-week, NO 2-exposed rats, the blood methemoglobin levels were not influenced by NO 2 exposure or the level of vitamin E in the diet. Tissues of the rats exposed to NO 2 for 18 weeks showed some histological changes; in the lung, increased atelectasis and alveolar thickening and in the liver, increased granular changes, karyolysis and karyorhexis. These differences were suppressed by increasing levels of dietary vitamin E. Tissue lipofuscin pigment (LFP) concentrations were not affected by NO 2 exposure or dietary vitamin E. Fatty acid distribution of lung lipid extracts showed no changes due to NO 2 exposure; however, some effects of dietary vitamin E could be seen. The results suggest that intermittent NO 2 exposure, under the described conditions, did not cause ultimate changes of the biochemical parameters measured.