Early changes in gene expression induced by tobacco smoke: Evidence for the importance of estrogen within lung tissue

Abstract

Lung cancer is the leading cause of cancer deaths in the United States, surpassing breast cancer as the primary cause of cancer-related mortality in women. The goal of the present study was to identify early molecular changes in the lung induced by exposure to tobacco smoke and thus identify potential targets for chemoprevention. Female A/J mice were exposed to either tobacco smoke or HEPA-filtered air via a whole-body exposure chamber (6 h/d, 5 d/wk for 3, 8, and 20 weeks). Gene expression profiles of lung tissue from control and smoke-exposed animals were established using a 15K cDNA microarray. Cytochrome P450 1b1, a phase I enzyme involved in both the metabolism of xenobiotics and the 4-hydroxylation of 17beta-estradiol (E(2)), was modulated to the greatest extent following smoke exposure. A panel of 10 genes were found to be differentially expressed in control and smoke-exposed lung tissues at 3, 8, and 20 weeks (P < 0.001). The interaction network of these differentially expressed genes revealed new pathways modulated by short-term smoke exposure, including estrogen metabolism. In addition, E(2) was detected within murine lung tissue by gas chromatography-coupled mass spectrometry and immunohistochemistry. Identification of the early molecular events that contribute to lung tumor formation is anticipated to lead to the development of promising targeted chemopreventive therapies. In conclusion, the presence of E(2) within lung tissue when combined with the modulation of cytochrome P450 1b1 and other estrogen metabolism genes by tobacco smoke provides novel insight into a possible role for estrogens in lung cancer.

Fig. 1. Genes differentially expressed following 3, 8 and 20 wk of smoke exposure

A. Venn diagram showing the distribution of differentially expressed genes (P < 0.001) between control lungs and lungs exposed to smoke for 3, 8 and 20 wk. The 10 genes depicted in Group A are modulated to all 3 time points. Groups B-D correspond to genes in common between 3 and 8 wk (B), 3 and 20 wk (C), and 8 and 20 wk (D), whereas Groups E-G represent genes identified only at 3 wk (E), 8 wk (F), or 20 wk (G). The genes represented in each group are listed in Table 1. B. A heat map representing the median normalized expression values for the 10 genes altered at all 3 time points. Data for technical (same letter, i.e. aa) and biological (different letters, i.e. ab) replicates are included.

Fig. 2. Western blot analysis of CYP1B1 in human pulmonary microsomes from nonsmokers (NS) and smokers (S)

Each sample (50 μg) contains a pool of microsomal protein from 4 individuals of mixed genders. HPRT was used as a loading control.

Fig. 3. Network of genes differentially expressed in common following 3, 8, and 20 wk of smoke exposure

Differentially expressed genes (N=7) are depicted as a network with overlayed functions and pathways according to Ingenuity Pathways Analysis Software. The green and red colors represent down- and upregulated genes, respectively. The remaining genes are involved in the network through direct or indirect interactions. Red lines connecting Cyp1b1 to other genes indicate a direct relationship with Cyp1b1 (protein-protein interaction or protein-DNA). For example, the Ahr-Arnt complex increases transcription of Cyp1b1 in mammals (47). Solid blue lines with balloons indicate gene function or a pathway in which a gene is involved.

Fig. 4. Detection of estrogens within murine lung tissue

Lung tissue from female A/J mice was subjected to immunohistochemical and GC/MS analyses. A. Detection of 17β-estradiol (E₂), Erα and Erβ in lung epithelial cells by immunostaining. The bronchioloalveolar epithelium (BAE) stained positive for all antigens evaluated. Subcellular staining was observed as follows: E₂, strong nuclear and cytoplasmic staining in the BAE and some pneumocytes; ERα, cytoplasmic staining of the BAE; and ERβ, nuclear staining in the BAE and some pneumocytes. B. Selective ion monitoring of trimethylsilyl derivatives of E₁ and E₂ (1.1 pmol each) and d5-E₂ (2.6 pmol) as standards (B1) and in the murine lung tissue (B2). Each trace represents different ions monitored. Deuterium-labeled E₂ represents the internal standard. Unmarked peaks in B2 denote unknowns; upper part of the chromatogram was cropped to enhance the visualization of small peaks.