Gene Expression in Normal Human Bronchial Epithelial (NHBE) Cells Following In Vitro Exposure to Cigarette Smoke Condensate

Wanda R. Fields1, Randi M. Leonard, Pamela S. Odom, Brian K. Nordskog, Michael W. Ogden and David J. Doolittle

Research and Development Department, R. J. Reynolds Tobacco Co., Winston-Salem, NC 27102

1 To whom correspondence should be addressed at R. J. Reynolds Tobacco Company, Research & Development, P.O. Box 1236, Winston-Salem, NC 27102–1236. Fax: 336-741-5019. E-mail: fieldsw{at}rjrt.com.

Received February 16, 2005; accepted April 18, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cigarettes that burn tobacco produce a complex mixture of chemicals, including mutagens and carcinogens. Cigarettes that primarily heat tobacco produce smoke with marked reductions in the amount of mutagens and carcinogens and demonstrate reduced mutagenicity and carcinogenicity in a battery of toxicological assays. Chemically induced oxidative stress, DNA damage, and inflammation may alter cell cycle regulation and are important biological events in the carcinogenic process. The objective of this study was to characterize and compare the effects of smoke condensates from cigarettes that burn tobacco and those that primarily heat tobacco on gene expression in NHBE cells. For this comparison, we used quantitative RT/PCR and further evaluated the effects on cell cycling using flow cytometry. Cigarette smoke condensates (CSCs) were prepared from Kentucky 1R4F cigarettes (a tobacco-burning product designed to represent the average full-flavor, low "tar" cigarette in the US market) and Eclipse (a cigarette that primarily heats tobacco) using FTC machine smoking conditions. The CSC from 1R4F cigarettes induced statistically significant increases in the mRNA levels of genes responsive to DNA damage (GADD45) and involved in cell cycle regulation (p21;WAF1/CIP1), compared to the CSC from Eclipse cigarettes. In addition, genes coding for cyclooxygenase-2 (COX-2) and interleukin 8 (IL-8), which are associated with oxidative stress and inflammation, respectively, were increased statistically significantly more by CSC from 1R4F than by that from Eclipse. Furthermore, a dose-dependent increase in IL-8 protein secretion into cell culture media was stimulated by 1R4F exposure, whereas minimal IL-8 protein was secreted after Eclipse treatment. The biological relevance of the differential effect on gene expression was reflected in differential cell cycle regulation, as cells exposed to 1R4F CSC exhibited more significant S phase and G2 phase accumulation than cells exposed to Eclipse CSC. These data indicate that the simplified smoke chemistry of the tobacco-heating Eclipse cigarette yields statistically significant reductions in the expression of key genes involved in DNA damage, oxidative stress, inflammatory response, and cell cycle regulation in normal human bronchial epithelial cells compared to a representative tobacco-burning cigarette.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bronchial epithelial cells are often exposed to airborne chemicals and irritants that may induce inflammatory responses and oxidative stress, as well as alter cellular proliferation (Baeza-Squiban et al., 1999Go). Although bronchial epithelial cells of smokers are exposed to relatively high concentrations of cigarette smoke, and although some constituents of cigarette smoke have been proposed to be important in the increased risk of lung cancer in smokers (Hecht, 1999Go), the specific smoke constituents associated with this risk have not been conclusively identified. The definitive identification of specific causative agent(s) is a challenge because cigarette smoke is a complex mixture of thousands of chemicals (Rodgman et al., 2000Go), which are likely to act in additive, synergistic, and antagonistic (Brown et al., 1999Go, 2001Go) manners. One approach to reducing the toxicity of tobacco smoke is to dramatically reduce the amount of tobacco burned. Consistent with this hypothesis, R. J. Reynolds Tobacco Company has developed Eclipse, a cigarette that primarily heats rather than burns tobacco. Smoke from Eclipse has been shown to be much simpler than smoke from cigarettes that burn tobacco, and CSC and whole-smoke from Eclipse are substantially less toxic than smoke from cigarettes than burn tobacco (Borgerding et al., 1998Go; Cline et al., 2000Go; Meckley et al., 2004Go).

Complex mixtures of chemicals such as CSC and diesel exhaust have been observed to affect gene expression. The majority of the in vitro investigations on smoke-induced changes in gene expression have focused on the inflammatory response. In particular, increases in IL-8 mRNA levels and secretion of the cytokine in culture media have been observed in bronchial epithelial cells (Hellermann et al., 2002Go; Shinji et al., 2000Go). Additionally, pulmonary lavage samples from smokers have elevated levels of mediators of inflammation (Mio et al., 1997Go). A recent report by Bosio et al. describes expression changes for genes encoding for antioxidant response, transcription factors, cell cycle regulation, and inflammatory response in Swiss 3T3 cells exposed to aqueous extracts of cigarette smoke (Bosio et al., 2002Go). Likewise, genes responsive to DNA damage and regulation of cell cycle control have been reported to be modulated in in vivo and in vitro respiratory cell models following exposure to environmental agents (Johnson et al., 1997Go). Finally, CSC exposure in primary cultures of human aortic endothelial cells altered a number of genes that affect cell cycle regulation, extracellular matrix degradation, and xenobiotic metabolism (Nordskog et al., 2003Go).

In our laboratory, primary cultures of normal human bronchial epithelial (NHBE) cells have been used as a model system for determining the changes in mRNA levels of the proto-oncogene c-myc following chemical treatment (Fields et al., 2001Go). Moreover, we have observed differential responses of NHBE cells exposed to B[a]P as compared to the k- and bay-region reactive metabolites (Fields et al., 2004Go). The objective of this study was to determine the effect of CSC from cigarettes that burn or primarily heat tobacco on gene expression in NHBE cells using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and to compare the effects on gene expression with cytotoxicity, cell cycle regulation, and inflammatory responses as determined by the neutral red assay, flow cytometric analysis, and the BioPlex assay, respectively.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of cigarette smoke condensate.
Kentucky Reference 1R4F (K1R4F or tobacco-burning; University of Kentucky) and Eclipse (primarily tobacco-heating; R. J. Reynolds Tobacco Company) cigarettes were used for the current studies. Smoke condensate from each cigarette type was prepared using modified Amesa smoke generators operated under Federal Trade Commission (FTC) conditions (35 ml puff volume, 2 s duration taken once per minute). Cigarette total particulate matter (TPM) was collected onto a Cambridge filter pad, and extracted with dimethylsulfoxide (DMSO) to yield a 10 mg TPM/ml stock solution.

Cell culture.
The NHBE donor cells were obtained from Cambrex, Inc. (Walkersville, MD). Cells were maintained in humidified incubators at 37°C and 5% CO2 and cultured in bronchial epithelial growth medium (BEGM) as previously described (Fields et al., 2001Go). Lung tissues from donors 1, 2, and 3 were disease free and all donors were non-smokers. The respective strain, lot number, age, and sex are as follows: donor 1—NHBE 4501, #17378, 10-year-old male; donor 2—NHBE 4653, #17684, 20-year-old male; and donor 3—NHBE 9179, #2F0513, 35-year-old male.

Cytotoxicity assay.
The cytototoxic potential of the respective condensates was determined by measuring the ability of exposed cells to incorporate neutral red (NR) into lysosomes. Cells were exposed to K1R4F or Eclipse at 3, 10, 30, 60, or 90 µg TPM/ml or vehicle control (0.9% DMSO) in growth medium continuously for 3 h and 24 h. Neutral red analysis was performed as previously described (Fields et al., 2001Go).

Cell treatment for gene expression analysis.
NHBE cells were seeded at 30,000 cells per well of 6-well dishes and grown to 50–70% confluence. In general, 2–3 wells were used per dose for each time point. Cells were exposed to CSC (K1R4F or Eclipse) at 3, 10, 30, 60, or 90 µg TPM/ml or vehicle control (0.9% DMSO) in growth medium for 3 h. The exposure medium was then removed, cells were lysed in TRIzol reagent (Gibco BRL, Gaithersburg, MD), and total RNA was isolated as recommended (Gibco BRL). Replicate exposures were generally performed for all treatments.

Real-time quantitative RT/PCR.
cDNA was prepared with 1 µg RNA using TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). Polymerase chain reaction was performed with 20 ng of cDNA; 1x Universal Master Mix/SYBR Green PCR Master Mix Reagents (Applied Biosystems); 300 nM each for GADD45, p21, COX2, and IL-8 primers and 50 nM for 18S primers, respectively. The cycling parameters used were per the manufacturer's recommendation and performed with the ABI Prism 7700 and ABI 5700 Sequence Detection Systems (Applied Biosystems). Arbitrary fluorescence units (AFU) were obtained from standard curve values for each target gene and housekeeping gene (18S) samples. The target gene AFU were divided by the 18S AFU and expressed as normalized fluorescence for mRNA accumulation. Subsequently, differences were determined by comparing the normalized fluorescence values of the solvent control sample to each treated sample. Statistical analysis of the data was performed by comparing the mean of the normalized fluorescence values or the percent change of control versus treated samples using one-way analysis of variance (ANOVA) with the Bonferroni adjustment. The level of statistical significance was expressed at p < 0.05.

Extracellular cytokine analysis.
Media samples were collected from each treatment group and frozen at –80°C. An aliquot was diluted and processed for analysis on a BioPlex protein analyzer (Bio-Rad Laboratories; Hercules, CA) as per manufacturer's protocol (Manual 411004 Rev C 0701). A Bio-Rad 8-plex human cytokine kit containing IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, INF-{gamma}, and TNF-{alpha} was used for these analyses.

Cell cycle analysis.
Cells were exposed to CSC (K1R4F or Eclipse) at 30, 60, or 90 µg TPM/ml or vehicle control (0.9% DMSO) in growth medium for 24 h. Cells were harvested by scraping into phosphate buffered saline (PBS), fixed with 3 ml of ice-cold 70% ethanol, and stored at –20°C until analysis. Cellular DNA was stained with a solution containing propidium iodide, 0.06% Nonidet P40, 37 µg/ml RNase A, 10 mM NaCl, and 4 mM citrate buffer (pH 7.4), and analyzed on a Coulter XL flow cytometer. The resulting DNA histograms were analyzed for cell cycle parameters by the ModFit program (Verity Software, Inc., Topsham, ME).

Primer and probe sequences.
The primer and probe sequences used in the present study were as follows: c-myc Forward Primer: 18S Forward Primer: 5'-CGG CTA CCA CAT CCA AGG AA-3'; 18S Reverse Primer: 5'-GCT GGA ATT ACC GCG GCT-3'; GADD45 Forward Primer 5'-GGGATGCCCTGGAGGAAGT-3'; GADD45 Reverse Primer 5'-GCAGGCACAACACCACGTTA-3'; p21 Forward Primer 5'-GGCAGACCAGCATGACAGATT-3'; p21 Reverse Primer 5'-GAAGATGTAGAGCGGGCCTTT-3'; COX-2 Forward Primer 5'-CCTTCCTCCTGTGCCTGATG-3'; COX-2 Reverse Primer 5'-ACAATCTCATTTGAATCAGGAAGCT-3'. IL-8 pre-developed primer/probe kits are commercially available, and were purchased from Applied Biosystems.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of CSCs from K1R4F and Eclipse on Neutral Red Uptake in NHBE Cells
Comparative analyses of the cytotoxic and/or growth inhibitory effects of CSCs from tobacco-burning versus tobacco-heating cigarettes were assessed with the neutral red assay. To ascertain the cellular state at early time points at which gene expression changes were occurring, NR comparisons were performed after 3-h and 24-h CSC exposures. Neither CSC induced a decrease in NR uptake following a 3-h exposure (data not shown). However, reductions in NR uptake were observed following 24 h of continuous exposure to K1R4F as compared to Eclipse (data not shown), a finding that is consistent with previously published research in Chinese hamster ovary cells (Bombick et al. 1998aGo, 1998bGo). In NHBE cells, K1R4F exhibited a 24.0% decrease in NR uptake (relative to control) at 60 µg/ml and 43.0% decrease in NR uptake at 90 µg/ml, whereas generally >100% NR uptake was observed in Eclipse-treated cells at 60 µg/ml and 90 µg/ml. Because of the altered growth process induced by K1R4F after the 24-h exposure, the comparative gene expression analyses were performed after 3 h of CSC exposure.

Comparative Gene Expression Analysis of NHBE Cells After Treatment with CSCs from K1R4F and Eclipse
K1R4F CSC induced statistically significant increases in the mRNA levels of GADD45 and p21 (donor 1, Fig. 1). Statistical significance in p21 mRNA levels was observed at 60 and 90 µg/ml K1R4F (p < 0.05) compared to the solvent control and at 90 µg/ml for Eclipse CSC. For GADD45, a statistically significant difference represented by a 4-fold change was observed in the 90 µg/ml K1R4F exposure compared to solvent control. K1R4F CSC also exhibited statistically significant differences from Eclipse CSC at 90 µg/ml (p < 0.05) in GADD45 mRNA levels. Likewise, statistically significant increases in COX-2 and IL-8 expression were induced by K1R4F compared to solvent controls (donor 1, Fig. 2). For COX-2, a statistically significant increase of 3.9-fold was observed by 60 µg/ml (p < 0.05), and the increase was 4.2-fold relative to control at 90 µg/ml (p < 0.05). Analyses by dose revealed statistically significant differences in K1R4F and Eclipse effects on COX-2 mRNA at 60 µg/ml and 90 µg/ml. Statistically significant increases of ≥5-fold were observed in IL-8 at 60 and 90 µg/ml K1R4F (p < 0.05) compared to the solvent control. Eclipse exposures yielded results that were comparable to the solvent control, and produced statistically significant (p < 0.05) differences from K1R4F-induced effects at 60 and 90 µg/ml.



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FIG. 1. Effect of K1R4F and Eclipse cigarette smoke condensates (CSC) on p21 and GADD45 mRNAs in NHBE cells. Cells were exposed to 3, 10, 30, 60, or 90 µg total particulate matter (TPM)/ml or dimethlysulfoxide (DMSO; 0.9%) in growth medium continuously for 3 h. The exposure medium was then removed, and the cells were harvested into TRIzol. First-strand cDNA synthesis, real-time quantitative PCR, and percent control calculations were performed as described. An asterisk (*) denotes statistical significance compared to the solvent control at p < 0.05, and the symbol ({ddagger}) denotes statistical significance between K1R4F and Eclipse at p < 0.05. (Data are represented as the mean ± SEM.)

 


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FIG. 2. Effect of K1R4F and Eclipse CSCs on COX-2 and IL-8 mRNAs in NHBE cells. Cells were exposed to 3, 10, 30, 60, or 90 µg TPM/ml or DMSO (0.9%) in growth medium continuously for 3 h. The exposure medium was then removed, and the cells were harvested into TRIzol. First-strand cDNA synthesis, real-time quantitative PCR, and percent control calculations were performed as described. An asterisk (*) denotes statistical significance compared to the solvent control at p < 0.05, and the symbol ({ddagger}) denotes statistical significance between K1R4F and Eclipse at p < 0.05. (Data are represented as the mean ± SEM.)

 
Subsequently, donor variability was assessed by comparing the response in two additional NHBE strains (data not shown). NHBE cells from donors 2 and 3 exhibited responses similar to those previously observed for GADD45, p21, and COX-2 mRNA levels following K1R4F exposures compared to Eclipse exposures. In these donors, K1R4F generally induced 2-fold to 4-fold increases for the respective mRNAs above solvent-control samples. However, less than 2-fold changes in IL-8 mRNA were induced by either condensate in cells from donors 2 and 3.

Cytokine Secretion After K1R4F and Eclipse Exposures
To further characterize the cellular response to gene expression changes, effects on cytokine secretion were determined with a multi-plex protein assay. Levels of IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-{gamma}, and TNF-{alpha} were evaluated in cell culture media following 3 h and 24 h of continuous exposure to K1R4F and Eclipse CSCs (0–90 µg TPM/ml). Of the 8 cytokines, detectable levels of secreted proteins were observed only for IL-6 and IL-8 at 24 h. Although there was a minimal response for IL-6, the level of secretion was similar for K1R4F and Eclipse. However, a dose-dependent and statistically significant increase in IL-8 protein secretion into the cell culture medium was stimulated by K1R4F exposure, reaching a maximum level of 1435 pg/ml (donor 1, Fig. 3). No differences compared to solvent-controls were observed in the Eclipse samples. Consistent with the gene expression (mRNA) data, statistically significant differences between K1R4F and Eclipse samples were observed at 60 and 90 µg TPM/ml exposures.



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FIG. 3. Effect of K1R4F and Eclipse CSCs on IL-8 excretion into NHBE cell culture medium. Cells were exposed to 3, 10, 30, 60, or 90 µg TPM/ml or DMSO (0.9%) in growth medium continuously for 24 h. The exposure medium was collected and stored at –80°C. An asterisk (*) denotes statistical significance compared to the solvent control at p < 0.05, and the symbol ({ddagger}) denotes statistical significance between K1R4F and Eclipse at p < 0.05. (Data are represented as the mean ± SEM.)

 
Effect of K1R4F and Eclipse CSCs on NHBE Cell Cycle Regulation
The biological relevance of inducing genes that affect cell cycle regulation was examined by flow cytometry analysis. The differential effect on gene expression was reflected in cell cycle regulation as K1R4F CSC–exposed cells from all three donors exhibited more significant effects on S and G2 phase accumulation than Eclipse-exposed cells (donor 1, Fig. 4). Specifically, statistically significant decreases in G1-phase cells in K1R4F exposed cells compared to control were observed at 60–90 µg/ml K1R4F CSC. The decline in G1 phase was accompanied by increased partitioning of cells to S phase beginning at 60 µg/ml K1R4F CSC. The S phase accumulation continued through 90 µg/ml K1R4F CSC exposure (p < 0.05). Likewise, G2 phase accumulation was observed for K1R4F exposed cells exhibiting statistical significance at 60 µg/ml. However, no differences in cell cycle profiles were observed in Eclipse-exposed cells. Furthermore, the cell cycle variations induced by K1R4F treatment also exhibited statistically significant differences from Eclipse at the G1 and G2 phase following 60 and 90 µg K1R4F/ml.



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FIG. 4. Effect of K1R4F and Eclipse CSCs on cell cycle in NHBE cells. Cells were exposed to 3, 10, 30, 60, or 90 µg TPM/ml or DMSO (0.9%) in growth medium continuously for 24 h. Flow cytometry analysis was performed on samples stained with propidium iodide as described in Materials and Methods. An asterisk (*) denotes statistical significance compared to the solvent control at p < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Complex mixtures of chemicals such as diesel exhaust and cigarette smoke contain numerous chemical constituents that could contribute to the human disease and health effects observed in epidemiological studies and research models (Lubin and Blot, 1984Go; Powell et al., 2003Go; Rom et al., 2000Go; Smith and Hansch, 2000Go). Evidence of inflammatory response, oxidative stress, hyperproliferation, and DNA damage are frequently associated with carcinogenesis, and lung tumors often show alterations in genes that control these events. Individual constituents of cigarette smoke (i.e., B[a]P and NNK) have been reported to alter inflammatory response molecules (Martin et al., 1998Go; Mio et al., 1997Go; Wistuba et al., 2002Go), and other studies have noted increased secretion of inflammatory response molecules in pulmonary lavage samples of smokers compared to nonsmokers (Kuschner et al., 1996Go). Inflammatory response is decreased when smokers of tobacco-burning cigarettes switch to Eclipse (Rennard et al., 2002Go).

As the progenitor cells for bronchogenic carcinomas, bronchial epithelial cells have been used in modeling the effect of chemicals on growth and morphological changes. For example, CSC has been shown to alter the following functions of NHBE cells: cell growth, epithelial growth factor binding, increased plasminogen activator activity, formation of single strand breaks, induction of terminal squamous differentiation, and gap junction intercellular communication (McKarns et al., 2000Go; Willey et al., 1987Go). Additionally, NHBE cells have been used to characterize the effect of cigarette smoke constituents, diesel exhaust particulates, and asbestos on inflammation-associated lung disease (Kawasaki et al., 2001Go; Loitsch et al., 2000Go; Takizawa et al., 1999Go).

We have previously investigated the response of NHBE cells to the exposure of several individual chemicals (Fields et al., 2001Go), and we have also observed divergent responses between B[a]P and its active metabolites (Fields et al., 2004Go). In the current study, we employed the use of a bronchial epithelial cell system for modeling lung epithelium responses to cigarette smoke. Specifically, the objective of this study was to evaluate and compare expression levels of genes involved in oxidative stress, DNA damage, cell cycle regulation, and inflammation after treatment of NHBE cells with CSC from K1R4F, representing the typical "low" tar tobacco-burning cigarette sold in the US market, and a tobacco heating product, Eclipse, which contains a 90% reduction in concentration of many chemicals and is less biologically active than tobacco-burning cigarettes (Cline et al., 2000Go).

GADD45 and p21 were significantly increased following treatment with smoke condensate from K1R4F cigarettes compared to treatment with CSC from Eclipse. These changes are consistent with the observed induction of oxidative stress by CSC in alveolar epithelial cells that was also accompanied with increases in p21 mRNA levels (Marwick et al., 2002Go). Because GADD45 and p21 are associated with growth arrest, DNA damage, and cell cycle regulation, we compared the effect of CSC on NR uptake to the gene expression data to correlate the effects on cellular proliferation. We determined that the gene expression changes were not associated with immediate toxicity as determined by NR analysis of NHBE cells exposed for 3 h. In contrast to the 3h NR data, significant reductions in NR uptake were observed following 24 h of continuous exposure to K1R4F as compared to Eclipse, suggesting that the early gene expression changes induced by K1R4F could be indicative of cellular responses that ultimately altered the proliferative state of the cells. Furthermore, the observed cell cycle delays in the S phase and G2/M phases of K1R4F-exposed cells is consistent with the altered proliferative state suggested by the decreased NR uptake. This finding also correlates with cigarette smoke inhibition of proliferation and chemotaxis in lung fibroblast, and inhibition in Chinese hamster ovary cells (Bombick et al., 1998bGo; Curvall et al., 1985Go; Nakamura et al., 1995Go; Putnam et al., 2002Go).

The cytotoxic response caused by cigarette smoke may be associated with free-radical induced oxidative damage (St. Clair et al., 1994Go). Therefore, inflammatory and oxidative stress responses were assessed by measuring COX-2 and IL-8 mRNA levels following condensate treatment. We observed dose-dependent and time-dependent increases in COX-2 mRNA after K1R4F exposure compared to controls, with no changes evident for Eclipse-exposed cells. Expression changes at the mRNA level were followed up at the protein level by cytokine analysis using the Bioplex 8-panel cytokine assay. A statistically significant increase in IL-8 protein secretion into media was observed in K1R4F-exposed cells, with no differences between any Eclipse exposures and the solvent-control. The K1R4F-induced changes are consistent with reports in which inflammatory response patterns were assessed following smoke, CSC, diesel exhaust, or benzo[a]pyrene exposures in bronchial epithelial cells and breath condensate (Dwyer, 2003Go; Kashyap et al., 2002Go; Kawasaki et al., 2001Go; Li et al., 2002Go; Takizawa et al., 1999Go).

Regarding a potential mechanism for smoke-induced changes in gene expression, it has been proposed that the mitogen-activated protein (MAP) kinase pathway mediates the inflammatory response through activation of either the nuclear transcription factor (NF-{kappa}B) or protein kinase C (PKC; Gebel and Muller, 2001Go; Mochida-Nishinura et al., 2001Go). The NF-{kappa}B hypothesis is supported by Hellermann et al. (2002)Go, who observed an increase in the phosphorylation of a specific MAP kinase, extracellular signal-regulated kinase (ERK1/2), within 30 min of CSC exposure in bronchial cells. Furthermore, this group observed increased activation of NF-{kappa}B and increased message levels for IL-8, granulocyte-macrophage colony-stimulating factor, intercellular adhesion molecule-1, and interleukin 1ß following CSC exposure. The NF-{kappa}B hypothesis was further supported by the observations of Anto et al. (2002)Go, which correlated the induction of COX-2 expression with the activation of NF-{kappa}B through degradation of I{kappa}B{alpha}, an inhibitor of NF-{kappa}B, after CSC exposure in U-937 lymphoma cells. Furthermore, the suppression of CSC-induced expression of COX-2, matrix metalloproteinase 9 (MMP-9), and cyclin D1 with the pretreatment of human lung cells with two anticarcinogens, curcumin and ursolic acid, was observed in conjunction with inhibition of I{kappa}B{alpha} kinase activity, thus inhibiting the NF-{kappa}B signal transduction pathway (Shishodia et al. 2003aGo, 2003bGo).

Alternatively, stimulation of IL-8 release in human and bovine bronchial epithelial cells following cigarette smoke extract treatment may be mediated by the PKC pathway (Kashyap et al., 2002Go; Wyatt et al., 1999Go), which has been shown to be stimulated by a volatile component of smoke, acetaldehyde (Wyatt et al., 2000Go). Cigarette smoke from tobacco-burning cigarettes was determined to produce 15–20 times greater concentrations of acetaldehyde, to induce greater levels of PKC activity, and to enhance inhibition of ciliary motility compared to Eclipse, a brand of primarily tobacco-heating cigarettes (Wyatt et al., 2000Go). Prolonged exposure to inflammatory mediators and cytokines can alter functions (i.e., mucin secretion, ion transport, or ciliary activity) that maintain normal lung and may lead to defective defense mechanisms; such exposure also has the potential to promote disease development (Adler et al., 1994Go; Hackett et al., 2003Go).

Gene expression assays are currently being applied to in vitro and in vivo sample analyses to assess toxicogenomic responses and to establish chemical fingerprints. Results of such studies with tissue-specific cell culture models are helpful for bridging in vitro responses with in vivo responses obtained from animal bioassays and human samples (e.g., pulmonary lavage and blood), and may help define biomarkers of effect for specific chemicals, including complex mixtures such as cigarette smoke. Here, we have described the use of NHBE cells to characterize the acute response of lung cells to CSC exposure. We observed distinct differences in the response of bronchial cells to condensates from tobacco-burning products versus tobacco-heating products. Previously published chemical and biological data (Cline et al., 2000Go) are consistent with the current data indicating that the simplified chemistry of tobacco-heating cigarettes yields statistically significant differences in gene expression in human bronchial epithelial cells as compared to tobacco-burning cigarettes. In particular, chemical analysis of whole smoke and smoke particulate matter detected 80% reductions in the concentrations for certain carcinogens and chemical classes (e.g., carbonyls, aromatic amines, tobacco-specific nitrosamines (TSNAs), polycyclic aromatic hydrocarbons (PAH), azaarenes, miscellaneous organic, hydroxybenzenes, inorganics, unsaturated hydrocarbons, and aromatic hydrocarbons) previously classified as carcinogens or reported as critical components in cigarette smoke associated with health hazards by the International Agency for Research on Cancer, the National Toxicology Program, and the Environmental Protection Agency (Cline et al., 2000Go; Borgerding et al., 1998Go). Additionally, mass balance analysis of mainstream smoke TPM indicates that the composition of the TPM from Eclipse is generally 75–80% water and glycerin, and about 20–25% "tar" (excluding glycerin) and nicotine. The TPM composition for cigarettes that burn tobacco are roughly the reverse—approximately 80% "tar" and nicotine, and 20% water and glycerin. Furthermore, the differential response of NHBE cells to condensate from burning cigarettes versus heating cigarettes is consistent with the substantial decreases in cytotoxicity and mutagenicity observed with Eclipse smoke compared to smoke from K1R4F (Bombick et al., 1998aGo, 1998bGo; Brown et al., 1998Go; Smith et al., 1996Go), and is consistent with the reduced tumorigenicity of Eclipse CSC compared to K1R4F CSC observed in animal models (Meckley et al., 2004Go). Therefore, the data observed in this in vitro lung model demonstrate the potential reduced risk characteristics of Eclipse.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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