Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. As a first step in applying functional genomic analysis to population studies, we have examined the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n = 20), smokers without lung cancer (SNC, n = 24), and never smokers (NS, n = 8). Functional enrichment analysis showed that the NRF2-mediated, antioxidant response element (ARE)-regulated genes, were significantly lower in SC, when compared with expression levels in SNC. Importantly, we found that the expression of MAFG (a binding partner of NRF2) was correlated with the expression of ARE genes, suggesting MAFG levels may limit target gene induction. Bioinformatically we identified single nucleotide polymorphisms (SNPs) in putative ARE genes and to test the impact of genetic variation, we genotyped these putative regulatory SNPs and other tag SNPs in selected NRF2 pathway genes. Sequencing MAFG locus, we identified 30 novel SNPs and two were associated with either gene expression or lung cancer status among smokers. This work demonstrates an analysis approach that integrates bioinformatics pathway and transcription factor binding site analysis with genotype, gene expression and disease status to identify SNPs that may be associated with individual differences in gene expression and/or cancer status in smokers. These polymorphisms might ultimately contribute to lung cancer risk via their effect on the airway gene expression response to tobacco-smoke exposure.
Cigarette smoke creates a molecular field of injury in epithelial cells that line the respiratory tract. We hypothesized that transcriptome sequencing (RNA-Seq) will enhance our understanding of the field of molecular injury in response to tobacco smoke exposure and lung cancer pathogenesis by identifying gene expression differences not interrogated or accurately measured by microarrays. We sequenced the high-molecular-weight fraction of total RNA (>200 nt) from pooled bronchial airway epithelial cell brushings (n = 3 patients per pool) obtained during bronchoscopy from healthy never smoker (NS) and current smoker (S) volunteers and smokers with (C) and without (NC) lung cancer undergoing lung nodule resection surgery. RNA-Seq libraries were prepared using 2 distinct approaches, one capable of capturing non-polyadenylated RNA (the prototype NuGEN Ovation RNA-Seq protocol) and the other designed to measure only polyadenylated RNA (the standard Illumina mRNA-Seq protocol) followed by sequencing generating approximately 29 million 36 nt reads per pool and approximately 22 million 75 nt paired-end reads per pool, respectively. The NuGEN protocol captured additional transcripts not detected by the Illumina protocol at the expense of reduced coverage of polyadenylated transcripts, while longer read lengths and a paired-end sequencing strategy significantly improved the number of reads that could be aligned to the genome. The aligned reads derived from the two complementary protocols were used to define the compendium of genes expressed in the airway epithelium (n = 20,573 genes). Pathways related to the metabolism of xenobiotics by cytochrome P450, retinol metabolism, and oxidoreductase activity were enriched among genes differentially expressed in smokers, whereas chemokine signaling pathways, cytokine–cytokine receptor interactions, and cell adhesion molecules were enriched among genes differentially expressed in smokers with lung cancer. There was a significant correlation between the RNA-Seq gene expression data and Affymetrix microarray data generated from the same samples (P < 0.001); however, the RNA-Seq data detected additional smoking- and cancer-related transcripts whose expression was were either not interrogated by or was not found to be significantly altered when using microarrays, including smoking-related changes in the inflammatory genes S100A8 and S100A9 and cancer-related changes in MUC5AC and secretoglobin (SCGB3A1). Quantitative real-time PCR confirmed differential expression of select genes and non-coding RNAs within individual samples. These results demonstrate that transcriptome sequencing has the potential to provide new insights into the biology of the airway field of injury associated with smoking and lung cancer. The measurement of both coding and non-coding transcripts by RNA-Seq has the potential to help elucidate mechanisms of response to tobacco smoke and to identify additional biomarkers of lung cancer risk and novel targets for chemoprevention.
Lung cancer is the most common cause of cancer-related deaths. Tobacco smoke exposure is the strongest aetiological factor associated with lung cancer. In this study, using serial analysis of gene expression (SAGE), we comprehensively examined the effect of active smoking by comparing the transcriptomes of clinical specimens obtained from current, former and never smokers, and identified genes showing both reversible and irreversible expression changes upon smoking cessation.
Twenty-four SAGE profiles of the bronchial epithelium of eight current, twelve former and four never smokers were generated and analyzed. In total, 3,111,471 SAGE tags representing over 110 thousand potentially unique transcripts were generated, comprising the largest human SAGE study to date. We identified 1,733 constitutively expressed genes in current, former and never smoker transcriptomes. We have also identified both reversible and irreversible gene expression changes upon cessation of smoking; reversible changes were frequently associated with either xenobiotic metabolism, nucleotide metabolism or mucus secretion. Increased expression of TFF3, CABYR, and ENTPD8 were found to be reversible upon smoking cessation. Expression of GSK3B, which regulates COX2 expression, was irreversibly decreased. MUC5AC expression was only partially reversed. Validation of select genes was performed using quantitative RT-PCR on a secondary cohort of nine current smokers, seven former smokers and six never smokers.
Expression levels of some of the genes related to tobacco smoking return to levels similar to never smokers upon cessation of smoking, while expression of others appears to be permanently altered despite prolonged smoking cessation. These irreversible changes may account for the persistent lung cancer risk despite smoking cessation.
Lung cancer is the worldwide leading cause of death from cancer. Tobacco usage is the major pathogenic factor, but all lung cancers are not attributable to smoking. Specifically, lung cancer in never-smokers has been suggested to represent a distinct disease entity compared to lung cancer arising in smokers due to differences in etiology, natural history and response to specific treatment regimes. However, the genetic aberrations that differ between smokers and never-smokers’ lung carcinomas remain to a large extent unclear.
Unsupervised gene expression analysis of 39 primary lung adenocarcinomas was performed using Illumina HT-12 microarrays. Results from unsupervised analysis were validated in six external adenocarcinoma data sets (n=687), and six data sets comprising normal airway epithelial or normal lung tissue specimens (n=467). Supervised gene expression analysis between smokers and never-smokers were performed in seven adenocarcinoma data sets, and results validated in the six normal data sets.
Initial unsupervised analysis of 39 adenocarcinomas identified two subgroups of which one harbored all never-smokers. A generated gene expression signature could subsequently identify never-smokers with 79-100% sensitivity in external adenocarcinoma data sets and with 76-88% sensitivity in the normal materials. A notable fraction of current/former smokers were grouped with never-smokers. Intriguingly, supervised analysis of never-smokers versus smokers in seven adenocarcinoma data sets generated similar results. Overlap in classification between the two approaches was high, indicating that both approaches identify a common set of samples from current/former smokers as potential never-smokers. The gene signature from unsupervised analysis included several genes implicated in lung tumorigenesis, immune-response associated pathways, genes previously associated with smoking, as well as marker genes for alveolar type II pneumocytes, while the best classifier from supervised analysis comprised genes strongly associated with proliferation, but also genes previously associated with smoking.
Based on gene expression profiling, we demonstrate that never-smokers can be identified with high sensitivity in both tumor material and normal airway epithelial specimens. Our results indicate that tumors arising in never-smokers, together with a subset of tumors from smokers, represent a distinct entity of lung adenocarcinomas. Taken together, these analyses provide further insight into the transcriptional patterns occurring in lung adenocarcinoma stratified by smoking history.
Lung cancer; Smoking; Gene expression analysis; Adenocarcinoma; EGFR; Never-smokers; Immune response
OBJECTIVE--To determine whether tobacco smoking causes increased DNA modification (adducts) in human cervical epithelium. DESIGN--Comparison of DNA adducts measured by the technique of postlabelling with phosphorus-32 in normal ectocervical epithelium of smokers and non-smokers. A questionnaire on smoking habit and a urinary cotinine assay were used to identify smokers and non-smokers. SETTING--Cytology unit in large teaching hospital. SUBJECTS--39 women (11 current smokers, seven former smokers, and 21 who had never smoked) undergoing gynaecological treatment (colposcopy or hysterectomy). Nineteen members of staff who did not smoke as controls. INTERVENTIONS--Biopsy of normal ectocervical epithelium. Urine sample. MAIN OUTCOME MEASURES--Measurement of DNA adducts in cervical epithelial tissue of smokers and non-smokers. Smoking habit derived from results of questionnaire and urinary cotinine:creatinine ratio. Proportion of adducts in women with abnormal and normal results of cervical smear test. RESULTS--DNA samples from smokers (identified from questionnaire) had significantly higher median proportions of DNA adducts that non-smokers (4.62 (95% confidence interval 4.04 to 7.74) v 3.47 (2.84 to 4.78) adducts/10(8) nucleotides; p = 0.048). Exclusion of women whose urinary cotinine:creatinine ratio did not confirm their self reported smoking habit (smoker or non-smoker) increased this difference (4.7 (3.85 to 8.08) v 3.52 (2.32 to 4.95) adducts/10(8) nucleotides; p = 0.03). Women who had abnormal results of cervical smear tests had significantly higher proportions of adducts than those with normal results (4.7 (3.90 to 8.13) v 3.47 (3.06 to 5.36) adducts/10(8) nucleotides; p = 0.03). CONCLUSIONS--Tobacco smoking by women leads to increased modification of DNA in cervical epithelium, suggesting biochemical evidence consistent with smoking as a cause of cervical cancer.
Rationale: Epidemiologic data demonstrate that individuals exposed to low levels of tobacco smoke have decrements in lung function and higher risk for lung disease compared with unexposed individuals. Although this risk is small, low-level tobacco smoke exposure is so widespread, it is a significant public health concern.
Objectives: To identify biologic correlates of this risk we hypothesized that, compared with unexposed individuals, individuals exposed to low levels of tobacco smoke have biologic changes in the small airway epithelium, the site of the first abnormalities associated with smoking.
Methods: Small airway epithelium was obtained by bronchoscopy from 121 individuals; microarrays were used to assess genome-wide gene expression; urine nicotine and cotinine were used to categorize subjects as “nonsmokers,” “active smokers,” and “low exposure.” Gene expression data were used to determine the threshold and induction half maximal level (ID50) of urine nicotine and cotinine at which the small airway epithelium showed abnormal responses.
Measurements and Main Results: There was no threshold of urine nicotine without a small airway epithelial response, and only slightly above detectable urine cotinine threshold with a small airway epithelium response. The ID50 for nicotine was 25 ng/ml and for cotinine it was 104 ng/ml.
Conclusions: The small airway epithelium detects and responds to low levels of tobacco smoke with transcriptome modifications. This provides biologic correlates of epidemiologic studies linking low-level tobacco smoke exposure to lung health risk, identifies the genes most sensitive to tobacco smoke, and defines thresholds at which the lung epithelium responds to low levels of tobacco smoke.
threshold; exposure; dose-dependant; ID50
Smokers weigh less and have less body fat than nonsmokers. Increased body fat and weight gain are observed following smoking cessation. To assess a possible molecular mechanism underlying the inverse association between smoking and body weight, we hypothesized that smoking may induce the expression of a fat depleting gene in the airway epithelium, the cell population that takes the brunt of the stress of cigarette smoke.
To assess if smoking up-regulates expression in the airway epithelium of genes associated with weight loss, microarray analysis was used to evaluate genes associated with fat-depletion in large airway epithelial samples obtained by fiberoptic bronchoscopy from normal smokers and normal nonsmokers. As a candidate gene we further evaluated the expression of alpha2-zinc-glycoprotein1 (AZGP1), a soluble protein that stimulates lipolysis, induces a reduction in body fat in mice, is associated with the cachexia related to cancer, and is known to be expressed in secretory cells of lung epithelium. AZGP1 protein expression was assessed by Western analysis and localization in the large airway epithelium by immunohistochemistry.
Both microarray and TaqMan analysis demonstrated that AZGP1 mRNA levels were higher in the large airway epithelium of normal smokers compared to normal nonsmokers (p<0.05, all comparisons). Western analysis of airway biopsies of smokers compared with non-smokers demonstrated upregulation of AZGP1 at the protein level, and immunohistochemical analysis demonstrated upregulation of AZGP1 in secretory as well as neuroendocrine cells of smokers.
In the context that AZGP1 is involved in lipolysis and fat loss, its overexpression in the airway epithelium of chronic smokers may represent one mechanism for the weight difference in smokers vs nonsmokers.
AZGP1; smoking; weight loss; airway epithelium; gene expression
The Wnt pathway mediates differentiation of epithelial tissues; depending on the tissue types, Wnt can either drive or inhibit the differentiation process. We hypothesized that key genes in the Wnt pathway are suppressed in the human airway epithelium under the stress of cigarette smoking, a stress associated with dysregulation of the epithelial differentiated state.
Microarrays were used to assess the expression of Wnt-related genes in the small airway epithelium (SAE) obtained via bronchoscopy and brushing of healthy nonsmokers, healthy smokers, and smokers with COPD. Thirty-three of 56 known Wnt-related genes were expressed in the SAE. Wnt pathway downstream mediators β-catenin and the transcription factor 7-like 1 were down-regulated in healthy smokers and smokers with COPD, as were many Wnt target genes. Among the extracellular regulators that suppress the Wnt pathway, secreted frizzled-related protein 2 (SFRP2), was up-regulated 4.3-fold in healthy smokers and 4.9-fold in COPD smokers, an observation confirmed by TaqMan Real-time PCR, Western analysis and immunohistochemistry. Finally, cigarette smoke extract mediated up-regulation of SFRP2 and down-regulation of Wnt target genes in airway epithelial cells in vitro.
Smoking down-regulates the Wnt pathway in the human airway epithelium. In the context that Wnt pathway plays an important role in differentiation of epithelial tissues, the down-regulation of Wnt pathway may contribute to the dysregulation of airway epithelium differentiation observed in smoking-related airway disorders.
Cigarette smoke disrupts the protective barrier established by the airway epithelium through direct damage to the epithelial cells, leading to cell death. Since the morphology of the airway epithelium of smokers does not typically demonstrate necrosis, the most likely mechanism for epithelial cell death in response to cigarette smoke is apoptosis. We hypothesized that cigarette smoke directly up-regulates expression of apoptotic genes, which could play a role in airway epithelial apoptosis.
Microarray analysis of airway epithelium obtained by bronchoscopy on matched cohorts of 13 phenotypically normal smokers and 9 non-smokers was used to identify specific genes modulated by smoking that were associated with apoptosis. Among the up-regulated apoptotic genes was pirin (3.1-fold, p < 0.002), an iron-binding nuclear protein and transcription cofactor. In vitro studies using human bronchial cells exposed to cigarette smoke extract (CSE) and an adenovirus vector encoding the pirin cDNA (AdPirin) were performed to test the direct effect of cigarette smoke on pirin expression and the effect of pirin expression on apoptosis.
Quantitative TaqMan RT-PCR confirmed a 2-fold increase in pirin expression in the airway epithelium of smokers compared to non-smokers (p < 0.02). CSE applied to primary human bronchial epithelial cell cultures demonstrated that pirin mRNA levels increase in a time-and concentration-dependent manner (p < 0.03, all conditions compared to controls).
Overexpression of pirin, using the vector AdPirin, in human bronchial epithelial cells was associated with an increase in the number of apoptotic cells assessed by both TUNEL assay (5-fold, p < 0.01) and ELISA for cytoplasmic nucleosomes (19.3-fold, p < 0.01) compared to control adenovirus vector.
These observations suggest that up-regulation of pirin may represent one mechanism by which cigarette smoke induces apoptosis in the airway epithelium, an observation that has implications for the pathogenesis of cigarette smoke-induced diseases.
Why do we observe a wage differential between smokers and non-smokers? Pooling reports of current and prior smoking activity across 15 years from the Panel Study of Income Dynamics (PSID) allows the reconstruction of individual smoking histories. Dividing the sample into smoking history groups, the four largest of which are: persistent smokers, never smokers, former smokers, and future quitters reveals that there is no observed wage gap between former smokers and those who have never smoked. There is, however, a wage gap between those smokers who will continue smoking and three other groups of individuals: (1) those smokers who will quit smoking in the future, (2) those smokers who have quit smoking already, and (3) those who never smoked. The wage gap between smokers and non-smokers, observed in the 1986 cross-section, is largely driven by those who persist as smokers, 1986–2001. These results support the hypothesis that the cross-sectional wage differential is not driven by smoking per se, but may be driven by a non-causal explanation. One plausible interpretation is that a common factor such as myopia, leads to reduced investment in both health capital or firm-specific or other human capital.
smoking; wages; health capital
The first changes associated with smoking are in the small airway epithelium (SAE). Given that smoking alters SAE gene expression, but only a fraction of smokers develop chronic obstructive pulmonary disease (COPD), we hypothesized that assessment of SAE genome-wide gene expression would permit biologic phenotyping of the smoking response, and that a subset of healthy smokers would have a “COPD-like” SAE transcriptome.
SAE (10th–12th generation) was obtained via bronchoscopy of healthy nonsmokers, healthy smokers and COPD smokers and microarray analysis was used to identify differentially expressed genes. Individual responsiveness to smoking was quantified with an index representing the % of smoking-responsive genes abnormally expressed (ISAE), with healthy smokers grouped into “high” and “low” responders based on the proportion of smoking-responsive genes up- or down-regulated in each smoker. Smokers demonstrated significant variability in SAE transcriptome with ISAE ranging from 2.9 to 51.5%. While the SAE transcriptome of “low” responder healthy smokers differed from both “high” responders and smokers with COPD, the transcriptome of the “high” responder healthy smokers was indistinguishable from COPD smokers.
The SAE transcriptome can be used to classify clinically healthy smokers into subgroups with lesser and greater responses to cigarette smoking, even though these subgroups are indistinguishable by clinical criteria. This identifies a group of smokers with a “COPD-like” SAE transcriptome.
Although prior studies have demonstrated a smoking-induced field of molecular injury throughout the lung and airway, the impact of smoking on the airway epithelial proteome and its relationship to smoking-related changes in the airway transcriptome are unclear.
Airway epithelial cells were obtained from never (n = 5) and current (n = 5) smokers by brushing the mainstem bronchus. Proteins were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE). After in-gel digestion, tryptic peptides were processed via liquid chromatography/ tandem mass spectrometry (LC-MS/MS) and proteins identified. RNA from the same samples was hybridized to HG-U133A microarrays. Protein detection was compared to RNA expression in the current study and a previously published airway dataset. The functional properties of many of the 197 proteins detected in a majority of never smokers were similar to those observed in the never smoker airway transcriptome. LC-MS/MS identified 23 proteins that differed between never and current smokers. Western blotting confirmed the smoking-related changes of PLUNC, P4HB1, and uteroglobin protein levels. Many of the proteins differentially detected between never and current smokers were also altered at the level of gene expression in this cohort and the prior airway transcriptome study. There was a strong association between protein detection and expression of its corresponding transcript within the same sample, with 86% of the proteins detected by LC-MS/MS having a detectable corresponding probeset by microarray in the same sample. Forty-one proteins identified by LC-MS/MS lacked detectable expression of a corresponding transcript and were detected in ≤5% of airway samples from a previously published dataset.
1D-PAGE coupled with LC-MS/MS effectively profiled the airway epithelium proteome and identified proteins expressed at different levels as a result of cigarette smoke exposure. While there was a strong correlation between protein and transcript detection within the same sample, we also identified proteins whose corresponding transcripts were not detected by microarray. This noninvasive approach to proteomic profiling of airway epithelium may provide additional insights into the field of injury induced by tobacco exposure.
A number of personality traits have been associated with cigarette smoking. Current smokers generally show higher levels of negative emotionality and lower levels of behavioral constraint than former smokers and those who never smoked. However, prior investigations have not examined thoroughly whether these smoking–personality associations are unique to smoking status or simply reflect the fact that these personality traits tend to be elevated across numerous forms of psychopathology. Likewise, prior studies have not addressed whether personality shows differential associations with smoking based on the presence or absence of lifetime psychiatric disorders.
The present study examined these questions using data from 472 current, 311 former, and 324 never-smokers aged 34–44 years.
Current smokers reported being more reactive to stress, more aggressive, more alienated, and less harm avoidant than both former smokers and never-smokers, whereas former smokers and never-smokers showed similar personality profiles overall. Psychiatric disorder history did not interact with smoking status in predicting personality. Controlling for differences in four major lifetime psychiatric disorders (major depression, alcohol dependence, drug dependence, and conduct disorder) reduced the differences in personality traits associated with smoking status. However, smoking status continued to relate uniquely and significantly to higher levels of negative emotionality and behavioral undercontrol with the most robust effect observed for trait alienation.
These results provide the most comprehensive depiction to date of interrelations among personality, psychopathology, and smoking and suggest an important role of personality in smoking that is not redundant with or conditional upon lifetime psychopathology.
In the work of Chari et al. entitled "Effect of active smoking on the human bronchial epithelium transcriptome" the authors use SAGE to identify candidate gene expression changes in bronchial brushings from never, former, and current smokers. These gene expression changes are categorized into those that are reversible or irreversible upon smoking cessation. A subset of these identified genes is validated on an independent cohort using RT-PCR. The authors conclude that their results support the notion of gene expression changes in the lungs of smokers which persist even after an individual has quit.
This correspondence raises questions about the validity of the approach used by the authors to analyze their data. The majority of the reported results suffer deficiencies due to the methods used. The most fundamental of these are explained in detail: biases introduced during data processing, lack of correction for multiple testing, and an incorrect use of clustering for gene discovery. A randomly generated "null" dataset is used to show the consequences of these shortcomings.
Most of Chari et al.'s findings are consistent with what would be expected by chance alone. Although there is clear evidence of reversible changes in gene expression, the majority of those identified appear to be false positives. However, contrary to the authors' claims, no irreversible changes were identified. There is a broad consensus that genetic change due to smoking persists once an individual has quit smoking; unfortunately, this study lacks sufficient scientific rigour to support or refute this hypothesis or identify any specific candidate genes. The pitfalls of large-scale analysis, as exemplified here, may not be unique to Chari et al.
Tobacco smoking is responsible for over 90% of lung cancer cases, and yet the precise molecular alterations induced by smoking in lung that develop into cancer and impact survival have remained obscure.
We performed gene expression analysis using HG-U133A Affymetrix chips on 135 fresh frozen tissue samples of adenocarcinoma and paired noninvolved lung tissue from current, former and never smokers, with biochemically validated smoking information. ANOVA analysis adjusted for potential confounders, multiple testing procedure, Gene Set Enrichment Analysis, and GO-functional classification were conducted for gene selection. Results were confirmed in independent adenocarcinoma and non-tumor tissues from two studies. We identified a gene expression signature characteristic of smoking that includes cell cycle genes, particularly those involved in the mitotic spindle formation (e.g., NEK2, TTK, PRC1). Expression of these genes strongly differentiated both smokers from non-smokers in lung tumors and early stage tumor tissue from non-tumor tissue (p<0.001 and fold-change >1.5, for each comparison), consistent with an important role for this pathway in lung carcinogenesis induced by smoking. These changes persisted many years after smoking cessation. NEK2 (p<0.001) and TTK (p = 0.002) expression in the noninvolved lung tissue was also associated with a 3-fold increased risk of mortality from lung adenocarcinoma in smokers.
Our work provides insight into the smoking-related mechanisms of lung neoplasia, and shows that the very mitotic genes known to be involved in cancer development are induced by smoking and affect survival. These genes are candidate targets for chemoprevention and treatment of lung cancer in smokers.
Cigarette smoking is a leading cause of preventable death and a significant cause of lung cancer and chronic obstructive pulmonary disease. Prior studies have demonstrated that smoking creates a field of molecular injury throughout the airway epithelium exposed to cigarette smoke. We have previously characterized gene expression in the bronchial epithelium of never smokers and identified the gene expression changes that occur in the mainstem bronchus in response to smoking. In this study, we explored relationships in whole-genome gene expression between extrathorcic (buccal and nasal) and intrathoracic (bronchial) epithelium in healthy current and never smokers.
Using genes that have been previously defined as being expressed in the bronchial airway of never smokers (the "normal airway transcriptome"), we found that bronchial and nasal epithelium from non-smokers were most similar in gene expression when compared to other epithelial and nonepithelial tissues, with several antioxidant, detoxification, and structural genes being highly expressed in both the bronchus and nose. Principle component analysis of previously defined smoking-induced genes from the bronchus suggested that smoking had a similar effect on gene expression in nasal epithelium. Gene set enrichment analysis demonstrated that this set of genes was also highly enriched among the genes most altered by smoking in both nasal and buccal epithelial samples. The expression of several detoxification genes was commonly altered by smoking in all three respiratory epithelial tissues, suggesting a common airway-wide response to tobacco exposure.
Our findings support a relationship between gene expression in extra- and intrathoracic airway epithelial cells and extend the concept of a smoking-induced field of injury to epithelial cells that line the mouth and nose. This relationship could potentially be utilized to develop a non-invasive biomarker for tobacco exposure as well as a non-invasive screening or diagnostic tool providing information about individual susceptibility to smoking-induced lung diseases.
The production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. Based on studies suggesting a role of the airway epithelial GABAergic system in asthma-related mucus overproduction, we hypothesized that cigarette smoking, another disorder associated with increased mucus production, may modulate GABAergic system-related gene expression levels in the airway epithelium.
We assessed expression of the GABAergic system in human airway epithelium obtained using bronchoscopy to sample the epithelium and microarrays to evaluate gene expression. RT-PCR was used to confirm gene expression of GABAergic system gene in large and small airway epithelium from heathy nonsmokers and healthy smokers. The differences in the GABAergic system gene was further confirmed by TaqMan, immunohistochemistry and Western analysis.
The data demonstrate there is a complete GABAergic system expressed in the large and small human airway epithelium, including glutamate decarboxylase, GABA receptors, transporters and catabolism enzymes. Interestingly, of the entire GABAergic system, smoking modified only the expression of GAD67, with marked up-regulation of GAD67 gene expression in both large (4.1-fold increase, p < 0.01) and small airway epithelium of healthy smokers (6.3-fold increase, p < 0.01). At the protein level, Western analysis confirmed the increased expression of GAD67 in airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05). There was a significant positive correlation between GAD67 and MUC5AC gene expression in both large and small airway epithelium (p < 0.01), implying a link between GAD67 and mucin overproduction in association with smoking.
In the context that GAD67 is the rate limiting enzyme in GABA synthesis, the correlation of GAD67 gene expression with MUC5AC expressions suggests that the up-regulation of airway epithelium expression of GAD67 may contribute to the increase in mucus production observed in association with cigarette smoking.
Use of tobacco is responsible for approximately 30% of all cancer-related deaths in the United States including cancers of the upper aerodigestive tract. In the current study, 40 current and 40 age- and gender-matched never smokers underwent buccal biopsies to evaluate the effects of smoking on the transcriptome. Microarray analyses were carried out using Affymetrix HGU 133 Plus2 arrays. Smoking altered the expression of numerous genes: 32 genes showed increased expression and 9 genes showed reduced expression in the oral mucosa of smokers vs. never smokers. Increases were found in genes involved in xenobiotic metabolism, oxidant stress, eicosanoid synthesis, nicotine signaling and cell adhesion. Increased numbers of Langerhans cells were found in the oral mucosa of smokers. Interestingly, smoking caused greater induction of aldo-keto reductases, enzymes linked to polycyclic aromatic hydrocarbon induced genotoxicity, in the oral mucosa of women than men. Striking similarities in expression changes were found in oral compared to the bronchial mucosa. The observed changes in gene expression were compared to known chemical signatures using the Connectivity Map database, and suggested that geldanamycin, an Hsp90 inhibitor, might be an anti-mimetic of tobacco smoke. Consistent with this prediction, geldanamycin caused dose-dependent suppression of tobacco smoke extract-mediated induction of CYP1A1 and CYP1B1 in vitro. Collectively, these results provide new insights into the carcinogenic effects of tobacco smoke, support the potential use of oral epithelium as a surrogate tissue in future lung cancer chemoprevention trials and illustrate the potential of computational biology to identify chemopreventive agents.
tobacco; smoking; microarray; aryl hydrocarbon receptor; heat shock protein 90
Data from the Tucson epidemiological study of airways obstructive disease on smoking of non-tobacco cigarettes such as marijuana were analysed to determine the effect of such smoking on respiratory symptoms and pulmonary function. Among adults aged under 40, 14% had smoked non-tobacco cigarettes at some time and 9% were current users. The prevalence of respiratory symptoms was increased in smokers of non-tobacco cigarettes. After tobacco smoking had been controlled for men who smoked non-tobacco cigarettes showed significant decreases in expiratory flow rates at low lung volumes and in the ratio of the forced expiratory volume in one second to the vital capacity. This effect on pulmonary function in male non-tobacco cigarette smokers was greater than the effect of tobacco cigarette smoking. These data suggest that non-tobacco cigarette smoking may be an important risk factor in young adults with respiratory symptoms or evidence of airways obstruction.
Nuclear factor erythroid 2–related factor 2 (Nrf2) is an oxidant-responsive transcription factor known to induce detoxifying and antioxidant genes. Cigarette smoke, with its large oxidant content, is a major stress on the cells of small airway epithelium, which are vulnerable to oxidant damage. We assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample the small airway epithelium in healthy-nonsmoker and healthy-smoker, and gene expression was assessed using microarrays. Relative to nonsmokers, Nrf2 protein in the small airway epithelium of smokers was activated and localized in the nucleus. The human homologs of 201 known murine Nrf2-modulated genes were identified, and 13 highly smoking-responsive Nrf2-modulated genes were identified. Construction of an Nrf2 index to assess the expression levels of these 13 genes in the airway epithelium of smokers showed coordinate control, an observation confirmed by quantitative PCR. This coordinate level of expression of the 13 Nrf2-modulated genes was independent of smoking history or demographic parameters. The Nrf2 index was used to identify two novel Nrf2-modulated, smoking-responsive genes, pirin (PIR) and UDP glucuronosyltransferase 1-family polypeptide A4 (UGT1A4). Both genes were demonstrated to contain functional antioxidant response elements in the promoter region. These observations suggest that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cells, and that there is variability within the human population in the Nrf2 responsiveness to oxidant burden.
Smoking remains the most common preventable cause of death. Very little tobacco exposure can increase cardiovascular disease risk. The relationship between smoking, sex, and weight remains unclear.
Between September 1992 and June 2007, 2582 consenting patients starting the Ottawa Hospital Weight Management program were grouped by sex and smoking status. “Former smokers” (771 females, 312 males) had quit for at least 1 year. “Smokers” (135 females, 54 males) smoked > 9 cigarettes daily. There were 979 females and 331 males who never smoked. Using SAS 9.2 statistical software, the prevalence of coronary artery disease (CAD), type 2 diabetes (T2DM), major depressive disorder (MDD), and medication use among the groups was compared (Chi-square [χ2]). Anthropometric measurements, lipid, glucose and thyroid levels were compared using analysis of variance (ANOVA). Interactions were assessed using 2-way ANOVA analysis for continuous data, and logistic regression for discrete data.
Smokers were more likely to have MDD (χ2), lower high-density lipoprotein levels and higher triglyceride levels than other groups. Former smokers had a greater prevalence of CAD, T2DM on pharmacotherapy, and impaired fasting glucose than other groups. They were also more likely to be taking lipid-lowering agents and antihypertensives (χ2). Never smokers had less MDD, CAD, and were less likely to be on antidepressants than the other groups. Males were more likely to have CAD and T2DM than females. Females were more likely to have MDD than males. Interactions between smoking status and sex were found for age, weight, fasting glucose and thyroid-stimulating hormone levels.
Obese never smokers suffer from the fewest chronic diseases. Obese former smokers have a greater prevalence of CAD, T2DM on pharmacotherapy, and impaired fasting glucose than other groups. Thus, clinicians and researchers should avoid combining former smokers with never smokers as “nonsmokers” in research and treatment decisions. The results of this study call for a longitudinal study comparing these groups over the weight management program.
smoking status; weight management; obesity
Cigarette smoking is associated with increased frequency and duration of viral respiratory infections, but the underlying mechanisms are incompletely defined. We investigated whether smoking reduces expression by human lung macrophages (Mø) of receptors for viral nucleic acids and, if so, the effect on CXCL10 production.
We collected alveolar macrophages (AMø) by bronchoalveolar lavage of radiographically-normal lungs of subjects undergoing bronchoscopies for solitary nodules (n = 16) and of volunteers who were current or former smokers (n = 7) or never-smokers (n = 13). We measured expression of mRNA transcripts for viral nucleic acid receptors by real-time PCR in those AMø and in the human Mø cell line THP-1 following phorbol myristate acetate/vitamin D3 differentiation and exposure to cigarette smoke extract, and determined TLR3 protein expression using flow cytometry and immunohistochemistry. We also used flow cytometry to examine TLR3 expression in total lung Mø from subjects undergoing clinically-indicated lung resections (n = 25). Of these, seven had normal FEV1 and FEV1/FVC ratio (three former smokers, four current smokers); the remaining 18 subjects (14 former smokers; four current smokers) had COPD of GOLD stages I-IV. We measured AMø production of CXCL10 in response to stimulation with the dsRNA analogue poly(I:C) using Luminex assay.
Relative to AMø of never-smokers, AMø of smokers demonstrated reduced protein expression of TLR3 and decreased mRNA for TLR3 but not TLR7, TLR8, TLR9, RIG-I, MDA-5 or PKR. Identical changes in TLR3 gene expression were induced in differentiated THP-1 cells exposed to cigarette smoke-extract in vitro for 4 hours. Among total lung Mø, the percentage of TLR3-positive cells correlated inversely with active smoking but not with COPD diagnosis, FEV1% predicted, sex, age or pack-years. Compared to AMø of never-smokers, poly(I:C)-stimulated production of CXCL10 was significantly reduced in AMø of smokers.
Active smoking, independent of COPD stage or smoking duration, reduces both the percent of human lung Mø expressing TLR3, and dsRNA-induced CXCL10 production, without altering other endosomal or cytoplasmic receptors for microbial nucleic acids. This effect provides one possible mechanism for increased frequency and duration of viral lower respiratory tract infections in smokers.
ClinicalTrials.gov NCT00281190, NCT00281203 and NCT00281229.
Lung; Cigarette smoking; Effects; Toll-like receptors; Macrophages; Alveolar
Smoking is a risk factor for cancer of the upper aerodigestive tract with recidivism rates high even after diagnosis. Nicotine, a major product in tobacco, is a complex drug with multiple characteristics including analalgesic properties. The goal of the study was to examine pain levels in the context of smoking status among patients recently diagnosed with cancer of the upper aerodigestive tract who have not yet received any treatment including radiation, surgery, or chemotherapy. A convenience sample of 112 newly diagnosed head and neck cancer patients (78 men and 34 women) were recruited from clinics at the University of Florida. Smoking rates were: 32% never smoked, 34% former smokers, 34% current smokers. Among current smokers, 62% reported plans to quit in the next 3 months and 38% had tried to quit more than three times in the past 5 years. Current smokers reported higher general (sensory and affective) and oral pain levels (spontaneous and functional) and pain-related interference than did never and former smokers (all F’s >8. and p’s <.0001) even after controlling for stage of diagnosis. In addition, current smokers reported significantly greater interference from the pain (F2,73=10.5 p<.0001).
This study highlights the importance of understanding self-reported pain in cancer patients who continue to smoke. When pain is elevated, smokers may be motivated to use tobacco as a means of reducing pain, which in turn reinforces smoking behavior. Tobacco cessation programs should include pain management as a component of treatment.
Cancer; Head and Neck Cancer; Smoking; Pain
Lectins are innate immune defense proteins that recognize specific bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with increased risk of bacterial infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey expression of lectin genes in large airway epithelium from 9 nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change, p<0.05) in lectin gene expression among healthy smokers compared to nonsmokers except for a striking down regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in the cell walls of bacteria (large airway epithelium, p<0.01; small airway epithelium, p<0.01). This was confirmed by TaqMan RT-PCR in both large (p<0.05) and small airway epithelium (p<0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared to healthy nonsmokers (p<0.02). Finally, compared to healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema with normal spirometry (n= 13, p<0.01) and smokers with established COPD (n= 14, p<0.01). In the context that intelectin 1 is an epithelial molecule that likely plays a role in defense against bacteria, its down regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers, including those with COPD.
OBJECTIVE: To estimate the risk of lung cancer in lifelong non-smokers exposed to environmental tobacco smoke. DESIGN: Analysis of 37 published epidemiological studies of the risk of lung cancer (4626 cases) in non-smokers who did and did not live with a smoker. The risk estimate was compared with that from linear extrapolation of the risk in smokers using seven studies of biochemical markers of tobacco smoke intake. MAIN OUTCOME MEASURE: Relative risk of lung cancer in lifelong non-smokers according to whether the spouse currently smoked or had never smoked. RESULTS: The excess risk of lung cancer was 24% (95% confidence interval 13% to 36%) in non-smokers who lived with a smoker (P < 0.001). Adjustment for the effects of bias (positive and negative) and dietary confounding had little overall effect; the adjusted excess risk was 26% (7% to 47%). The dose-response relation of the risk of lung cancer with both the number of cigarettes smoked by the spouse and the duration of exposure was significant. The excess risk derived by linear extrapolation from that in smokers was 19%, similar to the direct estimate of 26%. CONCLUSION: The epidemiological and biochemical evidence on exposure to environmental tobacco smoke, with the supporting evidence of tobacco specific carcinogens in the blood and urine of non-smokers exposed to environmental tobacco smoke, provides compelling confirmation that breathing other people's tobacco smoke is a cause of lung cancer.