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Brief, high-level nitrogen dioxide (NO(2)) exposures are major hazards during fires and heat-generating explosions. To characterize the lung response to a brief high-level NO(2) exposure, we exposed two groups (n = 5) of 325-375 g, male, Sprague-Dawley rats to either 200 +/- 5 ppm (376 +/- 9 mg/m(3)) NO(2) or room air for 15 min. The rats were nose-only exposed in a multiport exposure chamber fitted with pressure transducers to monitor their respiration during exposure. One hour after exposure, we euthanized the rats, collected blood samples, lavaged the lungs with warm saline, and then excised them. One lung lobe was cooled to -196 degrees C and used for low-temperature electron paramagentic resonance (EPR) analysis. The remainder was homogenized and used for biochemical analyses. Inspired minute ventilation (V(i)) during exposure decreased 59% (p < 0.05). Calculated total inspired dose was 0.880 mg NO(2). In lung lavage, both total and alveolar macrophage cell counts declined (approximately 75%, p < 0.05), but epithelial cell count increased 8.5-fold. Lung weight increased 40% (p < 0.05) after exposure. In the blood, potassium and methemoglobin increased 45 and 18% (p < 0.05), respectively; glucose, lactate, and total hemoglobin were not altered significantly. EPR analysis of lung tissue revealed hemoglobin oxidation and carbon-centered radical formation. Vitamins E and C and uric acid were depleted, and lipid peroxidation measured by three different methods (TBARS, conjugated dienes, and fluorescent peroxidation end products) was elevated, but total protein, DNA, and lipid contents were unchanged. These observations combined demonstrate that a brief (15 min) high-level (200 ppm) NO(2) exposure of rats was sufficient to cause significant damage. However, comparison of the exposure dose normalized to rat body weight with previously reported sheep and estimated human values revealed significant differences. This raises a question about interspecies dosimetry and species-specific responses when animal data are extrapolated to humans and used for safety standard setting, particularly with high-level brief exposures.

Exposure to nitrogen dioxide (NO2) has been shown to activate glutathione metabolism in lung and lung lavage. Since GGT is a key enzyme in glutathione metabolism and we have previously characterized GGT expression in distal lung epithelium and in lung surfactant, we examined the NO2 exposed lung for induction of gamma-glutamyl transferase (GGT) mRNA, protein, and enzyme activity. We found that the GGT gene product is induced in lung by NO2. The GGT mRNA level in lung increases 2-fold within 6 hr and 3-fold after 24 hr of exposure to this oxidant gas, and this 3-fold elevation persists even after 14 days of exposure. The pattern of GGT mRNA expression switches from the single GGT mRNA III transcript in the normal lung to the dual expression of GGT mRNA I and mRNA III. Enzyme activity in whole lung increases 1.6- to 2.5-fold while extracellular surfactant-associated GGT activity accumulates 5.5-fold and GGT protein accumulates in lung surfactant. Induction of GGT mRNA and protein is evident in cells of the bronchioles by in situ hybridization and immunolocalization, respectively. In contrast, alveolar type 2 cells lack an in situ hybridization signal and exhibit a reduction in the intensity of immunostaining with prolonged exposure. Our studies show that NO2 induces GGT mRNA expression, including GGT mRNA1, in lung and GGT protein and enzyme activity in lung and lung lavage in response to the oxidative stress of NO2 inhalation.

Various drugs have been shown to stimulate surfactant phospholipid metabolism. Particularly beta-adrenergic agonists play an important role under physiologic conditions. For the first time we have studied whether nitrogen dioxide (NO2) inhalation alters beta-adrenergic regulation of surfactant phospholipid metabolism in the model of the isolated lung. Rats were continuously exposed in vivo to a 5 ppm NO2-containing atmosphere for 48 hr. The lungs were isolated and perfused in presence of the beta-adrenergic agonist dopexamine and surfactant metabolism was studied in three lung compartments: (1) lung lavage, (2) lung tissue, and (3) lavagable free alveolar cells. We found that (1) in normal rat lungs dopexamine increased the incorporation of palmitate and choline from the perfusate into lung lavage phospholipids. In nitrogen dioxide exposed rat lungs beta-adrenergic stimulation did not cause an increase in precursor incorporation. No significant difference in unstimulated precursor incorporation was found for normal and NO2-exposed rat lungs. (2) Lung tissue from rats exposed to NO2 showed a decreased precursor incorporation into disaturated phosphatidylcholine due to an augmented cellular pool size. (3) Lavagable alveolar cells showed an increased palmitate uptake after nitrogen dioxide inhalation and by beta-adrenergic stimulation. From these data we conclude that nitrogen dioxide inhalation impairs the beta-adrenergic regulation of surfactant phospholipid metabolism. Moreover these data underline the importance of beta-adrenergic agonists in surfactant metabolism.

Rats were exposed for 6 hr per day to either ozone alone (0.2-0.8 ppm), nitrogen dioxide (NO2) alone (3.6-14.4 ppm), or to combinations of these two oxidant air pollutants. Their response was quantified by changes in the total protein content of lung lavage supernatants or by changes in the content of specific cell types in the lung lavage pellets. A concentration-dependent synergistic response was observed when rats were exposed to the combination of ozone and NO2. Apparent threshold concentrations for the observation of synergistic interaction between ozone and NO2 were assay specific, with epithelial cell content of lung lavage fluid being the most sensitive parameter evaluated, showing positive interaction (greater than additive response) at the lowest concentrations tested. Concurrent exposure to ozone and NO2 was necessary to elicit greater than additive responses; no such interactions were seen upon sequential exposure to ozone or NO2 in either order of presentation. Based upon apparent disappearance rates of ozone in the chambers during exposure of rats to ozone and NO2, we modelled the predicted outcomes based upon the assumption that the two oxidant gases were reacting to form nitrogen pentoxide (N2O5) in the chambers. Agreement between predicted concentrations of ozone and NO2 and those actually observed was excellent. Based upon such modelling estimates and our acute toxicological data, we conclude that synergistic toxicologic interactions between ozone and NO2 are found only at concentrations very much higher than would be encountered in environmental or occupational settings. It remains to be determined whether there are any chronic toxicological responses to exposure to combinations of ozone and NO2 at concentrations below the thresholds for observing acute responses.