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Published ahead of print on July 15, 2004, doi:10.1165/rcmb.2002-0300OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 483-490, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2002-0300OC

Reversible Cigarette Smoke Extract–Induced DNA Damage in Human Lung Fibroblasts

Huijung Kim, Xiangde Liu, Tetsu Kobayashi, Heather Conner, Tadashi Kohyama, Fu-Qiang Wen, Qiuhong Fang, Shinji Abe, Peter Bitterman and Stephen I. Rennard

Department of Internal Medicine, Seoul Adventist Hospital and WonKwang University Sanbon Medical Center, Seoul, Korea; University of Nebraska Medical Center, Omaha, Nebraska; Department of Respiratory Medicine, University of Tokyo, Tokyo, Japan; and University of Minnesota, Minneapolis, Minnesota

Address correspondence to: Stephen I. Rennard, M.D., University of Nebraska Medical Center, 985885 Nebraska Medical Center, Omaha, NE 68198-5885. E-mail: srennard{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cigarette smoke contains thousands of chemicals, many of which may contribute to cytotoxicity and carcinogenesis. Using assays detecting DNA strand breaks (terminal transferase dUTP nick end labeling [TUNEL]) and DNA content (flow cytometry), we evaluated the genotoxic effect of cigarette smoke extract (CSE) on human fetal lung fibroblasts (HFL-1) cultured in three-dimensional collagen gels as well as in monolayer culture. When HFL-1 cells were exposed to CSE, DNA strand breaks were detected in most, as determined by TUNEL. This effect was dependent on CSE concentration, duration of CSE exposure, and the density of HFL-1 cells cast into the collagen gels. Buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, significantly increased DNA damage induced by 1% CSE, and N-acetylcysteine, a glutathione precursor, blocked 5% CSE from inducing DNA damage. After CSE exposure, most cells were TUNEL-positive, but DNA quantification revealed no hypodiploid cells, indicating that apoptosis was not occurring during the CSE exposure. CSE-induced DNA damage was reversible, and cells proliferated when CSE was removed after 24 h exposure. These results demonstrate that cigarette smoke can induce DNA damage in HFL-1 cells cultured in both three-dimensional collagen gels and monolayer cultures, and that oxidants likely play a role in this damage. Moreover, this DNA damage is reversible, with cells surviving and TUNEL positivity reversing when CSE is removed within 24 h.

Abbreviations: buthionine sulfoximine, BSO • chronic obstructive pulmonary disease, COPD • cigarette smoke extract, CSE • Dulbecco's modified Eagle's medium, DMEM • fetal calf serum, FCS • human fetal lung cells, HFL-1 • N-acetylcysteine, NAC • phosphate-buffered saline, PBS • rat-tail tendon collagen, RTTC • terminal transferase dUTP nick end labeling, TUNEL


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cigarette smoke is associated with several lung diseases (1). In this context, smoke is able to induce lung damage by a variety of direct and indirect mechanisms. Smoke can directly damage the cellular and extracellular structural elements of the lung, and also initiate inflammatory processes that can secondarily damage the lung. By damaging the DNA of lung cells, smoke is believed to alter the genomic integrity of lung cells. Such alterations likely disrupt the mechanisms that regulate cellular proliferation and play key roles in the development of lung cancer. Interestingly, several lines of evidence suggest that acquired alterations in gene expression can also contribute to nonmalignant lung diseases, such as chronic obstructive pulmonary disease (COPD) (2, 3).

COPD is a collection of conditions characterized by the progressive development of airflow obstruction (4). Two structural lesions are important in causing loss of function: (i) destruction of alveolar parenchyma; i.e., emphysema, which results in loss of lung elastic recoil; and (ii) the narrowing of small airways by fibrosis, which increases airway resistance. COPD is currently the fourth leading cause of death in the United States and is increasing in prevalence. It is expected to be the third leading cause of death worldwide by the year 2020 (5). Cigarette smoking is the most common cause, accounting for more than 80% of cases. Although quitting early in the development of the disease slows the progressive loss of lung function (6), relentless progression is often seen in older individuals (7–9), suggesting that cigarette smoke may cause persistent or self-sustaining damage even after cessation.

Apoptosis, programmed cell death, is believed to be a key mechanism by which effete and other unnecessary cells are removed. This process is also believed to play a key role in removing damaged cells that might lead to functional abnormalities; that is, cells that have undergone DNA damage, and therefore are subject to mutations and alterations in function, frequently undergo apoptosis and are removed. Within this context, apoptosis of cells after cigarette smoke exposure has been suggested to be a key mechanism in the protection of lung tissue.

The current study, therefore, was designed to test the hypothesis that cigarette smoke extract (CSE) can induce DNA damage in lung fibroblasts, and to determine if this process initiates apoptosis. For this purpose, fibroblasts were cultured in three-dimensional collagen gels, a culture system that more closely models tissue than does the conventional dish culture system (10).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials and Cell Culture
Type I Collagen (rat tail tendon collagen [RTTC]) was extracted from rat-tail tendons by a previously published method (11). Protein concentration was determined by weighing a lyophilized aliquot from each batch of collagen. The RTTC was stored at 4°C until use.

N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) were purchased from Sigma (St. Louis, MO). Dulbecco's modified Eagle's medium (DMEM), Fetal calf serum (FCS), Trypsin/EDTA, penicillin G sodium, and streptomycin were purchased from Invitrogen (Life Technologies, Grand Island, NY). Amphotericin B was purchased from Pharma-Tek (Elmira, NY). The terminal transferase dUTP nick end labeling (TUNEL) assay kit was purchased from Roche Diagnostic Corporation (Indianapolis, IN). The TiterTACS Colorimetric Apoptosis Detection Kit was purchased from Trevigen (Gaithersburg, MD).

Human fetal lung fibroblasts (HFL-1; lung, diploid, human) were obtained from the American Type Culture Collection (#CCL-153; Rockville, MD). Cells were cultured in 100 mm tissue culture dishes (Falcon; Becton-Dickinson Labware, Lincoln Park, NJ) with DMEM supplemented with 10% FCS, 50 U/ml penicillin G sodium, 50 µg/ml streptomycin sulfate, and 1 µg/ml amphotericin B. Media were changed three times weekly, and cells subcultured weekly and maintained in 10% FCS–DMEM. Fibroblasts between passages 15 and 20 were used for all experiments.

Three-Dimensional Collagen Gel Culture
Prior to preparing collagen gels as described below, fibroblasts were detached by 0.05% trypsin in 0.53 mM EDTA and suspended in 10 ml serum-free DMEM containing soybean trypsin inhibitor. The cell number was then counted with Coulter Counter and the cell density was adjusted to 2 x 106/ml. Collagen gels were prepared, as previously described (11), by mixing RTTC, distilled water, 4 x DMEM and cells. The final concentration was 1 x DMEM, 0.75 mg/ml of collagen, and except for the cell density experiments, fibroblasts were present at 1 x 105 cells/ml. Following this, 500 µl of the mixture was cast into each well of a 24-well culture plate (Falcon). The solution was then allowed to polymerize at room temperature, generally completed in 20 min. After polymerization, the gels were either floated in the medium or attached in the plates. For most of the experiments, the gels were gently released from the plates in which they were cast and transferred into 60-mm tissue culture dishes (three gels in each dish), which contained 5 ml of 1% FCS–DMEM with indicated concentrations of CSE and/or various reagents. For the experiments of cell proliferation in three-dimensional culture, the gels remained attached, in which case the polymerized gels in 24-well plates were over-layered with 0.5 ml of 1% FCS–DMEM with indicated concentrations of CSE. All cultures were maintained at 37°C in a 5% CO2 atmosphere. For most experiments, 1% FCS–DMEM was used in the presence CSE. After removal of CSE, 10% FCS–DMEM was used in the experiments of DNA damage recovery and cell proliferation.

DNA Quantification
To estimate cell number in three-dimensional collagen gels, DNA was assayed fluorometrically with Hoechst dye no. 33,258 (Sigma) by a modification of a previously published method (12). Collagen gels were solubilized by heating to 65°C for 10 min and cell suspensions were collected by centrifugation at 2,000 x g for 5 min and resuspended in 1 ml of distilled water. After sonication, the suspensions were mixed with 2 ml of TNE buffer (3M NaCl, 10 mM Tris, and 1.5 mM EDTA, pH7.4) containing 2 µg/ml of Hoechst dye no. 33,258. Fluorescence intensity was measured with a fluorescence spectrometer (LS-5; Perkin-Elmer, Boston, MA) with excitation at 356 nm and emission at 458 nm.

Preparation of CSE
CSE was prepared by a modification of the method developed by Carp and Janoff (13). Briefly, one (100 mm) cigarette without filter (research cigarette; University of Kentucky) was combusted with a Peristaltic Pump (Model No. 7017; Cole-Parmer, Chicago, IL). The smoke was bubbled through 25 ml of RPMI at a speed of 50 cc/min. The resulting suspension was filtered through a 0.22-µm pore filter (Lida Manufacturing, Kenosha, WI) to remove bacteria and large particles. This solution, considered to be 100% CSE, was applied to fibroblast cultures within 30 min of preparation. CSE concentrations in the current study ranged from 1–10%. CSE concentrations prepared in this manner of up to 20% do not induce cytotoxicity (14).

Determination of DNA Damage and Cell Viability
For determination of DNA damage, both single cell staining with TUNEL assay kit and cell population staining with TiterTACS assay kit were performed following manufacturer's instructions. Briefly, collagen gels were transferred to Eppendorf tubes (Fisher, Pittsburgh, PA) and then solubilized with heating at 65°C for 10 min. This method effectively solubilized the collagen gels without resulting in further DNA damage, as assessed by TUNEL assay. Cell suspensions were collected by centrifugation at 2,000 x g for 5 min and resuspended in 150 µl of 10% FCS–DMEM. The resuspended cells were then used to prepare cytospins, 0.5 x 105 cells/spot, 1,000 x g for 5 min. Cytospin preparations were fixed with freshly prepared paraformaldehyde (4% in phosphate-buffered saline [PBS]; pH 7.4) for 1 h at room temperature. The cells were permeabilized with 0.1% Triton X-100 (in 0.1% sodium citrate) for 2 min at 4°C and rinsed with PBS. The TUNEL reaction process was then performed using the manufacturer's instructions (Roche). The number of cells stained by the TUNEL method was expressed as a percentage of the total number of cells stained with the counterstain propidium iodide. At least 500 nuclei were counted on each cytospin sample in 5–10 randomly selected viewing fields. For the cell population TiterTACS assay, cells were cultured in 6-well plates at a density of 2 x 105 cells/ml, 2 ml/well and treated with CSE or reagents as desired. Cells were then harvested by trypsinizing, fixed, permeabilized, and labeled following the manufacturer's instructions. Optimal density (OD) value for each sample in duplicate measurement was obtained and data expressed as percentage of positive control (DNase I–treated samples).

Cell viability was evaluated by ethidium homodimer-1 dye exclusion using the LIVE/DEAD Kit, following the manufacturer's instruction (Molecular Probes, Eugene, OR). Each gel was incubated with 200 µl sterile PBS containing Calcein AM (Green) and ethidium homodimer-1 (EthD-1, Red) at 37°C for 20 min followed by observation under fluorescence microscopy. Nuclear stained by EthD-1 in red counted as dead cells.

Profile of DNA Content by Flow Cytometry
For three-dimensional collagen gel culture, fibroblast-populated (2 ml of 105cells/ml) collagen gels were cast into 6-well tissue culture plates (Falcon). After polymerization, gels were gently released and incubated with varying concentrations of CSE in 1% FCS–DMEM for 24 h or with 1 µM staurosporine for 4 h (positive control). Gels were then transferred into 15-ml conical tubes and incubated with 0.05% Trypsin/0.53 mM EDTA-4Na (Invitrogen) for 10 min (500 µl/gel) at 37°C in a 5% CO2 atmosphere. Collagenase (1 mg/ml in DMEM) was then added (1 ml/gel) and incubated while shaking at 37°C in a 5% CO2 atmosphere for 30 min or until the gels were completely dissolved. DMEM containing 10% FCS was then added to stop the enzymatic reaction, and cells were pelleted by centrifugation. Cells were then fixed with Telford method and flow cytometry was performed as described below.

For monolayer culture, fibroblasts were plated in 6-well plates at a density of 2 x 105 cells/well in 2 ml 1% FCS–DMEM. Cells were allowed to attach overnight. On the following day, cells were treated with varying concentrations of CSE in 1% FCS–DMEM for 24 h or 1 nM staurosporine for 4 h. Cells were then trypsinized, suspended in 10% FCS–DMEM, and pelleted by centrifugation. Cells were fixed with Telford method and flow cytometry analysis was performed.

Flow cytometric analysis of DNA content was performed as previously described (15). Briefly, cells were fixed with cold 70% Ethanol in PBS for 30 min at 4°C. Cells were then pelleted by centrifugation and resuspended in the staining solution (50 µg propidium iodide, 100 µg RNAse A in 1 ml PBS for 106 cells) at 4°C for 1 h followed by flow cytometric analysis without washing.

Statistical Analysis
All data are expressed as mean ± SEM. Statistical comparison of paired data was performed using Student's t test, whereas multigroup data were analyzed by ANOVA followed by the Tukey's (one-way) or Bonferroni's (two-way) post-test using PRISM4 software (GraphPad Prism, San Diego, CA). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Cigarette Smoke on DNA Strand Breaks of Fibroblasts in Collagen Gels
To determine the time-dependent effect of cigarette smoke on fibroblast DNA strand breaks, cells in three-dimensional collagen gels were exposed to 5% CSE in 1% FCS–DMEM for 3, 4, 5, 6, 12, and 24 h, and were then harvested and TUNEL staining was performed. After 24 h, 9.3 ± 3.9% of control cells cultured in floating collagen gels were TUNEL-positive. In the presence of 5% CSE, however, 3 h exposure significantly increased DNA strand breaks (23.5 ± 6.8%, P < 0.05, compared with control at 24 h by one-way ANOVA followed by Tukey's test). Six-hour CSE exposure induced DNA strand breaks in 71.8 ± 1.0% of the cells, and DNA strand breaks occurred in 95.8 ± 1.3% of the cells after 24 h exposure (Figure 1A). To determine the concentration-dependent effect of CSE on DNA strand breaks, cells in contracting collagen gels were exposed to 0, 1, 2.5, or 5% CSE, respectively, for 24 h. One percent CSE significantly induced DNA strand breaks (10.1 ± 1.2% for control cells versus 22.7 ± 5.8% for 1% CSE–exposed cells, P < 0.05 compared with control by one-way ANOVA followed by Tukey's post-test). The percentage of DNA damaged cells increased as a function of CSE concentration (Figure 1B).





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  		Figure 1. CSE effect on the DNA integrity of fibroblasts. Fibroblasts cast into the collagen gels were exposed to 5% CSE for varying time periods (A). Data presented are from three separate experiments, each performed in triplicate. *P < 0.05 compared with control (0% CSE) by the Tukey's test. (B) Fibroblasts cast into collagen gels were exposed to indicated concentrations of CSE for 24 h. Data presented are from three separate experiments, each performed in triplicate. *P < 0.05 compared with control (0% CSE) by the Tukey's test. (C) Indicated densities of fibroblasts were cast into the gels and exposed to 5% CSE for 24 h. Data presented are from three separate experiments, each performed in triplicate. *P < 0.05 compared with 1 x 105 cells/ml group by the Tukey's test.

 
Cell Density and CSE-Induced DNA Strand Breaks
Because many responses of fibroblasts in three-dimensional gel culture depend on cell density, the relationship of cell density to CSE-induced DNA strand breaks was assessed. Varying numbers of cells were mixed into collagen gels and exposed to 5% CSE for 24 h (Figure 1C). At lower cell density (1–2 x 105 cells/ml), 5% CSE significantly induced DNA strand breaks (94.8 ± 2.7% and 93.6 ± 2.5%, respectively). At higher cell densities (3, 4, and 5 x 105 cells/ml), the number of TUNEL-positive cells progressively decreased (32.2 ± 4.9%, 16.7 ± 4.9%, and 12.8 ± 1.3%, respectively, P < 0.05 comparing to 1 x 105 cells/ml by one-way ANOVA followed by Tukey's test).

Effect of NAC and BSO on CSE-Induced DNA Strand Breaks in the Fibroblasts
Smoke can induce oxidant-mediated damage, and antioxidant defenses in fibroblasts may be protective. To investigate the role of the endogenous antioxidant glutathione in CSE-induced DNA strand breaks, two experiments were performed. First, the ability of the glutathione synthesis inhibitor buthionine sulfoximine (BSO) was assessed. BSO at 1 mM concentration very significantly increased the TUNEL positivity caused by 1% CSE (P < 0.001), whereas 0–0.1 mM BSO had no effect (P > 0.05 by two-way ANOVA followed by Bonferroni's test, Figure 2A). Second, the antioxidant NAC, which can enter cells and serve as a precursor for glutathione synthesis, significantly blocked the ability of 5% CSE to induce TUNEL positivity at concentrations of 1 and 10 mM (P < 0.05 and 0.01, respectively), whereas alone it was without effect (P > 0.05 by two-way ANOVA followed by Bonferroni's test, Figure 2B).





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  		Figure 2. Effect of N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) on DNA damage by CSE in fibroblasts cultured in monolayer as well as collagen gels. (A and B) Effect of BSO and NAC in monolayer culture. Fibroblasts were cultured in 6-well plates, treated with CSE (open bars, control; shaded bars, 1% CSE) with indicated concentrations of BSO or NAC and DNA damage was determined by TiterTASCs as described in MATERIALS AND METHODS. Horizontal axis: concentrations of BSO or NAC (mM). Vertical axis: percentage of TUNEL-positive versus DNase-treated control (%). *P < 0.05 and **P < 0.01 by two-way ANOVA followed by Bonferroni test. (C) Effect of BSO and NAC in collagen gel culture. Fibroblasts cast into collagen gels were incubated in 1% FCS–DMEM with either NAC (10 mM) ± 5% CSE or BSO (1 mM) ± 1% CSE. Cells were harvested after 24 h, and DNA damage was evaluated. Data presented are from three separate experiments, each performed in triplicate. *P < 0.001 by t test.

 
Reversibility of CSE-Induced DNA Strand Breaks
To determine the reversibility of CSE-induced DNA strand breaks, fibroblasts in collagen gels were treated with or without 5% CSE in 1% FCS–DMEM for 24 h (Figure 3A and 3B). Cells were then refed with 10% FCS–DMEM (Figures 3D, 3G, and 3J) or continuously exposed to 5% CSE with 1% FCS–DMEM for an additional 3 d (Figures 3E, 3H, and 3K). Few cells were TUNEL-positive in the control group (Figures 3C, 3F, and 3I), which was never exposed to CSE. When fibroblasts in the gels were continuously exposed to 5% CSE, a high percentage of TUNEL-positive cells persisted throughout the 3-d observation period (92.2 ± 3.5% on Day 1, 95.0 ± 2.6% on Day 2, and 96.3 ± 2.3% on Day 3). However, when the cells were exposed to 5% CSE for 24 h and then refed with 10% FCS–DMEM, the percent of TUNEL-positive cells decreased as a function of time (82.8 ± 6.8% on Day 1, 28.5 ± 10.0% on Day 2, and 13.3 ± 2.2% on Day 3). At no time were significant differences observed in cell viability between smoke-exposed and control cells by ethidium homodimer-1 dye exclusion (data not shown). Increasing concentrations of CSE not only resulted in a larger number of TUNEL-positive cells, but also required a longer time for complete recovery from DNA damage (Figure 4).



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  		Figure 3. Recovery from CSE-induced DNA damage of fibroblasts in collagen gel culture. Fibroblasts cast into the collagen gels were exposed to 0 or 5% CSE for 24 h in 1% FCS–DMEM in replicates. Cells then were refed with either 10% FCS–DMEM without CSE or continuously exposed to 5% CSE until Day 4. TUNEL assays were performed on Days 1, 2, 3, and 4. (A, C, F, and I) Control cells without CSE exposure. (B, D, G, and J) Cells were exposed to 5% CSE in 1% FCS–DMEM for 24 h, then cultured in 10% FCS–DMEM from Days 2–4. (B, E, H, and K) Cells were exposed to 5% CSE in 1% FCS-DMEM from Days 1–4.

 


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  		Figure 4. Effect of CSE concentration on recovery from DNA damage in fibroblasts in collagen gel culture. Fibroblasts in the gels were exposed to different concentrations of CSE for up to 24 h before cells were cultured in smoke-free, 10% FCS–DMEM for an additional 7 d. TUNEL staining was performed after 24 h exposure to CSE and on Days 1, 2, 3, and 7 after CSE removal. *P < 0.05 compared with control (Day 0) on each day by the ANOVA and Bonferroni test. Squares, control (0% CSE); triangles, 2.5% CSE; inverted triangles, 5% CSE; diamonds, 10% CSE.

 
Apoptosis Determination by Profiling DNA Content of Fibroblasts in Collagen Gel and Monolayer Cultures
That TUNEL positivity was not associated with a decrease in viable cells and was reversible suggests that cells underwent DNA strand breaks but not apoptosis. To determine whether TUNEL-positive cells following CSE exposure were undergoing apoptosis, profiling of DNA content was performed by flow cytometry. Under control conditions, i.e., when cells were cultured three-dimensionally in collagen gels and allowed to contract in 1% FCS–DMEM, 12–32% (20.7 ± 5.9%) of the cells contained hypodiploid amounts of DNA depending on the experiment (three separate occasions). When the cells were treated with 1 nM staurosporine for 4 h, 31–76% (54.3 ± 13.0%) of the cells contained hypodiploid amounts of DNA, indicating apoptosis (Figure 5A). When the cells were exposed to varying concentrations of CSE for 24 h, however, no increase above control in cells containing hypodiploid amounts of DNA was observed (Figure 5A). In contrast, when the cells were cultured under monolayer condition, there was very few cells containing hypodiploid amounts of DNA under control condition as well as cells exposed to 2.5–10% CSE (0.2–0.3%). Twenty percent CSE, however, significantly increased the number of cells with hypodiploid DNA content in monolayer culture (44.7 ± 5.0% versus 1.1 ± 0.5% of control, P < 0.01 by t test) but mildly in three-dimensional culture (21.4 ± 6.0% versus 20.7 ± 5.9% of control, P > 0.05 by t test).




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  		Figure 5. Representative profile of DNA content in fibroblasts exposed to different concentrations of CSE. (A) Three-dimensional culture. Collagen gels with fibroblasts were floated in 1% FCS–DMEM for 24 h, supplemented with 0, 1.25, 2.5, 5, or 10% CSE or, as positive control, treated with 1 nM staurosporine for 4 h before harvesting cells. (B) Monolayer culture. Cells were plated in 6-well plate (2 x 105/well/2 ml 1% FCS–DMEM) and allowed to attach overnight. Cells were then analyzed by flow cytometry. Vertical axis: cell number; horizontal axis: profile of DNA content. CSE concentration and percent of cells with hypodiploid DNA taken as an index of apoptosis are shown in each panel. Data presented are from one representative experiment of three experiments performed on separate occasions of three-dimensional collagen gel culture or monolayer culture.

 
Cell Proliferation after CSE Exposure: Time Course and CSE Concentration
To further confirm that DNA damage induced by CSE was both reversible and not associated with cell death, cell numbers were assessed in cultures refed with fresh 10% FCS–DMEM after 24 h exposure to 5% CSE. Cell numbers never appeared to decrease. In contrast, cells in all cultures were able to proliferate, and this effect was inversely dependent on CSE concentration (Figure 6). After treatment with higher concentrations of CSE (5%) for 24 h, cell numbers did not increase within 3 d, and only after 1 wk did the cell number increase significantly (P < 0.05, Figure 6A). In contrast, 1% CSE–treated cells started to proliferate rapidly, and cell numbers significantly increased after 2 d (P < 0.05, Figure 6B).




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  		Figure 6. Effect of CSE on fibroblast proliferation in collagen gel culture. (A) Time dependency. Fibroblasts embedded collagen gels were attached in 24-well plates. The cells were fed with either 1% FCS–DMEM alone (control, square) or 1% FCS–DMEM containing 5% CSE for 24 h. CSE was then either removed (triangle) or continuously applied (inverted triangle) in 10% FCS–DMEM, and cells were cultured for an additional 7 d, with refeeding every other day. *P < 0.05 compared with the group with CSE continuously by two-way ANOVA and Bonferroni's test. (B) CSE concentration effect. Fibroblast-embedded collagen gels were exposed to different concentrations of CSE in 1% FCS–DMEM for 24 h, and then cultured in smoke-free 10% FCS–DMEM for an additional 3 d. *P < 0.05, compared with 5% CSE–exposed cells by two-way ANOVA and Bonferroni's test. Square, control; triangle, 1.25% CSE; inverted triangle, 2.5% CSE; diamond, 5% CSE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using TUNEL assay and DNA content analysis, the current study assessed the toxic effect of cigarette smoke on HFL-1 cultured in three-dimensional collagen gels. CSE induced DNA damage, as assessed by TUNEL assay, but did not induce apoptosis in HFL-1 cells in gel culture, as assessed by the DNA content profiling assay. CSE-induced DNA damage was dependent on duration of exposure and the concentration of CSE. The effect of CSE on DNA damage was also inversely associated with the cell number cast into the collagen gels; that is, the higher the cell density, the fewer observable TUNEL-positive cells. NAC, which serves as a precursor for glutathione synthesis, significantly blocked the 5% CSE–induced DNA damage, whereas BSO, an inhibitor of glutathione synthesis, significantly increased DNA damage by 1% CSE. Following CSE removal from the culture system, the DNA damage was reversed; i.e., TUNEL-positive cells decreased, and the cells proliferated as a function of time.

Cytotoxic effects of cigarette smoke to lung resident cells may destroy lung tissues and lead to the development of emphysema (16). When exposed to cigarette smoke or other exogenous insult, cells may undergo cell membrane permeability changes and DNA strand breaks (17), but may also initiate repair mechanisms and survive (18). The current study demonstrates that CSE induced DNA damage, but this was not followed by apoptosis; i.e., after removal of the smoke, the cells recovered and proliferation was reinitiated.

Cigarette smoke–induced apoptosis has been reported in a variety of cell types (19–21). Recently, using flow cytometry analysis of annexin-V binding and 7-AAD dye uptake, Ishii and colleagues reported that CSE induced apoptosis at lower concentrations (10–25%) and necrosis at higher concentrations (50–100%) in HFL-1 (22). In the current study, however, severe DNA damage, but not apoptosis, was induced by 1–5% CSE. Several reasons could account for these differences in results, notably those related to experimental design. First, to make 100% CSE, Ishii and coworkers used ten 84-mm-long cigarettes, combusted through 10 ml of serum-free DMEM, whereas the current study used one 100-mm nonfiltered cigarette combusted through 25 ml of serum-free DMEM resulting in a much less concentrated preparation. Second, in contrast to the conventional monolayer cell culture used by Ishii and colleagues, the current study used the three-dimensional collagen gel culture system. The effect of cigarette smoke on fibroblasts in three-dimensional collagen gels may differ from that in the monolayer culture system. Finally, different detection techniques were used in the two studies. Ishii and coworkers determined cell membrane damage by flow cytometry analysis of annexin-V binding and 7-AAD dye uptake, a common method of detecting early apoptotic events because movement of phosphatidylserine from the inner membrane to the outer membrane may occur before cell membrane permeability changes or DNA damage occurs. One limitation with this method is that it cannot be used on tissues or fixed samples because of its low specificity. Enzymatic harvesting of adherent cell lines also may interfere with reliable detection of phosphatidylserine exposure, because trypsin or EDTA harvesting of cells before annexin-V labeling could induce changes in the plasma membrane, which could lead to false-positive results. Cells in the current study were embedded in collagen gels and treated with both trypsin and collagenase before staining. Attempts to use annexin-V staining revealed very high staining in control samples (data not shown), indicating that this method was not appropriate for the current study. Finally, it is possible that annexin-V positivity is not a perfect assay for commitment to apoptosis, but rather, like the TUNEL assay used in the current study, reflects a specific type of cell damage associated with apoptosis.

DNA fragmentation is considered to be a key event in apoptosis and can be detected as a typical DNA ladder on agarose gels. However, DNA fragmentation occurs in a late phase of apoptosis. Before fragmentation, single-strand DNA damage may occur, which cannot be detected by the DNA ladder method. This information can best be determined by enzymatic in situ labeling of 3'-OH ends of fragmented DNA, known as the TUNEL assay, which was used in the current study. Cells with TUNEL positivity may either survive through DNA repair or undergo apoptosis if the damage cannot be reversed (23). To detect genuine apoptotic cells, therefore, observation of the characteristic morphologic changes by electron microscopy or detection of cellular DNA loss by flow cytometry, known as profiling of DNA content, also should be performed. Quantitative studies by electron microscopy are problematic. Therefore, to determine whether apoptosis was present in cells with DNA damage due to CSE exposure, the TUNEL assay, followed by profiling of DNA content, was performed in the current study. This assay demonstrated that TUNEL positivity was present without evidence of hypodiploid DNA, thus supporting the concept that DNA strand breaks had occurred without induction of apoptosis. This result is consistent with the reversibility of the TUNEL positivity, the viability observed with ethidium dye exclusion, and by the ability of the smoke-exposed cells to proliferate. Interestingly, proliferation was delayed after smoke exposure in a concentration-dependent manner, consistent with the concept that DNA repair was initiated after smoke exposure.

Cigarette smoke contains thousands chemical components, many of which could contribute to cytotoxicity and carcinogenesis (24). Cigarette smoke also is a rich source of reactive oxygen species, which can induce DNA damage, lipid peroxidation, and protein oxidation. It also has been reported that NAC, an antioxidant and a free radical scavenger, reduces DNA damage or DNA adduct formation in a variety of cells (25, 26). Previous studies also reported that HFL-1 cells constitutively produce and release glutathione (27). NAC mitigated, whereas BSO potentiated, CSE inhibition on collagen gel contraction mediated by HFL-1 cells (27). Consistent with previous reports, the current study also showed that NAC blocked a higher concentration (5%) of CSE-induced DNA damage, whereas BSO, an inhibitor of glutathione synthesis, significantly enhanced DNA damage in the presence of a lower concentration of CSE (1%). These results suggested that oxidative stress by CSE may contribute to the DNA strand breaks. Whether this is a primary mechanism (NAC or BSO directly affect oxidative components of CSE) or secondary mechanism (NAC or BSO regulates intracellular level of redox state indirectly through the cells) remains to be determined.

Also in the current study, DNA damage appeared to decrease as the number of cells in the gels increased, suggesting that cells are able to protect themselves from CSE-induced insult. There are several potential mechanisms to account for the cell density dependence. One of them could be an antioxidant aggregation that results from the collective number of cells present within the system. Second, accumulative TGF-ß1 production in higher cell density may render the cells more resistant to CSE injury (28, 29).

Cytotoxic effects of cigarette smoke on mammalian cells such as the A549 alveolar epithelial cell line and human lung fibroblasts have been previously reported (30, 31). The current study, however, provides evidence of CSE-induced DNA damage without lethal cytotoxicity in HFL-1 cultured in three-dimensional collagen gels. Cells grown in this manner differ from those in monolayer culture in a variety of respects. They differ morphologically, extending long processes along the collagen fibers and maintaining cell–cell distances. These cells thus more closely resemble in vivo fibroblasts than do dish-cultured cells that form dense layers of cells characterized by close cell–cell contacts (10). Fibroblasts in gel culture also differ in their synthetic activity and sensitivity to growth regulators. It seems likely that they will also respond differently to exogenous insults than do fibroblasts in monolayer culture. Using this in vitro model, the current study demonstrates that CSE-induced DNA damage occurs at much lower concentrations than those used by previous investigators in monolayer culture (22). Consistently, the current study demonstrated that sensitivity of the cells to CSE-induced DNA damage and apoptosis is different in three-dimensional collagen gel comparing to the monolayer plastic dish culture. In three-dimensional collagen gel culture, DNA damage and apoptosis was detected under control condition. However, there was no significant increase in the cell number containing hypodiploid DNA content when the cells were cultured in the gels and exposed up to 20% CSE, although TUNEL-positive cell number was increased in a CSE concentration-dependent manner. In contrast, in monolayer culture, there were no cells containing hypodiploid DNA content under control condition while it was significantly increased in cells exposed to 20% CSE. DNA damage and apoptosis of the fibroblasts cultured in three-dimensional collagen gels may result from the mechanic strength produced during the contraction of the collagen gels (32), but not due to the cytotoxicity in that neither LDH release (14) nor dead cell number (LIVE/DEAD detection kit, data not shown) increased in these cells.

Cigarette smoke is associated with a number of lung diseases, and moreover, is capable of causing damage by a variety of mechanisms. The current study demonstrates that smoke can also induce reversible DNA strand breaks, which otherwise could lead to lung disease. By damaging DNA, smoke likely disrupts cell functions. Loss of control of cell proliferation by such mechanisms, for example, is believed to contribute to the development of cancer. Apoptosis following DNA damage is believed to be a mechanism to protect against such effects. By initiating repair rather than apoptosis, it is possible that DNA damage-induced alteration in the cellular genetic program may persist. Although fibroblasts, the target cells used in the current study, are not believed to be the most common cellular precursor for lung cancer, they may contribute to the development of lung cancer indirectly by producing co-factors required for cancer cell growth and support.

Apoptosis of pulmonary cells has been demonstrated to contribute to the development of emphysema in animal models (33, 34). Increased TUNEL staining has been reported in the lungs of patients with COPD (35), suggesting a role for apoptosis in human disease. Whether the DNA damage indicated by TUNEL positivity is reversible in vivo in the clinical setting remains to be determined. However, smokers exhibited the highest DNA repair capacity as compared with former or nonsmokers (36). Even without cell death, the DNA damage caused by smoke, by leading to alteration in the genetic program of lung cells, may contribute to nonmalignant disease. Altered gene expression has recently been associated with COPD (2, 3). The current study suggests the possibility that persistent, smoke-induced genetic alterations in structural cells in the lung also may be a mechanism that could contribute to the development of COPD.

In summary, the current study provides evidence that DNA damage occurs in HFL-1 cells in three-dimensional collagen gel culture in response to CSE exposure. The effect of this damage was dependent on CSE concentration and HFL-1 cell density, and likely was blocked by antioxidants. The DNA damage induced by CSE was reversible. Cell survival and proliferation after smoke removal provides a novel mechanism for smoke to cause persistent alterations in cellular function. Such a mechanism may be an important pathway for smoke-induced disease.


   Acknowledgments
 
The authors acknowledge the excellent secretarial support of Ms. Lillian Richards and the editorial assistance of Ms. Mary Tourek. This project was supported by the Larson Endowment, University of Nebraska Medical Center.


   Footnotes
 
Conflict of Interest Statement: S.I.R. has served on an advisory board of R.J. Reynolds, for which he received no compensation. No funds were received from R.J. Reynolds or any other tobacco company for the research represented in this article.

Received in original form December 12, 2002

Received in final form June 15, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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