Effects of exposure to 4 ppm nitrogen dioxide in healthy and asthmatic volunteers

Healthy and asthmatic volunteer subjects (N = 25 and N = 23, respectively) were exposed twice each to purified air (control) and to 4 ppm nitrogen dioxide (NO2) in a controlled-environment chamber. Exposures lasted 75 min, and included 15 min each of light exercise (ventilation rate near 25 L/min) and heavy exercise (near 50 L/min). Compared to control, NO2 exposure produced no statistically significant untoward effects on airway resistance, symptoms, heart rate, skin conductance, or self-reported emotional state in normal or asthmatic subjects. Exercise was associated with significantly (P less than .001) increased airway resistance in both subject groups, although the increase in normals was small. In both groups, systolic blood pressure showed small but significant (P less than .01) decreases with NO2 exposure, compared to control. This effect, if real, may relate to formation of a vasodilating nitrite or nitrate from inhaled NO2. The lack of respiratory response contrasts with previous findings elsewhere; at present, this inconsistency is unexplained.     

The effects of sulfur dioxide on pulmonary function in healthy nonsmoking male subjects aged 55 years and older

To determine whether normal nonsmoking individuals aged 55 years or greater have heightened bronchial reactivity to inhaled SO2, ten male subjects, 55 to 73 years of age, were exposed for 20 min at rest and 10 min during moderate exercise on a treadmill to the following: NaCl droplet aerosol, or 1.0 ppm of SO2 and NaCl droplet aerosol. Seven of the subjects also were exposed to 0.5 ppm SO2 and NaCl droplet aerosol. Significant decrease in forced expiratory volume in one sec (FEV1) were seen 2-3 min post-exercise following all three test modes. The reduction in FEV1 seen after NaCl aerosol + 1.0 ppm SO2 was significantly greater than that seen after NaCl aerosol alone. The results show that men aged 55 years or older are somewhat more sensitive to NaCl aerosol + 1.0 ppm SO2 than similarly exposed normal adolescents, but not nearly as sensitive as asthmatic subjects. This study also demonstrates that investigations of air pollutants and exercise can be undertaken in subjects of this age.     

Health effects of short-term inhalation of nitrogen dioxide and ozone mixtures.

The effects of single and multiple daily 3-hour exposures to nitrogen dioxide (NO₂) and ozone (O₃) mixtures on the resistance to streptococcal pneumonia were investigated. The concentrations of NO₂ ranged from 1.5 to 5.0 ppm, and those of O₃, from 0.05 to 0.5 ppm. The effect of a single exposure to the mixture was additive, whereby the excess mortality rates were equivalent to those induced by the inhalation of each individual pollutant. The ability to clear inhaled bacteria from the lungs was diminished in mice exposed to the NO₂O₃ mixtures for 3 hours. This impairment was manifested by the increased frequency of isolation of Streptococcus from the lungs for up to 6 days after the respiratory challenge. Excess mortalities observed after 20 daily 3-hour exposures suggested that a synergistic effect might be present upon repeated inhalation of pollutant mixtures, that made them more effective in reducing resistance to respiratory infection. The results emphasize the need for the establishment of primary air quality standards for short-term NO₂ exposures.     

Effects of 0.60 PPM nitrogen dioxide on circulating and bronchoalveolar lavage lymphocyte phenotypes in healthy subjects

Nitrogen dioxide (NO2) is a common oxidant air pollutant. Animal studies have suggested that NO2 exposure causes a decrease in the numbers of some splenic lymphocyte subtypes and impairs lymphocyte-dependent immune responses. To investigate whether ambient levels of NO2 alter circulating and bronchoalveolar lavage fluid (BALF) human lymphocytes, we studied five healthy nonsmoking adult volunteers. In each subject, blood and bronchoalveolar lavage fluid was obtained and then, more than 2 weeks later, volunteers were exposured to 0.60 ppm NO2 for 2 hr with intermittent light to moderate exercise on 4 separate days within a 6-day period. We measured standard tests of pulmonary function (airway resistance, thoracic gas volume, maximal expiratory flow) and had the subjects rate the severity of respiratory symptoms before and after each NO2 exposure. Circulating and BALF lymphocytes were labeled with fluorochrome-conjugated monoclonal antibodies to human lymphocyte antigens and a flow cytometer was used to count lymphocyte subtypes. Neither any single day's exposure nor all four exposures caused a change in symptoms or in the results of tests of pulmonary function. The total number of circulating lymphocytes obtained after NO2 exposure was slightly greater than at baseline (1792 +/- 544 vs 1598 +/- 549 cells/mm3 at baseline; P = not significant) but the proportions of lymphocyte subtypes did not differ. In the BALF obtained after NO2 exposure and in the baseline state, the total number of lymphocytes and the percentages of T cells (CD 3), B cells (CD 20), T cytotoxic-suppressor cells (CD 8), T helper-inducer cells (CD 4), and large granular lymphocytes (CD 57) also did not differ after NO2 exposure. A slightly but significantly greater proportion of natural killer cells (CD 16) was found in the BALF obtained after NO2 exposure (7.2 +/- 3.1 vs 4.2 +/- 2.4% of total lymphocytes). We conclude that repeated exposures of healthy nonsmoking adults to 0.60 ppm NO2 are not associated with clinically significant symptoms, changes in airway caliber, or alterations in circulating and BALF lymphocyte subtypes. We suggest that brief, daily exposures to NO2 at levels higher than those achieved in urban atmosphere are unlikely to provoke acute respiratory impairment in healthy, nonsmoking adults.     

Protective effects of dietary -tocopherol in rats exposed to toxic levels of ozone and nitrogen dioxide

The effect of dietary α-tocopherol at various levels, especially in the range 0–45 mg/kg, on the acute toxicity of O₃ and NO₂ to rats was studied. The highest level of α-tocopherol was 1500 mg/kg of diet in combination with ascorbic acid, dl-methionine and butylated hydroxytoluene; the level of O₃ ranged from 0.7 to 16 ppm and NO₂ from 20 to 25 ppm; and various exposure periods were used. α-Tocopherol alone and in combination with the other antioxidants exerted a protective effect in rats exposed to O₃ and NO₂ as measured by acute toxicity and animal weight change. During 1 ppm O₃ exposure voluntary activity decreased, while at lower levels of O₃ (0.7–0.8 ppm) α-tocopherol protection against lipid peroxidation in the lung, as measured by endogenous lung thiobarbituric acid reactants, was a reciprocal function of the logarithm of dietary α-tocopherol. Extracts of lung tissue from animals exposed to O₃ and O₂ showed no increase in fluorescent products associated with lipid peroxidation.     

The effect of duration of exposure on sulfuric acid-induced pulmonary function changes in asthmatic adolescent subjects: a dose-response study.

To evaluate the pulmonary effects of varying doses of sulfuric acid, adolescent subjects with asthma were exposed to 35 or 70 micrograms/m3 sulfuric acid for 45 or 90 min. Exposure was carried out during intermittent moderate exercise. The pulmonary functions measured before and after exposure were FEV1, FVC, and total respiratory resistance. The 45 min exposures were associated with larger decreases in FEV1 (-6% or -3%) than the 90 min exposures (-1% or +2%). Analysis of variance of the change in FEV1 among the exposures revealed that the 45 min exposure to 35 micrograms/m3 was significant (p = 0.03). The p value for 45 min exposure to 70 micrograms/m3 was not significant (p = 0.08). Using analysis of variance, neither of the 90 min exposures was associated with a significant decrease in FEV1 compared to air exposure. Also, none of the changes in FVC or RT was significant. When baseline to post-exposure changes were compared for each of the five test atmospheres using paired t tests, both of the 45 min exposures were associated with statistical significance (p < 0.001 for 35 micrograms/m3 and p < 0.005 for 70 micrograms/m3). This baseline to post exposure change was not statistically significant for the 90 min exposures. The reason for the lesser effect on pulmonary function at increased exposure duration is not known; it may be due to changes in either varying deposition patterns or changes in buffering capacity of the cells lining the airways. With respect to individual sensitivities to H2SO4, the data showed a significant consistency across test atmospheres.     

Effects of nitrogen dioxide on pulmonary function in human subjects: an environmental chamber study.

Twenty human subjects with asthma and chronic bronchitis and 10 normal, healthy adults were exposed to 0.5 ppm of nitrogen dioxide for 2 hr in an environmental chamber. Seven of the 13 subjects with asthma experienced symptoms with exposure, while only one each of the subjects with chronic bronchitis and the healthy, normal group experienced symptoms. Significant pulmonary function changes from control values with exposure to NO₂ were observed in decreased quasistatic compliance for the 10 normal subjects and the 20 subjects with asthma and chronic bronchitis. In addition, functional residual capacity increased significantly for the 20 subjects with asthma and chronic bronchitis. The subjects with asthma and the subjects with chronic bronchitis as separate groups, however, did not show any significant changes with exposure. With this study we are reasonably confident that exposure of subjects with asthma and chronic bronchitis to 0.5 ppm NO₂ for 2 hr does not produce a significant decrement in their pulmonary function.     

Nitrous acid, nitrogen dioxide, and ozone concentrations in residential environments

Nitrous acid (HONO) may be generated by heterogeneous reactions of nitrogen dioxide and direct emission from combustion sources. Interactions among nitrogen oxides and ozone are important for outdoor photochemical reactions. However, little is known of indoor HONO levels or the relationship between residential HONO, NO(2), and O(3) concentrations in occupied houses. Six-day integrated indoor and outdoor concentrations of the three pollutants were simultaneously measured in two communities in Southern California using passive samplers. The average indoor HONO concentration was 4.6 ppb, compared to 0.9 ppb for outdoor HONO. Average indoor and outdoor NO(2)concentrations were 28 and 20.1 ppb, respectively. Indoor O(3) concentrations were low (average 14.9 ppb) in comparison to the outdoor levels (average 56.5 ppb). Housing characteristics, including community and presence of a gas range, were significantly associated with indoor NO(2) and HONO concentrations. Indoor HONO levels were closely correlated with indoor NO(2) levels and were about 17% of indoor NO(2) concentrations. Indoor HONO levels were inversely correlated with indoor O(3) levels. The measurements demonstrated the occurrence of substantial residential indoor HONO concentrations and associations among the three indoor air pollutants.     

Estimated acute effects of ambient ozone and nitrogen dioxide on mortality in the Pearl River Delta of southern China

Background and objectives: Epidemiologic studies have attributed adverse health effects to air pollution; however, controversy remains regarding the relationship between ambient oxidants [ozone (O₃) and nitrogen dioxide (NO₂)] and mortality, especially in Asia. We conducted a four-city time-series study to investigate acute effects of O₃ and NO₂ in the Pearl River Delta (PRD) of southern China, using data from 2006 through 2008.Methods: We used generalized linear models with Poisson regression incorporating natural spline functions to analyze acute mortality in association with O₃ and NO₂, with PM₁₀ (particulate matter ≤ 10 μm in diameter) included as a major confounder. Effect estimates were determined for individual cities and for the four cities as a whole. We stratified the analysis according to high- and low- exposure periods for O₃.Results: We found consistent positive associations between ambient oxidants and daily mortality across the PRD cities. Overall, 10-μg/m³ increases in average O₃ and NO₂ concentrations over the previous 2 days were associated with 0.81% [95% confidence interval (CI): 0.63%, 1.00%] and 1.95% (95% CI: 1.62%, 2.29%) increases in total mortality, respectively, with stronger estimated effects for cardiovascular and respiratory mortality. After adjusting for PM₁₀, estimated effects of O₃ on total and cardiovascular mortality were stronger for exposure during high-exposure months (September through November), whereas respiratory mortality was associated with O₃ exposure during nonpeak exposure months only.Conclusions: Our findings suggest significant acute mortality effects of O₃ and NO₂ in the PRD and strengthen the rationale for further limiting the ambient pollution levels in the area.     

Glutathione peroxidase system and lysozyme in rats exposed to ozone or nitrogen dioxide

Exposure of rats to low concentrations of ozone (as low as 0.2 ppm) continuously for 8 days, or intermittently (8 hours/day) for 7 days, significantly increased the activities of the glutathione (GSH) peroxidase system in lung tissue. Linear regression analysis showed the increased enzymic activities to be a function of ozone concentration. Lysozyme activity was significantly increased in lung soluble and plasma, during continuous but not intermittent ozone exposure. The results indicated that lipid peroxidation damage occurred in lungs of rats exposed to relatively high levels of ozone. Rats were exposed to relatively higher levels of nitrogen dioxide for 4 days. The activities of GSH reductase and glucose-6-phosphate dehydrogenase were significantly increased during exposure to 2.3 ppm and 6.2 ppm nitrogen dioxide, respectively. There was no significant increase in the activity of GSH peroxidase in lung soluble, or of lysozyme in the plasma, during exposure to nitrogen dioxide. The results suggest that the mechanism of action of nitrogen dioxide is different from that of ozone.     

Airway responses to 2.0 ppm nitrogen dioxide in normal subjects

Nitrogen dioxide (NO2) is a common indoor air pollutant. To characterize the acute respiratory responses to this gas, 18 nonsmoking normal subjects (mean age +/- standard deviation [SD] = 25 +/- 4 yr) were exposed to filtered air or 2 ppm NO2 gas for 1 hr in a 30-m3 environmental chamber on different days, typically 1 wk apart, in a double-blind randomized fashion. Lung function tests included forced vital capacity, forced expiratory volume in one second, partial expiratory flow at 40% of vital capacity (Vp40), functional residual capacity, and specific airway conductance, and were measured before and after exposure. Airway reactivity to methacholine inhalation was determined within 45 min of each exposure. The dose of methacholine in mg/ml to cause a 40% decrease in specific airway conductance (PD40) was measured. Airway reactivity to methacholine aerosol increased significantly after NO2, which is shown by a decrease in the concentration of methacholine; PD40 (AIR) = 101 +/- 44, PD40 (NO2) = 81 +/- 45 mg/ml, p = .003. No significant changes were noted in the lung function tests after NO2 exposure. These findings indicate that normal nonsmokers exposed to 2.0 ppm NO2 for 1 hr develop an increase in airway reactivity to methacholine aerosol, which is not associated with changes in lung volumes, flow rates, or respiratory symptoms.     

Formation of strong airway irritants in mixtures of isoprene/ozone and isoprene/ozone/nitrogen dioxide.

We evaluated the airway irritation of isoprene, isoprene/ozone, and isoprene/ozone/nitrogen dioxide mixtures using a mouse bioassay, from which we calculated sensory irritation, bronchial constriction, and pulmonary irritation. We observed significant sensory irritation (approximately 50% reduction of mean respiratory rate) by dynamically exposing the mice, over 30 min, to mixtures of isoprene and O3 or isoprene, O3, and NO2. The starting concentrations were approximately 4 ppm O3 and 500 ppm isoprene (+ approximately 4 ppm NO2. The reaction mixtures after approximately 30 sec contained < 0.2 ppm O3. Addition of the effects of the residual reactants and the identified stable irritant products (formaldehyde, formic acid, acetic acid, methacrolein, and methylvinyl ketone) could explain only partially the observed sensory irritation. This suggests that one or more strong airway irritants were formed. It is thus possible that oxidation reactions of common unsaturated compounds may be relevant for indoor air quality.     

DNA strand breaks caused by exposure to ozone and nitrogen dioxide.

The present study demonstrates that exposure to ozone (O3) and nitrogen dioxide (NO2) can cause DNA single-strand breaks in alveolar macrophages. Three-month-old male Sprague-Dawley rats, specific pathogen free, were exposed to either 1.2 ppm NO2 or 0.3 ppm O3 alone or a combination of these two oxidants continuously for 3 days. The control group was exposed to filtered room air. The oxidant effects were substantiated by determining total and differential cell counts, lactate dehydrogenase activity, and total soluble protein in bronchoalveolar lavage. DNA damage was measured as single-strand breaks by alkaline elution assay. The results showed that, relative to control, NO2 exposure did not cause any significant change in the parameters studied. Exposure to O3 and combined exposure to NO2 and O3 caused significant changes in all parameters studied except cell viability. The rates of elution (Kc) of single-strand DNA from polycarbonate filter for O3 exposure and combined exposure were 73 and 79% faster than that of the control, respectively. The amounts of DNA single-strand breaks caused by O3 and combined exposure were significantly greater than the amounts detected for the NO2-exposed and control groups.     

Impairment of humoral immune responses in mice exposed to nitrogen dioxide and ozone mixtures

The relationship between immune defense mechanisms and environmental pollutants remains unknown because of uncertainty about the effects of combined or mixed pollutants. To investigate whether exposure to toxic gas mixtures change the effect of a single gas exposure on immune function, BALB/c mice were continuously exposed to 4.0 ppm nitrogen dioxide (NO2), 0.8 ppm ozone (O3), or the mixture of NO2 plus O3 for 3, 7, 14, and 56 days. Organ weights (lung, thymus, and spleen) and antibody responses to sheep red blood cells (SRBC), and to DNP-Ficoll were measured immediately after the exposure. Lung weights in mice exposed to O3 or the mixture were increased significantly in all exposure periods. The weights of thymus and spleen in mice exposed for 3, 7, and 14 days to the mixture were decreased. O3 exposure for 56 days showed significant decreases of the weights of both organs. Antibody response to SRBC in mice exposed for 3, 7, and 14 days to O3 or the mixture was markedly suppressed, but exposure to the mixture for 56 days did not show the suppression of anti-SRBC antibody response. No differences in anti-DNP antibody response between exposed and control mice were observed, except those exposed to O3 or the mixture for 14 days. These results suggest that mixed gas exposures variously modify the effects of a single gas exposure on antibody production in mice.     

Dose-response study of asthmatic volunteers exposed to nitrogen dioxide during intermittent exercise

Twenty-one mildly asthmatic volunteers were exposed to 0, 0.3, 1.0, and 3.0 ppm nitrogen dioxide (NO2) in purified background air in an environmental control chamber. Exposures were separated by 1-wk periods and occurred in random order. Each lasted 1 hr and included three 10-min bouts of moderately heavy exercise (mean ventilation rate 41 L/min). Exposure temperature was near 22 degrees C and relative humidity near 50%. Specific airway resistance and maximal forced expiratory performance were measured preexposure, after the initial exercise, and near the end of exposure. Bronchial reactivity was assessed immediately following exposure, by normocapnic hyperventilation with subfreezing air. Symptoms were recorded on questionnaires before, during, and for 1-wk after each exposure. Exercise induced significant bronchoconstriction regardless of NO2 level. No statistically significant untoward response to NO2 was observed at any exposure concentration. This negative finding agrees with our previous results, but contrasts with findings elsewhere of respiratory dysfunction after exposure to 0.3 ppm. The discrepancy is presently unexplained, but it may relate to different severity of asthma in different subject groups.     

Safety evaluation of pulmonary influenza vaccination in healthy and "asthmatic" mice

The present study reports animal immuno-toxicological data of pulmonary vaccination against inactivated seasonal influenza. Its aims were (i) to monitor the temporal kinetics of lung inflammation in normal mice over a period of 2 weeks following pulmonary vaccination in order to assess the risk of chronic lung inflammation, (ii) to evaluate the impact of pulmonary vaccination on the asthmatic phenotype in an established allergen-sensitized murine model of asthma. Both sets of experiments were performed using high doses of split influenza virus vaccine. In the first part of this study, we showed that pulmonary influenza vaccination induced a slight local inflammatory response which was limited in duration since it was no longer observed at 2 weeks post-vaccination. At this time point, it has previously been shown that the immunogenic efficacy was maintained. In the second part, we demonstrated that pulmonary influenza vaccination did not significantly exacerbate the cardinal features of asthma, i.e., allergen-specific IgE formation, the development of airway hyperreactivity (AHR) and eosinophilic airway inflammation. Our data therefore suggest that the overall immuno-toxicological profile of pulmonary vaccination against seasonal influenza was acceptable, even in an animal model of pulmonary hypersensitivity.