Cytochrome P450 in Nicotine Metabolism

描述（由申请人提供）：在美国，吸烟每年导致40多万人死亡。尼古丁是烟草中主要的成瘾性物质，是维持吸烟行为的关键因素。尼古丁代谢的个体差异影响吸烟行为和潜在的尼古丁成瘾。尼古丁主要通过5′-氧化代谢。P450 2A6是这个反应的主要肝脏催化剂，我们最近报道P450 2A13，一个肺P450，也是一个很好的催化剂。这些研究的一个意想不到和令人兴奋的结果是，尼古丁是一种基于机制的P450 2A6和P450 2A13的（自杀）抑制剂。确定这种抑制的机制是这项资助的主要目标。然而，为了开始研究尼古丁介导的失活对整个组织代谢的影响，我们将研究P450 2A3在灌注大鼠肺中的失活。P450 2A3（与P450 2A13相同89%）是一种极好的尼古丁代谢催化剂。我们的假设是P450 2A6、P450 2A13和P450 2A3的失活是通过共同的机制发生的，失活的任何差异都是由于这些酶的特异性和催化效率的差异。尼古丁的5'-氧化，以及2'-和甲基氧化的次要途径，产生活性的亚胺离子，所有可能的灭活分子。然而，初步数据支持？5'（1'）亚胺离子，作为灭活物质。这项拨款将研究尼古丁代谢的哪个途径是导致酶失活的，以及P450 2A3失活如何影响大鼠肺的代谢能力。具体目标是：1。表征尼古丁代谢的二次产物并确定其在灭活中的作用。2. 为了确定在尼古丁介导的P450 2A酶失活过程中是否发生了血红素和/或载脂蛋白的修饰，将进行质谱和核磁共振分析。3. 建立大鼠肺作为模型系统，研究尼古丁介导的P450 2A失活在体内的潜在影响。尼古丁介导的P450 2A6和2A13失活的特征对于准确和完整地了解吸烟者的尼古丁代谢至关重要。此外，确定这种情况发生的机制将有助于设计尼古丁类似物作为酶抑制剂和治疗尼古丁成瘾的潜在药物。同样重要的是，P450 2A酶的失活会影响烟草特异性致癌物NNK和NNN的代谢。

DESCRIPTION (provided by applicant): Tobacco smoking is responsible for more than 400,000 deaths annually in the United States. Nicotine is the major addictive agent in tobacco, the key ingredient that maintains smoking behavior. Individual differences in nicotine metabolism affect smoking behavior and potentially nicotine addiction. Nicotine is primarily metabolized by 5'-oxidation. P450 2A6 is the major hepatic catalyst of this reaction, and we recently reported that P450 2A13, a lung P450, is also a good catalyst. An unexpected and exciting outcome of these studies was that nicotine was a mechanism-based (suicide) inhibitor of P450 2A6 and P450 2A13. Determining the mechanism of this inhibition is the primary goal of this grant. However, to begin to investigate the impact of nicotine-mediated inactivation on whole tissue metabolism, we will study P450 2A3 inactivation in the perfused rat lung. P450 2A3 (89% identical to P450 2A13) is an excellent catalyst of nicotine metabolism. Our hypothesis is that inactivation of P450 2A6, P450 2A13 and P450 2A3 occurs through a common mechanism and that any differences in inactivation are due to differences in the specificity and catalytic efficiency of these enzymes. Nicotine 5'-oxidation, as well as minor pathways of 2'- and methyl oxidation, generates reactive iminium ions, all possible inactivating molecules. However, preliminary data support a metabolite of the ?5'(1') iminium ion, as the inactivating species. This grant will investigate what pathway of nicotine metabolism is responsible for enzyme inactivation and how P450 2A3 inactivation affects the metabolic capacity of the rat lung. The Specific aims are: 1. To characterize the secondary products of nicotine metabolism and determine their role in inactivation. 2. To determine if modification of the heme and/or apoprotein occurs during nicotine-mediated inactivation of P450 2A enzymes, both Mass Spectrometry and NMR analyses will be carried out. 3. To establish the rat lung as a model system in which to investigate potential in vivo effects of nicotine-mediated P450 2A inactivation. The characterization of nicotine-mediated inactivation of P450 2A6 and 2A13 is critical to an accurate and complete understanding of nicotine metabolism in smokers. In addition, identifying the mechanism by which this occurs will aid in the design of nicotine analogs as enzyme inhibitors and potential medications for treatment of nicotine addiction. Also of key importance, is that the inactivation of P450 2A enzymes will impact the metabolism of the tobacco specific carcinogens, NNK and NNN.