Project 2 - 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) α-Hydroxy Glucuronides, Metabolic Profiling and Activation

摘要 烟草的使用导致了90%的肺癌。然而，个人吸烟者和 不同族裔/种族群体的成员差异很大。致癌物暴露、激活的差异 而排毒也会导致这种可变的风险。在这个提案中，我们将描述新陈代谢途径 烟草特有的肺癌致癌物NNK在日裔美国人(JA)、夏威夷原住民(NH)和白人中的分布 吸烟者。这三组人关注NNK代谢的理由是：1)CYP2A6活性和 2)CYP2A6基因拷贝数变异和其他基因缺失与肺癌的发生有关 JA和NH的功能等位基因相对较高。3)CYP2A6是NNK生物活化的催化剂；4)α- 在吸烟者中发现的羟甲基NNK葡萄糖醛酸(α-OHNNK Gluc)是一种潜在的 NNK活化的生物标志物。CYP2A6活性和基因与肺癌风险的关系是 部分原因是由于CYP2A6介导的尼古丁代谢对烟草剂量的影响。CYP2A6还 催化新陈代谢NNK。因此，P4502A6活性缺乏的个体不仅可能吸烟更少 但也可防止NNK致癌。在这个项目中，我们将检验致癌物质的假设 激活因种族而异，这种差异部分是由于P450 2A6活性的变化。 Nnk通过α-羟基化被激活，此外它被还原为nNAL，这也是一种致癌物质：激活 α-羟基化。NNK和NNALα羟化的最终产物是尼古丁的次要代谢物。 因此，不可能通过代谢物分析来量化吸烟者NNK的生物活性。这个 给吸烟者服用D4-NNK可以消除尼古丁代谢物的并发症；然而，它仍然 不能区分NNAL和NNK的α-羟化代谢产物。α-OH-NNK Gluc是一种 吸烟者体内NK型α-羟基化的独特产物及其水平应依赖于其活性。这个 以下目标的目标是：将α-OH-NNK Gluc表征为NNK激活的衡量标准，以开发 建立了NNK代谢产物图谱分析方法，并测定了细胞色素P450 2A6对NNK生物活化的贡献。目标1 将确定CYP2A6基因对服用JA吸烟者的NNKα羟化水平的影响 D4 NNK，AIM 2将在JA，NH和白人吸烟者中定量α-OH NNK Gluc，AIM 3将开发高 基于分辨率质谱学的代谢谱方法测量吸烟者的nnk生物活性。 该方法将在服用NNK和D4-NNK的大鼠以及添加D4-NNK的吸烟者的尿液中得到验证 香烟。目标4将描述接受D4-NNK(目标1)和JA的吸烟者的NNK代谢特征。 NH和白人吸烟者。

ABSTRACT Tobacco use is responsible for 90% of all lung cancers. Yet, the risk of lung cancer for individual smokers and members of different ethnic/racial groups varies significantly. Differences in carcinogen exposure, activation and detoxification contribute to this variable risk. In this proposal we will characterize the metabolic pathways of the tobacco-specific lung carcinogen, NNK, in Japanese American (JA), Native Hawaiians (NH) and White smokers. The rationale for focusing on NNK metabolism in these three groups is: 1) CYP2A6 activity and genotype is associated with lung cancer, 2) the prevalence of CYP2A6 copy number variants and other loss of function alleles is relatively high in JA and NH. 3) CYP2A6 is a catalyst of NNK bioactivation and 4) the α- hydroxymethyl NNK glucuronide (α-OHNNK Gluc) identified in smokers (prior grant period) is a potential biomarker of NNK activation. The association of CYP2A6 activity and genotype with the risk of lung cancer is in part due to the influence of CYP2A6-mediated nicotine metabolism on tobacco dose. CYP2A6 also catalyzes the metabolism NNK. Therefore, individuals deficient in P450 2A6 activity may not only smoke less but may also be protected from NNK carcinogenesis. In this project we will test the hypothesis that carcinogen activation varies by ethnicity and that this variation is in part due to variable P450 2A6 activity. NNK is activated by α-hydroxylation, in addition it is reduced to NNAL, which is also a carcinogen: activated by α-hydroxylation. The final products of NNK and NNAL α-hydroxylation are minor metabolites of nicotine. Therefore, it is not possible to quantify the NNK bioactivation in smokers by metabolite analysis. The administration of D4-NNK to smokers eliminates the complication with nicotine metabolites; however, it is still not possible to distinguish the metabolites of NNAL and NNK α-hydroxylation. The α-OH-NNK Gluc is a unique product of NNK α-hydroxylation and its level in smokers should be dependent on CYP2A6 activity. The goals of the below aims are: to characterize the α-OH-NNK Gluc as a measure of NNK-activation, to develop an NNK metabolite profiling method and to determine the contribution of CYP2A6 to NNK bioactivation. Aim 1 will determine the effect of CYP2A6 genotype on the level of NNK α–hydroxylation in JA smokers administered D4 NNK, Aim 2 will quantify α-OH NNK Gluc in JA, NH and White smokers, Aim 3 will develop a high resolution mass spectrometry based metabolic profiling method to measure NNK bioactivation in smokers. The method will be validated in urine from rats administered NNK and D4-NNK and smokers of D4-NNK spiked cigarettes. Aim 4 will characterize the NNK metabolic profile in smokers receiving D4-NNK (Aim 1) and in JA, NH and White smokers.