Humans are exposed to a large variety of pneumotoxic or carcinogenic chemicals that exert their toxicities after bioactivation of the parent compound by enzymes in the cytochrome P450 supergene family. Lung diseases cause significant morbidity and mortality, and specific P450 enzymes are at least partially responsible for many of these diseases. Our laboratory has demonstrated that pulmonary cytochrome P450 enzymes bioactive one important pneumotoxin, 3-methylindole, to highly reactive electrophilic intermediates in specific lung cells of animals and man. Selective toxicity to lung tissues is primarily related to the selective expression of P450 genes in bronchiolar or alveolar epithelial cells, without concomitant expression in liver or other tissues. Thus, the expression of P450 genes in certain cells leads to the selective damage of these cells by reactive intermediates produced by the enzymes. It is also likely that lung-specific expression of certain members of the P450 gene superfamily by different individuals leads to differential human susceptibilities to pneumotoxicants, but the biochemical mechanisms responsible for the selective expression of these genes in lung cells are not known. The hypothesis of this research is that the selective expression of certain cytochrome P450 genes in human lung epithelial cells is driven by unique, previously uncharacterized transcription factors; these factors act in concert to regulate the expression of two important P450 genes, CYP2F1 and CYP4B1. The major goal of this research is to precisely determine the factors that regulate the expression of P450 genes in lung cells. This goal will be realized through the following objectives: 1) clone the human pulmonary P450 genes CYP2F1 and CYP4B1, characterize the 5' regulatory regions of the genes, and screen the sequences for known cis-regulatory domains; 2) utilize deletion analysis with luciferase constructs in human bronchial epithelial lung cells and in vitro DNA binding assays to analyze and identify unique, new cis-regulatory domains; and 3) employ DNA affinity chromatography techniques and the yeast one-hybrid system to characterize the trans-regulatory proteins that regulate expression of these genes. These studies will provide vital, unique knowledge concerning the specific mechanisms that control expression of P450 genes in lung cells and will most likely have significant impact on human lung diseases that are related to bioactivation of toxicants by cytochrome P450 enzymes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL060143-04
Application #
6629000
Study Section
Alcohol and Toxicology Subcommittee 4 (ALTX)
Program Officer
Gail, Dorothy
Project Start
2000-02-01
Project End
2004-06-30
Budget Start
2003-02-01
Budget End
2004-06-30
Support Year
4
Fiscal Year
2003
Total Cost
$338,475
Indirect Cost
Name
University of Utah
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Behrendorff, James B Y H; Moore, Chad D; Kim, Keon-Hee et al. (2012) Directed evolution reveals requisite sequence elements in the functional expression of P450 2F1 in Escherichia coli. Chem Res Toxicol 25:1964-74
Sun, Hao; Moore, Chad; Dansette, Patrick M et al. (2009) Dehydrogenation of the indoline-containing drug 4-chloro-N-(2-methyl-1-indolinyl)-3-sulfamoylbenzamide (indapamide) by CYP3A4: correlation with in silico predictions. Drug Metab Dispos 37:672-84
Kartha, Jaya S; Yost, Garold S (2008) Mechanism-based inactivation of lung-selective cytochrome P450 CYP2F enzymes. Drug Metab Dispos 36:155-62
Biggs, Jason S; Wan, Jie; Cutler, N Shane et al. (2007) Transcription factor binding to a putative double E-box motif represses CYP3A4 expression in human lung cells. Mol Pharmacol 72:514-25
Wan, Jie; Carr, Brian A; Cutler, N Shane et al. (2005) Sp1 and Sp3 regulate basal transcription of the human CYP2F1 gene. Drug Metab Dispos 33:1244-53
Poch, Mark T; Cutler, N Shane; Yost, Garold S et al. (2005) Molecular mechanisms regulating human CYP4B1 lung-selective expression. Drug Metab Dispos 33:1174-84
Kassahun, Kelem; Skordos, Konstantine; McIntosh, Ian et al. (2005) Zafirlukast metabolism by cytochrome P450 3A4 produces an electrophilic alpha,beta-unsaturated iminium species that results in the selective mechanism-based inactivation of the enzyme. Chem Res Toxicol 18:1427-37
Simmonds, Andrea C; Reilly, Christopher A; Baldwin, R Michael et al. (2004) Bioactivation of 1,1-dichloroethylene to its epoxide by CYP2E1 and CYP2F enzymes. Drug Metab Dispos 32:1032-9
Sheets, Patrick L; Yost, Garold S; Carlson, Gary P (2004) Benzene metabolism in human lung cell lines BEAS-2B and A549 and cells overexpressing CYP2F1. J Biochem Mol Toxicol 18:92-9
Carr, Brian A; Ramakanth, Swayampakula; Dannan, Ghazi A et al. (2003) Characterization of pulmonary CYP4B2, specific catalyst of methyl oxidation of 3-methylindole. Mol Pharmacol 63:1137-47

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