Approximately 1.3 billion people smoke cigarettes worldwide contributing to almost 5 million preventable deaths per year 1. Smoking is a significant risk factor for lung cancer, the leading cause of cancer-related death in the United States, and chronic obstructive pulmonary disease (COPD), the fourth leading cause of death overall. Approximately 90% of lung cancer can be attributed to cigarette smoking, with 10–15% of smokers developing this disease 2. Despite the well-established causal role of cigarette smoke in lung cancer and COPD, the molecular mechanisms by which these diseases arise are poorly understood and there are no tools currently available to determine individual variations in response to smoking.

Previous work has demonstrated that cigarette smoke creates a field of injury in epithelial cells that line the respiratory tract. Several studies have shown that histologically normal large airway epithelial cells of current and former smokers with and without lung cancer display allelic loss 34, p53 mutations 5, changes in promoter methylation 6 and increased telomerase activity 7. Using epithelial cells collected from brushings of the mainstem bronchus at the time of bronchoscopy, we have previously characterized the effect of smoking on the bronchial airway epithelial transcriptome and found that smoking induces expression of genes involved in regulation of oxidant stress, xenobiotic metabolism, and oncogenesis while suppressing those involved in regulation of inflammation and tumor suppression 8. In addition, we recently developed a profile of bronchial airway gene expression that can distinguish smokers with and without lung cancer and serve as an early diagnostic biomarker for disease 9. Although these studies of intrathoracic airway epithelium obtained via bronchoscopy have successfully identified candidate biomarkers of smoking-related lung damage, there remains significant impetus to develop biomarkers of these events from tissue obtained via less invasive collection procedures. Use of material from a less invasive collection site would allow for the use of larger cohorts for developing and validating biomarkers of tobacco exposure and susceptibility to tobacco-related disease.

Oral and nasal epithelium are attractive candidate tissues for assaying the host response to tobacco-smoke exposure since, like the bronchial airway, they are exposed to high concentrations of compounds contained within cigarette smoke. We have previously shown that it is feasible to obtain sufficient RNA from buccal mucosa for gene expression analysis 10 despite the high level of RNAses in saliva 1112. Few studies have characterized global gene expression in either buccal or nasal mucosa, and none have attempted to establish a link between extra- and intrathoracic airway gene expression changes that occur with smoking. Smith et al. used brush biopsies of buccal mucosa from smokers and non-smokers to obtain RNA for cDNA microarrays and found approximately 100 genes that could distinguish the two groups in training and test sets 13. While this study provided evidence that buccal gene expression changes with smoking, it did not address the relationship between the gene expression response to tobacco smoke in the mouth and bronchial airways. Using real-time PCR, Spivack et al. found a qualitative relationship between matched buccal mucosa and laser-dissected lung epithelial cell samples across nine carcinogen or oxidant-metabolizing genes in 11 subjects being evaluated for lung cancer 14. Smoking has also been implicated in the formation of DNA adducts in nasal mucosa 15, and the correlation between adduct formation in bronchial and nasal epithelium has been previously reported 16. While global gene expression profiling of nasal epithelial brushing has been recently reported in children with asthma 17 and cystic fibrosis 18, there are no studies that address the effects of smoking on global gene expression in nasal epithelium or explore relationships in gene expression between epithelial cell types throughout the respiratory tract in response to cigarette smoke.

In this study, we examined relationships in gene expression between bronchial, nasal, and buccal epithelial cells in current and never smokers. Using previously published microarray data, we evaluated the relationship of buccal, nasal, and bronchial epithelial gene expression in never smokers compared to other epithelial and non-epithelial tissues. We subsequently compared gene expression consequences of smoking in bronchial epithelial cells to those seen in buccal and nasal epithelium. Our results suggest that gene expression changes occurring in bronchial epithelium in response to cigarette smoke are reflected in buccal and nasal epithelium. As a result, we believe that gene expression biomarkers of host response tobacco exposure may ultimately either be applied to or derived from these tissues.

We have studied global gene expression in bronchial, nasal, and buccal epithelial cells in never and current smokers. Our findings suggest that similar functional categories of genes are expressed in nasal and bronchial epithelial cells of healthy never smokers. We have shown that there are similarities between the effect of smoking on bronchial epithelial gene expression and the gene expression response to smoking in buccal and nasal epithelium. This implies the potential to study disease-relevant responses to tobacco smoke in any of these tissues. This represents a significant advantage as buccal and nasal mucosa can be readily collected from large study cohorts as a result of their ability to be collected non-invasively. Given the burden of smoking-related disease, there is a need for non-invasive biomarkers of the individual-level variability in host responses to cigarette smoke.

The similar pattern of gene expression in bronchial and nasal epithelial cells of never smokers suggests a shared architecture and function. The nasal passage and bronchus are both lined with ciliated pseudostratified columnar epithelial cells, and some of the functions of genes that are highly expressed in both nasal and bronchial epithelial cells are likely due to this common cellular architecture. For example, cilia-related genes such as DNAH7, DNAH9, and DNAI2 were highly expressed in both bronchial and nasal airway epithelium. Consistent with this finding, previous studies have shown that normal ciliated airway epithelial cells express these genes 202122. Other dynein light chain genes such as DYNLRB1 which have been characterized in other non-epithelial tissues 23 were found to be specifically expressed in bronchial epithelial cells, while dynein light chain genes such as DYNLL1 shared relatively high expression specifically in nasal epithelium as well as non-epithelial tissues. Many genes involved in host defense are also expressed at high levels in extra- and intrathoracic airway epithelial cells. Glutathione expression has been previously well characterized in bronchial epithelium as well as in the lung 24. Our data show high expression of glutathiones such as GSTM1 in bronchial, nasal, and buccal epithelium relative to other non-airway epithelial cells. We also observed high expression of mucins such as MUC1, MUC4, and MUC5AC in bronchial and nasal epithelium and somewhat lower expression of MUC1 in buccal mucosa and lung tissue. Expression of these genes has been well documented in respiratory tract epithelium 2526272829. We found the genes belonging to the cytochrome P450 family and several aldehyde dehydrogenase genes 30 are highly expressed the bronchial and nasal epithelium. Cytochrome P450 genes have previously been shown to be expressed highly in both bronchial epithelial cells 313233 and nasal mucosa 34. Our global analysis of gene-expression of the airway epithelium of healthy never smokers recapitulates gene expression patterns previously observed within these tissue types, thereby lending support to the similarities and differences between tissues that are suggested by our analysis of gene expression in the normal intrathoracic airway transcriptome.

Smoking altered the expression of a set of genes in bronchial epithelial cells which were also commonly altered in nasal and buccal epithelial cells. Gene set enrichment analysis of 361 smoking-induced bronchial genes yielded a subset of genes that were among the most up-regulated by smoking in the mouth (74 leading edge genes), as well as a subset of genes that are among the most up-regulated by smoking in the nose (120 leading edge genes). Forty-five genes were common to both sets, representing genes that share similar smoking-related expression patterns across all three airway epithelial tissues. This analysis demonstrates a common response to cigarette smoke exposure in cells lining the respiratory tract. Within this set are multiple genes involved in each of several processes including detoxification, cell cycle progression and cell adhesion. In addition, a common set of genes was down-regulated in response to smoking in both bronchial and nasal epithelium.

Several CYP450 genes were commonly up-regulated by smoking in only the nose and airway including CYP1A1 and CYP1B1, as well as cell cycle (CCNG2, RAB2) and cell adhesion genes (CEACAM5, CEACAM6). The presence of mutations in CYP1A1 in nasal and bronchial mucosa has been shown previously in smokers 16, and cytochrome P450 genes are known to be involved in xenobiotic metabolism in bronchial mucosa 35. Exposure of alveolar epithelial cells to environmental toxins has been shown to promote cell cycle progression 36, which could explain the increased expression of cell cycle genes in the nasal epithelial cells of smokers. Glutathiones such as GPX2 were up-regulated in both bronchial and buccal epithelial cells. Aldo-keto reducatse genes which are activated in response to cigarette smoke in human oral squamous cell lines 37, are also up-regulated in both bronchial and buccal epithelial samples. Oxidoreductase genes are up-regulated in all three airway epithelium including other CYP450 genes (CYP4F3, CYP4F11), aldehyde dehydrogenases (ALDH3A1), and aldo-keto reductases (AKR1B1), suggesting that smoking activates common detoxification pathways in exposed airway epithelial cells.

Gene set enrichment analysis also identified genes that are among the most down-regulated in both nasal and bronchial epithelium in response to tobacco exposure. SLIT2, which is a known tumor suppressor that is down-regulated in lung cancer 3839 is among these genes. We found HNMT, which is expressed highly in both bronchial and nasal mucosa 4041, and has been shown to be down-regulated with smoking in other mucosal cells 42, to be down regulated in smoking in bronchial and nasal epithelium. The genes that are among the most down regulated by smoking in both bronchial and nasal epithelium were enriched for those with functions in cellular localization, migration, and motility genes. These data suggest that smoking results in the down-regulation of structural genes in these tissues.

Based on the data presented here, we suggest that aspects of the bronchial gene expression response to smoking are also changed by smoking in nasal epithelium, with certain of these genes also being perturbed by smoking in buccal mucosa. This suggests that there are common features in the field of injury caused by cigarette smoke throughout the airway. Our data also suggest that the gene-expression consequences of smoking are less pronounced in buccal mucosa (see Figure 3). This could be due to a number of factors: 1) the effects of smoking on buccal mucosa might indeed be less pronounced; 2) there may be more inter-subject variability in buccal mucosa gene expression; 3) the partial degradation of the RNA in the buccal mucosa samples may contribute to variability in gene expression estimates. Due to the high concentration of RNAses found in saliva, RNA obtained from buccal epithelial cells was subject to degradation, and relatively small amounts of RNA were extracted from these cells. This required us to pool samples collected from the same individual serially over several weeks. Previous studies report similar issues with salivary RNA run on microarrays 43. Consistent with the low yield of partially degraded RNA from buccal samples, we detected sequence-specific hybridization intensity for fewer probesets in the buccal samples than in the nasal or bronchial samples. Despite these technical limitations, there was considerable overlap between genes that are among the most altered in response to smoking in the nose and bronchus and those that are most differentially expressed in the mouth based on the overlap between the leading edge subsets from gene set enrichment analysis. Differential expression of several genes seen to be induced by smoking in buccal mucosa was validated in buccal mucosa samples from independent volunteers using real competitive PCR. Taken together, these findings indicate that gene expression is perturbed by smoking in buccal mucosa and suggest that techniques for assaying gene expression in the context of partially degraded RNA will facilitate further studies to determine if buccal-mucosa specific factors contribute to the apparent differences in the magnitude of the smoking response of buccal mucosa relative to that seen in other airway epithelia.

This study highlights the relationships between gene expression profiles in epithelial cells that line the intra- and extra-thoracic airway and identifies a common set of genes that are induced by tobacco smoke in buccal, nasal and bronchial epithelium, supporting the concept that smoking induces a common field of injury throughout the airway. These similarities suggest that easily collected buccal and nasal epithelium can be used to measure an individual's physiologic response to tobacco smoke.

All statistical analyses described were performed using R v. 2.2.0 47. The gene annotations used for each probe set were from the December 2004 NetAffx HG-U133A annotation file. Additional information from this study, including expression levels from all genes in all samples, and relevant clinical data on all subjects are available from our interactive database http://pulm.bumc.bu.edu/MouthDB/index.html which supports user-defined statistical and graphical analyses of data. Data from nasal and buccal epithelial microarray experiments have also been submitted to the National Center for Biotechnology Information Gene Expression Omnibus (GSE8987).

SS contributed to the design of the analytic strategy and was responsible for the computational analysis and its interpretation. FS contributed to the gene-expression analysis, performed the mass spectrometry gene-expression assays, and contributed to analysis of mass-spectrometry data. JZ AMG and VS contributed to gene-expression analysis of non-diseased tissue datasets. KS contributed to study coordination and analysis of gene-expression data. GL and XZ performed the buccal and nasal microarray experiments and GL contributed to running mass spectrometry assays. YMD was responsible for coordinating all patient recruitment and sample collection. JB contributed to the design of the study and biological interpretation of the data. MEL conceptualized aspects of the analytic strategy. AS was responsible for the conception and design of this study and oversaw all aspects of the study including patient recruitment, experimental protocols and data analysis. All authors have read and approved the final manuscript.