Effects of nitrogen dioxide on fatty acid compositions of red cell membranes, sera, and livers in rats

Male Wistar rats were exposed to nitrogen dioxide (NO₂) at concentrations of 4 and 10 ppm for 10 and 7 days, respectively, in order to obtain evidence of changes in fatty acids of red cell membranes, sera, and liver supernatants. A significant increase in the percentage of arachidonic acid (20:4) to the total fatty acid was observed in red cell membranes during exposure to NO₂ at both concentrations. This increase accompanied a decrease in palmitic acid (16:0) and/or stearic acid (18:0), so that the ratio of major unsaturated fatty acids to major saturated fatty acids was raised to 1.25- and 1.33-fold at concentrations of 4 and 10 ppm, respectively. Consistently, the percentage of 20:4 in serum showed a highly significant increase simultaneously with a less significant decrease in oleic acid (18:1) and linoleic acid (18:2) upon 7-day exposure to 10 ppm NO₂. It must be noted that a decrease in 16:0 and an increase in 18:0 were also observed with high significance. In addition, the fatty acid composition of serum phosphatidylcholine (PC) paralleled the fatty acid composition of sera, but these changes were more pronounced. The percentage of 20:4 was increased to 1.53-fold that of the control. Exposure to 4 ppm NO₂ for 10 days resulted in the same profile of changes in serum PC as that of 10 ppm NO₂ inhalation, although the magnitude of changes was smaller. These results may support the assumption that 20:4 in serum is preferentially incorporated into red cell membranes. During exposure to 4 ppm NO₂ the percentage of 20:4 in serum PC increased progressively in striking contrast to a decrease in 18:2. A precursor—product relationship of these fatty acids was also suggested by the alteration profile in liver supernatant fraction (S₁₀₅). It is, therefore, assumed that synthesis of 20:4 molecules might be stimulated in the liver upon exposure to 4 ppm NO₂. Exposure to 10 ppm NO₂ caused completely different and more complex alterations of the percentages of 18:2 and 20:4 in serum PC and liver S₁₀₅. In serum PC the percentage of 20:4 stayed at a higher level than that of the control and that of 18:2 remained at a lower level in the whole period of exposure. On the other hand, the percentage of 20:4 in liver S₁₀₅ showed a value rather lower than that of the control, while that of 18:2 stayed at a higher level. From these results differences between effects of 4 and 10 ppm NO₂ are discussed.     

Nitrogen Dioxide Inhalation and Human Blood Biochemistry

Blood from ten young adult male humans, exposed to 1 ppm or 2 ppm nitrogen dioxide (NO₂) for 2.5–3.0 hr, was examined for evidence of biochemical changes. The experiments lasted three days. The subjects entered an environmental chamber, performed mild exercise, and completed a series of measurements of pulmonary physiology while breathing filtered air. Blood samples were then taken and analyzed. This regimen was repeated on the second and third day, except that the chamber atmosphere now contained 1 ppm or 2 ppm NO₂.Paired group analyses were performed on the data. A statistically significant decrease was observed in the activity of the erythrocyte membrane enzyme acetylcholinesterase at both NO₂ levels. Levels of peroxidized red blood cell lipids showed statistically significant elevations after inhalation of 2 ppm NO₂ but not 1 ppm. Glucose-6-phosphate dehydrogenase was significantly elevated only after the second 2-ppm NO₂ exposure. Small but statistically significant decreases were observed in both hemoglobin and hematocrit values after exposure to both NO₂ levels.

The experiment was repeated with NO₂, (i.e., three days of filtered air) to detect possible effects of the experimental procedure. Decreases were again seen in hemoglobin and hematocrit, and acetyecholinesterase, although of smaller magnitude than when NO₂ was inhaled. Other data showed random variations that were not additive over the three-day sham exposure period.

It was concluded that significant blood biochemical changes resulted from NO₂ inhalation, although the three-day experimental regimen independently produced changes that account for some of the apparent response.     

In vivo effects of nitrogen dioxide on membrane constituents in lung and liver of rats

When rats were exposed to 10 ppm NO₂ for 7 days, the succinate-cytochrome c reductase activity, the rate-limiting step of mitochondrial succinoxidase, of the liver decreased progressively, reaching 77% (P < 0.01) of the control level by the fifth day. By contrast, this activity was gradually increased by a 10-day exposure to 4 ppm NO₂ and reached 1.14-fold that of the control level at the seventh day. A reduction in the components of electron-transport systems in liver microsomes was found during exposure to NO₂ at both concentrations. This reduction was preferential for components of drug-metabolizing systems. The NADPH-cytochrome c reductase activity and the cytochrome P-450 content were decreased to 82% (P < 0.05) and 76% (P < 0.05), respectively, at the fifth day of exposure to 10 ppm NO₂. Exposure to 4 ppm NO₂ also caused a significant decrease in the NADPH-cytochrome c reductase and the cytochrome P-450 content, which were lowered to 84% (P < 0.05) of the control level at the fourth and seventh days, respectively. Reduction in these components appears to occur even at the concentration of 0.4 ppm at the seventh day. Alterations in the components of mitochrondria and microsomes of the lung were considerably different from those of the liver. The protein content of the lung increased to 1.18 (P < 0.05)- and 1.14 (P < 0.05)-fold that of the control during exposure to 10 and 4 ppm NO₂, respectively. The succinate-cytochrome c reductase activity showed a reduced value, which was 73% (P < 0.01) of the control level, 1 day after exposure to 10 ppm NO₂. Subsequently, the activity increased to 1.14-fold that of the control during exposure to 10 (at the fifth day) and 4 ppm (at the fourth day) NO₂. Components of microsomal electron-transport systems also showed a slightly elevated value during exposure to 4 ppm NO₂. Seven days after exposure, the cytochrome P-450 content was decreased to 61% (P < 0.01) of the control level, while other components were retained at control levels. A significant reduction in the cytochrome P-450 content was also observed at the exposure concentration of 1.2 ppm, but not of 0.4 ppm, at the seventh day.     

Cardiopulmonary effects of short-term nitrogen dioxide exposure in conscious sheep

We used conscious sheep to (a) evaluate the airway effects of 2-hr exposures to 7.5 and 15 ppm NO₂, and (b) to measure pulmonary hemodynamics after a 4-hr exposure to 15 ppm NO₂. Bronchial reactivity to aerosolized carbachol (BR), tracheal mucous velocity (TMV), and mechanics of breathing were determined to assess whether NO₂ induced airway effects immediately and 24 hr after exposure. The hemodynamic measurements included arterial blood gases, estimates of diffusing capacity, combined capillary blood and tissue volume, cardiac output, and pulmonary vascular pressures. In 10 sheep 7.5 ppm NO₂ did not significantly alter mean pulmonary resistance prior to carbachol challenge (Rpul), BR, or TMV. However, in 5 of these 10 animals, enhanced BR was apparent immediately after the exposure. In 10 animals, 15 ppm NO₂ did not produce significant changes in mean Rpul or BR, although in 7 animals there was a mean increase of 113% in Rpul immediately after the exposure, while BR was evident in 2 other sheep. TMV was, however, significantly depressed immediately after this exposure. In six sheep, who breathed 15 ppm NO₂ for 4 hr, Rpul was significantly elevated immediately after the exposure. In the same animals, there were no changes in arterial blood gases, cardiac output, pulmonary vascular pressures, systemic pressure, diffusing capacity, and combined pulmonary tissue plus capillary blood volume. We conclude that in conscious sheep (a) exposures to 7.5 and 15 ppm NO₂ for 2 hr may induce BR or elevations in Rpul, (b) exposure to 15 ppm NO₂ for 2 hr depresses TMV, while exposure to 7.5 ppm NO₂ for 2 hr has no effect on TMV, and (c) 15 ppm NO₂ for 4 hr has no effects on pulmonary hemodynamics.     

Cell Renewal in the Lungs of Rats Exposed to Low Levels of NO2

Effects of continuous exposure to low concentrations of NO₂ on cell proliferation in bronchiolar and alveolar tissues were determined. Young rats were exposed continuously for as long as 360 days, removed at intervals, and killed. Dividing cells were labeled with tritiated thymidine. Animals exposed to 17 ppm NO₂ showed an Increased labeling index in terminal bronchioles and alveoli after two to three days of exposure; the index returned to control levels by five days. Similar results were found in animals exposed to 2 ppm NO₂, but the increase was not as great. This resulted in epithelial hyperplasia of terminal bronchioles and increased cellularity of alveoli associated with the distal ends of the terminal bronchioles. In peripheral alveoli, there was an increase in turnover rate of type 2 alveolar cells. All of these changes occurred within three days of continuous exposure. After this time, the areas of cellularity did not increase In size and the labeling indexes returned to normal for the remainder of the study.     

Effect of subacute exposure to NO2 on lymphocytes required for antibody responses

$\text{BALB}\text{c}$ mice were continuously exposed to 0.4 and 1.6 ppm NO₂ for 4 weeks and the effects on lymphocytes which are required for primary and secondary antibody responses to sheep red blood cells were examined in vitro. The primary antibody response was significantly suppressed by both concentrations of NO₂, whereas the secondary antibody response was slightly stimulated by 1.6 ppm NO₂ exposure. In reconstitution experiments no significant differences were observed in the activities of T and B lymphocytes from mice exposed to 1.6 ppm NO₂.     

Immunologic response in vaccinated mice during long-term exposure to nitrogen dioxide.

Swiss albino mice were continuously exposed to either 2 ppm NO₂, 0.5 ppm NO₂ with daily 1-hour peaks of 2 ppm NO₂, 5 days a week, or to filtered air. After a 12-week exposure the mice were vaccinated with A₂/Taiwan influenza virus vaccine and the exposures to the various environmental conditions continued for an additional 28 weeks. At 2 weeks after vaccination, the serum neutralizing (SN) antibody titers and SN seroconversion rates were depressed to a various extent in mice exposed to NO₂. At 4 weeks after the vaccination and later, the SN antibody titers did not differ significantly among the various groups.Nonvaccinated mice exposed to NO₂ for 12 weeks showed a marked decrease in concentrations (mg/ml) of serum IgA, and an increase in serum IgM, IgG₁ and IgG₂ immunoglobulins. When concentrations of immunoglobulins were adjusted for age of mice after vaccination, a significant elevation was observed in serum IgM, IgG₁ and IgG₂ of mice exposed to NO₂ as compared to controls. During the 2-week period immediately following the vaccination, exposure to NO₂ appeared to be the predominant factor influencing the percentage concentration of the four serum immunoglobulins.     

Effect of lung damage by acute exposure to nitrogen dioxide on lung immunity in the rat

Exposure to nitrogen dioxide (NO₂) damages Type I alveolar epithelial cells and the epithelium of the terminal bronchiole. Because an intact epithelium may help control the number of inhaled particles that are cleared by the pulmonary lymphatics, damage to the alveolar epithelium could alter the antigen load to the lung-associated lymph nodes (LALN). To determine the effects of lung damage by NO₂ inhalation on lung immunity, we exposed adult, male rats to 26 ppm NO₂ for 24 hr at various time intervals before and after intratracheal immunization with 10⁸ sheep red blood cells (SRBC). Seven days after immunization, we determined the number of anti-SRBC antibody-forming cells (AFC) in the LALN, cervical lymph nodes, and spleen. The immunologic response to SRBC was limited to the LALN, with few or no AFC in either the cervical lymph nodes or spleen. A fivefold increase in the number of IgG anti-SRBC $\text{AFC}\text{10}{}^6$ LALN cells was evident in rats immunized 1 day after NO₂ exposure. The increase was followed by a slight suppression of the IgG response when rats were immunized 3 days after exposure, returning to normal levels by 7 days after exposure. Histopathological examination of lung tissues showed a slight respiratory bronchiolitis which was followed by a bronchiolar epithelial cell hyperplasia and Type II cell hyperplasia in the adjacent alveoli. Based on these observations, it appears that the fluctuations observed in the LALN response may be the result of damage and subsequent repair of bronchiolar and alveolar epithelium following NO₂ inhalation.     

Chronic Toxicity of NO2 in Squirrel Monkeys

Continuous exposure to 10 and 5 ppm nitrogen dioxide (NO₂) for one and two months, respectively, increased the susceptibility of squirrel monkeys to airborne Klebsiella pneumoniae as demonstrated by mortality and reduced lung clearance of viable bacteria. The respiratory function was also affected by NO₂ exposure. Infectious challenge with influenza virus 24 hours before exposure to 10 ppm was fatal to all monkeys within three days. Infected controls showed symptoms of viral infection but did not succumb to the infection. Exposure to 5 ppm after viral infection produced death in one of three monkeys.     

Long-Term Nitrogen Dioxide Exposure

Male Long Evans hooded rats were chronically exposed to an average 2.9 ppm (±0.71 SD) of nitrogen dioxide 24 hours each day, five days a week for nine months. The major effects of NO₂ were a significant 12.7% increase in lung wet weight, a 13.0% decrease in lung compliance, and a significant reduction in surface-active properties of the lung wash. The NO₂-exposed lungs also revealed a significant 8.7% decrease in lung lipid content and a marked decrease in percentage of total saturated phospholipid fatty acids. This reduction in saturation was due almost entirely to a decrease in the percentage of palmitic acid. The suggestion is made that alterations in lung lipid metabolism may be the underlying mechanism which leads to some of the pulmonary effects following long-term exposure to subacute levels of NO₂.     

Biochemical effects of acute and subacute nitrogen dioxide exposure in rat lung and bronchoalveolar lavage fluid

The pulmonary inflammatory response to NO₂ exposure was measured by evaluating a series of biochemical and cellular parameters in rat bronchoalveolar lavage fluid. Animals were exposed to 9 mg/m³ (5 ppm) or 18 mg/m³ (10 ppm) of the gas for 24 h or 7 days. After bronchoalveolar lavage collection, a differential count of polymorphonuclear leukocytes, macrophages, and lymphocytes was done. A significant increase in polymorphonuclear leukocytes was found after 24 h of exposure, and after 7 days the number of macrophages increased significantly. After 7 days of exposure to 9 mg/m³ of NO₂ (a dose that under our conditions did not induce migration of cells in the bronchoalveolar spaces) the ex vivo phorbol myristate acetate-induced superoxide anion production by resident cells was inhibited. After 24 h and 7 days of exposure to 18 mg/m³ of NO₂, phorbol myristate acetate-induced superoxide anion production was lower than in the control group. The migration of polymorphonuclear leukocytes in the bronchoalveolar lavage fluid was not associated with any real increase in elastase. However, there was a dose- and time-dependent increase in 1-proteinase inhibitor in response to both 9 and 18 mg/m³ of NO₂. Total glutathione was significantly increased in blood by 24 h treatment with 9 or 18 mg/m³ of NO₂, whereas blood oxidized glutathione was not affected. In lung tissue we observed only a significant increase of oxidized glutathione after 24 h of exposure to 9 and 18 mg/m³ of NO₂. These data suggest that many biochemical and cellular parameters are altered after acute or subacute exposure to relatively high doses of NO₂, especially in the first 24 h. The increase of 1-proteinase inhibitor and blood glutathione can be considered a prompt protective response to the toxic injury.     

Experimental suppression of tolerance to ozone and of cross-tolerance (NO2O3) in rats by actinomycin D and colchicine

Rats were initially exposed to 2 ppm ozone or 16–20 ppm NO₂ for 3 hr in order to induce a certain tolerance to ozone during 3 days after the initial exposure. After the initial exposure, the y were intraperitoneally administered actinomycin D or colchicine at a scheduled interval. Twenty-four hours after the administration, the induction of tolerance was assayed by the response of lung weight to challenge exposure to 5.6 ppm ozone for 3 hr. Either actinomycin D or colchicine administered immediately after the initial exposure suppressed the induction of tolerance (O₃O₃) and of cross-tolerance (NO₂O₃), while the tolerance was not suppressed by the inhibitors administered 12 to 24 hr following the initial exposure. Normal tolerance to ozone was induced 12 to 24 hr after the initial exposure. It was thus shown that the induction of tolerance could not be suppressed by the inhibitors administered after the lungs became fully tolerant; we tentatively interpret these results to suggest that no tolerance is induced without pulmonary cell proliferation stimulated by the initial exposure.     

Effect of NO2 inhalation and vitamin C deficiency on protein and lipid accumulation in the lung

Vitamin C-deficient and normal guinea pigs were exposed to various concentrations of NO₂ or air, and lavage fluid was obtained and analyzed for protein and lipid content. Exposure of normal animals to 752, 1880, 5640, or 9400 μg NO₂/m³ (0.4, 1.0, 3.0, or 5.0 ppm) for 72 hr did not alter the protein or lipid content of lung lavage fluid. However, exposure of vitamin C-deficient animals to the same concentrations of NO₂ caused marked increases in lavage proteins and lipids at all but the 752 μg/m³ (0.4 ppm) level. At 9400 μg NO₂/m³ (5.0 ppm), 50% of the exposed vitamin C-deficient animals died, and pathologic study of the lungs showed proteinaceous edema fluid in the alveoli. Lungs from air-exposed animals and normal animals exposed to NO₂ appeared healthy. No effects were seen at 752 μg NO₂ (0.4 ppm) in either normal or deficient animals even when the time of exposure was extended to 1 week. At 9400 μg NO₂/m³ (5 ppm) effects could be seen in vitamin C-deficient animals even when the exposure period was shortened to 3 hr. Assessment of protein and lipid content of lavage fluid provided a sensitive method for determining subtle changes in the lung following NO₂ exposure.     

Suppression of antibody response in mice by acute exposure to nitrogen dioxide: In vitro study

The effect of acute exposure to nitrogen dioxide (NO₂) on in vitro primary and secondary antibody responses to sheep red blood cells (SRBC) was studied in $\text{BALB}\text{c}$ mice. After the in vivo exposure to 20 ppm NO₂ for 12 hr, spleen cells were separated and then cultured with SRBC. Cell reconstitution experiments indicated that in the primary antibody response B cells were more strongly suppressed than T cells, while in the secondary response suppression of primed T cells was greater than that of primed B cells.     

Early Response of Lungs to Low Levels of Nitrogen Dioxide

Significant changes in the lungs of rats exposed to nitrogen dioxide occur during the first 72 hours in animals exposed to 17 ppm. These changes appear in focal areas at the level of the terminal bronchiole: loss of cilia; thickening of tissue; injury to the epithelium lining the adveoli adjacent to the terminal bronchioles, when whole type 1 cells slough away leaving the basement membrane exposed to the air; between 24 and 48 hours of continuous exposure, these areas are repaired with a low cuboidal cell type that tolerates NO₂ and thickens the air-blood barrier; and eventual formation of crystalloids and interruption of ciliogenesis. Changes at 2 ppm include loss of cilia, hypertrophy and focal hyperplasia in the epithelium of the terminal bronchiole, and “apparent” return to normal after 21 days of continuous exposure.     

Experimental Studies on Human Health Effects of Air Pollutants. IV. Short-Term Physiological and Clinical Effects of Nitrogen Dioxide Exposure

Adult male volunteers were exposed to nitrogen dioxide (NO₂) at 1.0 ppm in purified air under conditions simulating ambient photochemical smog exposures (2-hr exposure with intermittent light exercise at 31°C and 35% relative humidity). Sham exposures to purified air alone served as controls. Exposure effects were assessed by pulmonary physiological tests and by a standardized clinical evaluation. No statistically significant physiological changes attributable to NO₂ exposure were found except for a marginal loss in forced vital capacity after exposure on two successive days (1.5%mean decrease p<.05). Reported respiratory and other symptoms were slightly increased with exposure as compared to control, but the change was not significant. Short-term toxicity of NO₂ at peak ambient concentrations appears to be substantially less than that of ozone in healthy people, but adverse NO₂ effects in diseased people or in long-term exposures cannot be ruled out at present.     

Pulmonary NO2 toxicity: Phosphatidylcholine levels and incorporation of [3H]thymidine into DNA

Lungs from rats exposed to 30 ppm NO₂(μl/liter) for either 1 or 5 hr had higher incorporation rates of [³H]thymidine into DNA and greater amounts of phosphatidylcholine than the lungs from control animals. Although there was no difference between the pulmonary phosphatidylcholine levels in rats exposed to NO₂ for 1 or 5 hr, the incorporation of [³H]thymidine into DNA was considerably higher in the lungs of animals exposed to NO₂ for 5 hr than in those exposed for 1 hr. Analyses of autoradiograms of lung tissue showed essentially the same labeling index for the alveoli from animals exposed to NO₂ for 1 or 5 hr; however, the animals exposed to NO₂ for 5 hr had a greater number of labeled cells in the lower airways and alveolar ducts than in the lungs from rats exposed for only 1 hr. These results suggest that the increased levels of phosphatidylcholine after exposure to NO₂ are directly related to damage of the alveolar cells, whereas the incorporation of [³H]thymidine into lung DNA represents more widespread damage to areas involving the lower airways. Second and third exposures (24 hr apart) of rats to 30 ppm NO₂ for either 1 or 5 hr caused little increase in the incorporation of [³H]thymidine into pulmonary DNA and no further increase in lung phosphatidylcholine levels. Decreased incorporation of thymidine into the DNA of pulmonary cells after second and third exposures to NO₂ is referred to as “adaptation” in this report. When NO₂-adapted animals were again exposed to NO₂ 7 days after the previous NO₂ treatment, the lungs again exhibited increases in both [³H]thymidine incorporation and levels of phosphatidylcholine.     

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 (O ₂ ^– ) 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 O ₂ ^– 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 O ₂ ^– production by AM from 8 ppm NO₂-exposed rats at rest during zymosan phagocytosis and PMA stimulation on and after day 3. The O ₂ ^– 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 O ₂ ^– production by PMA-stimulated AM from 4 ppm NO₂-exposed rats decreased on day 3. The results suggest that such diminution in phagocytosis and O ₂ ^– production by AM from NO₂-exposed rats demonstrated the adverse effects on the pulmonary early defense systems and potentially causes bacterial infections.     

In vitro methemoglobin formation in human blood exposed to NO2

The in vitro formation of methemoglobin in human blood was determined for various NO₂ concentrations and exposure times. Blood was exposed either to measured amounts of NO₂ in air or to a continuous flow of known concentrations of NO₂ in air. CO₂ was added to the gas phase to maintain pH and PCO₂ in a normal range. Exposure to 45 ppm NO₂ oxidized 95% of the total hemoglobin (THb) in 5 hr. Six ppm NO₂ oxidized 17% of THb in 3 hr. Differences between in vitro and in vivo NO₂ results are discussed.     

Effect of 0.62 ppm NO2 on cardiopulmonary function in young male nonsmokers

Cardiopulmonary and metabolic responses of three groups, each consisting of five adult males (aged 20–25), were determined before, during, and after a 2-hr exposure to 0.62 ± 0.12 ppm NO₂ at 25°C and 45% RH. The three groups exercised during exposure at 40% of V?_{O2 max} for either 15, 30, or 60 min for Groups A, B, and C, respectively. During the exercise periods the ventilation was about 33 liter/min, a fourfold increase over the resting level. There were no physiologically significant cardiovascular, metabolic, or pulmonary function changes which could be attributed to exposure to this level of NO₂ (0.62 ppm). There were no differences between the groups in their response despite the fact that Groups A and B received more NO₂ as a result of 28% and 84% greater ventilations, respectively.