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 271021236. Fax: 336-741-5019. E-mail: fieldsw{at}rjrt.com.
Received February 16, 2005; accepted April 18, 2005
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ABSTRACT |
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INTRODUCTION |
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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., 2002; Shinji et al., 2000
). Additionally, pulmonary lavage samples from smokers have elevated levels of mediators of inflammation (Mio et al., 1997
). 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., 2002
). 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., 1997
). 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., 2003
).
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., 2001). 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., 2004
). 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.
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MATERIALS AND METHODS |
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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., 2001). 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 1NHBE 4501, #17378, 10-year-old male; donor 2NHBE 4653, #17684, 20-year-old male; and donor 3NHBE 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., 2001).
Cell treatment for gene expression analysis.
NHBE cells were seeded at 30,000 cells per well of 6-well dishes and grown to 5070% confluence. In general, 23 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-, and TNF-
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.
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RESULTS |
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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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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-, and TNF-
were evaluated in cell culture media following 3 h and 24 h of continuous exposure to K1R4F and Eclipse CSCs (090 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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DISCUSSION |
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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., 2000; Willey et al., 1987
). 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., 2001
; Loitsch et al., 2000
; Takizawa et al., 1999
).
We have previously investigated the response of NHBE cells to the exposure of several individual chemicals (Fields et al., 2001), and we have also observed divergent responses between B[a]P and its active metabolites (Fields et al., 2004
). 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., 2000
).
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., 2002). 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., 1998b
; Curvall et al., 1985
; Nakamura et al., 1995
; Putnam et al., 2002
).
The cytotoxic response caused by cigarette smoke may be associated with free-radical induced oxidative damage (St. Clair et al., 1994). 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, 2003
; Kashyap et al., 2002
; Kawasaki et al., 2001
; Li et al., 2002
; Takizawa et al., 1999
).
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-B) or protein kinase C (PKC; Gebel and Muller, 2001
; Mochida-Nishinura et al., 2001
). The NF-
B hypothesis is supported by Hellermann et al. (2002)
, 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-
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-
B hypothesis was further supported by the observations of Anto et al. (2002)
, which correlated the induction of COX-2 expression with the activation of NF-
B through degradation of I
B
, an inhibitor of NF-
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
B
kinase activity, thus inhibiting the NF-
B signal transduction pathway (Shishodia et al. 2003a
, 2003b
).
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., 2002; Wyatt et al., 1999
), which has been shown to be stimulated by a volatile component of smoke, acetaldehyde (Wyatt et al., 2000
). Cigarette smoke from tobacco-burning cigarettes was determined to produce 1520 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., 2000
). 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., 1994
; Hackett et al., 2003
).
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., 2000) 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., 2000
; Borgerding et al., 1998
). Additionally, mass balance analysis of mainstream smoke TPM indicates that the composition of the TPM from Eclipse is generally 7580% water and glycerin, and about 2025% "tar" (excluding glycerin) and nicotine. The TPM composition for cigarettes that burn tobacco are roughly the reverseapproximately 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., 1998a
, 1998b
; Brown et al., 1998
; Smith et al., 1996
), and is consistent with the reduced tumorigenicity of Eclipse CSC compared to K1R4F CSC observed in animal models (Meckley et al., 2004
). Therefore, the data observed in this in vitro lung model demonstrate the potential reduced risk characteristics of Eclipse.
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