Pulmonary response to exposure to ozone of emphysematous rats

ENVIRONMENTAL RESEARCH 42, 114-120 (1987)

Pulmonary Response to Exposure to Ozone of Emphysematous Rats E . YOKOYAMA, Z . N A M B U , I. ICHIKAWA, I. UCHIYAMA, AND H . A R A K A W A

Department of Industrial Health, Institute of Public Health, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan Received March 13, 1984 Rats were treated with a single intratracheal instillation of 6.5 units elastase or normal saline. Seven weeks after treatment, the animals were exposed for 24 hr to filtered air or 1 ppm 03, and their lung functions were measured. The exposure to 03 resulted in functional changes depending mainly on peripheral airway obstruction, and the direction and degree of those functional changes were in general similar between the saline- and elastase-treated animals. Another group of saline- or elastase-treated rats were exposed to 3 ppm 03 for 3 hr and the edematous response of their lungs was again similar. These results indicate that elastase-treated lungs responded to the exposure to 0 3 in a fashion similar to normal lungs in rats, but lung damage caused by the exposure to 03 superimposed over preexisting emphysematous damage, resulted in an additional lessening of the margin of pulmonary reserve capacity. © 1987AcademicPress, Inc.

INTRODUCTION

Individuals who are at increased risk to the effects of atmospheric air pollutants in industry or the community are thought to exist among populations (Calabrese, 1978), and the identification and quantification of such high-risk groups may be essential for establishing air pollution control plans at the present time. Among suspected high-risk groups are most probably patients with chronic lung disease, because the defense mechanism of their lungs may be more or less impaired (Wanner, 1977), in addition to the fact that the lungs are certainly the first target of various air pollutants. In fact, surveys on the episode of acute air pollution in London (Logan, 1953) and Donora (Schrenk et al., 1949) revealed that patients with chronic lung disease were highly susceptible to the effects of smog. New areas in research for identifying high-risk groups may be opened if suitable animal models are developed. From this point of view, we recently established an elastase-induced model of emphysema in rats which was physiologically and morphologically judged to be suitable for studies evaluating interaction with air pollutants (Yokoyama et al., submitted). The present study was performed to learn if this model of emphysema might show an increased susceptibility to acute exposure to relatively high concentrations of ozone. MATERIALS AND METHODS

Intratracheal instillation ofelastase. Sixteen male Wistar rats, approximately 6 weeks of age and weighing from 210 to 230 g were anesthetized by inhalation of 114 0013-9351/87 $3.00 Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any formreserved.

RESPONSE TO 0 3 OF E M P H Y S E M A T O U S RATS

115

ethyl ether and 0.25 ml of 0.15 N saline containing 6.5 units of porcine pancreatic elastase (ESFF, Millipore Corp., Freehold, N.J.) injected into the surgically ex, posed trachea as described elsewhere (Yokoyama et al., submitted). Twelve animals injected with 0.25 ml of saline alone served as the control. The rats were kept in an air-conditioned room and weighed twice a week before the beginning of the studies. Food and water were provided ad libitum. Pulmonary function study. Seven weeks after elastase treatment, half of the saline- or elastase-treated animals were exposed to 1 ppm 03 for 24 hr (groups S-O 3 and E-O 3, respectively) and the controls were exposed to filtered air for 24 hr (groups S-A and E-A, respectively). Exposure to 03 was performed in a stainless steel chamber, 100 x 100 x I00 cm. The chamber was supplied with an unidirectional (top to bottom) flow of filtered air at the rate of eight to nine changes per hour. The 03 was produced by passing oxygen through a silent discharge 03 generator and it joined the air stream at the top of chamber. Concentration of 03 in the chamber was monitored with a Mast 03 meter which was calibrated by the neutral-buffered KI method. Exposure to air was performed at similar flow rates and chamber conditions. Food and water were provided during the exposure. Immediately after the end of exposure, the animals were subjected to pulmonary function study. The method for measuring pulmonary function was exactly the same as reported previously (Yokoyama, 1983). In brief, the anesthetized animals were placed in prone position in a body chamber and breathed through a tracheal cannula. Intrathoracic air volume at the level of end expiration, defined as functional residual capacity (FRC), was measured by an application of Boyle's law. The animals were subsequently paralyzed and ventilated by means of a Harvard pump: frequency was 80 per minute and the tidal volume 3 ml. Dynamic lung compliance (Cayn) and pulmonary flow resistance (RE) were calculated from the records of transpulmonary (cannula to esophagus) pressure (Ptp), the rate of airflow into and out of the body chamber, and the volume with electrical integration of flow signal. Frequency dependence of dynamic respiratory compliance was examined at pump frequencies varied over a range of 40 to 200 per minute. The static deflation volume-pressure (V/P) curve of the lung was constructed from the records of Ptp during a stepwise deflation from total lung capacity (TLC) to residual volume (RV). TLC was defined as volume of air retained in the lungs at Ptp of 30 cm H20 , and RV at a point where no further air withdrawal was attained. Their absolute values were calculated from a combination of the V/P curve thus obtained and the FRC determined during spontaneous breathing on the assumption that muscle paralysis did not significantly alter the resting level of breathing. Static lung compliance (Cst) was obtained as the slope of the V/P curve over the tidal volume range from the level of FRC. Expiratory flow-volume (V/V) curve during forced deflation was simulated by venting the lungs maintained at the level of TLC to a negative pressure reservoir ( - 3 cm Hg), and the peak flow rate (l/p) and the flow rate at 50% TLC (Vma~50%TLC)were obtained from the ¢¢/V curve recorded on a storage oscilloscope. Study on edematous response. An additional 40 rats of the same age and sex as previously described were similarly treated with elastase or saline alone. Seven

116

YOKOYAMA ET AL.

weeks after treatment, half of the rats was exposed to 3 ppm 0 3 for 3 hr. Immediately after the end of exposure, the rats were sacrificed by exsanguination from the abdominal aorta after anesthesia with intraperitoneal pentobarbital sodium. The remaining 20 rats were treated similarly. The lungs were then removed from the thorax, rinsed with saline, blotted, and weighed. Dry weight of the lungs was estimated after drying for 2 days in an oven at 110°C. Statistical analysis. The results were presented as means _ SD. The results were evaluated using Student's t test and a P value less than 0.05 was considered to be significant.

RESULTS

Comparisons of lung functions between the S-A group and the E-A group. As shown in Table 1, increases in the fractions of lung volume (TLC, FRC, and RV) and Cst and decreases in f'p and CVmaxS0%TLc were noted in the E-A group as compared to those in the S-A group, although all of the differences were not necessarily significant. Similar trends in the differences were also obtained in these parameters after dividing by TLC. N o practical difference in Cdyn and R L was observed between the groups, but R L multiplied by FRC in the E-A group was significantly larger than that in the S-A group. Functional effects o f the exposure to 1 ppm 03for 24 hr. The present exposure to 1 ppm 03 for 24 hr caused the loss of body weight: it reached 14 _+ 6 and 15 + TABLE 1

PULMONARY FUNCTIONS OF RATS EXPOSED TO AIR OR 1 ppm O 3 FOR 24 HR IN THE SALINE- AND ELASTASE-TREATED GROUPS

Saline

Bodywt(g) T L C (ml) F R C (ml) RV (ml) ~p(ml/s) VmaxS0%TLC (ml/s) Cst (ml/cm H20) Cayn (ml/cm H20) R L (cm H20/ml/s) F R C / T L C (%) R V / T L C (%) .Vp/TLC (TLC/s) Vm~x5o%TLc/TLC (TLC/s) Cst/TLC ( T L C / c m H20) Cayn/FRC ( F R C / c m H20) R L × F R C (cm HzO/FRC/S)

Note. Values are means

Elastase

Air

03

Air

(n = 6)

(n = 6)

(n = 8)

423 13.9 3.2 1.5 119 53 1.11 0.39 0.16 22.7 11.1 8.6 3.8 0.079 0.127 0.52

± 17 _--- 1.1 ± 0.5 ± 0.3 ± 12 ± 6 ± 0.07 ± 0.08 ± 0.03 ± 2.6 -- 2.6 ± 0.3 -+ 0.4 _4- 0.006 ± 0.032 ± 0.12

407 13.4 4.2 2.6 109 38 0.92 0.30 0.16 31.3 19.4 8.1 2.8 0.069 0.074 0.68

+ ± ± ± ± ± _+ ± ± ± ± ± ± ± ±

11 0.9 0.8* 1.0" 5 8* 0.08** 0.05 0.04 5.4** 6.9* 0.7 0.6* 0.008* 0.019"* 0.23

415 14.8 4.3 2.3 107 46 1.43 0.43 0.16 29.6 16.2 7.3 3.1 0.094 0.102 0.71

± ± ± ± ± ± _+ ± ± ± ± ± ~ -+ ± ±

11 2.2 1.0 ° 0.7 ° 11 8 0.37 ° 0.15 0.03 5.0 °° 7.0 0.7 °o 0.8 ° 0.015 ° 0.040 0.21 °°

03 (n = 8) 401 15.0 5.2 3.2 100 32 1.32 0.45 0.15 34.2 21.1 6.6 2.1 0.088 0.090 0.78

± ± ± ± ± ± ± ± -+ ± ± ± ± -+ ± ±

17 0.8 0.7* 0.9* 15 13" 0.29 0.12 0.02 5.8 6.9 0.8 0.5* 0.019 0.028 0.16

-+ SD. °°,° Significantly different from the S-A group, P < 0.01, and P < 0.05, respectively. **,* Significantly different from the respective air-exposed group, P < 0.01, and P < 0.05, respec-

tively.

RESPONSE TO 0 3 OF EMPHYSEMATOUS RATS

117

4 g in the S-O 3 and E - O 3 groups, respectively, and consequently, the body weights differed, though not significantly, between the S-A and S-O3 groups and the E-A and E - O 3 groups, as shown in Table 1. The S-A group versus the S-O3 group: Differences of mean values between the S-A and S-O3 groups were significant (P < 0.05 or P < 0.01) in FRC, RV, ~/ma~50%TLC and Cst. The increase in FRC and RV in the S-O3 group was not accompanied by any noticeable change in TLC, and the decrease in VmaxS0%TLc by no significant change in Vp. Differences between the groups were still significant in these parameters divided by TLC. No significant difference was noted in Cdyn and R E, but Cdyn divided by FRC (specific compliance) was significantly (P < 0.01) smaller in the S-O 3 group. As shown in Fig. 1, dynamic respiratory compliance of the S-A group tended to increase at frequencies higher than 160 per minute, but that of the S-O3 group decreased at the frequency of 200 per minute and its difference from the S-A group was significant (P < 0.05). The E-A group versus the E - O 3 group: Qualitative and quantitative differences in functional parameters noted between the E-A and E - O 3 groups were in general similar to those between the S-A and S-O 3 groups, although there were some divergencies in the presence or absence of statistical significance due probably to relatively wide scattering of the measured values in the elastase-treated animals. As shown in Fig. 1, dynamic respiratory compliance of the E-A group decreased at the frequency of 200 per minute, but that of the E - O 3 group further decreased at this frequency by an amount which attained significant difference (P < 0.05) from the former group. Edematous effect of the exposure to 3 ppm 03for 3 hr. As shown in Fig. 2, the exposure to 3 ppm 03 for 3 hr caused increases in lung wet and dry weights of the saline-treated animals, but the increase in dry weight did not attain statistical

,.i[

1.0

:E (3. LL

o

-

0.9

"'""

(n=6)

Saline- treated :

: ....

AIR

~ 03

1.1

-o L} 1.0

"•:@•,, , ~

,3

(n=8)

0.9 Elastase- treated

" ~" (n=8)

0.6

10

4

L

BO

i

120

i

160

2 0

Frequency, RPM

FIG. 1. Dynamic respiratory compliance (Cdyn(R)) at varying respiratory frequency of the rats exposed for 24 hr to air or 1 ppm 03 in the saline- and elastase-treated groups. Cdyn(R)at each frequency is expressed as a relative value to Cdyn(m at the frequency of 40 per minute. Data are means ± SD. Numbers in parentheses represent the numbers of animals tested.

118

YOKOYAMA

ETAL.

Saline 2.o

C

Elastase

03

C

T

T

03

1.5

3

I.

:>-

c3

0.2

T

J 85,

80 Q= BW, g Number of rats

400 +-53

406 -*60

401 398 +-42 -4-50

(9)

(10)

(10)

(11)

Fro. 2. Lung wet and dry weights and relative lung water content of the control rats (C) and the rats exposed to 3 ppm 03 for 3 hr (03) in the saline- and elastase-treated groups. Data are means _+ SD. **,* Significantly different from the respective control, P < 0.01 and P < 0.05, respectively. significance. Consequently, the relative lung water content (wet weight - dry weight/wet weight x t00) was also significantly increased by the e x p o s u r e to 03. The degree of e d e m a t o u s r e s p o n s e of lungs of the elastase-treated rats was nearly similar to that of the saline-treated rats. DISCUSSION

We d e m o n s t r a t e d in a recent report (Yokoyama et al., submitted) that the lungs of rats treated with single intratracheal instillation of elastase of the same dose as used in the present e x p e r i m e n t r e s e m b l e d morphologically h u m a n panlobular emp h y s e m a and its elastic recoil lowered, and these lesions were m o r e evident app r o x i m a t e l y 7 to 10 w e e k s after treatment. The characteristics of their lung function w e r e an increased lung volume and compliance and a d e c r e a s e d flow rate during forced expiration. The same trend in change was shown by comparing the lung functions of the E - A group with those of the S-A group. The tendency was for an increase in d y n a m i c respiratory compliance of the S-A group at higher frequencies. This pattern was c o m m o n l y o b s e r v e d in normal rats in our laboratory and might be due to inertial error involved in the pressure m e a s u r e m e n t at higher frequencies (Yokoyama, 1983). Differed r e s p o n s e of the dynamic compliance u p o n changing the respiratory f r e q u e n c y was noted in the E-A group, and

R E S P O N S E TO 0 3 O F E M P H Y S E M A T O U S RATS

119

might have resulted from uneven distribution of ventilation due to the lowered elastic recoil of the lungs of these animals. The changes in lung functions observed in the S-O3 group, that is, the increase in FRC and RV and the decrease in ~/maxS0%a'Lcnot accompanied by noticeable changes in either ~/p or RI., suggested that exposure to 1 ppm 03 for 24 hr produced obstruction of the peripheral airways. The response of the respiratory compliance of this group upon changing respiratory frequency might be compatible with these results. Our recent measurement of pulmonary flow resistance at different elastic recoil pressures in postmortem lungs of rats exposed to the same dose of 03 (1 ppm x 24 hr) also suggested the increased flow resistance of peripheral airways (Yokoyama et al., 1984). The S-O 3 grou p also demonstrated a decrease in Cst, which might be due to the increased trapped air volume in the lungs at FRC-level, but not to the change in elastic properties of the lungs. The static volume-pressure curve of postmortem lungs of the rats cited above was found to shift upwards at elastic recoil pressures below 5 cm H20. Whatever the mechanisms involved in the respiratory effect of the exposure to 1 ppm 03 for 24 hr were, it is more interesting to note that not only the direction, but also the degree of changes in lung functions observed in the E-O 3 group were in general similar to those of the S-O 3 group. Exposure to 3 ppm 03 for 3 hr was performed in order to see if the response of the alveolar capillary network of elastase-treated lungs might differ from that of the saline-treated lungs. Again, the degree of edematous response was almost similar in these lungs. Several reports are now available concerning the effects of exposure to gaseous insults on emphysematous animals. Goldring et al. (1970) reported no significant changes in the mechanical properties of the lungs in either saline- or elastasetreated hamsters exposed to 650 ppm SO 2 for 19-74 days. No marked difference was observed in the histopathologic response of their lungs either. Harkema et al. (1982) reported that in rats elastase-treated lungs responded functionally to hyperoxia similarly to normal lungs and there was no significant difference in their survival curves. A similar result was also reported by Busch et al. (1984) for the structural response of alveoli of elastase-treated rats and guinea pigs to shortterm (NH4)2SO 4 exposure. On the other hand, Raub et al. (1983) reported a slightly suppressed response of diffusing capacity for carbon monoxide of elastase-treated hamsters to the exposure to a complex mixture of olefin-ozonesulfur dioxide for 28 days compared with that of saline-treated animals, and there was a report showing an increased survival of emphysematous hamsters upon exposure to hyperoxia (Kovnat et al., 1975). Direct comparisons among these results, including ours, are difficult because of differences not only in the conditions of exposure to the insults, but also in the conditions of elastase treatment. All the above authors exposed the animals to the insults about 3-4 weeks after treatment, but our exposure was 7 weeks after treatment. We believe that at least 7 weeks are needed to produce a suitable model of mild emphysema in rat lung with the dose of elastase used in the current study. We may not be justified in discussing the susceptibility of the lungs to 03 without knowing the inhaled dose, butJt was clearly shown by the present results

120

YOKOYAMA ET AL.

that emphysematous lungs of the rat responded to 03 in a fashion similar to the normal organ and consequently, the lung damage caused by 03 was superimposed over the preexisting emphysematous damage. Therefore, the margin of reserve capacity of the lung is evidently less in emphysematous rats than in normal rats after the exposure to 03 . These results cannot be applied directly to humans, but might a similar effect have resulted in victims of acute air pollution episodes in the past? ACKNOWLEDGMENT This study was partially supported by a grant-in-aid from the Environment Agency, Japan.

REFERENCES Busch, R. H., Buschbom, R. L., Cannon, W. C., Lauhala, K. E., Miller, E J., Graham, J. A., and Smith, L. G. (1984). Effects of ammonium sulfate aerosol exposure on lung structure of normal and elastase-impaired rats and guinea pigs. Environ. Res. 33,454-472. Calabrese, E. J. (1978). "Pollutants and High-Risk Groups: The Biological Basis of Increased Human Susceptibility to Environmental and Occupational Pollutants." Wiley-Interscience, New York. Goldring, I. R, Greenburg, L., Parks, S., and Ratner, [. M. (1970). Pulmonary effects of sulfur dioxide exposure in the Syrian hamster. II. Combined with emphysema. Arch. Environ. Health 21, 32-37. Harkema, J. R., Mauderly, J. L., and Graham, J. A. (1982). The effects of emphysema on oxygen toxicity in rats. Amer. Rev. Respir. Dis. 126, 1058-1065. Kovnat, D. M., Lory, R, Snider, G. L., and Brody, J. S. (1975). Susceptibility of the emphysematous lung to oxygen toxicity. Chest 68, 417. Logan, W. R D. (1953). Mortality in the London fog incident, 1952. Lancet 14, 336-338. Raub, J. A., Miller, E J., Graham, J. A., Gardner, D. E., and O'Neil, J. J. (1983). Pulmonary function in normal and elastase-treated hamsters exposed to a complex mixture of olefin-ozone-sulfur dioxide reaction products. Environ. Res. 31, 302-310. Schrenk, H. H., Heimann, H., Clayton, G. D., Gefafer, W. M., and Wexler, H. (1949). Air pollution in Donora, Pa. Public Health Bull., no. 306, Federal Security Agency, PHS, Division of Industrial Hygiene, Washington, D.C. Wanner, A. (1977). Clinical aspects of mucociliary transport. Amer. Rev. Respir. Dis. 116, 73-125. Yokoyama, E. (1983). Methods for measuring respiratory mechanics in rats. Japan. J. Thorac. Dis. 21, 357-363. Yokoyama, E., Ichikawa, I., Nambu, Z., Kawai, K., and Kyono, Y. (1984). Respiratory effects of intermittent exposure to ozone on rats. Environ. Res. 33, 271-283. Yokoyama, E., Nambu, Z., Uchiyama, 1., and Kyono, H. (1987). An emphysema model in rats treated with intratracheal elastase. Environ. Res., in press.