Acute effects of cigarette smoking on pulmonary function

Regulatory Toxicology and Pharmacology 57 (2010) 241–246

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Acute effects of cigarette smoking on pulmonary function q M. Unverdorben a,*, A. Mostert b, S. Munjal a, A. van der Bijl b, L. Potgieter b, C. Venter b, Q. Liang a, B. Meyer b, H.-J. Roethig a a b

Altria Client Services, Research Development & Engineering, 401 Commerce Road, Richmond, VA 23234, United States Farmovs-Parexel: Kampuslaan Suid, UFS, Bloemfontein 9301, South Africa

a r t i c l e

i n f o

Article history: Received 1 November 2009 Available online 15 March 2010 Keywords: Reduced cigarette smoke exposure Electrically heated cigarette smoking system (EHCSS) Body plethysmography Pulmonary function

a b s t r a c t Introduction: Chronic smoking related changes in pulmonary function are reflected as accelerated decrease in FEV1 although histologic changes occur in the peripheral bronchi earlier. More sensitive pulmonary function parameters might mirror those early changes and might show a dose response. Methods: In a randomized three-period cross-over design 57 male adult conventional cigarette (CC)smokers (age: 45.1 ± 7.1 years) smoked either CC (tar:11 mg, nicotine:0.8 mg, carbon monoxide:11 mg [Federal Trade Commission (FTC)]), or used as a potential reduced-exposure product the electrically heated smoking system (EHCSS) (tar:5 mg, nicotine:0.3 mg, carbon monoxide:0.45 mg (FTC)) or did not smoke (NS). After each 3-day exposure period, hematology and exposure parameters were determined preceding body plethysmography. Results: Cigarette smoke exposure was significantly (p < 0.0001) higher in CC than in EHCSS and in NS: (carboxyhemoglobin: CC: 6.4 ± 1.9%; EHCSS: 1.3 ± 0.6%; NS: 0.5 ± 0.3%; serum nicotine: CC: 18.9 ± 7.4 ng/ml; EHCSS: 8.4 ± 4.3 ng/ml; NS: 1.2 ± 1.6 ng/ml). Significantly lower in CC than in EHCSS and NS were specific airway conductance (0.22 ± 0.09; 0.25 ± 0.12; 0.25 ± 0.1 1/cmH2O  s; CC vs EHCSS: p < 0.05; CC vs NS: p < 0.01), forced expiratory flow 25% (7.6 ± 1.7; 7.8 ± 1.7; 7.9 ± 1.7 L/s; CC vs EHCSS or NS: p < 0.01). Thoracic gas volume (5.1 ± 1; 5 ± 1.1; 5 ± 1.1 L/min) changed insignificantly. Conclusion: The data indicate acute and reversible effects of cigarette smoke exposures and no-smoking on mid to small size pulmonary airways in a dose dependent manner. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction The health effects of cigarette smoking have been established and are a component cause of the development of myocardial infarction, stroke, lung cancer, and chronic obstructive pulmonary disease (COPD) (McBride, 1992; Stratton et al., 2001). At least 15% of cigarette smokers have been reported to develop COPD (Fletcher and Peto, 1976; Lundback et al., 2003) as reflected mainly by the reduction of the forced expiratory volume in 1 s (FEV 1). Changes of FEV 1, which is predominantly related to the mid size and larger airways, have been reported to take 1 year to develop depending on factors such as the intensity and duration of cigarette smoke exposure (Simmons et al., 2005; Tockman et al., 1995). Thus, FEV1 seems to reflect the longterm rather than the acute effects of smoking (Burrows et al., 1977). It has also been shown, that cigarette smoking, by way of particle deposition in the periphery of the bronchial tree and in the centers of the acini, i.e. the small airways, induces subclinical inflammatory q Sources of support in the form of grants, equipment or drugs: The study was supported by Philip Morris USA, Inc. * Corresponding author at: Center for Cardiovascular Diseases, Heinz-MeiseStrasse 100, 36199 Rotenburg an der Fulda, Germany. Fax: +49 6623 88 5976. E-mail address: [email protected] (M. Unverdorben).

0273-2300/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.yrtph.2009.12.013

response several years before COPD ensues (Cosio et al., 1977; Guy et al., 1994; Pinkerton et al., 2000; Takizawa et al., 2000; Verbanck et al., 2004). In a landmark study, Cosio et al. (1977) related pulmonary function to histologic findings obtained from open chest biopsy specimens of 34 smokers and 2 non-smokers. The researchers demonstrated a quantitative relationship between histologic changes in small airways such as mucosal ulcers, goblet-cell and squamous-cell metaplasia, and inflammatory-cell bronchial wall infiltration with pulmonary function parameters reflecting the small airways using room air and a mixture of 80% helium and 20% oxygen. However, it is unclear yet how soon cigarette smoking triggers those changes, whether they occur dose dependently, and whether they are reversible. A period of a few days is the likely time window. As we demonstrated recently, significant improvements in dyspnea and maximum oxygen uptake occur after 3 days already, when adult smokers of conventional cigarettes stopped smoking (Unverdorben et al., 2007). 1.1. Objective To evaluate the extent and potential reversibility of changes of pulmonary function in adult smokers of conventional cigarettes

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after 3 days of smoking conventional cigarettes or the potential reduced exposure electrically heated cigarette smoking system (EHCSS) (Frost-Pineda et al., 2008) or no-smoking. 2. Materials and methods 2.1. Ethical considerations The study was conducted in accordance with the Declaration of Helsinki (Scotland Revision, 2000), the ICH-Guidelines for Good Clinical Practice (1997), and the guidelines of the Department of Health, South Africa (Clinical Trials Guidelines, 2000). The study protocol was approved by an independent local Ethics Committee. 2.2. Study design The pilot study was designed as a single-blind (technicians and laboratory staff), randomized, controlled, three-period, cross-over study (Fig. 1) conducted at a single research center (FARMOVSPAREXEL, Bloemfontein, Republic of South Africa [altitude 1395, m = 4577 ft). 2.3. Study products The products used in the study were conventional test cigarettes (CC) (Philip Morris USA, Richmond, VA, USA) and as a potential reduced-exposure product the third generation electrically heated cigarette smoking system (EHCSS series K, Philip Morris USA, Richmond, VA, USA) (Frost-Pineda et al., 2008). According to the Federal Trade Commission (FTC) method the conventional cigarettes delivered 11 mg tar, 0.8 mg nicotine, and 11 mg carbon monoxide while EHCSS delivered 5 mg tar, 0.3 mg nicotine, and 0.45 mg carbon monoxide. The EHCSS has been shown to reliably reduce the delivery of selected smoke constituents and smokers’ exposure to particulateand gas-phase smoke constituents by 40–95% compared to conventional cigarettes and was therefore used as the reducedexposure product (Frost-Pineda et al., 2008; Roethig et al., 2005, 2007). The EHCSS is not intended to being marketed. EHCSS consists of a cigarette containing a column of standard cigarette tobacco filler, wrapped in a tobacco mat and paper overwrap, which is inserted into a Puff Activated Lighter™. One of the lighter’s eight blades at a time heats the cigarette only while the smoker takes a puff, thereby avoiding smoldering of the cigarette between puffs. Using this design, the tobacco reaches a peak temperature of approximately 500 °C during puffing (Patskan and Reininghaus,

Table 1 Demographic data of participants. Subject demographics at enrollment Number of participants evaluable/enrolled Age (years) Height (cm) Weight (kg) Body mass index (kg/m2) Caucasian

49/57 (86%)a 45.1 ± 7.1b 179.2 ± 6.7b 83.3 ± 11.7b 25.9 ± 3.3b 57

a Six participants withdrew from the study for personal reasons, in two subjects the data set was incomplete. b Mean ± SD.

2003). This is in contrast to the burning cone of a lit-end cigarette, which reaches approximately 900 °C during puffing (Keith and Newsome, 1958). 2.4. Subject selection To likely achieve the target of 48 evaluable subjects 57 adult male smokers in good general health without any history or clinical signs of pulmonary disease were recruited through the database of volunteers at the clinical research unit to evaluate cigarette smoking related influences on pulmonary function. Each volunteer gave written informed consent and was compensated for time and inconvenience. Forty-nine of the 57 (86%) male adult smokers were evaluable (Table 1). Subjects (6/57 (10.5%)) withdrew from the study for personal reasons during the course of the clinical study period and in 2/57 (1.7%) subjects the data set was incomplete. The main inclusion criteria encompassed men aged 35–60 years with a smoking history of 20–40 cigarettes/day for at least 10 years, and with brand and daily cigarette use stable for at least 3 months prior to enrollment. The carboxyhemoglobin concentration in blood had to be P2.5% at the initial screening visit. The main exclusion criteria comprised a body mass index of P35 kg/m2, the use of any other non-cigarette tobacco or nicotine product within 2 weeks prior to enrollment, any pulmonary or cardiovascular disease, and the use of any pulmonary or cardiovascular medication. 2.5. Study conduct 2.5.1. Screening and familiarization During the 7 weeks prior to the clinical conduct, each subject was familiarized with the research unit and study procedures. 2.5.2. Clinical setting Qualified subjects were admitted to the clinical research unit within 7–21 days after completion of the screening procedures and randomized to one of the six exposure sequences (Fig. 1). The subjects were not allowed to leave the unit for 10 days unless they withdrew from the study. Meals, beverages, and leisure activities were standardized in the clinical research unit in order to minimize nutritional influence on the test results.

Fig. 1. Sequences of exposure of the participants. The subjects were randomly assigned to the sequences (six subjects per each sequence). Whole body plethysmography was performed at the end of each 3-day exposure period.

2.5.3. Smoking exposure The subjects were allowed to smoke up to 20% more than the daily maximum number of cigarettes (either CC or EHCSS) reported in their smoking history at designated smoking times between 7 a.m. and 11 p.m. (‘‘controlled smoking” conditions (Roethig et al., 2005)) with an upper limit of 30 cigarettes per day. Subjects were never forced to smoke by the study staff and were allowed to reduce their daily cigarette consumption or to quit smoking at any

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time during the study. The participants were also counseled for smoking cessation. Subjects smoked outdoors in areas widely apart from each other according to the product (CC, EHCSS) they used. The air between the two spaces could not mix (airflow separation). On the third day of the CC or EHCSS exposure period, the time from the last cigarette until pulmonary function testing had to be 1 h minimum to avoid the acute bronchoconstrictory effects of smoking (Higenbottam et al., 1980; Rees et al., 1982; Taveira Da Silva and Hamosh, 1980, 1981). 2.5.4. Blood sampling Before performing whole body plethysmography at the end of each 3-day exposure period, blood was drawn for the analysis of full blood cell count, the concentrations of carboxyhemoglobin, and the levels of nicotine and cotinine in serum. The analyzes were performed by the certified clinical laboratory at FARMOVS-PAREXEL, Bloemfontein, South Africa. 2.5.5. Whole body plethysmography Whole body plethysmography was performed in the sitting position using the Elite™ D Body Plethysmography System (Medical Graphics Corporation, St. Paul, MN, USA). In the well-ventilated pulmonary function laboratory temperature and humidity were controlled within a range of 18–24 °C (64–75 F) and 25–65% humidity. The laboratory technicians were blinded as to the subject’s exposure. Following the 25 min supine resting period all body plethysmographies of a specific subject were performed by the same technician at the same time of the day to avoid the reported chronobiological influences (Ciappi et al., 1982). The parameters listed below were measured or computed by the spiroergometry system. Airways resistance Specific conductance (1/cmH2O  s) Conductance (L/s/cmH2O) Specific resistance (cmH2O  s) Resistance (cmH2O/L/s)

sGaw Gaw sRaw Raw

Spirometry FEV1 FEF 25% FEF 50% FEF 25– 75% PEF PIF

Forced expiratory volume after one second (L) Forced expiratory flow after the first 25% of the vital capacity (L/s) Forced expiratory flow after the first 50% of the vital capacity (L/s) Forced mid expiratory flow (L/s) Peak expiratory flow (L/s) Peak inspiratory flow (L/s)

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Palo Alto, CA, USA). For the measurement of carboxyhemoglobin (lower limit of quantification 0.3%) a blood gas oximeter was used (Roche AVL OMNI 9, Basel, Switzerland). 2.7. Statistical analyzes Descriptive statistics (N, mean, standard deviation, minimum, maximum, median, range) were provided for continuous variables. All categorical variables were summarized as frequency distributions. An analysis of covariance was performed on the pulmonary function measurements with the third screening pulmonary function test values as covariate and period as the main effect. The model terms included type of exposure, period, sequence, subject, and exposure by stage interaction terms. All 49 subjects who completed the study were included in the data analysis. The statistical tests were two-sided with a 5% Type I error rate. SAS (Version 6.12, SASÒ Institute Inc., Cary, NC) was used. 3. Results 3.1. Exposure The mean number of cigarettes the participants smoked during the exposure periods was similar throughout the study. During the CC period, the subjects smoked 22.4 ± 3.9 cigarettes/day (mean ± SD) and during the EHCSS period they used 24.3 ± 4.8 cigarettes/day (p < 0.001). Carboxyhemoglobin concentrations in whole blood were 6.4 ± 1.9% during the period when the participants smoked the conventional cigarettes (CC), 1.3 ± 0.6% when the EHCSS was smoked, and 0.5 ± 0.3% during the no-smoking period (p < 0.0001 for all pair-wise comparisons). Mean (±SD) nicotine serum concentrations were 18.9 ± 7.4 ng/ml (CC), 8.2 ± 4.3 ng/ml (EHCSS), and 1.2 ± 1.6 ng/ml (NS) (p < 0.0001 for all pair-wise comparisons). Mean (±SD) cotinine serum levels were 338.9 ± 100 ng/ml (CC), 159.9 ± 57 ng/ml (EHCSS), and 16.1 ± 19 ng/ml (NS) (p < 0.0001 for all pair-wise comparisons). The subjects significantly increased weight when switching from CC (83.78 ± 11.18 kg) to EHCSS (84.24 ± 11.38 kg) and to NS (84.73 ± 11.48 kg) (p < 0.0001 for all pair-wise comparisons) with a corresponding increase in body mass index from 26.12 ± 3.17 kg/m2 (CC) to 26.26 ± 3.22 kg/m2 (EHCSS) and to 26.42 ± 3.26 kg/m2 (NS) (p < 0.0001 for all pair-wise comparisons). 3.2. Hematology With reduced exposure (EHCSS, NS) the following changes in hematology parameters were observed (Table 2): 3.3. Whole body plethysmography

Lung volumes VC FIVC TGV

Vital capacity (L) Forced inspiratory vital capacity (L) Thoracic gas volume (L)

2.6. Bioanalysis Serum nicotine (lower limit of quantification 0.953 ng/mL) and cotinine (lower limit of quantification 7.68 ng/mL) were determined by gas chromatography (Agilent Model 6890 N, Hewlett–Packard,

With reduced smoke exposure the small airway resistance declined with a corresponding increase in conductance (Table 3). This reduction was more pronounced in NS than in EHCSS. The early (FEF 25%) forced expiratory flow showed a significant improvement with reduced exposure while mid forced midexpiratory flows FEF 50% and FEF 25–75% and the forced inspiratory flow FIF 50% exhibited trends towards improvement (Fig. 2). The peak flows at inspiration and expiration tended to increase with reduced exposure. The forced expiratory volume after one second (FEV1) remained nearly unchanged while the FEV1 relative to the vital capacity (FEV1/VC) tended to be lower during the highest exposure

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Table 2 Hematology parameters during the different exposures.

Hematocrit (%) Hemoglobin (g/dL) Erythrocyte count (1012/L) White blood cell count (109/L)

CC

EHCSS

NS

NS vs EHCSS

CC vs EHCSS

CC vs NS

48.9 ± 2.4 16.42 ± 0.91 5.31 ± 0.35 9.67 ± 2.08

48.8 ± 2.4 16.27 ± 0.83 5.3 ± 0.34 9.27 ± 2.05

48.6 ± 2.3 16.18 ± 0.82 5.26 ± 0.32 9.06 ± 2.12

ns ns 0.05 ns

ns 0.01 ns ns

0.04 <0.0001 0.01 0.005

Table 3 Airways resistance (Raw), specific airway resistance (sRaw), conductance (Gaw) and specific conductance (sGaw) in the three exposure groups. Resistance and conductance

a

Parameter

Exposure

N

CCa

EHCSSa

NSa

CC vs EHCSS

p-value CC vs NS

EHCSS vs NS

Raw (cmH2O/L/s) sRaw (cmH2O  s) Gaw (L/s/cmH2O) sGaw (1/cmH2O  s)

1.027 ± 0.338 5.203 ± 1.899 1.081 ± 0.341 0.221 ± 0.087

1.005 ± 0.408 4.997 ± 2.209 1.188 ± 0.539 0.246 ± 0.12

0.957 ± 0.404 4.623 ± 1.813 1.224 ± 0.47 0.253 ± 0.103

0.68 0.36 0.1 0.05

0.11 0.006 0.02 0.009

0.23 0.07 0.49 0.5

All parameters are given as mean ± SD.

Pulmonary Flow Parameters 12 10

[L/s]

8 6 4 2 0

FEF25%

FEF50%

mean ±SD

FEF25-75%

CC

PEF

FIF50%

EHCSS

PIF

NS

Fig. 2. Forced expiratory flow at 25%, at 50%, and from 25% to 75% of the expired vital capacity, at 50% of inspiratory flow, and the peak flows at expiration (PEF) and at inspiration (PIF) tend to increase with reduced cigarette smoke exposure. p = 0.1 for CC vs EHCSS; p = 0.01 for CC vs EHCSS and p = 0.002 for CC vs NS. All other pair-wise comparisons p > 0.2.

(CC) (Table 4). The thoracic gas volume did not differ between nosmoking and EHCSS but was somewhat higher during conventional cigarette exposure (Table 4). It is important to emphasize, that for none of the parameters analyzed neither a sequence nor a period effect occurred.

4. Discussion The present study demonstrates that reduced exposure to cigarette smoke for 3 days improves pulmonary function. The parameters derived from whole body plethysmography indicate improved

Table 4 Additional parameters derived from whole body plethysmography (FEV1 = forced expiratory volume after 1 s, VC = vital capacity, FVC = forced vital capacity, FIVC = forced inspiratory vital capacity, TGV = thoracic gas volume). Pulmonary volume parameters Parameter

a

Exposure

p-value

CCa

EHCSSa

NSa

CC vs EHCSS

CC vs NS

EHCSS vs NS

FEV1 (L) FEV1/VC (%)

3.70 ± 0.56 77.4 ± 5.7

3.72 ± 0.54 77.7 ± 5.5

3.68 ± 0.58 77.7 ± 5.6

0.43 0.21

0.29 0.2

0.07 0.98

FVC (L) FIVC (L) TGV (L)

4.8 ± 0.69 4.04 ± 0.81 5.11 ± 0.96

4.79 ± 0.67 4.16 ± 0.87 5.01 ± 1.07

4.74 ± 0.69 4.08 ± 0.69 5.01 ± 1.13

0.95 0.05 0.19

0.07 0.57 0.18

0.08 0.16 0.95

All parameters are given as mean ± SD.

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pulmonary function when switching from a conventional cigarette to no-smoking. Reduced exposure during EHCSS use falls between these two extremes. The most sensitive parameters for detection of differences between the three exposures were the conductance and resistance measurements reflecting changes mainly in the small to mid size airways. This finding is in line with a significantly lower forced expiratory flow at 25% of the vital capacity (FEF 25%) and a trend towards lower forced flows at 50% of the vital capacity during expiration (FEF 50%, FEF 25–75%) and inspiration (FIF 50%) during conventional cigarette exposure. From a pathophysiologic point of view, these findings appear plausible: Cigarette smoking induced subclinical early inflammatory changes occur not only in the larger airways (Rennard et al., 2002) but to a considerable extent in the periphery of the bronchial tree (particle deposition in the centers of the acini) and, thus, within the small to mid size airways (Cosio et al., 1977; Guy et al., 1994; Pinkerton et al., 2000; Takizawa et al., 2000; Verbanck et al., 2004). This epithelial reaction in the small airways with thickening (‘‘remodeling”) of the bronchial walls occurs independently from similar reactions in the larger airways (Hogg et al., 2004) thereby reducing the bronchial lumen to a relatively greater extent than in the larger airways. Therefore, it is not too surprising that during the conventional cigarette smoking period compared to the potential reduced-exposure product and no-smoking the values for FEV1/VC and the peak flows at expiration and inspiration, which mirror the larger airways, were insignificantly lower. These results are in line with those reported by other groups (Macklem, 1998; Takizawa et al., 2000). The thoracic gas volume tended to be the highest while the subjects were assigned to smoking conventional cigarettes possibly reflecting trapped air. Since cigarette smoking has bee reported to induce inflammation (Cosio et al., 1977; Kilburn, 1984; Roth et al., 1998) it seems likely that the associated processes such as inflammatory-cell infiltration, goblet- and squamous-cell metaplasia, and swelling of the epithelium along with enhanced production of mucus rich in neutrophils may lead to complete obstruction of small peripheral airways with the ensuing impaired ventilation (Cosio et al., 1977; Pinkerton et al., 2000; Takizawa et al., 2000; Thompson et al., 1989; Verbanck et al., 2004). Our study was not designed to analyze which of the mechanisms mentioned above contributed to the improvements in pulmonary function with reduced cigarette smoke exposure or no-smoking. However, the significant decline in white blood cell count and neutrophils in particular are strong indicators of reduced inflammation that in the lung is typically associated with reductions in epithelial swelling, a decline of mucus secretion, and a normalization in surfactant composition (Carel et al., 1988; Cosio et al., 1977; Finley and Ladman, 1972; Sparrow et al., 1984; Turato et al., 2002; Yeung and Buncio, 1984). As the above changes occurred within 3 days only, direct effects of smoke constituents independent from inflammation have to be considered too. The study results may have been confounded by the fact that smoking a single cigarette increases the tone of the muscular bronchi in the upper respiratory tract immediately (Higenbottam et al., 1980; McDermott and Collins, 1965; Nadel and Comroe, 1961; Rees et al., 1982; Taveira Da Silva and Hamosh, 1980, 1982) with the associated impairments of pulmonary function. This bronchial reaction decays over a period of about 40 min (Nadel and Comroe, 1961; Rieben, 1992; Taveira Da Silva and Hamosh, 1981). Hence, it is very unlikely that these acute smoking induced effects on bronchial tone may have influenced the study results since a minimum time interval of 1 h was scheduled between the last cigarette and whole body plethysmography. The trends and significant changes were consistent across the parameters with conventional cigarettes showing the poorest pulmonary functionß EHCSS exhibiting a slight improvement while no-smoking was associated with the best pulmonary function,

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indicating a dose dependent effect. In line with our previous work, apparently, reduced exposure/no-smoking relate to clinical parameters, some of which are associated with morbidity and mortality (Munjal et al., 2009; Unverdorben et al., 2007, 2008). The small differences between the exposures observed in this study might be considered as a limitation. However, given the fact that small airways account for only 10% of the total bronchial resistance the observed changes are relatively small (Hogg et al., 1968; Macklem and Mead, 1967). It might well be that with longer reduced exposure periods and, hence more time for the inflammatory response to subside, the improvements in pulmonary function parameters may become more pronounced. The results of this study may also be different in chronic smokers with a longer smoking history and in those who suffer from COPD due to the more advanced and mainly irreversible pulmonary changes (Verbanck et al., 2004). 5. Conclusion The data indicate acute and reversible effects of different cigarette smoke exposures and no-smoking on mid to small size pulmonary airways in a dose dependent manner. Disclaimers M. Unverdorben, S. Munjal, and H.-J. Roethig were formerly employed by Philip Morris USA Inc./Altria Client Services. Acknowledgments The authors are grateful for the support of this work by the technical staff of Farmovs-Parexel, Bloemfontein, South Africa, and the PM USA library. References Burrows, B., Knudson, R.J., Cline, M.G., Lebowitz, M.D., 1977. Quantitative relationships between cigarette smoking and ventilatory function. Am. Rev. Respir. Dis. 115, 195–205. Carel, R.S., Tockman, M.S., Baser, M., 1988. Smoking, leukocyte count, and ventilatory lung function in working men. Chest 93, 1137–1143. Ciappi, G., De Benedetto, F., D’Ilario, F., Fuciarelli, R., Luciano, A., Sborgia, M., Cervone, M., Sensi, S., 1982. Chronobiological aspects of bronchial tone. Chronobiologia 9, 163–172. Cosio, M., Ghezzo, H., Hogg, J.C., Corbin, R., Loveland, M., Dosman, J., Macklem, P.T., 1977. The relations between structural changes in small airways and pulmonary-function tests. N. Engl. J. Med. 298, 1277–1281. Finley, T.N., Ladman, A.J., 1972. Low yield of pulmonary surfactant in cigarette smokers. N. Engl. J. Med. 286, 223–227. Fletcher, C., Peto, R., 1976. The natural history of chronic airflow obstruction. Br. Med. J. 25, 1645–1648. Frost-Pineda, K., Zedler, B., Oliveri, D., Feng, S., Laing, Q., Roethig, H., 2008. Shortterm clinical exposure evaluation of a third-generation electrically heated cigarette smoking system (EHCSS) in adult smokers. Regul. Toxicol. Pharmacol. 52, 104–110. Guy, H.J., Prisk, G.K., Elliott, A.R., Deutschman 3rd, R.A., West, J.B., 1994. Inhomogeneity of pulmonary ventilation during sustained microgravity as determined by single-breath washouts. J. Appl. Physiol. 76, 1719–1729. Higenbottam, T., Hamilton, D., Feyerband, C., Clark, T.J., 1980. Acute effects of smoking a single cigarette on the airway resistance and the maximal and partial forced expiratory flow volume curves. Br. J. Dis. Chest 74, 37–46. Hogg, J.C., Chu, F., Utokaparch, S., Woods, R., Elliott, W.M., Buzatu, L., Cherniack, R.M., Rogers, R.M., Sciurba, F.C., Coxson, H.O., Pare, P.D., 2004. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N. Engl. J. Med. 350, 2645–2653. Hogg, J.C., Macklem, P.T., Thurlbeck, W.M., 1968. Site and nature of airway obstruction in chronic obstructive lung disease. N. Engl. J. Med. 278, 1355–1360. Keith, C., Newsome, C., 1958. Quantitative studies on cigarette smoke. III. Methods of analysis of filter cigarettes. Tob. Sci. 2, 14–19. Kilburn, K.H., 1984. Particles causing lung disease. Environ. Health Perspect. 55, 97– 109. Lundback, B., Lindberg, A., Lindstrom, M., Ronmark, E., Jonsson, A.C., Jonsson, E., Larsson, L.G., Andersson, S., Sandstrom, T., Larsson, K., 2003. Not 15 but 50% of

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