Nitric oxide (NO) in exhaled air after experimental ozone exposure in humans

RESPIRATORY MEDICINE (2001) 95, 491–495 doi:10.1053/rmed.2001.1076, available online at http://www.idealibrary.com on

Nitric oxide (NO) in exhaled air after experimental ozone exposure in humans A.-C. OLIN*, N. STENFORS{, K. TORE´N*{, A. BLOMBERG{, R. HELLEDAY{, M.-C. LEDIN{, G. LJUNGKVIST*, A. EKMAN* AND T. SANDSTRO¨M{ *Section of Occupational and Environmental Medicine, Sahlgrenska University Hospital, Go¨teborg, {Department for Respiratory Medicine and Allergy, University Hospital and Medical Division, National Institute for Working Life, Umea˚ and {Section of Respiratory Medicine and Allergology, Sahlgrenska University Hospital, Go¨teborg, Sweden

We hypothesized that ozone, a common air pollutant, potent in producing airway inflammation, would increase the production of exhaled nitric oxide (NO). If so, measurement of exhaled NO could potentially be a valuable tool in population studies of air pollution effects. Eleven healthy non-smoking volunteers were exposed to 0?2 ppm ozone (O3) and filtered air for 2 h on two separate occasions. Exhaled NO and nasal NO were measured before and on five occasions following the exposures. Changes in exhaled and nasal NO after ozone exposure were adjusted for changes after air exposure. There was a slight decrease in exhaled NO (70?6; 73?1–1?2 ppb) (median and 95% confidence interval) and of nasal NO (757; 7173–75 ppb) directly after the ozone exposure. No significant changes in exhaled or nasal NO were however found 6 or 24 h after the exposure. Within the examined group, an O3 exposure level proven to induce an airway inflammation caused no significant changes in exhaled or nasal NO levels. Hence, the current study did not yield support for exhaled NO as a useful marker of ozone-induced oxidative stress and airway inflammation after a single exposure. This contrasts with data for workers exposed to repeated high peaks of ozone. The potential for exhaled NO as a marker of oxidative stress therefore deserves to be further elucidated. Key words: nitric oxide, ozone, exhaled air, humans. RESPIR. MED. (2001) 95, 491–495

Introduction Nitric oxide (NO) can be detected in exhaled air of healthy individuals. NO is generated from L-arginine by the enzyme NO-synthase, of which at least three different iso-forms exist (1). It has also been proposed that NO could be formed non-enzymatically during oxidative stress (2, 3). The main sources of NO production in human airways are the paranasal sinuses where very high concentrations have been detected (more than 20 ppm) (4). A low basal level of NO is also formed in the epithelial cells of the respiratory tract and this might be further induced by iNOS (inducible, Ca2+-independent, type II NO synthase) by various inflammatory cytokines (5). Increased exhaled NO levels Recevied 28 August 2000 and accepted in revised form 12 March 2001. Correspondence should be addressed to: Dr Anna-Carin Olin, Section of Occupational and Environmental Medicine, Sahlgrenska University Hospital, St Sigfridsgatan 85B, S-412 66 Go¨teborg, Sweden. Fax: +4631409728; E-mail: [email protected]

0954-6111/01/060491+05 $35?00/0

# 2001 HARCOURT PUBLISHERS LTD

and increased iNOS in epithelial cells have been found in asthmatics. Both are down-regulated by inhaled glucocorticoids (6,7). Hence NO in exhaled air has been proposed as a marker of airway inflammation (8). Ozone is a major component of urban air pollution. It is a well-known respiratory irritant causing both nasal and distal airway inflammation, predominantly involving polymorphonuclear leukocytes (PMNs) (9). After ozone exposure, the PMNs are believed to alter their oxidantgenerating capability in the lung, further contributing to lung injury (10). Superoxide anion produced by neutrophils and macrophages might react with NO to form peroxynitrite, an even more potent cytotoxic oxidant (11). Little is yet known about how ozone interacts with NO formation in humans and how exhaled NO levels are affected. In rats, exposure to 2 ppm of ozone has been shown to induce iNOS expression and NO production in airway type II cells and macrophages (12,13). Superoxide has been shown to induce iNOS expression in human epithelial cells in vivo (14). Further, in iNOS knock-out mice, no peroxynitrite is formed after ozone exposure and these animals also seem to # 2001 HARCOURT PUBLISHERS LTD

492

A.-C. OLIN ET AL.

be protected against ozone induced epithelial damage (15). Hence it has been suggested that NO might be an important mediator of ozone-induced lung injury. We have previously found increased NO levels in exhaled air (16) of pulp mill workers exposed to high peak levels of ozone (1–2 ppm). In that study, the peak ozone exposure preceded the NO measurements by months, thus possibly reflecting chronic inflammatory changes in the airways. In this study, we aimed to investigate whether measurement of NO in exhaled air could be a useful biomarker for monitoring the effects of ozone at ambient levels on the respiratory tract.

Subjects and methods

exhalation (the plateau-phase) was registered. The mean of three successive recordings, at an interval of 1 min, has been used in the analysis. Nasal NO (nNO) was measured by letting the analyser sample nasal air directly from one nostril. The sampling probe was connected to a soft polystyrene nasal olive and gently introduced into the vestibulum of one nostril. The contralateral nostril was left open. The subjects were instructed to keep their breath for 20 sec with the soft palate voluntarily closed, while the analyser sampled nasal air at a constant rate of 0?06 l min71 (20). The NO concentration during the plateau-phase was registered and the mean of one measurement in each nostril is presented. Whenever the NO concentration in ambient air exceeded 20 ppb the air concentration of NO was subtracted from the result.

STUDY SUBJECTS Eleven healthy non-smoking volunteers (six males and five females; mean age 24 years, range 20–29 years) without any history of asthma or other pulmonary diseases, participated in the study. One subject had a positive skin prick test against moulds, but no symptoms. The other subjects were non-atopic. All had normal lung function with a mean forced expiratory volume in 1 sec (FEV1) of 112% (range 96–137%) (17). None had a history of upper respiratory tract infection 3 weeks prior to the first exposure or during the study. The local ethics committee approved the study and informed consent was obtained from all volunteers prior to inclusion in the study.

BLOOD SAMPLES Peripheral blood samples were drawn prior to the exposures, after 6 and after 24 h. Analyses of neutrophil counts were performed with an auto-analyser at the Department of Clinical Chemistry, University Hospital, Umea˚, according to clinical routine. For the analyses of myeloperoxidase (MPO) the blood was allowed to clot for 60–120 min, and serum was separated by centrifugation and kept in freezer (7208C) until analysed. The analyses were carried out with a commercial radio-immunoassay kit (Pharmacia Upjohn, Uppsala, Sweden).

STATISTICS EXPOSURE The subjects were exposed twice, once to 0?2 ppm ozone (O3) and once to filtered air, for 2 h at least 2 weeks apart, in an exposure chamber, as previously described (18). Half of the subjects were randomized to first be exposed to filtered air and the other half were first exposed to ozone. During the 2-h exposure, mild exercise was alternated with rest at 15-min intervals. Exercise was performed using a cycle ergometer situated within the chamber, with a sufficient loading to produce an average minute ventilation of 20 l min71 m72 body surface area. The exposures were single-blinded in that the subjects were blinded with regard to the exposure of either ozone or air.

EXHALED AND NASAL NITRIC OXIDE NO was measured with a chemiluminescence analyser (Ecophysics CDL 700 AL, Du¨rnten, Switzerland) before and at 0, 0?5, 1?0, 1?5, 6 and 24 h after exposure. The NO measurements were performed in accordance with ERS guidelines (19), as previously described (20). After inhaling NO-free air the subjects were asked to performed a single slow exhalation with a constant expiratory flow rate of 0?150 l sec71 against an oral pressure of 10 cm H2O, using online visual monitoring. The mean NO concentration (ppb) of exhaled NO (eNO) during the latter part of the

Statistical analyses were carried out with SAS, version 6?12 (SAS Statistical package, Cary, NC, U.S.A). The results have been given as median exhaled NO concentration (ppb) (Table 1) and median nasal NO concentration (ppb) (Table 2) after air and ozone exposures respectively. For each subject, air-adjusted values were calculated as [(after ozone exposure7before ozone exposure)7(after air exposure7 before air exposure)] at 0, 30 min, 60 min, 90 min, 6 h and 24 h (Tables 1 and 2). Since these values (NO, neutrophils and MPO) were not normally distributed, the medians and the 95% confidence intervals (CI) for medians are presented (21).

Results The exhaled and nasal NO concentrations after exposure to air and ozone are shown in Tables 1 and 2. The individual changes of the ozone-induced effects on exhaled NO (ppb), adjusted for changes after air exposure at 0, 6 and 24 h are shown in Fig. 1. When adjusted for filtered air exposure, there was a slight but non-significant decrease in exhaled NO concentration (70?3; 73?1–1.2 ppb) (median; 95% CI) and nasal NO (756; 7173–75 ppb) immediately after the exposure (time 0) (Tables 1 and 2). In the peripheral blood, the number of neutrophils increased after ozone exposure (2?0; 1?5–2?5 109 l71), as

NITRIC OXIDE AND OZONE 493 TABLE 1. Median exhaled NO concentration (ppb) at different time points before and after exposures to air and 0?2 ppm ozone. The medians of the differences for each subject, calculated as [(after ozone exposure7before ozone exposure)7(after air exposure7before air exposure)], at each time point are also shown, as well as the 95% confidence intervals for the difference Exposure Time

Air

Ozone

Difference

95% CI

Before After 0 30 min 60 min 90 min 6h 24 h

7?7

7?2

—

—

7?8 7?0 7?2 7?2 8?8 6?8

6?6 6?5 7?7 7?6 7?4 6?6

70?6 71?0 0?5 0?7 0?3 70?4

73?1–1?2 72?1–3?9 71?7–4?0 72?6–3?8 70?5–2?3 73?0–3?8

TABLE 2. The median nasal NO concentration (ppb) at different time points before and after exposures to air and 0?2 ppm ozone. The medians of the differences for each subject, calculated as [(after ozone exposure7before ozone exposure)7(after air exposure7before air exposure)], at each time point are also shown, as well as the 95% confidence intervals for the difference Exposure Time

Air

Ozone

Difference

95% CI

Before After 0 30 min 60 min 90 min 6h 24 h

444

520

—

—

572 566 535 559 570 494

531 497 507 487 554 477

756?6 715?0 5?3 9?5 5?0 716?5

7173–75 7198–60 7183–114 7184–65 7202–65 7221–107

well as after air exposure (1?5; 0?7–2?3 109 l71). When adjusting for air exposure, the increase in neutrophils after ozone exposure was not statistically significant (0?5; 70?4– 1?4 109 l71). Similarly, MPO increased after both air (30; 733–207 mg l71) and ozone exposure (114; 79–431 mg l71) and when adjusted for the increase after air exposure the change after ozone exposure was not significant.

Discussion No major effects of exhaled or nasal NO after experimental exposure to ozone in ambient levels could be detected in

this study. Exhaled and nasal NO decreased slightly, but non significantly, directly after exposure. One hour after the ozone exposure, the nasal and exhaled NO levels were normalized and remained so for the rest of the follow-up period. Our hypothesis was however to find an increase in exhaled NO due to airway inflammation. We have examined a small number of subjects and there is a considerable variation of changes in exhaled NO between subjects, which limits our possibilities to detect small changes after the ozone exposure. Hypothetically, if we could assume the changes to be normally distributed, then the power to detect a 50% increase of exhaled NO would be 99% 6 h after the exposure, when we know from earlier studies that airway inflammation is present. We do not believe that lack of internal validity is the reason for the negative results. NO measurements were performed with high accuracy according to ERS guidelines and the results can hardly be biased. It is also difficult to comprehend how selection bias could influence the results. Similar results have also recently been published in a study by Nightingale et al. (22). There may be different explanations for the lack of effects on exhaled NO-levels following ozone exposure. Firstly, the airway inflammation after this relatively low exposure level might be mild. Even though an increased number of neutrophils has been described in BAL after exposure to lower ozone levels (23) it is unknown whether the neutrophils are activated and really take part in an inflammatory process or whether they serve a protective purpose. No increase of myeloperoxidase, a marker for neutrophilic activation, has been shown in healthy volunteers after equivalent exposure (24). The increased NOlevels we described in pulp mill workers were related to markedly higher ozone exposure levels, 1–2 ppm (16). In the same study, we found a significant correlation between eNO and MPO among never-smokers (unpublished results) indicating that these workers had activated neutrophils. Secondly, it could be that an increased production of NO occurs in the most distal airways and the terminal bronchioli–alveolar junction, but with the current method of measuring exhaled NO we were not able to detect it. Animal studies and mathematical modelling of ozone uptake show distal airway inflammation, primarily at the broncho-alveolar junction, to be most likely after ozone exposure (25,26). The increased levels of exhaled NO among patients with asthma are, in contrast, believed to originate in the central airways, and the contribution of NO from distal parts of the lungs is assumed to be small (27). This might imply that even if a more distal airway inflammation were present, we would not have been able to detect it by the technique for NO measurements used in this study. Thirdly, NO is a free radical, which reacts rapidly with ozone to form peroxynitrite or other reactive oxygen intermediates. If the produced NO were scavenged in this way, the levels of NO would decrease in exhaled air. This might explain the tendency towards decreased eNO directly after the ozone exposure, although the study size was too small to detect any significant changes. In a similar way would superoxide anion, produced by neutrophils, react

494

A.-C. OLIN ET AL.

FIG. 1. Individual change of exhaled NO (ppb) after ozone exposure, adjusted for changes of exhaled NO after exposure for filtered air.

with NO to form peroxynitrite. This would explain why we did not find any increase of eNO 6 or 24 h after the exposure, when we would assume airway neutrophilia to be present. Animal models have shown an increased formation of peroxynitrite by alveolar macrophages after inhalation of high doses of nitric oxide (28). An in vitro study (29) has shown that PMNs, induced to produce O27, and added to a murine lung cell culture, reduced NO formation. To conclude, the current study did not yield support for exhaled NO as a useful marker of ozone-induced oxidative stress and airway inflammation after a single exposure to ozone. This contrasts with data for workers chronically exposed to ozone. The potential for exhaled NO deserves therefore to be further elucidated.

Acknowledgements The authors wish to thank Gerd Granung, Jamshid Pourazar, Annika Hagenbjo¨rk-Gustafsson, Ann-Britt Lundstro¨m, Lena Skedebrant and Hele´n Bertilsson for their excellent technical assistance. The study was performed with financial support from the Swedish Heart and Lung Foundation, Torsten and Ragnar So¨derbergs Stiftelse and from the Swedish Council for Worklife Research.

References 1. Moncada S, Higgs EA. Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest 1991; 21: 361–374. 2. Nagase S, Takemura K, Ueda A, et al. A novel nonenzymatic pathway for the generation of nitric oxide by the reaction of hydrogen peroxide and D- or L-arginine. Biochem Biophys Res Commun 1997; 233: 150–153. 3. Chowienczyk P, Ritter J. Arginine: NO more than a simple amino acid? Lancet 1997; 350: 901–902.

4. Lundberg JO. Airborne nitric oxide: inflammatory marker and aerocrine messenger in man. Acta Physiol Scand Suppl 1996; 633 (Suppl.): 1–27. 5. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43: 109–142. 6. Alving K, Weitzberg E, Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J 1993; 6: 1368–1370. 7. Kharitonov SA, Yates D, Robbins RA, et al. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994; 343: 133–135. 8. Barnes PJ, Liew FY. Nitric oxide and asthmatic inflammation. Immunol Today 1995; 16: 128–130. 9. Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society. Health effects of outdoor air pollution. Am J Respir Crit Care Med 1996; 153: 3–50. 10. Esterline RL, Bassett DJ, Trush MA. Characterization of the oxidant generation by inflammatory cells lavaged from rat lungs following acute exposure to ozone. Toxicol Appl Pharmacol 1989; 99: 229–239. 11. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 1990; 87: 1620– 1624. 12. Laskin DL, Pendino KJ, Punjabi CJ, Rodriguez del Valle M, Laskin JD. Pulmonary and hepatic effects of inhaled ozone in rats. Environ Health Perspect 1994; 102 (Suppl. 10): 61–64. 13. Pendino KJ, Laskin JD, Shuler RL, Punjabi CJ, Laskin DL. Enhanced production of nitric oxide by rat alveolar macrophages after inhalation of a pulmonary irritant is associated with increased expression of nitric oxide synthase. J Immunol 1993; 151: 7196–7205. 14. Adcock IM, Brown CR, Kwon O, Barnes PJ. Oxidative stress induces NF kappa B DNA binding and inducible NOS mRNA in the human epithelial cell line. Biochem Biophys Res Commun 1994; 199: 1515–1524.

NITRIC OXIDE AND OZONE 495 15. Fakhrzadeh L, et al. Role of peroxynitrite in ozoneinduced lung-injury. Am J Resp Crit Care Med 1999; 159: A272. 16. Olin AC, Ljungkvist G, Bake B, Hagberg S, Henriksson L, Toren K. Exhaled nitric oxide among pulpmill workers reporting gassing incidents involving ozone and chlorine dioxide. Eur Respir J 1999; 14: 828–831. 17. Berglund E, et al. Spirometric studies in normal subjects. Acta Med Scand 1963; 173: 185–191. 18. Krishna MT, Blomberg A, Biscione GL, et al. Shortterm ozone exposure upregulates P-selectin in normal human airways. Am J Respir Crit Care Med 1997; 155: 1798–1803. 19. Kharitonov S, Alving K, Barnes PJ. Exhaled and nasal nitric oxide measurements: recommendations. Eur Respir J 1997; 10: 1683–1693. 20. Olin AC, Aldenbratt A, Ekman A, et al. Increased nitric oxide in exhaled air after intake of a nitrate-rich meal. Respir Med 2001; 95: 153–158. 21. Altman DG. Practical Statistics for Medical Research. 1st ed. London: Chapman and Hall, 1999. 22. Nightingale JA, Rogers DF, Barnes PJ. Effect of inhaled ozone on exhaled nitric oxide, pulmonary function, and induced sputum in normal and asthmatic subjects. Thorax 1999; 54: 1061–1069. 23. Devlin RB, McDonnell WF, Mann R. Exposure of humans to ambient levels of ozone for 6.6 hours causes

24.

25.

26.

27.

28.

29.

cellular and biochemical changes in the lung. Am J Respir Cell Mol Biol 1991; 4: 72–81. Blomberg A, Mudway IS, Nordenhall C, et al. Ozoneinduced lung function decrements do not correlate with early airway inflammatory or antioxidant responses. Eur Respir J 1999; 13: 1418–1428. Chitano P, Hosselet JJ, Mapp CE, Fabbri LM. Effect of oxidant air pollutants on the respiratory system: insights from experimental animal research. Eur Respir J 1995; 8: 1357–1371. Ultman JS, Ben-Jebria A, Hu SC. Noninvasive determination of respiratory ozone absorption: the bolus-response method. Res Resp Health Eff Inst 1994; 69: 1–27. Silkoff PE, McClean PA, Caramori M, Slutsky AS, Zamel N. A significant proportion of exhaled nitric oxide arises in large airways in normal subjects. Respir Physiol 1998; 113: 33–38. Weinberger B, Fakhrzadeh L, Heck DE, Laskin JD, Gardner CR, Laskin DL. Inhaled nitric oxide primes lung macrophages to produce reactive oxygen and nitrogen intermediates. Am J Respir Crit Care Med 1998; 158: 931–938. Jones KL, Bryan TW, Jinkins PA, et al. Superoxide released from neutrophils causes a reduction in nitric oxide gas. Am J Physiol 1998; 275: L1120–L1126.