CYTOCHROME P450 GENE REGULATION IN LUNG

人类暴露在多种有毒或致癌的化学物质中，这些化学物质在细胞色素P450超基因家族中的酶生物激活母体化合物后产生毒性。肺部疾病会导致严重的发病率和死亡率，而特定的P450酶至少是许多这些疾病的部分原因。本实验室已证实，肺细胞色素P450酶对一种重要的肺毒素3-甲基吲哚具有生物活性，可在动物和人的特定肺细胞中产生高活性的亲电中间体。对肺组织的选择性毒性主要与P450基因在细支气管或肺泡上皮细胞中的选择性表达有关，而在肝脏或其他组织中不伴随表达。因此，P450基因在某些细胞中的表达导致这些细胞通过酶产生的活性中间产物选择性地损伤。P450基因超家族某些成员的肺特异性表达也可能导致不同的人对肺毒物的敏感性不同，但导致这些基因在肺细胞中选择性表达的生化机制尚不清楚。这项研究的假设是，某些细胞色素P450基因在人肺上皮细胞中的选择性表达是由独特的、以前未确定的转录因子驱动的；这些因子协同作用，调节两个重要的P450基因的表达，即CYP2F1和CYP4B1。这项研究的主要目标是精确确定调节肺细胞中P450基因表达的因素。这一目标将通过以下目标实现：1)克隆人肺P450基因CYP2F1和CYP4B1，鉴定这些基因的5‘调控区，并筛选已知顺式调控区的序列；2)利用人支气管上皮细胞荧光素酶结构的缺失分析和体外DNA结合试验来分析和鉴定独特的新的顺式调控区；以及3)利用DNA亲和层析技术和酵母单杂交系统来鉴定调控这些基因表达的反式调控蛋白。这些研究将提供重要的、独特的关于控制肺细胞中P450基因表达的具体机制的知识，并很可能对与细胞色素P450酶激活毒物有关的人类肺部疾病产生重大影响。

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.