Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effects of repeated exposures to peak concentrations of nitrogen dioxide and ozone on resistance to streptococcal pneumonia Richard Ehrlich , J.C. Findlay & D. E. Gardner To cite this article: Richard Ehrlich , J.C. Findlay & D. E. Gardner (1979) Effects of repeated exposures to peak concentrations of nitrogen dioxide and ozone on resistance to streptococcal pneumonia, Journal of Toxicology and Environmental Health, 5:4, 631-642, DOI: 10.1080/15287397909529775 To link to this article: http://dx.doi.org/10.1080/15287397909529775
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 37 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 14 November 2015, At: 04:26
EFFECTS OF REPEATED EXPOSURES TO PEAK CONCENTRATIONS OF NITROGEN DIOXIDE AND OZONE ON RESISTANCE TO STREPTOCOCCAL PNEUMONIA Richard Ehrlich, J. C. Findlay Life Sciences Division, IIT Research Institute, Chicago, Illinois
Downloaded by [University of Florida] at 04:26 14 November 2015
D. E. Gardner Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Exposures to various mixtures of nitrogen dioxide (NO1) and ozone (O3) reduced the resistance of mice to streptococcal pneumonia as evidenced by increased mortality rates and shortened survival time. Daily 3-h exposures (5 d/wk) for 2-6 mo to an air pollutant mixture consisting of 940 µg/m3 (0.5 ppm) NO2 and 196 µg/m3 (0.1 ppm) O3 were most effective in reducing the resistance to infection. The decrease in resistance to the infection occurred sooner when the mice continued to be exposed to the air pollutants instead of clean air for 14 d after the respiratory challenge with Streptococcus pyogenes aerosol. After 3 mo of exposure to the pollutant mixture, there was some decrease in the ability of mice to clear inhaled streptococci from their lungs. At the same time the total cell count in the fluid lavaged from the lungs of mice was markedly reduced, as were the viability and phagocytic activity of the alveolar macrophages. Exposure to the pollutants combined with challenge with Streptococcus aerosol resulted in marked morphological changes in lung tissues as seen by scanning electron microscopy.
INTRODUCTION A large portion of the nitrogen oxides present in the urban environment is generated by human activity, and one of the major sources of these pollutants is vehicular traffic. Accordingly, nitric oxide concentrations are high during morning and evening rush hours. In the presence of sunlight nitric oxide is converted to nitrogen dioxide (NO2), resulting in peak concentrations of this pollutant. Thereafter concentrations of ozone (O3) The authors are indebted to Mr. J. Hingeveld for his technical assistance, to Ms. K. V. Ketels for the scanning electron microscopy evaluations, and to Ms. C. Aranyi for studies of the alveolar macrophages. These studies were supported by funds provided under U.S. Environmental Protection Agency contract 68-02-2274. Requests for reprints should be sent to Richard Ehrlich, Life Sciences Division, IIT Research Institute, 10 West 35th Street, Chicago, Illinois 60616. 631 Journal of Toxicology and Environmental Health, 5:631-642, 1979 Copyright © 1979 by Hemisphere Publishing Corporation 0098-4108/79/030631 -12$2.25
Downloaded by [University of Florida] at 04:26 14 November 2015
632
R. EHRLICH ETAL.
increase to a maximum near the middle of the day, and this cycle is repeated in the late afternoon. Thus peak concentrations of NO 2 and O 3 on clear days can be directly related to automotive traffic. Human populations in an urban environment can therefore be exposed over extended periods to low concentrations of NO 2 with superimposed peak concentrations of the same pollutant, frequently mixed with O 3 . Prolonged exposures to NO 2 reduce the resistance of mice to bacterial pneumonia initiated by inhalation of Klebsiella pneumoniae aerosols (Ehrlich and Henry, 1968). Continuous exposure to 940 Mg/m3 (0.5 ppm) NO 2 for 24 h/d, 7 d/wk, produced this change in resistance after 3 mo, whereas with intermittent (6 or 18 h/d, 7d/wk) exposures similar changes were not observed until 6 mo. The decreased resistance to infection was manifested by an enhanced mortality rate, decreased survival time, and a reduced rate of clearance of inhaled bacteria from the lungs compared with mice challenged with the infectious microorganism but exposed to clean air. Changes in resistance to bacterial pneumonia caused by challenge with airborne Streptococcus pyogenes were observed after a single 3-h exposure to 3760 jug/m3 (2.0 ppm) NO 2 or 196 Mg/m3 (0.1 ppm) O 3 . Moreover, a single 3-h exposure to NO 2 and O 3 mixtures produced an additive effect (Ehrlich et al., 1977). For a more realistic simulation of the exposure of urban populations to air pollutants, we studied the effects of continuous exposures to NO 2 with superimposed peak concentrations of NO 2 or NO 2 -O 3 mixtures on resistance of mice to respiratory infections.
MATERIALS AND METHODS Animals Female C D 2 F ! mice (Murphy Laboratory, Plainfield, Ind.) wore used in most of the experiments. In limited experiments female CD-1 mice (Charles River, Wilmington, Mass.) were also used; their response to the infectious challenge and to the pollutants paralleled that of the C D 2 F , mice. The 6- to 8-wk-old mice were housed in groups of 3-10 in suspended wire cages and were quarantined for 14 d before being used. Exposure Chambers Five identical 1 . 2 X 1 . 8 X 2 . 0 m (4420 I) aluminum-lined chambers were used for exposure to the pollutants or filtered air. The mean temperature in the exposure chambers was 23 ± 2°C at ambient humidity (~ 30% RH). Randomly selected mice were housed in suspenced wire cages and provided with food and water ad libitum. The cages were rotated biweekly to various positions on the cage racks to ensure unbiased exposure to the experimental environment. To prevent a buildup of ammonia, sheets of treated paper (DACB, Upjohn, Kalamazoo, Mich.) were
N0 2 AND O3 EFFECTS ON BACTERIAL PNEUMONIA
633
Downloaded by [University of Florida] at 04:26 14 November 2015
placed on the pans of each cage row. Mice were removed from the chambers for 1 h three times a week for maintenance. The pollutants, diluted in a stream of charcoal-filtered air, entered the chambers at a rate that provided approximately 20 air changes per hour. An identical air flow was maintained in the control chamber, where charcoal-filtered ambient air was used. To verify the concentration and homogeneity of the pollutants, air samples were taken from different sections of the chamber. The exposure chambers were equipped with a manifold system that provided automatic sampling; the sampling time for each chamber was 15 min. Nitrogen Dioxide A mixture of 5% NO 2 in air (99.5% pure N O 2 ; Matheson, Joliet, III.) was diluted with filtered air in a mixing glass chamber and then passed into the animal exposure chambers. The NO 2 concentration was monitored by a NO-NO 2 -NO X chemiluminescent analyzer (model 8101 B, Bendix, Ronceverte, W.Va.) and was expressed as parts per million and micrograms per cubic meter (ppm X 1880 = Mg/m 3 ). Ozone A high-voltage generator (IITRI) was used to convert O 2 to O 3 . To provide the desired concentration O 3 was diluted with filtered air in a mixing glass chamber, and the mixture was then passed into the animal exposure chambers. The O 3 concentration was monitored with an O 3 chemiluminescent analyzer (model OA 310, Meloy Laboratories, Springfield, Va.) and was expressed as parts per million and micrograms per cubic meter (ppm X 1960 = jug/m 3 ). Nitrogen Dioxide-Ozone Mixture The gases were introduced into separate glass vessels and were then combined in a mixing glass chamber. The appropriately diluted NO 2 O 3 mixture was then passed into the animal exposure chamber. The concentration of each gas in the' exposure chamber was monitored with the NO* and O 3 chemiluminescent analyzers. Infectious Challenge Mice were challenged with airborne 5. pyogenes (group C) as previously described (Ehriich et al., 1977). Immediately after the challenge, which lasted approximately 10 min, there were 750-2000 inhaled viable bacteria per lung. Clearance of Inhaled Bacteria from Lungs Mice were challenged with Streptococcus aerosol, and groups of 5 mice each were killed immediately and at 1, 2, 3, 4, and 5 h after the challenge. The lungs were removed aseptically, weighed, homogenized, and
634
R. EHRLICH ETAL.
cultured quantitatively. The initial counts, expressed as the number of viable streptococci per gram of lung, were considered as unity (100%). Counts obtained at the other time intervals were calculated as percentage recovery. The rate of clearance was expressed as the time in hours required for 50% of the inhaled viable streptococci to clear the lun£;s (t50). This value was determined on a semilogarithmic regression after converting the percentage recovery to logarithmic values. Exposure Protocol
Downloaded by [University of Florida] at 04:26 14 November 2015
The mice were exposed for 1, 2, 3, and 6 mo to the following environmental conditions.
Continuous exposure (24 h/d, 7 d/wk) Filtered air Filtered air 188Mg/m3 (0.1 ppm) NO2 Filtered air 188Mg/m3 (0.1 ppm) NO2
Peak exposure (3 h/d, 5 d/wk) Filtered air 940 Mg/m3 (0.S ppm) NO2 196/jg/m 3 (0.1 ppm) O3 940 jig/m 3 (0.S ppm) NO2 196iug/m3 (0.1 ppm) O3 940jig/m 3 (O.Sppm) NO2 940 /jg/m 3 (0.5 ppm) NO2
and mixture and mixture
The mice were subjected to the peak exposures Monday through Friday between 8:30 and 11:30 a.m. Within 1 h after termination of the final exposure, groups of 24 mice each, representing all treatments, were simultaneously infected by the respiratory route with Streptococcus aerosol. In one group of experiments the mice were held in a clean-air isolated animal room for 14 d after the infectious challenge. In another group of experiments the mice were reexposed for an additional 14 d to the respective pollutants. Deaths were recorded daily during these 14-d observation periods. Mortality rates were reported as either the number of mice that died out of the total number of mice used [D/T), percentage mortality (%), or percentage mortality change (% dead among pollutant-exposed mice minus % dead among filtered-air controls). The mean survival time was estimated as described by Pindak et al. (1971). As appropriate, chi-square analysis, Student Mest, and linear regression analysis ware used to determine the statistical significance of the observed differences. RESULTS Mortalities and mean survival times of mice exposed for up to 6 mo to the various environmental conditions, challenged with Streptococcus aerosol, and held for 14 d in clean air are shown in Table 1.
N0 2 AND O3 EFFECTS ON BACTERIAL PNEUMONIA
635
TABLE 1. Mortality and Survival Time of Mice Exposed to NO2 and O 3 , Challenged with Streptococcus Aerosol, and Kept for 14 d in Clean Air Concentration |>g/m 3 ) 3 h/d 6
24 h/d" NO 2
Survival time
Mortality %
Days
Change
24/126 5/99 21/127 28/127 18/127
19.1 5.1 16.5 22.1 14.2
12.3 13.6 12.5 11.7 12.7
_ + 1.3C +0.2 -0.6 +0.4
940 940
0 196 196 0 0
21/87 20/72 14/80 24/96 28/117
24.1 27.8 17.5 27.1 23.9
12.2 12.0 12.9 12.0 12.1
_ -0.2 +0.7 -0.2 -0.1
0 940 940 940 940
0 196 196 0 0
62/156 94/155 38/156 86/169 39/172
39.7 60.7 24.4 50.9 22.7
11.3 8.8 11.9 9.5 12.4
-2.5 C +0.6 -1.8e +0.9
0 940 940 940 940
0 196 196 0 0
9/44 21/44 18/51 13/46 14/42
20.5 47.7 35.3 28.3 33.3
12.8 11.0 11.2 11.8 11.5
-1.8C -1.6C -1.0c -1.3C
NO 2
o3
0 940
0 196 196 0 0
D/T
Downloaded by [University of Florida] at 04:26 14 November 2015
1 mo 0 0 188 0 188
940 940 940
2 mo 0 0 188 0 188
0 940 940
3 mo 0 0 188 0 188
_
6 mo 0 0 188 0 188
_
"24 h/d , 7d/wk. 3 h/d, 5 d/wk. c Significant change from corresponding infected mice exposed to filtered air Ip < 0.05). 6
The 1- or 2-mo exposure did not induce any remarkable changes in mortality or survival time. The one exception was a significant (p < 0.05) decrease in mortality accompanied by an increase in survival time after 1 mo of daily 3-h exposures to the NO 2 -O 3 mixture. The 3-mo exposure resulted in a significant ( p < 0 . 0 5 ) increase in mortality after daily 3-h exposures to either 940 Mg/m3 NO 2 alone or the NO 2 -O 3 mixture. The corresponding mean survival times were reduced by 1.8 and 2.5 d, respectively. On the other hand, a significant decrease in mortality was seen among mice continuously exposed to 188 pg/m 3 NO 2 with superimposed daily 3-h peaks of either NO 2 alone or the NO 2 -O 3 mixture. These decreases in mortality were not accompanied by significant changes
R. EHRLICH ETAL.
Downloaded by [University of Florida] at 04:26 14 November 2015
636
in the mean survival time. After 6 mo of exposure, increased mortality and decreased survival time were observed in all pollutant treatment groups compared with mice exposed to filtered air. The statistical significance of the differences in mortality was ascertained for mice exposed daily for 3 h to the NO 2 -O 3 mixture (+27.2%) and those continuously exposed to 188 ng/m3 NO 2 with superimposed 3-h daily NO 2 and O 3 peaks (+14.8%). Decreases in mean survival time were significant in all treatment groups. Mortalities and survival times of mice exposed for up to 3 mo to the various pollutant treatments, challenged with the Streptococcus aerosol, and then reexposed for 14 d to the same pollutant treatments are shown in Table 2. After 1 mo of exposure the increases in mortality were small in all treatment groups, even though one was statistically significant. After 2 mo there was a significant increase in mortality paralleled by a decrease in survival time in all treatment groups compared with mice kept in filtered TABLE 2. Mortality and Survival Time of Mice Exposed to NO2 and O 3 , Challenged with Streptococcus Aerosol, and Reexposed for 14 d to the Pollutants Concentration (Mg/m3) 3h/d6
24 h/d° NO 2
Mortality
Survival time
NO2
O3
D/T
%
Days
Change
0 940 940 940 940
0 196 196 0 0
0/78 2/78 3/77 2/78 1/84
0 2.6 3.9 2.6 1.2
14.0 13.9 13.8 13.8 13.9
-0.1 -0.2 -0.2 -0.1
0 940 940 940 940
0 196 196 0 0
5/50 18/52 11/45 12/52 14/67
10.0 34.6 24.4 23.1 20.9
13.2 12.0 12.1 12.5 12.5
— -1.2 C -1.1C -0.7 -0.7
0
4/60 24/60 16/60 8/60 9/60
6.7 40.0 26.7 13.3 15.0
13.4 10.8 11.9 13.2 12.7
— -2.6 C -1.5 C -0.2 -0.7
1 mo 0 0
188 0 188 2 mo 0 0 188 0 188
_
3 mo 0 0 188 0 188
0 940 940 940 940
196 196 0 0
°24h/d, 7 d/wk. °3 h/d, 5 d/wk. Significant change from corresponding infected mice exposed to filtered air (p < 0.05).
Downloaded by [University of Florida] at 04:26 14 November 2015
NO, AND O3 EFFECTS ON BACTERIAL PNEUMONIA
637
air after the infectious challenge. The 3-mo exposure also resulted in significant changes in mortality and survival time. A significant increase in mortality and decrease in survival time was seen in mice continuously exposed to 188 Mg/m3 NO 2 with superimposed daily 3-h peaks of the NO 2 -O 3 mixture, and in those exposed to the NO 2 and O 3 peaks only and then reexposed to the same pollutants after the infectious challenge. An increase in mortality and decrease in survival time, although not statistically significant, also occurred with the two other exposure regimens. The rates of clearance (t50) of inhaled streptococci from the lungs of control mice exposed to filtered air for 1 and 3 mo were 1.33 h [±0.14 C.L. (confidence limits)] and 1.32 h (±0.20 C.L), respectively. After 1 mo of exposure to the pollutants tso ranged from 1.28 to 1.55 h, and after 3 mo of exposure it ranged from 1.26 to 1.86 h. At both times the greatest delay in the clearance of inhaled bacteria was seen in mice exposed daily for 3 h to the NO 2 -O 3 mixture (t50 = 1.84 h) and those continuously exposed to 188 Mg/m3 NO 2 with daily 3-h peaks of 940 jug/m3 NO 2 (tso = 1.86 h). The damage to lung tissues in mice exposed for 3 mo to the pollutants, challenged with Streptococcus aerosol, and reexposed to the pollutants for 2 d was determined by scanning electron microscopy (Ketels et al., 1977). At low magnification, the alveoli of mice exposed to filtered air only and those exposed to air and challenged with Streptococcus appeared round and cup-shaped, with a normal honeycomb appearance (Fig. 1A). A t higher magnifications a slight thickening around the edges of the alveolar walls was apparent and alveolar pores were enlarged throughout the lung (Fig. 1B). Lungs of mice exposed daily for 3 h to 940 Mg/m3 NO 2 and challenged with the infectious agent showed similar changes. The lungs of mice that were exposed for 3 mo to daily 3-h NO 2 -O 3 peaks or to this mixture superimposed on a continuous exposure to 188 /ug/m3 NO 2 and were then challenged with Streptococcus showed marked changes. At low magnifications the usual honeycomb appearance of the alveoli was absent; instead, areas of fused alveoli next to alveoli with greatly enlarged pores were present (Fig. 1C). At higher magnifications the alveolar walls appeared to have thickened and in some areas had fused together, giving the appearance of a single large alveolus (Fig. 1D). Because of this thickening the size of the pores was markedly reduced. Alveolar pores in adjacent areas were enlarged and in some instances formed filamentous septa (Fig. 1E). The activity of alveolar macrophages lavaged from lungs of mice (Coffin et al., 1968) exposed for 1 , 2, and 3 mo to the various pollutants was studied to determine alterations in the pulmonary cellular defense mechanisms (Table 3). The percentage of alveolar macrophages present in the lavage fluid ranged from 96.7 to 99.3 and generally was not affected by exposure to the pollutants. The only exception was a small but statistically significant ( p < 0 . 0 5 ) decrease in the percentage of
R. EHRLICHETAL.
Downloaded by [University of Florida] at 04:26 14 November 2015
638
FIGURE 1. Scanning electron micrographs challenged with Streptococcus aerosol, and mice exposed for 3 mo to daily 3-h peaks aerosol, and reexposed to the pollutants for
of lungs of (A and B) mice kept in clean air for 3 mo, kept in clean air for 2 d after the challenge, and (C-E) of the NO 2 -O 3 mixture, challenged with Streptococcus 2 d. Scale bars, 20 /jm.
639
NO2 AND O3 EFFECTS ON BACTERIAL PNEUMONIA
TABLE 3. Effect of Exposure to NO2 and O3 on Alveolar Macrophages Concentration (Mg/m3) 24h/d
Downloaded by [University of Florida] at 04:26 14 November 2015
NO, 1 mo 0 0 188 0 2 mo 0 0 188 0 3 mo 0 0 188 0
3h/d
Macrophage
Cell count Total (X 10 6 )
Macrophage (%)
Viability (%)
Mean
SE
Mean
SE
Mean
SE
Phagocytosis (%)"
NO2
O3
Mean
SE
0 940 940 940
0 196 196 0
1.68 1.50 1.046 1.02*
0.21 0.30 0.27 0.10
98.8 99.3 98.8 98.6
0.8 0.5 0.6 0.2
93.2 92.3 94.9 89.6
1.6 1.2 0.7 1.7
87.2 87.2 87.8 89.5
0.6 0.7 0.6 0.7
0 940 940 940
0 196 196 0
1.37 1.54 1.53 1.30
0.23 0.21 0.17 0.09
98.6 96.8 6 97.5 97.8
0.4 0.9 0.7 0.6
93.8 95.2 95.0 93.8
1.0 1.1 1.1 1.1
89.1 82.1 b 86.5 6 84.0 6
0.9 0.6 0.8 1.9
0 940 940 940
0 196 196 0
0.94 0.60 6 0.78 1.04
0.11 0.04 0.09 0.11
96.7 97.4 97.1 97.4
0.7 0.6 0.4 0.8
87.3 78.9 6 84.9 88.1
2.2 1.1 2.5 1.4
85.8 83.0 6 83.7 86.2
0.7 1.2 1.0 1.0
Percentage of alveolar macrophages engulfing one or more latex spheres after 1 h of incubation at 37°C (ratio of macrophages to latex = 1:200). Significant change from corresponding control mice exposed to filtered air (p < 0.05).
macrophages seen after 2 mo of daily 3-h exposures to the NO 2 -O 3 mixture (96.8% compared with 98.6% in controls). The viability of the alveolar macrophages was not affected by exposure for 1 or 2 mo to the pollutants. After 3 mo there was a significant (p < 0 . 0 5 ) decrease in viability in macrophages isolated from mice exposed to the NO 2 and O 3 peaks (78.9% compared with 87.3% in controls). Although not significant, a decrease in viability was also seen in macrophages of mice exposed continuously to 188 jug/m3 NO 2 with superimposed daily peaks of the NO 2 -O 3 mixture (84.9%). Phagocytic activity, expressed as the percentage of macrophages that engulfed at least one latex sphere after 1 h of incubation at 37°C (Gardner et al., 1973), was not affected by 1 mo of exposure to the pollutants. After 2 and 3 mo, however, there was a significant (p < 0.05) decrease in the phagocytic activity of macrophages from mice exposed daily for 3 h to the NO 2 -O 3 mixture (83.0%) and those continuously exposed to 188 Mg/m3 NO 2 and the superimposed NO 2 -O 3 peaks (83.7%) compared with control animals exposed to filtered air (85.8%). After 2 mo but not after 3 mo a significant decrease in phagocytosis was also seen in mice exposed daily to the 3-h peaks of 940 Mg/m3 NO 2 .
640
R. EHRL1CH ET AL.
Limited experiments were conducted to determine the effects of 1, 3, and 6 mo of exposure to the pollutants on the activity of selected blood serum enzymes in mice. In general, there was an increase in the activity of lactate dehydrogenase (LDH), serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamic-pyruvic transaminase (SGPT) and a depression of acid phosphatase (AP) and cholinesterase (CHE) activity. Continuous exposure to 188 Mg/m3 NO 2 with superimposed 3-h daily peaks of the NO 2 -O 3 mixture appeared to be most effective in inducing these changes.
Downloaded by [University of Florida] at 04:26 14 November 2015
DISCUSSION The results of this study indicate that the sequence of exposures to the pollutants is of major importance in reducing the resistance to streptococcal pneumonia. More specifically, the combination of extended exposure to the pollutants and continuation of exposure after the respiratory challenge with Streptococcus, aerosol was most deleterious. After such a sequence of exposures, significantly increased mortalities and shortened survival times were seen in mice exposed to almost all pollutant regimens. Moreover, decreased resistance to bacterial pneumonia appeared sooner in mice reexposed to the pollutants than- in those allowed to breathe clean air after the infectious challenge. Another important factor in the decreased resistance to infection was the stress of daily 3-h peak exposures to a mixture of 940 Mg/m3 NO 2 and 196 Mg/m3 O 3 . Increased mortality and reduced survival time were evident after 2, 3, and 6 mo of such exposures, whether the mice were kept in clean air or were reexposed to the pollutant mixture after the infectious challenge. After 2 and 3 mo of exposure the reduced resistance to infection was accompanied by significant decreases in phagocytic activity, and after 3 mo by reduced viability of alveolar macrophages. Lungs of mice exposed for 3 mo to this pollutant mixture, challenged with Streptococcus aerosol, and reexposed to the pollutants showed widespread damage when examined by scanning electron microscopy. Instead of the honeycomb appearance of alveoli seen in lungs of infected mice exposed to filtered air, the alveolar walls had thickened and in some areas walls of several alveoli appeared to have fused, creating a single large alveolus. Exposure to peak concentrations of the NO 2 -O 3 mixture also appeared to be most effective in inducing changes in blood serum enzymesdecreases in AP and CHE and increases in LDH, SGOT, and SGPT. Elevations of SGOT, SGPT, and LDH are seen in many pathological conditions, and these enzymes are released as the result of damage to various tissues. Thus increased levels of these enzymes could be indicative of damage to the lung resulting from exposure to air pollutants. Similar decreases in AP and CHE and increases in LDH and SGOT were observed by S. Kosmider (personal communication) and by Menzel et al. (1977) in
641
N0 2 AND 0 3 EFFECTS ON BACTERIAL PNEUMONIA
Downloaded by [University of Florida] at 04:26 14 November 2015
guinea pigs exposed to 940 Mg/m3 NO 2 for 8 h/d, 7 d/wk, for 4 mo. Despite the differences in species and in the regimen and duration of exposure, the trends of the changes closely resembled those seen in mice after 6 mo of exposure to daily 3-h peaks of the NO 2 -O 3 mixture. Daily 3-h exposures to the mixture of 940 Mg/m3 NO 2 and 196 ng/m3 O 3 superimposed on the continuous exposure to 188 Mg/m3 NO 2 were also effective in reducing the resistance to bacterial pneumonia. Significant increases in mortality and shortened survival times were seen in mice exposed to this pollutant regimen for 1, 2, and 3 mo before the infectious challenge and for 14 d after the challenge. However, reduced mortality was seen after 1, 2, and 3 mo of exposure when mice were maintained in
POLLUTANT
24 l i r . / d a , : AIR
Jhr./dayj
>
CHALLENGE
188*g/m'N0 2
AI
.
FIGURE 2. Changes in mortality from streptococcal pneumonia in mice subjected to various NO2 and O3 exposure regimens.
Downloaded by [University of Florida] at 04:26 14 November 2015
642
R. EHRLICH ETAL.
clean air for 14 d after the infectious challenge. This trend was reversed after 6 mo of exposure, at which time there was a significant ( p < 0 . 1 ) increase in mortality (Fig. 2). A similar response, namely an initial decrease followed by an increase in mortality, was observed during 6-mo continuous exposures to 188 Aig/m3 NO 2 with 3-h daily peaks of 940 Mg/m3 NO 2 . Thus, continuous exposure to the low concentration of NO 2 for 3 mo might have protected against the daily peak exposures to the higher concentrations of NO 2 or the NO 2 -O 3 mixture. This effect was seen only in animals removed from the polluted atmosphere after 1, 2, and 3 mo of exposure and kept in clean air for 14 d following the respiratory challenge with Streptococcus aerosol. The protective response was not observed when the same exposure regimens continued during the 14-d period after the infectious challenge or when the exposure to the pollutants extended from 3 to 6 mo. In general, these results confirm the results of previous experiments that showed the importance of intermittent exposure to air pollutants as a factor in altering the host's resistance to respiratory infection. Thus, this study confirms the conclusion that primary air quality standards for short-term NO 2 exposures must be established to fully protect the health of populations at risk. REFERENCES Coffin, D. L., Gardner, D. E., Holzman, R. S., and Wolock, F. J. 1968. Influence of ozone on pulmonary cells. Arch. Environ. Health 16:633-636. Ehrlich, R. and Henry, M. C. 1968. Chronic toxicity of nitrogen dioxide. I. Effect on resistance to bacterial penumonia. Arch. Environ. Health 17:860-865. Ehrlich, R., Findlay, J. C., Fenters, J. D., and Gardner, D. E. 1977. Health effects of short-term inhalation of nitrogen dioxide and ozone mixtures. Environ. Res. 14:223-231. Gardner, D. E., Graham, J. A., Miller, F. J., Illing, J. W., and Coffin, D. L. 1973. Technique for differentiating particles that are cell associated or ingested by macrophages. Appl. Microbiol. 25:471-475. Ketels, K. V., Bradof, J. N., Fenters, J. D., and Ehrlich, R. 1977. SEM studies of the respiratory tract of mice exposed to sulfuric acid mist-carbon particle mixtures. Scanning Electron Microsc. 2:519-526. Menzel, D. B., Abou-Donia, M. D., Roe, C. R., Ehrlich, R., Gardner, D. E., and Coffin, D. L. 1977. Biochemical indices of nitrogen dioxide intoxication of guinea pigs following low level long term exposure. In Proceedings of the International Conference on Photochemical Oxidant Pollution and Its Control, EPA Publ. EPA-600/3-77-001b, vol. 2, pp. 577-587. Research Triangle Park, N.C.: Environmental Protection Agency. Pindak, F. F., Schmidt, J. P., Gibon, D. J., and Allen, P. T. 1971. Interferon levels and resistance to viral infection associated with selected interferon inducers. Proc. Soc. Exp. Biol. Med. 138:317-321. Received September 25, 1978 Accepted November 20, 1978
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Effects of repeated exposures to peak concentrations of nitrogen dioxide and ozone on resistance to streptococcal pneumonia Richard Ehrlich , J.C. Findlay & D. E. Gardner To cite this article: Richard Ehrlich , J.C. Findlay & D. E. Gardner (1979) Effects of repeated exposures to peak concentrations of nitrogen dioxide and ozone on resistance to streptococcal pneumonia, Journal of Toxicology and Environmental Health, 5:4, 631-642, DOI: 10.1080/15287397909529775 To link to this article: http://dx.doi.org/10.1080/15287397909529775
Published online: 20 Oct 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 37 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=uteh20 Download by: [University of Florida]
Date: 14 November 2015, At: 04:26
EFFECTS OF REPEATED EXPOSURES TO PEAK CONCENTRATIONS OF NITROGEN DIOXIDE AND OZONE ON RESISTANCE TO STREPTOCOCCAL PNEUMONIA Richard Ehrlich, J. C. Findlay Life Sciences Division, IIT Research Institute, Chicago, Illinois
Downloaded by [University of Florida] at 04:26 14 November 2015
D. E. Gardner Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina
Exposures to various mixtures of nitrogen dioxide (NO1) and ozone (O3) reduced the resistance of mice to streptococcal pneumonia as evidenced by increased mortality rates and shortened survival time. Daily 3-h exposures (5 d/wk) for 2-6 mo to an air pollutant mixture consisting of 940 µg/m3 (0.5 ppm) NO2 and 196 µg/m3 (0.1 ppm) O3 were most effective in reducing the resistance to infection. The decrease in resistance to the infection occurred sooner when the mice continued to be exposed to the air pollutants instead of clean air for 14 d after the respiratory challenge with Streptococcus pyogenes aerosol. After 3 mo of exposure to the pollutant mixture, there was some decrease in the ability of mice to clear inhaled streptococci from their lungs. At the same time the total cell count in the fluid lavaged from the lungs of mice was markedly reduced, as were the viability and phagocytic activity of the alveolar macrophages. Exposure to the pollutants combined with challenge with Streptococcus aerosol resulted in marked morphological changes in lung tissues as seen by scanning electron microscopy.
INTRODUCTION A large portion of the nitrogen oxides present in the urban environment is generated by human activity, and one of the major sources of these pollutants is vehicular traffic. Accordingly, nitric oxide concentrations are high during morning and evening rush hours. In the presence of sunlight nitric oxide is converted to nitrogen dioxide (NO2), resulting in peak concentrations of this pollutant. Thereafter concentrations of ozone (O3) The authors are indebted to Mr. J. Hingeveld for his technical assistance, to Ms. K. V. Ketels for the scanning electron microscopy evaluations, and to Ms. C. Aranyi for studies of the alveolar macrophages. These studies were supported by funds provided under U.S. Environmental Protection Agency contract 68-02-2274. Requests for reprints should be sent to Richard Ehrlich, Life Sciences Division, IIT Research Institute, 10 West 35th Street, Chicago, Illinois 60616. 631 Journal of Toxicology and Environmental Health, 5:631-642, 1979 Copyright © 1979 by Hemisphere Publishing Corporation 0098-4108/79/030631 -12$2.25
Downloaded by [University of Florida] at 04:26 14 November 2015
632
R. EHRLICH ETAL.
increase to a maximum near the middle of the day, and this cycle is repeated in the late afternoon. Thus peak concentrations of NO 2 and O 3 on clear days can be directly related to automotive traffic. Human populations in an urban environment can therefore be exposed over extended periods to low concentrations of NO 2 with superimposed peak concentrations of the same pollutant, frequently mixed with O 3 . Prolonged exposures to NO 2 reduce the resistance of mice to bacterial pneumonia initiated by inhalation of Klebsiella pneumoniae aerosols (Ehrlich and Henry, 1968). Continuous exposure to 940 Mg/m3 (0.5 ppm) NO 2 for 24 h/d, 7 d/wk, produced this change in resistance after 3 mo, whereas with intermittent (6 or 18 h/d, 7d/wk) exposures similar changes were not observed until 6 mo. The decreased resistance to infection was manifested by an enhanced mortality rate, decreased survival time, and a reduced rate of clearance of inhaled bacteria from the lungs compared with mice challenged with the infectious microorganism but exposed to clean air. Changes in resistance to bacterial pneumonia caused by challenge with airborne Streptococcus pyogenes were observed after a single 3-h exposure to 3760 jug/m3 (2.0 ppm) NO 2 or 196 Mg/m3 (0.1 ppm) O 3 . Moreover, a single 3-h exposure to NO 2 and O 3 mixtures produced an additive effect (Ehrlich et al., 1977). For a more realistic simulation of the exposure of urban populations to air pollutants, we studied the effects of continuous exposures to NO 2 with superimposed peak concentrations of NO 2 or NO 2 -O 3 mixtures on resistance of mice to respiratory infections.
MATERIALS AND METHODS Animals Female C D 2 F ! mice (Murphy Laboratory, Plainfield, Ind.) wore used in most of the experiments. In limited experiments female CD-1 mice (Charles River, Wilmington, Mass.) were also used; their response to the infectious challenge and to the pollutants paralleled that of the C D 2 F , mice. The 6- to 8-wk-old mice were housed in groups of 3-10 in suspended wire cages and were quarantined for 14 d before being used. Exposure Chambers Five identical 1 . 2 X 1 . 8 X 2 . 0 m (4420 I) aluminum-lined chambers were used for exposure to the pollutants or filtered air. The mean temperature in the exposure chambers was 23 ± 2°C at ambient humidity (~ 30% RH). Randomly selected mice were housed in suspenced wire cages and provided with food and water ad libitum. The cages were rotated biweekly to various positions on the cage racks to ensure unbiased exposure to the experimental environment. To prevent a buildup of ammonia, sheets of treated paper (DACB, Upjohn, Kalamazoo, Mich.) were
N0 2 AND O3 EFFECTS ON BACTERIAL PNEUMONIA
633
Downloaded by [University of Florida] at 04:26 14 November 2015
placed on the pans of each cage row. Mice were removed from the chambers for 1 h three times a week for maintenance. The pollutants, diluted in a stream of charcoal-filtered air, entered the chambers at a rate that provided approximately 20 air changes per hour. An identical air flow was maintained in the control chamber, where charcoal-filtered ambient air was used. To verify the concentration and homogeneity of the pollutants, air samples were taken from different sections of the chamber. The exposure chambers were equipped with a manifold system that provided automatic sampling; the sampling time for each chamber was 15 min. Nitrogen Dioxide A mixture of 5% NO 2 in air (99.5% pure N O 2 ; Matheson, Joliet, III.) was diluted with filtered air in a mixing glass chamber and then passed into the animal exposure chambers. The NO 2 concentration was monitored by a NO-NO 2 -NO X chemiluminescent analyzer (model 8101 B, Bendix, Ronceverte, W.Va.) and was expressed as parts per million and micrograms per cubic meter (ppm X 1880 = Mg/m 3 ). Ozone A high-voltage generator (IITRI) was used to convert O 2 to O 3 . To provide the desired concentration O 3 was diluted with filtered air in a mixing glass chamber, and the mixture was then passed into the animal exposure chambers. The O 3 concentration was monitored with an O 3 chemiluminescent analyzer (model OA 310, Meloy Laboratories, Springfield, Va.) and was expressed as parts per million and micrograms per cubic meter (ppm X 1960 = jug/m 3 ). Nitrogen Dioxide-Ozone Mixture The gases were introduced into separate glass vessels and were then combined in a mixing glass chamber. The appropriately diluted NO 2 O 3 mixture was then passed into the animal exposure chamber. The concentration of each gas in the' exposure chamber was monitored with the NO* and O 3 chemiluminescent analyzers. Infectious Challenge Mice were challenged with airborne 5. pyogenes (group C) as previously described (Ehriich et al., 1977). Immediately after the challenge, which lasted approximately 10 min, there were 750-2000 inhaled viable bacteria per lung. Clearance of Inhaled Bacteria from Lungs Mice were challenged with Streptococcus aerosol, and groups of 5 mice each were killed immediately and at 1, 2, 3, 4, and 5 h after the challenge. The lungs were removed aseptically, weighed, homogenized, and
634
R. EHRLICH ETAL.
cultured quantitatively. The initial counts, expressed as the number of viable streptococci per gram of lung, were considered as unity (100%). Counts obtained at the other time intervals were calculated as percentage recovery. The rate of clearance was expressed as the time in hours required for 50% of the inhaled viable streptococci to clear the lun£;s (t50). This value was determined on a semilogarithmic regression after converting the percentage recovery to logarithmic values. Exposure Protocol
Downloaded by [University of Florida] at 04:26 14 November 2015
The mice were exposed for 1, 2, 3, and 6 mo to the following environmental conditions.
Continuous exposure (24 h/d, 7 d/wk) Filtered air Filtered air 188Mg/m3 (0.1 ppm) NO2 Filtered air 188Mg/m3 (0.1 ppm) NO2
Peak exposure (3 h/d, 5 d/wk) Filtered air 940 Mg/m3 (0.S ppm) NO2 196/jg/m 3 (0.1 ppm) O3 940 jig/m 3 (0.S ppm) NO2 196iug/m3 (0.1 ppm) O3 940jig/m 3 (O.Sppm) NO2 940 /jg/m 3 (0.5 ppm) NO2
and mixture and mixture
The mice were subjected to the peak exposures Monday through Friday between 8:30 and 11:30 a.m. Within 1 h after termination of the final exposure, groups of 24 mice each, representing all treatments, were simultaneously infected by the respiratory route with Streptococcus aerosol. In one group of experiments the mice were held in a clean-air isolated animal room for 14 d after the infectious challenge. In another group of experiments the mice were reexposed for an additional 14 d to the respective pollutants. Deaths were recorded daily during these 14-d observation periods. Mortality rates were reported as either the number of mice that died out of the total number of mice used [D/T), percentage mortality (%), or percentage mortality change (% dead among pollutant-exposed mice minus % dead among filtered-air controls). The mean survival time was estimated as described by Pindak et al. (1971). As appropriate, chi-square analysis, Student Mest, and linear regression analysis ware used to determine the statistical significance of the observed differences. RESULTS Mortalities and mean survival times of mice exposed for up to 6 mo to the various environmental conditions, challenged with Streptococcus aerosol, and held for 14 d in clean air are shown in Table 1.
N0 2 AND O3 EFFECTS ON BACTERIAL PNEUMONIA
635
TABLE 1. Mortality and Survival Time of Mice Exposed to NO2 and O 3 , Challenged with Streptococcus Aerosol, and Kept for 14 d in Clean Air Concentration |>g/m 3 ) 3 h/d 6
24 h/d" NO 2
Survival time
Mortality %
Days
Change
24/126 5/99 21/127 28/127 18/127
19.1 5.1 16.5 22.1 14.2
12.3 13.6 12.5 11.7 12.7
_ + 1.3C +0.2 -0.6 +0.4
940 940
0 196 196 0 0
21/87 20/72 14/80 24/96 28/117
24.1 27.8 17.5 27.1 23.9
12.2 12.0 12.9 12.0 12.1
_ -0.2 +0.7 -0.2 -0.1
0 940 940 940 940
0 196 196 0 0
62/156 94/155 38/156 86/169 39/172
39.7 60.7 24.4 50.9 22.7
11.3 8.8 11.9 9.5 12.4
-2.5 C +0.6 -1.8e +0.9
0 940 940 940 940
0 196 196 0 0
9/44 21/44 18/51 13/46 14/42
20.5 47.7 35.3 28.3 33.3
12.8 11.0 11.2 11.8 11.5
-1.8C -1.6C -1.0c -1.3C
NO 2
o3
0 940
0 196 196 0 0
D/T
Downloaded by [University of Florida] at 04:26 14 November 2015
1 mo 0 0 188 0 188
940 940 940
2 mo 0 0 188 0 188
0 940 940
3 mo 0 0 188 0 188
_
6 mo 0 0 188 0 188
_
"24 h/d , 7d/wk. 3 h/d, 5 d/wk. c Significant change from corresponding infected mice exposed to filtered air Ip < 0.05). 6
The 1- or 2-mo exposure did not induce any remarkable changes in mortality or survival time. The one exception was a significant (p < 0.05) decrease in mortality accompanied by an increase in survival time after 1 mo of daily 3-h exposures to the NO 2 -O 3 mixture. The 3-mo exposure resulted in a significant ( p < 0 . 0 5 ) increase in mortality after daily 3-h exposures to either 940 Mg/m3 NO 2 alone or the NO 2 -O 3 mixture. The corresponding mean survival times were reduced by 1.8 and 2.5 d, respectively. On the other hand, a significant decrease in mortality was seen among mice continuously exposed to 188 pg/m 3 NO 2 with superimposed daily 3-h peaks of either NO 2 alone or the NO 2 -O 3 mixture. These decreases in mortality were not accompanied by significant changes
R. EHRLICH ETAL.
Downloaded by [University of Florida] at 04:26 14 November 2015
636
in the mean survival time. After 6 mo of exposure, increased mortality and decreased survival time were observed in all pollutant treatment groups compared with mice exposed to filtered air. The statistical significance of the differences in mortality was ascertained for mice exposed daily for 3 h to the NO 2 -O 3 mixture (+27.2%) and those continuously exposed to 188 ng/m3 NO 2 with superimposed 3-h daily NO 2 and O 3 peaks (+14.8%). Decreases in mean survival time were significant in all treatment groups. Mortalities and survival times of mice exposed for up to 3 mo to the various pollutant treatments, challenged with the Streptococcus aerosol, and then reexposed for 14 d to the same pollutant treatments are shown in Table 2. After 1 mo of exposure the increases in mortality were small in all treatment groups, even though one was statistically significant. After 2 mo there was a significant increase in mortality paralleled by a decrease in survival time in all treatment groups compared with mice kept in filtered TABLE 2. Mortality and Survival Time of Mice Exposed to NO2 and O 3 , Challenged with Streptococcus Aerosol, and Reexposed for 14 d to the Pollutants Concentration (Mg/m3) 3h/d6
24 h/d° NO 2
Mortality
Survival time
NO2
O3
D/T
%
Days
Change
0 940 940 940 940
0 196 196 0 0
0/78 2/78 3/77 2/78 1/84
0 2.6 3.9 2.6 1.2
14.0 13.9 13.8 13.8 13.9
-0.1 -0.2 -0.2 -0.1
0 940 940 940 940
0 196 196 0 0
5/50 18/52 11/45 12/52 14/67
10.0 34.6 24.4 23.1 20.9
13.2 12.0 12.1 12.5 12.5
— -1.2 C -1.1C -0.7 -0.7
0
4/60 24/60 16/60 8/60 9/60
6.7 40.0 26.7 13.3 15.0
13.4 10.8 11.9 13.2 12.7
— -2.6 C -1.5 C -0.2 -0.7
1 mo 0 0
188 0 188 2 mo 0 0 188 0 188
_
3 mo 0 0 188 0 188
0 940 940 940 940
196 196 0 0
°24h/d, 7 d/wk. °3 h/d, 5 d/wk. Significant change from corresponding infected mice exposed to filtered air (p < 0.05).
Downloaded by [University of Florida] at 04:26 14 November 2015
NO, AND O3 EFFECTS ON BACTERIAL PNEUMONIA
637
air after the infectious challenge. The 3-mo exposure also resulted in significant changes in mortality and survival time. A significant increase in mortality and decrease in survival time was seen in mice continuously exposed to 188 Mg/m3 NO 2 with superimposed daily 3-h peaks of the NO 2 -O 3 mixture, and in those exposed to the NO 2 and O 3 peaks only and then reexposed to the same pollutants after the infectious challenge. An increase in mortality and decrease in survival time, although not statistically significant, also occurred with the two other exposure regimens. The rates of clearance (t50) of inhaled streptococci from the lungs of control mice exposed to filtered air for 1 and 3 mo were 1.33 h [±0.14 C.L. (confidence limits)] and 1.32 h (±0.20 C.L), respectively. After 1 mo of exposure to the pollutants tso ranged from 1.28 to 1.55 h, and after 3 mo of exposure it ranged from 1.26 to 1.86 h. At both times the greatest delay in the clearance of inhaled bacteria was seen in mice exposed daily for 3 h to the NO 2 -O 3 mixture (t50 = 1.84 h) and those continuously exposed to 188 Mg/m3 NO 2 with daily 3-h peaks of 940 jug/m3 NO 2 (tso = 1.86 h). The damage to lung tissues in mice exposed for 3 mo to the pollutants, challenged with Streptococcus aerosol, and reexposed to the pollutants for 2 d was determined by scanning electron microscopy (Ketels et al., 1977). At low magnification, the alveoli of mice exposed to filtered air only and those exposed to air and challenged with Streptococcus appeared round and cup-shaped, with a normal honeycomb appearance (Fig. 1A). A t higher magnifications a slight thickening around the edges of the alveolar walls was apparent and alveolar pores were enlarged throughout the lung (Fig. 1B). Lungs of mice exposed daily for 3 h to 940 Mg/m3 NO 2 and challenged with the infectious agent showed similar changes. The lungs of mice that were exposed for 3 mo to daily 3-h NO 2 -O 3 peaks or to this mixture superimposed on a continuous exposure to 188 /ug/m3 NO 2 and were then challenged with Streptococcus showed marked changes. At low magnifications the usual honeycomb appearance of the alveoli was absent; instead, areas of fused alveoli next to alveoli with greatly enlarged pores were present (Fig. 1C). At higher magnifications the alveolar walls appeared to have thickened and in some areas had fused together, giving the appearance of a single large alveolus (Fig. 1D). Because of this thickening the size of the pores was markedly reduced. Alveolar pores in adjacent areas were enlarged and in some instances formed filamentous septa (Fig. 1E). The activity of alveolar macrophages lavaged from lungs of mice (Coffin et al., 1968) exposed for 1 , 2, and 3 mo to the various pollutants was studied to determine alterations in the pulmonary cellular defense mechanisms (Table 3). The percentage of alveolar macrophages present in the lavage fluid ranged from 96.7 to 99.3 and generally was not affected by exposure to the pollutants. The only exception was a small but statistically significant ( p < 0 . 0 5 ) decrease in the percentage of
R. EHRLICHETAL.
Downloaded by [University of Florida] at 04:26 14 November 2015
638
FIGURE 1. Scanning electron micrographs challenged with Streptococcus aerosol, and mice exposed for 3 mo to daily 3-h peaks aerosol, and reexposed to the pollutants for
of lungs of (A and B) mice kept in clean air for 3 mo, kept in clean air for 2 d after the challenge, and (C-E) of the NO 2 -O 3 mixture, challenged with Streptococcus 2 d. Scale bars, 20 /jm.
639
NO2 AND O3 EFFECTS ON BACTERIAL PNEUMONIA
TABLE 3. Effect of Exposure to NO2 and O3 on Alveolar Macrophages Concentration (Mg/m3) 24h/d
Downloaded by [University of Florida] at 04:26 14 November 2015
NO, 1 mo 0 0 188 0 2 mo 0 0 188 0 3 mo 0 0 188 0
3h/d
Macrophage
Cell count Total (X 10 6 )
Macrophage (%)
Viability (%)
Mean
SE
Mean
SE
Mean
SE
Phagocytosis (%)"
NO2
O3
Mean
SE
0 940 940 940
0 196 196 0
1.68 1.50 1.046 1.02*
0.21 0.30 0.27 0.10
98.8 99.3 98.8 98.6
0.8 0.5 0.6 0.2
93.2 92.3 94.9 89.6
1.6 1.2 0.7 1.7
87.2 87.2 87.8 89.5
0.6 0.7 0.6 0.7
0 940 940 940
0 196 196 0
1.37 1.54 1.53 1.30
0.23 0.21 0.17 0.09
98.6 96.8 6 97.5 97.8
0.4 0.9 0.7 0.6
93.8 95.2 95.0 93.8
1.0 1.1 1.1 1.1
89.1 82.1 b 86.5 6 84.0 6
0.9 0.6 0.8 1.9
0 940 940 940
0 196 196 0
0.94 0.60 6 0.78 1.04
0.11 0.04 0.09 0.11
96.7 97.4 97.1 97.4
0.7 0.6 0.4 0.8
87.3 78.9 6 84.9 88.1
2.2 1.1 2.5 1.4
85.8 83.0 6 83.7 86.2
0.7 1.2 1.0 1.0
Percentage of alveolar macrophages engulfing one or more latex spheres after 1 h of incubation at 37°C (ratio of macrophages to latex = 1:200). Significant change from corresponding control mice exposed to filtered air (p < 0.05).
macrophages seen after 2 mo of daily 3-h exposures to the NO 2 -O 3 mixture (96.8% compared with 98.6% in controls). The viability of the alveolar macrophages was not affected by exposure for 1 or 2 mo to the pollutants. After 3 mo there was a significant (p < 0 . 0 5 ) decrease in viability in macrophages isolated from mice exposed to the NO 2 and O 3 peaks (78.9% compared with 87.3% in controls). Although not significant, a decrease in viability was also seen in macrophages of mice exposed continuously to 188 jug/m3 NO 2 with superimposed daily peaks of the NO 2 -O 3 mixture (84.9%). Phagocytic activity, expressed as the percentage of macrophages that engulfed at least one latex sphere after 1 h of incubation at 37°C (Gardner et al., 1973), was not affected by 1 mo of exposure to the pollutants. After 2 and 3 mo, however, there was a significant (p < 0.05) decrease in the phagocytic activity of macrophages from mice exposed daily for 3 h to the NO 2 -O 3 mixture (83.0%) and those continuously exposed to 188 Mg/m3 NO 2 and the superimposed NO 2 -O 3 peaks (83.7%) compared with control animals exposed to filtered air (85.8%). After 2 mo but not after 3 mo a significant decrease in phagocytosis was also seen in mice exposed daily to the 3-h peaks of 940 Mg/m3 NO 2 .
640
R. EHRL1CH ET AL.
Limited experiments were conducted to determine the effects of 1, 3, and 6 mo of exposure to the pollutants on the activity of selected blood serum enzymes in mice. In general, there was an increase in the activity of lactate dehydrogenase (LDH), serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamic-pyruvic transaminase (SGPT) and a depression of acid phosphatase (AP) and cholinesterase (CHE) activity. Continuous exposure to 188 Mg/m3 NO 2 with superimposed 3-h daily peaks of the NO 2 -O 3 mixture appeared to be most effective in inducing these changes.
Downloaded by [University of Florida] at 04:26 14 November 2015
DISCUSSION The results of this study indicate that the sequence of exposures to the pollutants is of major importance in reducing the resistance to streptococcal pneumonia. More specifically, the combination of extended exposure to the pollutants and continuation of exposure after the respiratory challenge with Streptococcus, aerosol was most deleterious. After such a sequence of exposures, significantly increased mortalities and shortened survival times were seen in mice exposed to almost all pollutant regimens. Moreover, decreased resistance to bacterial pneumonia appeared sooner in mice reexposed to the pollutants than- in those allowed to breathe clean air after the infectious challenge. Another important factor in the decreased resistance to infection was the stress of daily 3-h peak exposures to a mixture of 940 Mg/m3 NO 2 and 196 Mg/m3 O 3 . Increased mortality and reduced survival time were evident after 2, 3, and 6 mo of such exposures, whether the mice were kept in clean air or were reexposed to the pollutant mixture after the infectious challenge. After 2 and 3 mo of exposure the reduced resistance to infection was accompanied by significant decreases in phagocytic activity, and after 3 mo by reduced viability of alveolar macrophages. Lungs of mice exposed for 3 mo to this pollutant mixture, challenged with Streptococcus aerosol, and reexposed to the pollutants showed widespread damage when examined by scanning electron microscopy. Instead of the honeycomb appearance of alveoli seen in lungs of infected mice exposed to filtered air, the alveolar walls had thickened and in some areas walls of several alveoli appeared to have fused, creating a single large alveolus. Exposure to peak concentrations of the NO 2 -O 3 mixture also appeared to be most effective in inducing changes in blood serum enzymesdecreases in AP and CHE and increases in LDH, SGOT, and SGPT. Elevations of SGOT, SGPT, and LDH are seen in many pathological conditions, and these enzymes are released as the result of damage to various tissues. Thus increased levels of these enzymes could be indicative of damage to the lung resulting from exposure to air pollutants. Similar decreases in AP and CHE and increases in LDH and SGOT were observed by S. Kosmider (personal communication) and by Menzel et al. (1977) in
641
N0 2 AND 0 3 EFFECTS ON BACTERIAL PNEUMONIA
Downloaded by [University of Florida] at 04:26 14 November 2015
guinea pigs exposed to 940 Mg/m3 NO 2 for 8 h/d, 7 d/wk, for 4 mo. Despite the differences in species and in the regimen and duration of exposure, the trends of the changes closely resembled those seen in mice after 6 mo of exposure to daily 3-h peaks of the NO 2 -O 3 mixture. Daily 3-h exposures to the mixture of 940 Mg/m3 NO 2 and 196 ng/m3 O 3 superimposed on the continuous exposure to 188 Mg/m3 NO 2 were also effective in reducing the resistance to bacterial pneumonia. Significant increases in mortality and shortened survival times were seen in mice exposed to this pollutant regimen for 1, 2, and 3 mo before the infectious challenge and for 14 d after the challenge. However, reduced mortality was seen after 1, 2, and 3 mo of exposure when mice were maintained in
POLLUTANT
24 l i r . / d a , : AIR
Jhr./dayj
>
CHALLENGE
188*g/m'N0 2
AI
.
FIGURE 2. Changes in mortality from streptococcal pneumonia in mice subjected to various NO2 and O3 exposure regimens.
Downloaded by [University of Florida] at 04:26 14 November 2015
642
R. EHRLICH ETAL.
clean air for 14 d after the infectious challenge. This trend was reversed after 6 mo of exposure, at which time there was a significant ( p < 0 . 1 ) increase in mortality (Fig. 2). A similar response, namely an initial decrease followed by an increase in mortality, was observed during 6-mo continuous exposures to 188 Aig/m3 NO 2 with 3-h daily peaks of 940 Mg/m3 NO 2 . Thus, continuous exposure to the low concentration of NO 2 for 3 mo might have protected against the daily peak exposures to the higher concentrations of NO 2 or the NO 2 -O 3 mixture. This effect was seen only in animals removed from the polluted atmosphere after 1, 2, and 3 mo of exposure and kept in clean air for 14 d following the respiratory challenge with Streptococcus aerosol. The protective response was not observed when the same exposure regimens continued during the 14-d period after the infectious challenge or when the exposure to the pollutants extended from 3 to 6 mo. In general, these results confirm the results of previous experiments that showed the importance of intermittent exposure to air pollutants as a factor in altering the host's resistance to respiratory infection. Thus, this study confirms the conclusion that primary air quality standards for short-term NO 2 exposures must be established to fully protect the health of populations at risk. REFERENCES Coffin, D. L., Gardner, D. E., Holzman, R. S., and Wolock, F. J. 1968. Influence of ozone on pulmonary cells. Arch. Environ. Health 16:633-636. Ehrlich, R. and Henry, M. C. 1968. Chronic toxicity of nitrogen dioxide. I. Effect on resistance to bacterial penumonia. Arch. Environ. Health 17:860-865. Ehrlich, R., Findlay, J. C., Fenters, J. D., and Gardner, D. E. 1977. Health effects of short-term inhalation of nitrogen dioxide and ozone mixtures. Environ. Res. 14:223-231. Gardner, D. E., Graham, J. A., Miller, F. J., Illing, J. W., and Coffin, D. L. 1973. Technique for differentiating particles that are cell associated or ingested by macrophages. Appl. Microbiol. 25:471-475. Ketels, K. V., Bradof, J. N., Fenters, J. D., and Ehrlich, R. 1977. SEM studies of the respiratory tract of mice exposed to sulfuric acid mist-carbon particle mixtures. Scanning Electron Microsc. 2:519-526. Menzel, D. B., Abou-Donia, M. D., Roe, C. R., Ehrlich, R., Gardner, D. E., and Coffin, D. L. 1977. Biochemical indices of nitrogen dioxide intoxication of guinea pigs following low level long term exposure. In Proceedings of the International Conference on Photochemical Oxidant Pollution and Its Control, EPA Publ. EPA-600/3-77-001b, vol. 2, pp. 577-587. Research Triangle Park, N.C.: Environmental Protection Agency. Pindak, F. F., Schmidt, J. P., Gibon, D. J., and Allen, P. T. 1971. Interferon levels and resistance to viral infection associated with selected interferon inducers. Proc. Soc. Exp. Biol. Med. 138:317-321. Received September 25, 1978 Accepted November 20, 1978
