Xenobiotic-metabolizing cytochrome P450 enzymes in human lung

Janne Hukkanen

Department of Pharmacology and Toxicology, University of oulu, P.O. Box 5000, FIN-90014 University of Oulu, Finland

Abstract

The cytochrome P450 (CYP) enzyme system in human lung is an essential component in the pulmonary carcinogenicity of several inhaled xenobiotic compounds. The aim of this study was to elucidate the expression and regulation of xenobiotic-metabolizing CYP enzymes in human lung.

To evaluate which of the two is a better surrogate cell model for lung tissue, the expression patterns of CYP enzymes in alveolar macrophages and peripheral blood lymphocytes were clarified by reverse transcriptase-polymerase chain reaction (RT-PCR) and compared to the expression in lung tissue. The pattern of CYP expression in alveolar macrophages was found to closely resemble the expression pattern in human lung tissue, while the pattern in lymphocytes was markedly different. The expression of CYP2B6, CYP2C, CYP3A5, and CYP4B1 mRNAs in alveolar macrophages was demonstrated for the first time.

To facilitate mechanistic studies on human pulmonary CYP induction, the A549 lung adenocarcinoma cell line was characterized by RT-PCR, and the CYP expression pattern was found to compare reasonably well to human lung epithelial cells. The induction of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) behaved as predicted, and CYP1B1 and CYP3A5 were also inducible by TCDD and dexamethasone, respectively. TCDD elevated the level of CYP1A1 mRNA (56-fold), while the induction of CYP1B1 mRNA was more modest (2.5-fold). The tyrosine kinase inhibitor genistein and the protein kinase C inhibitor staurosporine blocked CYP1A1 induction by TCDD, but did not affect CYP1B1 induction. The serine/threonine protein phosphatase inhibitor calyculin A and okadaic acid enhanced CYP1B1 induction slightly, but did not alter CYP1A1 induction.

The expression of CYP3A forms in human pulmonary tissues was studied with RT-PCR and immunohistochemistry, and both methods established CYP3A5 as the main CYP3A form. CYP3A4 was expressed in only about 20% of the cases. In A549 cells, CYP3A5 was induced about 4-fold by the glucocorticoids budesonide, beclomethasone dipropionate, and dexamethasone. Maximal induction was achieved by concentrations as low as ~100 nM, suggesting that CYP3A5 could be induced in vivo in patients using inhaled glucocorticoids. However, there were no differences in CYP3A5 expression in alveolar macrophages in current glucocorticoid users, ex-users, and non-users. Cigarette smoking had a marked decreasing effect on CYP3A5 levels in alveolar macrophages. The presence and possible induction of CYP3A5 by glucocorticoids in human lung could have consequences for the maintenance of physiological steroid hormone balance in lung and the individual susceptibility to lung cancer of patients using glucocorticoids.

Table of Contents
Acknowledgments
Abbreviations
List of original articles
1. Introduction
2. Review of the literature
2.1. Cytochrome P450 (CYP) enzymes
2.2. Human xenobiotic-metabolizing CYP enzymes
2.2.1. CYP1 family
2.2.2. CYP2 family
2.2.3. CYP3 family
2.2.4. CYP4B1
2.2.5. Novel CYPs
2.3. Regulation of human xenobiotic-metabolizing CYP expression
2.3.1. Aryl hydrocarbon receptor (AHR)
2.3.2. Pregnane X receptor (PXR)
2.3.3. Constitutively active receptor (CAR)
2.3.4. Glucocorticoid receptor (GR)
2.3.5. Other regulatory mechanisms for xenobiotic-metabolizing CYPs
2.4. CYP enzymes in human lung
2.4.1. General aspects of the metabolic capacity of human lung
2.4.2. Expression of xenobiotic-metabolizing CYP enzymes in human pulmonary tissues
2.4.3. Localization of individual CYP forms in human lung
2.5. Regulation of human pulmonary xenobiotic-metabolizing CYP expression
3. Aims of the present study
4. Materials and methods
4.1. Human tissue samples
4.2. Cell culture
4.3. Chemicals
4.4. Extraction of mRNA, mRNA blot analysis and cDNA synthesis
4.5. Qualitative RT-PCR
4.6. Quantitative RT-PCR
4.7. Immunohistochemistry
4.8. Statistical analysis
5. Results
5.1. Expression of xenobiotic-metabolizing CYP mRNAs in human lung tissues
5.1.1. Whole lung tissue
5.1.2. Alveolar macrophages
5.1.3. Lymphocytes
5.1.4. A549 cells
5.2. Expression of CYP3A4 and CYP3A5 in human lung
5.3. Localization of CYP3A4 and CYP3A5 in human lung
5.4. Induction of CYP1 and CYP3A mRNAs in A549 cells
5.5. Modulation of TCDD-induced CYP1A1 and CYP1B1 in A549 cells
5.6. Characterization of the mechanism of CYP3A5 induction by glucocorticoids in A549 cells
5.7. Effect of inhaled glucocorticoids and smoking on the alveolar macrophage CYP3A5 mRNA levels
6. Discussion
7. Conclusions
References
List of Tables
1. Human CYP families and their main functions. Data adapted from (Gonzalez 1992, Nelson et al. 1996, White et al. 1997, Nelson 1999, Lund et al. 1999).
2. Summary of expression of CYPs in human lung. -- moderate negative evidence, - weak negative evidence, +/- conflicting evidence, + weak positive evidence, ++ moderate positive evidence, +++ strong positive evidence. For references, see chapter 2.4.2.
3. Expression of CYP mRNAs in lung tissue, alveolar macrophages, peripheral lymphocytes, and A549 cells (articles I and III).
List of Figures
1. Schematic presentation of the mechanism of AHR-mediated CYP1 induction.
2. Schematic presentation of the mechanism of PXR-mediated CYP3A induction.
3. Schematic presentation of the proposed mechanism of CAR-mediated CYP2B6 induction.
4. Schematic presentation of the possible role of phenobarbital on CYP2B6 induction.
5. Schematic presentation of the mechanism of GR-mediated CYP3A5 induction.
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