Journal of Toxicology and Environmental Health
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Induction of transient airway hyperresponsiveness by exposure to 4 ppm nitrogen dioxide in guinea pigs Takahiro Kobayashi & Yumi Shinozaki To cite this article: Takahiro Kobayashi & Yumi Shinozaki (1992) Induction of transient airway hyperresponsiveness by exposure to 4 ppm nitrogen dioxide in guinea pigs, Journal of Toxicology and Environmental Health, 37:3, 451-461, DOI: 10.1080/15287399209531683 To link to this article: http://dx.doi.org/10.1080/15287399209531683
Published online: 15 Oct 2009.
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Citing articles: 3 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: 06 November 2015, At: 10:45
INDUCTION OF TRANSIENT AIRWAY HYPERRESPONSIVENESS BY EXPOSURE TO 4 PPM NITROGEN DIOXIDE IN GUINEA PIGS
Downloaded by [University of Florida] at 10:45 06 November 2015
Takahiro Kobayashi Department of Basic Medical Sciences, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan Yumi Shinozaki
Department of Sciences, University of Toho, Funabashi, Chiba, Japan.
In the present study, we investigated (1) whether airway responsiveness to inhaled histamine-aerosol could be induced during 7-d exposure of guinea pigs to 4 ppm NO2 and, if so, (2) whether thromboxane A2 may be involved in such increase. Female Hartley guinea pigs were divided into 6 groups (n - 15/group). Three groups were exposed to filtered air and the other 3 groups were exposed to NO2 for 1, 3, or 7 d (24 h/d). Baseline specific airway resistance (SRaw0) did not change significantly after exposure to 4 ppm NO2 or air. Airway responsiveness was determined 1 wk before the beginning of exposure and on the day of termination of the exposure. Prior to exposure to NO2 the EC200His, the concentrations of inhaled histamine necessary to double SRawNaCl (SRaw after inhalation of 0.9% NaCI), were 1.07 ± 0.20, 1.30 ± 0.20, and 1.01 ± 0.18 mM for the 3 groups later given NO2 for 1, 3, and 7 d, respectively. Following exposure to NO2 for 1, 3, or 7 d, EC200His values were 1.42 ± 0.25, 0.66 ± 0.10 (p < .05), and 1.05 ± 0.22 mM, respectively. These results show that 7-d exposure to 4 ppm NO2 induced a significant increase in airway responsiveness on d 3. Exposure to air had no significant effect on the airway responsiveness. This transient hyperresponsiveness was inhibited by a specific inhibitor of thromboxane synthetase, OKY 046. These results indicated that (1) a lower concentration (4 ppm) of NO2 than that previously reported can induce transient hyperresponsiveness in guinea pigs during appropriate long-term exposure, and (2) thromboxane A2 may play an important role in this transient airway hyperresponsiveness.
This work was supported in part by a grant-in-aid (62570354) C for Research from the Ministry of Education, Science, and Culture of Japan. The writers thank Dr. Shyunsuke Suzuki, Yokohama City University, and Dr. Seiichiro Hirano, National Institute for Environmental Studies (NIES), for their help in making our apparatus for measurement of specific airway resistance; Dr. Ryozou Fujii (Toho University) for encouragement; and Mrs. Kumiko Terakado for her secretarial assistance. OKY 046 was generously donated by Ono Pharmaceuticals Co. Ltd. (Osaka, Japan). Requests for reprints should be sent to T. Kobayashi, Ph.D., Department of Basic Medical Sciences, National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan.
451 Journal of Toxicology and Environmental Health, 37:451-461, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
452
T. KOBAYASHI AND Y. SHINOZAKI
Downloaded by [University of Florida] at 10:45 06 November 2015
INTRODUCTION Nitrogen dioxide (NO2), which is produced by a variety of combustion processes, is a major component of air pollution in urban areas. This gas is found in both indoor as well as outdoor environments (Speizer et al., 1980). Epidemiological studies have shown that there is a possible relationship between NO2 and alteration of pulmonary function and airway responsiveness (Monstardi et al., 1981). In the study on experimental animals, Silbaugh and co-workers (1981) demonstrated that a 1-h exposure of NO2 at >40 ppm to guinea pigs induces an increase in airway reactivity in inhaled histamine. Abraham and co-workers (1980) also showed that exposure to 7.5 and 15 ppm NO2 for 2 h may induce hyperresponsiveness in some sheep. Therefore, in the case of short-term exposure to NO2, high concentrations of NO2 induce airway hyperresponsiveness. However, the effects of relatively long-term exposure to NO2 on airway responsiveness and the mechanism of airway hyperresponsiveness induced by NO2 have remained ill defined. Previous studies showed that thromboxane A2 may be an important mediator of airway hyperresponsiveness during the inflammatory process (Aizawa et al., 1985; Chung et al., 1986a, 1986b; O'Byrne et al., 1985). Therefore, it is important to determine whether thromboxane A2 is involved in airway hyperresponsiveness induced during relatively long-term exposure to NO2. In the present study, in order to elucidate the relationship between exposure to NO2 and asthma, we investigate (1) whether airway responsiveness to inhaled histamine-aerosol could be induced during 7-d exposure of guinea pigs to 4 ppm NO2 and, if so, (2) whether thromboxane A2 may be involved in the airway hyperresponsiveness induced during 7-d exposure to this gas. MATERIALS AND METHODS
Animals One hundred and thirty female Hartley strain guinea pigs (about 250 g body weight and 4 wk of age, Sic) were used in this study and were fed standard guinea pig feed (RC4 Oriental Yeast Co. Ltd., Tokyo) and given water ad libitum. Ninety animals were used to study the effect of NO2 on airway responsiveness. Forty animals were used to study the effect of OKY 046 on airway hyperresponsiveness induced by NO2 exposure. Protocol The animals weighed 302-424 g (6 wk of age) at the time of measurement of baseline specific airway resistance and airway responsiveness to inhaled histamine aerosol (d 0). On d 7, the animals were exposed to nitrogen dioxide or filtered air after having been divided into 6 groups (n = 15 each). Three groups were exposed to filtered air for 1, 3, or 7 d
N O 2 EXPOSURE A N D AIRWAY HYPERRESPONSIVENESS
453
(24 h/d), and the other 3 to NO2 for 1, 3, or 7 d (24 h/d). On the day of termination of exposure to filtered air or NO2, airway responsiveness to inhaled histamine aerosol was determined immediately after exposure. Immediately after measurement of airway responsiveness, guinea pigs were stunned and killed by exsanguination. Lung wet weight was measured.
Downloaded by [University of Florida] at 10:45 06 November 2015
Nitrogen Dioxide Exposure
Animal exposure to NO2 has been described (Kobayashi et al., 1983). Briefly, the guinea pigs were exposed to nitrogen dioxide in one of the two identical chambers with a volume of 1.39 m3 in all experiments. Only filtered room air flowed through one chamber, and in the other chamber for NO2 exposure, filtered room air was mixed with -4000-5000 ppm NO2/N2 from a bomb before it entered that chamber. The chambers were operated under dynamic conditions at 25 ± 1 °C, 55 ± 5% humidity, and - 5 mm H2O relative to atmospheric pressure with an air flow of 110 m3/ h. The concentrations oí NO2 and NO were feedback controlled by continuous monitoring with an NOX analyzer (Monitor Labs model 8440, San Diego, Calif.) that operates on a chemiluminescence principle. NO was undetectable in either chamber. Measurement of Specific Airway Resistance
Specific airway resistance (SRaw) was measured in a constant volume (-12,000 cm3, 17 x 43 x 16.5 cm) body plethysmograph based on the design of Agrawal (1981). An intact, unanesthetized, spontaneously breathing guinea pig was placed in a two-chamber restrainer that kept the head fixed and isolated from the body behind the neck. Airflow and volume changes at the snout were measured with a pneumotachograph (number 0, Fleish Instruments, Lausanne, Switzerland) connected to a differential pressure transducer (model MP45-14, Validyne, Northridge, Calif.), a carrier demodulator (model CD72, Validyne), and an integrator (AR-601G, Nihonkohden, Tokyo, Japan). Box pressure changes were determined with a differential pressure transducer (model MP45-14) and a carrier demodulator (model CD72). Airflow and box pressure were recorded on a recorder (RJC-4124, Nihonkohden). Airflow (ordinate) and box pressure signals (abscissa) were also displayed simultaneously on an X-Y digital storage oscilloscope (SS-5802, Iwatsu Electric, Tokyo, Japan). The signal was stored every 0.5 ms. Loops, formed from the signals, were recorded on an X-Y recorder (RY-11A, Rikadenki Kogyo, Tokyo, Japan). The rising limb of the loop corresponds to the transition period from exhalation to inhalation. During this period, change in lung volume is minimal. Therefore temperature-humidity artifacts are negligible. The slope of the rising limb provided the ratio of air flow and the corresponding change in box pressure. SRaw is a reciprocal of specific airway conductance (SGaw), SRaw = 1/SGaw. Therefore, according to previous re-
454
T. KOBAYASHI AND Y. SHINOZAKI
port (Agrawal, 1981), SRaw was calculated by dividing the barometric pressure (Pb) minus water vapor pressure at body temperature (PJ by the average value of the slopes in the rising limb of the loops (tan 0) as followings, SRaw = (Pb — PJ/tan 6. The airflow was calibrated by use of a rotometer (R-2-15D, Ueshima, Tokyo, Japan) through which known airflows were passed. The box pressure was calibrated by rapid delivery of boluses of air from a calibrated syringe.
Downloaded by [University of Florida] at 10:45 06 November 2015
Airway Responsiveness
Airway responsiveness was assessed by measurement of SRaw as a function of increasing concentration of histamine aerosol. Saline and histamine aerosols were generated by nebulizers (NE-U11B, Omron, Tokyo) containing 0.0, 0.1, 0.25, 0.5,1.0, 2.5, 5.0, and 10.0 mJW histamine in saline (0.9% NaCI). A constant flow of room air at 3 ml/s was provided at the nares in the anterior chamber of the restrainer by an air pump (AP032Z, Iwaki, Tokyo, Japan). To provide the aerosol, we passed 3 ml/s of room air through the nebulizer using a 3-way stopcock connected to the inlet of the anterior chamber. At this airflow, each nebulizer was adjusted to deliver 1.55 mg/min wet weight of the 0.9% NaCI aerosol. At this air flow, aerodynamic mass median diameter determined by a Marple personal cascade impactor (series 290, Sierra Instruments, Carmel Valley, Calif.) was 1.2 ¡xm {ag = 1.25). To protect the rotometer, we connected a filter (2500QAT-UP, Pallflex Products Corp., Putnam, Conn.) between the outlet of the anterior chamber and the air pump. At first, SRaw (SRaw0) was determined before inhalation of the aerosols. After inhalation of each dose for 30 s, SRaw was determined within 1 min. A challenge dose was given every 5 min until SRaw increased to twice the value of SRawNac, (SRaw after inhalation of 0.9% NaCI aerosol). Concentration (histamine)-response (SRaw) curves were constructed by plotting on semilogarithmic paper. The effective molar concentration of histamine (EC200His) that produced a doubling of SRawNaC, was determined by interpolation from these concentration-response curves. Effect of OKY 046 on NO2-lnduced Airway Hyperresponsiveness
To investigate whether the NO2-induced increase in airway responsiveness was due to thromboxane A2, we studied the effect of a specific thromboxane synthetase inhibitor, OKY 046, on airway hyperresponsiveness. Animals were divided into 4 groups (n = 10 each), and SRaw0, SRawNaC„ and airway responsiveness to inhaled histamine aerosol were determined on d 0. On d 7, group 1 and group 3 were exposed to filtered air and group 2 and group 4 were exposed to 4 ppm NO2 for 3 d. Immediately after the termination of the exposure, groups 1 and 2 were exposed to aerosol from 0.9% NaCI water solution, and groups 3 and 4 were exposed to OKY 046 (4 mg/ml in 0.9% NaCI) aerosol, for 5 min. The aerosol was generated by an ultrasonic nebulizer (model 65, Devilbiss
N O , EXPOSURE A N D AIRWAY HYPERRESPONSIVENESS
455
Co., Somerset, Pa.). Air at 10 ml/s was passed through the nebulizer and delivered to the nare. Thirty minutes after inhalation of the saline or the inhibitor, airway responsiveness to inhaled histamine aerosol was determined. Statistical Analysis The EC200 and SRaw0 were expressed as means ± SE and were compared by Student's f-test for paired data of the preexposure and postexposure to NO2 or filtered air and pretreatment and posttreatment with OKY 046. A p value
ISSN: 0098-4108 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/uteh19
Induction of transient airway hyperresponsiveness by exposure to 4 ppm nitrogen dioxide in guinea pigs Takahiro Kobayashi & Yumi Shinozaki To cite this article: Takahiro Kobayashi & Yumi Shinozaki (1992) Induction of transient airway hyperresponsiveness by exposure to 4 ppm nitrogen dioxide in guinea pigs, Journal of Toxicology and Environmental Health, 37:3, 451-461, DOI: 10.1080/15287399209531683 To link to this article: http://dx.doi.org/10.1080/15287399209531683
Published online: 15 Oct 2009.
Submit your article to this journal
View related articles
Citing articles: 3 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: 06 November 2015, At: 10:45
INDUCTION OF TRANSIENT AIRWAY HYPERRESPONSIVENESS BY EXPOSURE TO 4 PPM NITROGEN DIOXIDE IN GUINEA PIGS
Downloaded by [University of Florida] at 10:45 06 November 2015
Takahiro Kobayashi Department of Basic Medical Sciences, National Institute for Environmental Studies, Tsukuba, Ibaraki, Japan Yumi Shinozaki
Department of Sciences, University of Toho, Funabashi, Chiba, Japan.
In the present study, we investigated (1) whether airway responsiveness to inhaled histamine-aerosol could be induced during 7-d exposure of guinea pigs to 4 ppm NO2 and, if so, (2) whether thromboxane A2 may be involved in such increase. Female Hartley guinea pigs were divided into 6 groups (n - 15/group). Three groups were exposed to filtered air and the other 3 groups were exposed to NO2 for 1, 3, or 7 d (24 h/d). Baseline specific airway resistance (SRaw0) did not change significantly after exposure to 4 ppm NO2 or air. Airway responsiveness was determined 1 wk before the beginning of exposure and on the day of termination of the exposure. Prior to exposure to NO2 the EC200His, the concentrations of inhaled histamine necessary to double SRawNaCl (SRaw after inhalation of 0.9% NaCI), were 1.07 ± 0.20, 1.30 ± 0.20, and 1.01 ± 0.18 mM for the 3 groups later given NO2 for 1, 3, and 7 d, respectively. Following exposure to NO2 for 1, 3, or 7 d, EC200His values were 1.42 ± 0.25, 0.66 ± 0.10 (p < .05), and 1.05 ± 0.22 mM, respectively. These results show that 7-d exposure to 4 ppm NO2 induced a significant increase in airway responsiveness on d 3. Exposure to air had no significant effect on the airway responsiveness. This transient hyperresponsiveness was inhibited by a specific inhibitor of thromboxane synthetase, OKY 046. These results indicated that (1) a lower concentration (4 ppm) of NO2 than that previously reported can induce transient hyperresponsiveness in guinea pigs during appropriate long-term exposure, and (2) thromboxane A2 may play an important role in this transient airway hyperresponsiveness.
This work was supported in part by a grant-in-aid (62570354) C for Research from the Ministry of Education, Science, and Culture of Japan. The writers thank Dr. Shyunsuke Suzuki, Yokohama City University, and Dr. Seiichiro Hirano, National Institute for Environmental Studies (NIES), for their help in making our apparatus for measurement of specific airway resistance; Dr. Ryozou Fujii (Toho University) for encouragement; and Mrs. Kumiko Terakado for her secretarial assistance. OKY 046 was generously donated by Ono Pharmaceuticals Co. Ltd. (Osaka, Japan). Requests for reprints should be sent to T. Kobayashi, Ph.D., Department of Basic Medical Sciences, National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan.
451 Journal of Toxicology and Environmental Health, 37:451-461, 1992 Copyright © 1992 by Hemisphere Publishing Corporation
452
T. KOBAYASHI AND Y. SHINOZAKI
Downloaded by [University of Florida] at 10:45 06 November 2015
INTRODUCTION Nitrogen dioxide (NO2), which is produced by a variety of combustion processes, is a major component of air pollution in urban areas. This gas is found in both indoor as well as outdoor environments (Speizer et al., 1980). Epidemiological studies have shown that there is a possible relationship between NO2 and alteration of pulmonary function and airway responsiveness (Monstardi et al., 1981). In the study on experimental animals, Silbaugh and co-workers (1981) demonstrated that a 1-h exposure of NO2 at >40 ppm to guinea pigs induces an increase in airway reactivity in inhaled histamine. Abraham and co-workers (1980) also showed that exposure to 7.5 and 15 ppm NO2 for 2 h may induce hyperresponsiveness in some sheep. Therefore, in the case of short-term exposure to NO2, high concentrations of NO2 induce airway hyperresponsiveness. However, the effects of relatively long-term exposure to NO2 on airway responsiveness and the mechanism of airway hyperresponsiveness induced by NO2 have remained ill defined. Previous studies showed that thromboxane A2 may be an important mediator of airway hyperresponsiveness during the inflammatory process (Aizawa et al., 1985; Chung et al., 1986a, 1986b; O'Byrne et al., 1985). Therefore, it is important to determine whether thromboxane A2 is involved in airway hyperresponsiveness induced during relatively long-term exposure to NO2. In the present study, in order to elucidate the relationship between exposure to NO2 and asthma, we investigate (1) whether airway responsiveness to inhaled histamine-aerosol could be induced during 7-d exposure of guinea pigs to 4 ppm NO2 and, if so, (2) whether thromboxane A2 may be involved in the airway hyperresponsiveness induced during 7-d exposure to this gas. MATERIALS AND METHODS
Animals One hundred and thirty female Hartley strain guinea pigs (about 250 g body weight and 4 wk of age, Sic) were used in this study and were fed standard guinea pig feed (RC4 Oriental Yeast Co. Ltd., Tokyo) and given water ad libitum. Ninety animals were used to study the effect of NO2 on airway responsiveness. Forty animals were used to study the effect of OKY 046 on airway hyperresponsiveness induced by NO2 exposure. Protocol The animals weighed 302-424 g (6 wk of age) at the time of measurement of baseline specific airway resistance and airway responsiveness to inhaled histamine aerosol (d 0). On d 7, the animals were exposed to nitrogen dioxide or filtered air after having been divided into 6 groups (n = 15 each). Three groups were exposed to filtered air for 1, 3, or 7 d
N O 2 EXPOSURE A N D AIRWAY HYPERRESPONSIVENESS
453
(24 h/d), and the other 3 to NO2 for 1, 3, or 7 d (24 h/d). On the day of termination of exposure to filtered air or NO2, airway responsiveness to inhaled histamine aerosol was determined immediately after exposure. Immediately after measurement of airway responsiveness, guinea pigs were stunned and killed by exsanguination. Lung wet weight was measured.
Downloaded by [University of Florida] at 10:45 06 November 2015
Nitrogen Dioxide Exposure
Animal exposure to NO2 has been described (Kobayashi et al., 1983). Briefly, the guinea pigs were exposed to nitrogen dioxide in one of the two identical chambers with a volume of 1.39 m3 in all experiments. Only filtered room air flowed through one chamber, and in the other chamber for NO2 exposure, filtered room air was mixed with -4000-5000 ppm NO2/N2 from a bomb before it entered that chamber. The chambers were operated under dynamic conditions at 25 ± 1 °C, 55 ± 5% humidity, and - 5 mm H2O relative to atmospheric pressure with an air flow of 110 m3/ h. The concentrations oí NO2 and NO were feedback controlled by continuous monitoring with an NOX analyzer (Monitor Labs model 8440, San Diego, Calif.) that operates on a chemiluminescence principle. NO was undetectable in either chamber. Measurement of Specific Airway Resistance
Specific airway resistance (SRaw) was measured in a constant volume (-12,000 cm3, 17 x 43 x 16.5 cm) body plethysmograph based on the design of Agrawal (1981). An intact, unanesthetized, spontaneously breathing guinea pig was placed in a two-chamber restrainer that kept the head fixed and isolated from the body behind the neck. Airflow and volume changes at the snout were measured with a pneumotachograph (number 0, Fleish Instruments, Lausanne, Switzerland) connected to a differential pressure transducer (model MP45-14, Validyne, Northridge, Calif.), a carrier demodulator (model CD72, Validyne), and an integrator (AR-601G, Nihonkohden, Tokyo, Japan). Box pressure changes were determined with a differential pressure transducer (model MP45-14) and a carrier demodulator (model CD72). Airflow and box pressure were recorded on a recorder (RJC-4124, Nihonkohden). Airflow (ordinate) and box pressure signals (abscissa) were also displayed simultaneously on an X-Y digital storage oscilloscope (SS-5802, Iwatsu Electric, Tokyo, Japan). The signal was stored every 0.5 ms. Loops, formed from the signals, were recorded on an X-Y recorder (RY-11A, Rikadenki Kogyo, Tokyo, Japan). The rising limb of the loop corresponds to the transition period from exhalation to inhalation. During this period, change in lung volume is minimal. Therefore temperature-humidity artifacts are negligible. The slope of the rising limb provided the ratio of air flow and the corresponding change in box pressure. SRaw is a reciprocal of specific airway conductance (SGaw), SRaw = 1/SGaw. Therefore, according to previous re-
454
T. KOBAYASHI AND Y. SHINOZAKI
port (Agrawal, 1981), SRaw was calculated by dividing the barometric pressure (Pb) minus water vapor pressure at body temperature (PJ by the average value of the slopes in the rising limb of the loops (tan 0) as followings, SRaw = (Pb — PJ/tan 6. The airflow was calibrated by use of a rotometer (R-2-15D, Ueshima, Tokyo, Japan) through which known airflows were passed. The box pressure was calibrated by rapid delivery of boluses of air from a calibrated syringe.
Downloaded by [University of Florida] at 10:45 06 November 2015
Airway Responsiveness
Airway responsiveness was assessed by measurement of SRaw as a function of increasing concentration of histamine aerosol. Saline and histamine aerosols were generated by nebulizers (NE-U11B, Omron, Tokyo) containing 0.0, 0.1, 0.25, 0.5,1.0, 2.5, 5.0, and 10.0 mJW histamine in saline (0.9% NaCI). A constant flow of room air at 3 ml/s was provided at the nares in the anterior chamber of the restrainer by an air pump (AP032Z, Iwaki, Tokyo, Japan). To provide the aerosol, we passed 3 ml/s of room air through the nebulizer using a 3-way stopcock connected to the inlet of the anterior chamber. At this airflow, each nebulizer was adjusted to deliver 1.55 mg/min wet weight of the 0.9% NaCI aerosol. At this air flow, aerodynamic mass median diameter determined by a Marple personal cascade impactor (series 290, Sierra Instruments, Carmel Valley, Calif.) was 1.2 ¡xm {ag = 1.25). To protect the rotometer, we connected a filter (2500QAT-UP, Pallflex Products Corp., Putnam, Conn.) between the outlet of the anterior chamber and the air pump. At first, SRaw (SRaw0) was determined before inhalation of the aerosols. After inhalation of each dose for 30 s, SRaw was determined within 1 min. A challenge dose was given every 5 min until SRaw increased to twice the value of SRawNac, (SRaw after inhalation of 0.9% NaCI aerosol). Concentration (histamine)-response (SRaw) curves were constructed by plotting on semilogarithmic paper. The effective molar concentration of histamine (EC200His) that produced a doubling of SRawNaC, was determined by interpolation from these concentration-response curves. Effect of OKY 046 on NO2-lnduced Airway Hyperresponsiveness
To investigate whether the NO2-induced increase in airway responsiveness was due to thromboxane A2, we studied the effect of a specific thromboxane synthetase inhibitor, OKY 046, on airway hyperresponsiveness. Animals were divided into 4 groups (n = 10 each), and SRaw0, SRawNaC„ and airway responsiveness to inhaled histamine aerosol were determined on d 0. On d 7, group 1 and group 3 were exposed to filtered air and group 2 and group 4 were exposed to 4 ppm NO2 for 3 d. Immediately after the termination of the exposure, groups 1 and 2 were exposed to aerosol from 0.9% NaCI water solution, and groups 3 and 4 were exposed to OKY 046 (4 mg/ml in 0.9% NaCI) aerosol, for 5 min. The aerosol was generated by an ultrasonic nebulizer (model 65, Devilbiss
N O , EXPOSURE A N D AIRWAY HYPERRESPONSIVENESS
455
Co., Somerset, Pa.). Air at 10 ml/s was passed through the nebulizer and delivered to the nare. Thirty minutes after inhalation of the saline or the inhibitor, airway responsiveness to inhaled histamine aerosol was determined. Statistical Analysis The EC200 and SRaw0 were expressed as means ± SE and were compared by Student's f-test for paired data of the preexposure and postexposure to NO2 or filtered air and pretreatment and posttreatment with OKY 046. A p value
