Reversible and permanent effects of tobacco smoke exposure on airway epithelial gene expression

Abstract

Background: Tobacco use remains the leading preventable cause of death in the US. The risk of dying from smoking-related diseases remains elevated for former smokers years after quitting. The identification of irreversible effects of tobacco smoke on airway gene expression may provide insights into the causes of this elevated risk.

Results: Using oligonucleotide microarrays, we measured gene expression in large airway epithelial cells obtained via bronchoscopy from never, current, and former smokers (n = 104). Linear models identified 175 genes differentially expressed between current and never smokers, and classified these as irreversible (n = 28), slowly reversible (n = 6), or rapidly reversible (n = 139) based on their expression in former smokers. A greater percentage of irreversible and slowly reversible genes were down-regulated by smoking, suggesting possible mechanisms for persistent changes, such as allelic loss at 16q13. Similarities with airway epithelium gene expression changes caused by other environmental exposures suggest that common mechanisms are involved in the response to tobacco smoke. Finally, using irreversible genes, we built a biomarker of ever exposure to tobacco smoke capable of classifying an independent set of former and current smokers with 81% and 100% accuracy, respectively.

Conclusion: We have categorized smoking-related changes in airway gene expression by their degree of reversibility upon smoking cessation. Our findings provide insights into the mechanisms leading to reversible and persistent effects of tobacco smoke that may explain former smokers increased risk for developing tobacco-induced lung disease and provide novel targets for chemoprophylaxis. Airway gene expression may also serve as a sensitive biomarker to identify individuals with past exposure to tobacco smoke.

Figure 1

Methodology for gene classification by degree of reversibility upon smoking cessation. For each probeset, the relationship between gene expression in log₂ scale (ge), age, current smoking status (x_curr), former smoking status (x_form), and the interaction between former smoking status and months elapsed since quitting smoking (x_tq) was examined with the linear regression model. Genes differentially expressed between current (C) and never (N) smokers were categorized based on their behavior in former smokers (F) relative to never smokers as a function of time since smoking cessation. Genes were classified as 'rapidly reversible' if there was not a significant difference between former and never smokers. Genes were classified as 'indeterminate' if there was a significant difference between former and never smokers, but the age-adjusted fold change between former and never smokers was not greater than or equal to 1.5. If the fold change criterion was met, genes were classified as 'slowly reversible' if there was a significant relationship between gene expression and time since quitting smoking or as 'irreversible' if there was not a significant relationship with time.

Figure 2

Characteristics of genes classified as irreversible, slowly reversible, or rapidly reversible based on their behavior in former smokers. (a) Numbers of genes up-regulated (red) or down-regulated (blue) in current smokers compared to never smokers. The percentage of genes up-regulated in smoking decreases from the most to the least reversible tertile of rapidly reversible genes and is lowest in the slowly reversible and irreversible genes. (b) The age-adjusted fold change between never versus former smokers (y-axis) is plotted as a function of time since quitting smoking (x-axis) for the genes classified as slowly reversible. All the slowly reversible genes are down-regulated in smoking. The time point that the fold change equals 1.5 (see dotted line) is defined as the time that the genes become reversible. The time point at which this occurs is greater than 78 months (6.5 years) after smoking cessation for all of the slowly reversible genes.

Figure 3

Quantitative real time PCR results for select genes across never, former, and current smokers. For each graph sample identifiers for never (orange), former (purple), and current (green) smokers are listed along the x-axis. The sample identifications P1, P2, and P3 refer to three samples collected prospectively from never smokers that do not have corresponding microarrays. The months since smoking cessation are listed below each former smoker. The relative expression level on the y-axis is the ratio of the expression level of a particular sample versus that of a dummy reference sample. (a) Plots of two rapidly reversible genes, CYP1B1 and ALDH3A1. (b) Plots of two irreversible genes, CEACAM5 and NQO1.

Figure 4

Relationship between samples according to the expression of genes with different reversibility characteristics. PCAs are shown on the left for (a) the slowly reversible and irreversible genes (n = 34) and (b) the most rapidly reversible genes (n = 46). (c) False-color heatmaps are shown on the right for the slowly reversible and irreversible genes (top) and the most reversible tertile of rapidly reversible genes (bottom). Never, former, and current smokers are colored in orange, purple, and green respectively. The PCA and heatmaps were constructed using gene expression data normalized to a mean of zero and a standard deviation of 1. Never and current smokers are organized according to increasing age and former smokers are ordered by decreasing time since quitting smoking (denoted by the gradient) along the sample axis in the heatmap. Affymetrix identifications and HUGO gene symbols are listed for each gene as well as membership in two over-represented functional categories by EASE analysis.

Figure 5

Similarities and differences between our dataset and other bronchial airway datasets. (a) GSEA was used to determine if there was a gene expression relationship between other airway datasets (see Table 3 for a description of conditions 1 and 2) and our dataset based on the genes we identified to be regulated by smoking. The normalized enrichment score is plotted for datasets that had a FDR < 0.25. (b) Gene lists derived from functional categories and chromosomal locations found to be over-represented by EASE analysis in our dataset were tested for enrichment in our dataset and the other ten datasets using GSEA. A false-color heatmap of the positive (red) and negative (blue) normalized enrichment scores (with a FDR < 0.25) is shown for each category. An asterisk indicates the results passed a stricter FDR < 0.05. The nine datasets and conditions that yielded significant results in either (a) or (b) are indicated in Table 3 by the presence of a single asterisk.