Estrogen metabolism in the human lung: impact of tumorigenesis, smoke, sex and race/ethnicity

Abstract

Previous data from this group demonstrate that the murine lung metabolizes estrogen. Production of the putative carcinogen 4-hydroxyestrogen (4-OHE) is elevated within the lungs of female vs. male mice and accelerated by tobacco smoke. The goal of this study was to determine if the human lung metabolizes estrogen and evaluate the impact of tumor formation, smoke, sex and race/ethnicity on metabolism. Urine and lung tissue (normal, tumor) were obtained from 49 non-small cell lung cancer patients. Healthy postmenopausal Caucasian (n = 19) and Chinese (n = 20) American women (never-smokers) donated urine. Quantitative RT-PCR analyses indicate that multiple estrogen synthesis and metabolism genes are expressed in human bronchoalveolar cells. Estrogen and its metabolites were measured in lung tissue and urine using liquid chromatography/tandem mass spectrometry. Wilcoxon rank tests were used for statistical comparisons. E₁, E₂, E₃ and estrogen metabolites 2-OHE₁, 2-OHE₂, 4-OHE₁, 4-OHE₂, 2-OME₁ and 2-OME₂ were detected at higher levels in tumor vs. adjacent normal tissue and in women vs. men (P < 0.05). The proportion of 4-OHEs was higher in tumors than in normal lung tissue (P < 0.05), and elevated in normal tissue from current- vs. never-smoking women (P = 0.006); similar trends were observed in urine. The proportion of 4-OHEs in the urine of postmenopausal Chinese American women was 1.8-fold higher than that of Caucasian women (P = 0.015). These data indicate that estrogen metabolites are present in the human lung. A shift towards 4-hydroxylation during lung tumorigenesis may contribute to the risk conferred by smoking, sex or race/ethnicity.

Keywords: 4-hydroxy estrogen; estrogen metabolism; never-smoking Chinese women; non-small cell lung cancer; tobacco smoke.

Figure 1. Expression of estrogen-related genes in human bronchoalveolar cells

Cells were microdissected from the tumor and adjacent normal lung of NSCLC patients. RNA was extracted and analyzed by quantitative real-time RT-PCR. The percentage of samples with detectable transcripts (Ct values ≤ 35) is plotted for each gene.

Figure 2. Comparison of estrogen and estrogen metabolites in lung tumors vs. adjacent normal lung tissue from NSCLC patients

(A) Fold difference (tumor/normal ratio) in estrogen and estrogen metabolites in tumor vs. paired normal lung tissue. The median of the independent values (open circles) is shown as a solid horizontal line. (B) The level of 2-OHEs, 4-OHEs and 2-OMEs (% total estrogens and estrogen metabolites) and the ratio of 4-OHEs/2-OHEs in lung tumors vs. paired normal lung tissue were calculated. The fold difference between tumor and paired normal lung tissue (tumor/normal ratio) is plotted. The box plots denote the median and the 75th and 25th percentiles (inter-quartile range, IQR). The “whiskers” represent the most extreme points (≤ 1.5 times the IQR). Outliers are plotted outside the box. Values above 1 indicate the levels of estrogen or estrogen metabolites are higher in lung tumors. Comparisons between normal and tumor tissue were performed using the Wilcoxon signed rank test. The FDR was calculated to adjust for multiple comparisons (^*P < 0.05; ^**P < 0.01; ^***P < 0.001).

Figure 3. Impact of sex and smoking status on levels of estrogen metabolites

(A) Comparison of estrogen metabolites in normal lung tissue from women vs. men. (B) Comparison of 4-OHEs and 2-OHEs in normal lung tissue, lung tumors and urine from women with NSCLC who are never-smokers (NS) or current-smokers (CS). Box plots denote the median and the 75th and 25th percentiles (inter-quartile range, IQR). The “whiskers” represent the most extreme points (≤ 1.5 times the IQR). Outliers are outside the box. P values are based on the Wilcoxon rank sum test (^**P < 0.01).

Figure 4. Level of urinary catechol estrogens (% total estrogens and estrogen metabolites) in healthy postmenopausal Chinese (n = 20) vs. Caucasian (n = 19) American women who never smoked

The box plots denote the median and the 25th and 75th percentiles (inter-quartile range, IQR). The “whiskers” represent the most extreme points (≤ 1.5 times the IQR). Outliers are outside the boxes. P values are based on the Wilcoxon rank sum test (^*P < 0.05; ^***P < 0.001).