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

摘要 吸烟是导致90%肺癌的原因。然而，个别吸烟者患肺癌的风险和 不同族裔/种族群体的成员差别很大。致癌物暴露、活化的差异 和解毒作用会增加这种可变风险。在本提案中，我们将描述代谢途径 在日裔美国人（JA）、夏威夷土著人（NH）和白色人中， 吸烟者。在这三个组中关注NNK代谢的基本原理是：1）CYP 2A 6活性， 基因型与肺癌相关，2）CYP 2A 6拷贝数变异和其他缺失的患病率， 功能等位基因在JA和NH中相对较高。3)CYP 2A 6是NNK生物活化的催化剂，4）α- 在吸烟者中发现的羟甲基NNK葡萄糖醛酸苷（α-OHNNK Gluc）（前授权期）是一种潜在的 NNK活化的生物标志物。CYP 2A 6活性和基因型与肺癌风险的关系是 部分原因是CYP 2A 6介导的尼古丁代谢对烟草剂量的影响。CYP 2A 6也 催化代谢NNK。因此，缺乏P450 2A 6活性的个体不仅吸烟量减少， 但也可保护其免于NNK致癌作用。在这个项目中，我们将测试致癌物质 激活因种族而异，这种变化部分是由于可变的P450 2A 6活性。 NNK通过α-羟基化活化，此外还还原为NNAL，NNAL也是一种致癌物质： α-羟基化。NNK和NNAL α-羟基化的最终产物是尼古丁的次要代谢产物。 因此，不可能通过代谢物分析定量吸烟者中的NNK生物活化。的 向吸烟者施用D4-NNK消除了尼古丁代谢物的并发症;然而， 无法区分NNAL和NNK α-羟基化的代谢产物。α-OH-NNK Gluc是一种 NNK α-羟基化的独特产物，其在吸烟者中的水平应取决于CYP 2A 6活性。的 以下目标的目标是：表征α-OH-NNK Gluc作为NNK活化的量度， NNK代谢物分析方法，并确定CYP 2A 6对NNK生物活化的贡献。要求1 将确定CYP 2A 6基因型对JA吸烟者中NNK α-羟基化水平的影响， D4 NNK，Aim 2将定量JA、NH和白色吸烟者中的α-OH NNK Gluc，Aim 3将产生高水平的 基于分辨率质谱的代谢谱分析方法来测量吸烟者中的NNK生物活化。 该方法将在给予NNK和D4-NNK的大鼠以及加标D4-NNK的吸烟者的尿液中进行验证 香烟目的4将表征接受D4-NNK的吸烟者（目的1）和JA中的NNK代谢谱， NH和白色吸烟者。

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.