FUNCT. CHARACTERIZATION OF HUMAN CYP2A6 GENETIC VARIANTS

功能。

• 批准号: 6206980
• 负责人: Jun-Yan Hong
• 金额: $ 27.61万
• 依托单位: UNIV OF MED/DENT NJ-SCH OF PUBLIC HEALTH
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2003-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6206980
• 关键词: African American; alpha benzopyrone; cancer risk; caucasian American; chemical stability; clinical research; cytochrome P450; cytotoxicity; drug metabolism; genetic markers; genetic polymorphism; genetic screening; genotype; human subject; neoplasm /cancer genetics; nicotine; nitrosamines; northern blottings; point mutation; site directed mutagenesis; smoking; tobacco abuse; urinalysis

DESCRIPTION: (Adapted from the Investigator's Abstract) Cigarette smoking is a major risk factor of human cancers. Human cytochrome P4502A6 (CYP2A6) plays a predominant role in the metabolic activation of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK), two major carcinogenic tobacco-specific nitrosamines, as well as in the metabolism of nicotine which is responsible for smoking addiction. Existence of functional genetic polymorphism of CYP2A6 is strongly suggested by the reports on large inter-individual variations (up to 170-fold) in CYP2A6 activity (assayed as coumarin 7-hydroxylase) in general populations, and on bimodal activity distribution. There area a total of 6 genetic variants on human CYP2A6, including 3 missense variants we recently identified. One of the reported missence variants (2A6v1, leu160His) lacked coumarin 7-hydroxylase activity. However, the activities of 2A6v1 and other variants in metabolizing NNN, NNK, and nicotine as well as their importance in tobacco-related carcinogenesis have not been investigated. Our working hypothesis is that functional genetic polymorphisms of CYP2A6, such as 2A6v1, significantly affects an individual's ability to metabolize NNN, NNK, and nicotine. Therefore, it could be an important determinant in human susceptibility to tobacco-related carcinogenesis. As an essential step to our working hypothesis, the present proposal focuses on the functional characterization of CYP2A6 genetic variants with the following specific aims: 1. To characterize the functional significance of CYP2A6 missense genetic variants through: (a) generation of the variant proteins by site-directed mutagenesis and heterologous expression; (b) comparison of the enzyme kinetics of the variants with the wild-type protein in metabolizing NNN, NNK and nicotine; (c) comparison of NNN- or NNK-induced cytotoxicity in cells transfected with either wild-type or variant CYP2A6 cDNAs; and (d) determination of protein stability of the missense variants. 2. To determine the allelic frequency distribution of functional CYP2A6 polymorphisms in different ethnic populations. This study will provide background information for future epidemiological studies, and provide the genotyped subjects for the human study proposed in specific aim 3. 3. To determine the impact of functional YCP2A6 genetic polymorphisms on the in vivo metabolism of nicotine and coumarin, both probe drugs of CYP2A6, by correlating the urinary metabolic ratio (MR) of nicotine and coumarin with the CYP2A6 polymorphic genotypes in volunteer subjects. Individuals with significant MR alterations not without known CYP2A6 polymorphisms will be used to identify new CYP2A6 variants. Our proposed studies are critical to elucidate the functional importance of CYP2A6 genetic polymorphism and help asses the possibility of using CYP2A6 genetic polymorphism as a susceptibility marker of tobacco-related human carcinogenesis, which could contribute significantly to cancer prevention by identifying susceptible subpopulations.

描述：（改编自研究者摘要）吸烟是一种 人类癌症的主要危险因素。人细胞色素P4502 A6（CYP 2A 6）在细胞内发挥重要作用。 在N '-亚硝基去甲烟碱（NNN）的代谢活化中起主要作用， 4-（甲基亚硝胺基）-1-（3-吡啶基）-1-丁酮（NNK），两种主要致癌物质 烟草特有的亚硝胺，以及尼古丁的代谢， 是导致吸烟成瘾的原因 CYP 2A 6的功能遗传多态性的存在是强烈建议， 关于CYP 2A 6个体间变异较大（高达170倍）的报告 活性（测定为香豆素7-羟化酶）在一般人群中， 双峰活性分布共有6个基因变异， 人CYP 2A 6，包括我们最近发现的3个错义变体。之一 报道的错义变体（2A 6v 1，leu 160 His）缺乏香豆素7-羟化酶 活动然而，2A 6v 1和其他变体在代谢中的活性 NNN、NNK和尼古丁以及它们在烟草相关疾病中的重要性 致癌作用尚未研究。我们的假设是 CYP 2A 6的功能基因多态性，如2A 6v 1， 影响个体代谢NNN、NNK和尼古丁的能力。 因此，它可能是人类易感性的一个重要决定因素， 烟草相关的致癌作用。作为我们工作假设的重要一步， 目前的建议集中在CYP 2A 6的功能特性 具有以下特定目的的遗传变异： 1.研究CYP 2A 6错义基因的功能意义。 变体通过：（a）通过定点突变产生变体蛋白质， 诱变和异源表达;（B）比较酶动力学 具有野生型蛋白质的变体在代谢NNN、NNK和 （c）比较NNN或NNK诱导的细胞毒性 用野生型或变体CYP 2A 6 cDNA转染;和（d） 确定错义变体的蛋白质稳定性。2.以确定 CYP 2A 6功能性多态性等位基因频率分布 不同的种族人口。这项研究将提供背景资料， 为未来的流行病学研究，并提供基因分型的主题， 在具体目标3中建议进行人体研究。3.为了确定 功能性YCP 2A 6基因多态性对尼古丁体内代谢的影响 CYP 2A 6探针药物香豆素与尿代谢的相关性 尼古丁和香豆素的比值（MR）与CYP 2A 6多态性基因型， 志愿者受试者。具有显著MR改变的个体， 已知的CYP 2A 6多态性将用于鉴定新的CYP 2A 6变体。 我们提出的研究对于阐明 CYP 2A 6基因多态性，帮助评估使用CYP 2A 6的可能性 遗传多态性作为烟草相关人群易感性标记 致癌作用，这可能有助于显着预防癌症， 识别易感亚群。