American Journal of Respiratory and Critical Care Medicine

Rationale: Cigarette smoke extract inhibits chloride secretion in human bronchial epithelial cells. Oxidants decrease gene expression, protein expression, and function of the cystic fibrosis transmembrane conductance regulator (CFTR).

Objectives: Because cigarette smoke is a rich source of oxidants, we verified the hypothesis that CFTR may be suppressed by exposure to cigarette smoke in vitro and in vivo.

Methods: The effects of cigarette smoke exposure on Calu-3 and T84 cell CFTR expression and function were observed. Also studied were the nasal potential differences (PDs) in 26 men (9 smokers, 17 nonsmokers) who had no detectable CFTR gene mutations as determined during investigations for infertility. CFTR expression and function were determined by Northern blotting, Western blotting, and cAMP-dependent 125I efflux assays. Extensive CFTR genotyping was performed in each subject. Nasal PD measurements were made at baseline and during amiloride, chloride-free buffer, and isoproterenol perfusions.

Main Results: Cigarette smoke decreased CFTR expression and function in Calu-3 and T84 cell lines. Furthermore, the nasal PDs of cigarette smokers showed a pattern typical of CFTR deficiency with a blunted response to chloride-free buffer and isoproterenol compared with nonsmokers (−9.6 ± 4.0 vs. −22.3 ± 10.1 mV; p < 0.001).

Conclusions: We conclude that cigarette smoke decreases the expression of CFTR gene, protein, and function in vitro and that acquired CFTR deficiency occurs in the nasal respiratory epithelium of cigarette smokers. We suggest that acquired CFTR deficiency may contribute to the physiopathology of cigarette-induced diseases such as chronic bronchitis.

The cystic fibrosis transmembrane conductance regulator (CFTR) is essential to prevent cystic fibrosis (CF), a disease characterized by the obstruction of cylindric epithelial tissues that produce mucin-rich secretions (1). The absence of functional CFTR leads to a recognizable disease complex that is dominated by increased airway resistance, excessive cough, sputum production, and repeated bouts of infectious bronchitis (2, 3). These are also characteristics that can be found to a lesser degree in millions of cigarette smokers throughout the world, most of whom do not have CF (4).

Kreindler and colleagues have recently reported that cigarette smoke extract (CSE) exposure in vitro suppresses human bronchial epithelial cell chloride secretion without affecting sodium absorption (5). Exposure of epithelial cells to CSE led to an abnormally high ratio of sodium absorption to chloride secretion, a situation similar to that of patients with CF. CSE was also shown to suppress cAMP-stimulated chloride secretion, suggesting that the suppression of chloride secretion was mediated at least in part by a decrease in CFTR function.

Cigarette smokers show evidence of oxidative stress in their respiratory tract as reflected by high levels of exhaled pentane, a marker of lipid peroxidation (6, 7). Smokers also show signs of systemic oxidation, such as elevated levels of isoprostanes in their plasma and urine, and 8-hydroxy-2′-deoxyguanosine (8-OHdG, a marker of oxidative DNA damage) in their white blood cells and sperm (810).

The gas phase of cigarette smoke contains over 1015 radicals per puff (11). The particulate phase contains 1017 radicals per gram as determined by electron spin resonance, with high concentrations of quinones and semiquinones. Quinones are hydrocarbon rings that contain two ketone groups in any position. The single electron reduction of quinones produces the semiquinone radical that reacts readily with oxygen to produce superoxide and hydrogen peroxide. We have recently observed that the quinone tert-butylhydroquinone decreases CFTR gene expression, protein expression, and function in the human respiratory and intestinal cell lines Calu-3 and T84. We therefore hypothesized that cigarette smoke may induce similar effects on CFTR at the gene, protein, and functional levels.

The goal of this study was to observe the effects of cigarette smoke on CFTR expression and function. The physiologic response of epithelial cells to cigarette smoke in vitro and in vivo is to increase glutathione (GSH) synthesis through transcription of the genes encoding glutamate-cysteine ligase, the rate-limiting enzyme in GSH synthesis (12, 13). To ensure that we observed smoke-induced changes under physiologic conditions that induce GSH synthesis in vitro, we simultaneously studied GSH and glutamate-cysteine ligase catalytic subunit (GCLC) induction during cigarette smoke exposure. We also assessed the effects of cigarette smoking on CFTR function in vivo as determined by the nasal transepithelial potential differences (PDs) measured in healthy smokers and nonsmokers undergoing genetic and functional CFTR testing for infertility. Some of the results of these studies have been previously presented in the form of an abstract (14).

Study Population

Institutional review board approval was obtained from the Toronto Hospital for Sick Children. Data were obtained from 26 infertile and 30 fertile men who were enrolled in studies of infertility, CFTR gene mutations, and CFTR protein function as determined by nasal transepithelial PD. We included only subjects in whom no CFTR mutations were detected by extensive genotyping (see below). None of these subjects had congenital absence of the vas deferens. Of the infertile subjects, nine were regular smokers. Seven of the infertile subjects reported smoking one pack per day, and two subjects reported smoking half a pack per day. All other infertile and fertile men were nonsmokers. The mean ages of the subjects were as follows: smokers, 33.6 yr (SD, 6.0); infertile nonsmokers, 34.3 yr (SD, 3.7); and fertile nonsmokers, 30.6 yr (SD, 5.9). None of the subjects involved in the study reported any respiratory symptoms. Nasal transepithelial PDs were measured as described below.

Sweat Chloride

Measurement of sweat chloride concentration was performed by the quantitative pilocarpine iontophoresis method of Gibson and Cooke (15). Based on recommendations from a consensus statement by the U.S. Cystic Fibrosis Foundation, a sweat chloride concentration greater than 60 mmol/L was classified as abnormal, and a concentration of 40 to 60 mmol/L was borderline (16). All subjects underwent two measurements of sweat chloride concentration, and the two values were averaged.

Extensive CFTR Genotyping

Total human genomic DNA was isolated from peripheral blood cells by standard methods (17). Blood samples were coded, and genotyping was performed in a blinded fashion. Polymerase chain reaction–based multiplex heteroduplex gel shift analysis on MDE gel matrix (BMA, Rockland, ME) was used for the detection of CFTR mutations (18). The analysis included all CFTR exons, their flanking intron sequences, and the promoter region (∼ 1 kb upstream of exon 1). Fragments displaying aberrant migration patterns were further characterized by direct sequencing analysis with the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (Amersham-Life Science, Cleveland, OH). This screening protocol detects approximately 95% of mutations (18). Three variants (9T, 7T, and 5T) of the polythymidine tract (T-tract) in intron 8 were also tested.

Nasal Transepithelial PD

Nasal transepithelial PD measurements were performed by using the technique described by Knowles and colleagues (19). A baseline maximum PD was obtained under the inferior turbinate to reflect the activity of inwardly directed sodium transport at the apical surface of respiratory epithelial cells. Amiloride, an inhibitor of sodium reuptake, was perfused to depolarize the membrane and assess the change in PD and percentage change in PD from the baseline maximum PD. A chloride-free solution was used to measure basal chloride secretion, which, in normal epithelial cells, creates a more negative PD. Isoproterenol, a stimulator of cAMP–mediated chloride secretion through CFTR, was then added to chloride-free media. The change in PD after chloride-free media plus isoproterenol (ΔClf + Iso) directly represents the assessment of CFTR chloride secretion. Genotyping data was not known at the time of nasal PD testing.

Cell Lines and Reagents

The human airway epithelial Calu-3 (ATCC; HTB 55) cells were cultured in minimum essential medium supplemented with minimum essential medium nonessential amino acids (0.1 mM) and sodium pyruvate (1 mM), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.5 μg/ml fungizone, and 15% fetal bovine serum at 37°C in 5% CO2. Calu-3 cells were seeded at 2 × 106 cells/well in six-well petri dishes to confluency. The human colon adenocarcinoma T84 cells (American Type Culture Collection [ATCC] cell line CCL248; Rockville, MD) were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 (Gibco, Invitrogen Life Technologies, Burlington, ON) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.5 μg/ml fungizone, and 5% fetal bovine serum at 37°C in 5% CO2. The cells were seeded at 6 × 106 cells/well in six-well tissue culture plates (Linbro Chemical Co., New Haven, CT) to confluence. The antibody used for Western blots of CFTR, mAb-450, was a generous gift from Tim Jensen and Jack Riordan. This antibody recognizes the R domain of CFTR. Forskolin, 3-isobutyl-1-methylxanthine (IBMX), and dibutyryl cAMP were from Sigma-Aldrich. 125I was from Perkin Elmer Life Sciences (Boston, MA).

Cigarette Smoke Gaseous Phase and Condensate Exposure

Confluent cells in 6-well tissue culture plates were washed three times with Hanks' balanced salt solution and placed in a 5-liter chamber. Cigarette smoke was drawn into a syringe and injected into the chamber at a rate of 35 ml/min for 10 min. Calu-3 cells were exposed to cigarette smoke for 10 min every 2 h for a total of four times. Between exposures, Hanks' balanced salt solution was replaced with fresh media. RNA extraction for Northern blot analysis was done 6 h after the first smoke exposure. Cell harvesting for GSH assays, CFTR Western blot, and cAMP-dependent 125I-efflux assays was done 24 h after the first smoke exposure. The T-84 cells were exposed once for 10 min to the gaseous phase of cigarette smoke, and the supernatant was replaced with fresh media. To determine the effects of cigarette smoke condensate (CSC) on GCLC and CFTR mRNA expression, we used the methods described by Rahman and colleagues (12, 20). Briefly, cigarette smoke condensate was prepared by drawing 35 ml/min cigarette smoke for 5 min into a syringe and injecting this smoke into a tonometer containing 10 ml culture media. The resulting smoke-exposed media was defined as 100% CSC. The cells were then exposed to 0 to 100% CSC for 6 h before being harvested for Northern blot analysis as described below.

Glutathione Assays

Cellular glutathione was measured using a model DU7 spectrophotometer (Beckman Instruments Canada, Inc., Mississauga, ON, Canada) to determine the reduction of DTNB in the presence of glutathione reductase and NADPH as previously described (21).

RNA Extraction and Northern Blot Analysis

Calu-3 and T-84 cells were harvested for Northern blot analysis as previously described (22). Total cell RNA was isolated with a one-step guanidinium-phenol-chloroform extraction procedure (23). RNA was separated by electrophoresis on 1% agarose and transferred onto a hybond-N+ membrane (Amersham, Oakville, ON, Canada) for analysis. Membranes were prehybridized for 4 h in a mixture containing 120 mM Tris, 600 mM NaCl, 0.1% Na4P2O7, 8 mM ethylenediaminetetraacetic acid, 0.2% SDS, 625 μg/ml heparin, and 10% dextran sulfate at pH 7.4. Hybridization was performed overnight at 68°C in the same buffer. The human GCLC probe was obtained from the ATCC (GenBank/EMBL: M90656). The CFTR PcDNA3 was a gift from Dr. Gergely Lukacs of the Hospital for Sick Children, Toronto, Ontario. The probe was prepared by using a 1.1-kb fragment after EcoR1 digestion. Each probe was labeled with the multiprime DNA labeling system (Amersham) using α-32PdCTP (specific activity > 3,000 Ci/mmol/L). The membrane was washed once at room temperature for 20 min in 2 × saline sodium citrate and for 1 h at 68°C in 0.1% SDS, 0.1 × saline sodium citrate and was rinsed at room temperature in 0.1 × saline sodium citrate. The membrane was exposed to X-OMAT film (Kodak, Rochester, NY) with intensifying screens at −80°C. As a control for RNA integrity, the blot was hybridized with a 1 kb Pstl cDNA probe (ATCC) of the housekeeping gene glyceraldehyde phosphate dehydrogenase (GAPDH). Signal intensity was quantitated densitometrically with a PowerLook II scanner (UMAX Technologies, Inc., Dallas, TX). Densitometric values are expressed as the ratio of GCLC/GAPDH or CFTR/GAPDH densitometric quantifications.

Western Blot

Calu-3 cells were harvested by scraping and suspended in 0.5 ml radioimmunoprecipitation assay buffer composed of 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 0.08% deoxycholic acid, 20 mM Tris-HCl (pH 8.0), and one protease inhibitor cocktail tablet/50 ml (Roche, Mannheim, Germany) before being placed on ice for 10 min to lyse the cells. The lysate was centrifuged at 9,000 × g for 10 min at 4°C, and the supernatant was collected. The protein concentration of the lysate was estimated using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). Samples containing 50 μg protein were electrophoresed on a 10% SDS–polyacrylamide gel. The separated proteins were electro-transferred to a nitrocellulose membrane (Bio-Rad Laboratories) for Western blot analysis.

cAMP-Dependent Iodide Efflux Assay

The cAMP-dependent anion efflux was determined in Calu-3 cells. Cells were cultured to 90% confluence in six-well plates and labeled with 15 μCi/well 125I for 1 h at room temperature. The cells were washed six times in efflux buffer (119 mM Na gluconate, 1.2 mM K2HPO4, 0.6 mM KH2PO4, 25 mM NaHCO3). The cells were then incubated in efflux buffer. Supernatants were collected, and the buffer was replaced with fresh efflux buffer every minute for 4 min. At 4 min, efflux buffer containing 0.5 mM 2′O-dibutyryl cAMP, 10 μM 3-isobutyl-1 methylxanthine, and 10 μM forskolin (cAMP buffer) was added. Supernatants were harvested, and fresh cAMP buffer was added to the cells every minute for 8 min. The radioactivity of the supernatants was determined in a γ counter.


The results are presented as the mean ± SE unless stated otherwise. Data presented in Figures 1, 2, 4A, and 4B were analyzed using Student's t test, and data in Table 1 were analyzed with the Student's t test for unequal variances. For data including multiple comparisons, two-way analysis of variance (ANOVA; Figure 3) or repeated-measures ANOVA (Figure 4) were applied followed by Bonferroni post hoc tests. A p value of less than 0.05 was considered significant.








ΔClf + Iso

Total ΔPD
Infertile smokers (n = 9)22.1 ± 12.2*−21.7 ± 4.513.8 ± 4.666.0 ± 18.2−1.5 ± 3.8−9.6 ± 4.04.2 ± 5.9
Control nonsmokers (n = 30)20.3 ± 10.7−23.2 ± 7.712.5 ± 4.458.1 ± 10.5−13.8 ± 9.0−26.8 ± 10.2−14.3 ± 11.6
Infertile nonsmokers (n = 17)22.9 ± 9.8−25.0 ± 6.514.3 ± 3.968.0 ± 14.5−10.3 ± 7.2−22.3 ± 10.1−8.0 ± 8.8
Infertile smokers versus control nonsmokersp = 0.7p = 0.5p = 0.4p = 0.2p < 0.0001p < 0.0001p < 0.0001
Infertile smokers versus infertile nonsmokersp = 0.9p = 0.19p = 0.8p = 0.8p = 0.0004p = 0.0001p = 0.001
Infertile nonsmokers versus control nonsmokers
p = 0.4
p = 0.4
p = 0.2
p = 0.02
p = 0.2
p = 0.2
p = 0.04

Definition of abbreviations: ΔAmil = change in transepithelial nasal potential difference in amiloride solution; %Amil = percent change in transepithelial nasal potential difference in amiloride solution; ΔClf = change in transepithelial nasal potential difference in chloride-free solution; ΔClf + Iso = change in transepithelial nasal potential difference in chloride-free plus isoproterenol solution; MaxPD = maximum potential difference; ΔPD = change in potential difference.

*Mean ± SD.

Cigarette Smoke Increases Glutathione

Exposure of Calu-3 cells to the gaseous phase of cigarette smoke increased cellular GSH levels at 24 h (control GSH, 94.6 ± 0.9 nmol/mg protein; smoke GSH, 167.1 ± 6.3 nmol/mg protein; n = 10; p < 0.0001; Figure 1A). There was no evidence of cytotoxicity as determined by chromium release assays (data not shown).

Cigarette Smoke Decreases CFTR mRNA Levels in Calu-3

Although low-level expression of mRNA for GCLC was detectable in control cells, exposure to the gaseous phase of cigarette smoke increased GCLC expression without affecting the level of expression of the housekeeping gene GAPDH (GCLC/GAPDH mRNA ratio: control, 0.86 ± 0.12; smoke, 1.58 ± 0.07 units; n = 8; p = 0.0002; Figures 1B and 1C). In contrast, mRNA for CFTR was robustly expressed in control cells but markedly decreased in the smoke-exposed cells (CFTR/GAPDH mRNA ratio: control, 0.87 ± 0.03; smoke, 0.47 ± 0.03 units; n = 11; p < 0.0001; Figures 1B and 1D).

Cigarette Smoke Decreases CFTR Protein Levels in Calu-3 Cells

Exposure of Calu-3 cells to the gaseous phase of cigarette smoke resulted in a clear decrease of CFTR protein as determined by Western blotting (CFTR/actin protein ratio: control, 2.07 ± 0.24; smoke, 1.24 ± 0.14 units; n = 6; p = 0.011; Figure 2B).

125I Efflux Is Decreased in Calu-3 Cells by Cigarette Smoke

Calu-3 cells previously loaded with 125I and stimulated with IBMX, forskolin, and dibutyryl cAMP secreted high levels of 125I (Figure 3). In contrast, exposure to the gaseous phase of cigarette smoke markedly suppressed Calu-3 cell cAMP-dependent 125I secretion (two-way ANOVA for smoke exposure as the source of variation, p < 0.0001, n = 5; Bonferroni post-test: control vs. smoke at 7, 8, and 9 min, p < 0.01).

Cigarette Smoke Decreases CFTR Expression and Function in T84 Cells

Exposure of T84 cells to the gaseous phase of cigarette smoke for 10 min was followed 24 h later by a clear increase in GSH (GSH: control, 41.7 ± 1.0; smoke exposure, 89.8 ± 1.5 nmol/mg protein; n = 6; p < 0.001; data not shown). The increase in GSH was preceded at 6 h postexposure by a marked increase in GCLC mRNA and a decrease in CFTR mRNA within the same cells (GCLC/GAPDH mRNA ratio: control, 0.69 ± 0.08; smoke, 2.44 ± 0.25; p < 0.0001 [Figure 4A] CFTR/GAPDH mRNA ratio: control = 0.57 ± 0.04 vs. smoke = 0.21 ± 0.02, p < 0.0001 [Figure 4B]). Similarly cigarette smoke condensate induced a dose-dependent increase in GCLC and a decrease in CFTR mRNA (Figure 4C), with a significant difference between 100% cigarette smoke condensate and control for GCLC/GAPDH and CFTR/GAPDH ratios (p < 0.01, n = 3; Figure 4D).

CFTR-mediated Nasal Transepithelial PD Is Decreased in Cigarette Smokers

Typical recordings of the nasal transepithelial differences in a healthy nonsmoker, a healthy smoker, and a patient with CF are shown in Figure 5. The presence of functional CFTR is characterized by an increase in transepithelial nasal PD in chloride-free (ΔClf) and isoproterenol (ΔClf + Iso) solutions. No such increase is seen in the tracing from a patient with CF. Nasal PD testing was completed in 56 subjects without CF. After extensive analysis, no CFTR gene mutations were detected in any of the subjects, and all subjects had sweat chloride concentrations within the normal range (0–40 mmol/L). There were no differences in sweat chloride concentrations between infertile smokers, infertile nonsmokers, and fertile nonsmokers (Table 1). The amiloride response expressed as change in PD and percentage change in PD was also similar in all three groups. In contrast, the ΔCl + Iso response in millivolts, expressed as mean ± SD, was −9.6 ± 4.0 in infertile smokers (n = 9), −22.3 ± 10.1 in infertile nonsmokers (n = 17), and −26.8 ± 10.2 (n = 30) in fertile nonsmokers (infertile smokers vs. infertile nonsmokers, p = 0.0001; infertile smokers vs. fertile nonsmokers, p < 0.0001; infertile nonsmokers vs. fertile nonsmokers, p = 0.2; Table 1).

Cigarette smoke exposure increased cellular GSH and GCLC expression and decreased CFTR expression at the gene, protein, and functional levels in the human respiratory epithelial cell line Calu-3. The gaseous phase and condensate of cigarette smoke also increased cellular GSH and GCLC gene expression while decreasing CFTR mRNA expression in the human intestinal cell line T84. Because Calu-3 cells are derived from the human airways and T84 from the human intestine (24, 25), the effects of cigarette smoke on CFTR expression were not cell or tissue specific. Furthermore, cigarette smokers without genetic evidence of CFTR alterations showed decreased CFTR-dependent changes in nasal transepithelial PDs.

The in vivo component of this study represents a retrospective analysis of a population of infertile men. This population was chosen because we had extensive CFTR genotyping data for these subjects. We do not have such data in a general population of cigarette smokers. It is possible that the nasal transepithelial PDs could be specific to infertile smokers, and extrapolation of the current data to the general population must be done with caution. However, the control group of infertile nonsmokers had normal CFTR function, indicating that there was a smoking-specific effect on CFTR function in this population.

Sustained epithelial cell oxidative stress induces GSH synthesis (12, 21, 26). Transcription of the GCLC gene markedly increases in cells exposed to cigarette smoke (12, 27). Cigarette smokers have increased GSH levels in their lung epithelial lining fluid (21). This latter observation suggests that our in vitro conditions may accurately reflect the in vivo responses observed in cigarette smokers. We found that cigarette smoke exposure sufficient to induce GCLC gene expression in Calu-3 and T84 markedly suppressed CFTR expression and function. Cigarette smoke is a rich source of quinones (28). The current study is consistent with the observations of Kreindler and colleagues, indicating that CSE can suppress chloride secretion in human bronchial epithelial cells, and, with our recent findings, that a quinone oxidative stress induced by t-butylhydroquinone accelerates CFTR mRNA degradation, decreases CFTR protein, and inhibits CFTR function in vitro (29). We have also observed that cigarette smoke tends to decrease CFTR band C more than band B as seen by Western blot, raising the possibility that increased proteosome degradation of CFTR may be occurring, as has been previously reported for oxidatively modified proteins and for α-tubulin in cigarette smoke–exposed cells (30, 31). Most importantly, we were able to confirm the relevance of our in vitro observations of cigarette smoke–mediated suppression of CFTR expression by observing that cigarette smokers have clearly decreased nasal transepithelial PDs in response to ΔClf + isoproterenol. However, because in vitro smoke experiments represent acute rather chronic exposures as occur in smokers, several important questions about the kinetics of in vivo CFTR suppression remain. Further studies are required to determine the intensity and duration of cigarette smoking that leads to CFTR deficiency and the time needed after the last cigarette to recover normal nasal transepithelial PDs.

Our observations raise the possibility that cigarette smoking may induce an acquired form of intermittent CFTR deficiency that contributes to some of the clinical manifestations in smokers. Many cigarette smokers develop chronic obstructive pulmonary disease, a condition with several features that recall CF, such as increased airway bacterial infections and, in the more severe cases, bronchiectasis (32). CFTR deficiency has been linked to periciliary liquid depletion and compromised antimicrobial properties of airway surface liquid (3, 33). Furthermore, the absence of CFTR function decreases anion-mediated fluid secretion from submucosal glands and is associated with defective mucus secretion from these glands known to play a key role in airway innate immune defenses (3436).

Evidence that cigarette smoke may contribute to accentuate an existing CFTR deficiency stems from a study of 9,141 individuals to determine the fecundity of ΔF508 heterozygotes and cigarette smokers (37). Nonsmokers who were heterozygous for the ΔF508 CFTR mutation had a slightly higher fecundity than noncarriers, indicating that heterozygotes have sufficient residual CFTR function to maintain fecundity. In contrast, a clear decrease in fecundity was observed in smokers who were heterozygous for the ΔF508 CFTR mutation. The decrease in fecundity was not observed in smokers who were noncarriers. Because carriers of the ΔF508 mutation have decreased CFTR expression, they also have half the normal CFTR function. Infertility, at least in men, is the most sensitive manifestation of CFTR deficiency (38). Therefore, a plausible explanation of the reduced fecundity in ΔF508 heterozygote smokers is that cigarette smoke induced a further decrease in CFTR function in reproductive tissues, bringing it below the critical level needed to ensure normal fecundity.

In summary, we have shown that the gaseous phase and the aqueous extract of cigarette smoke can suppress CFTR expression in vitro while simultaneously increasing the cellular antioxidant GSH. Furthermore, cigarette smokers have altered nasal transepithelial PDs, which is suggestive of decreased CFTR function. We submit that at the risk of impairing mucus clearance, transient CFTR suppression during an oxidant stress such as cigarette smoke exposure represents an antioxidant response to help protect epithelial tissues by increasing the viscosity of the mucus barrier. Because sustained CFTR suppression as observed in the extreme situation of CF leads to substantial respiratory manifestations that have similarities to those of smokers, it is possible that some of the disease manifestations of persistent heavy cigarette smokers may be explained by an acquired form of CFTR deficiency. The potential importance of cigarette smoke-induced CFTR abnormalities in the physiopathology of smoking-related diseases should be further investigated.

The authors thank Dr. Gergely Lukacs for the CFTR cDNA and Tim Jensen and John Riordan for the CFTR antibody. The authors also thank Dr. Keith Jarvi for contributions to patient ascertainment and Dr. Mary Corey for help with the CF research database.

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Correspondence and requests for reprints should be addressed to André M. Cantin, M.D., Pulmonary Research Unit, Faculty of Medicine, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Quebec, J1H 5N4 Canada. E-mail:


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