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.
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