LC-MS Analysis of DNA Adducts of Aromatic Amines: Relationship to Bladder Cancer

描述（由申请人提供）：基因毒素与 DNA 共价结合形成 DNA 加合物被认为是化学诱导致癌的第一步。细胞和组织中 DNA 加合物的测量提供了遗传损伤的直接证据以及评估暴露造成的人类健康风险的方法。然而，受影响器官中加合物的水平较低，而且可用的 DNA 量有限，这使得精确测量成为一项极具挑战性的任务。该项目中开发的基于微毛细管液相色谱法与纳米电喷雾质谱法相结合的方法使我们能够实现加合物的检测和定量能力，其水平类似于使用低微克量 DNA 的人类暴露中经常遇到的水平。芳香胺、香烟烟雾、染发剂和其他环境来源中发现的化合物与包括膀胱癌在内的多种癌症有关。 4-氨基联苯 (4-ABP) 是此类化合物中的主要致癌物，最近的证据已经确定了人类膀胱癌中 p53 基因的突变热点与 4-ABP 内收位点之间的潜在关系。将开发一种高度灵敏的分析方案，对参与吸烟/戒烟临床研究的人类志愿者的尿路上皮细胞中 4-ABP DNA 加合物进行定量，以评估吸烟与致癌风险之间的联系。使用尿液作为生理介质提供了一种非侵入性程序，更适合纳入人体研究以评估环境化学物质导致膀胱癌的风险。抑制芳香胺-DNA 加合物形成的药物将在人膀胱细胞系中进行测试，从而为它们在诱导膀胱癌中的生物学意义提供更广泛的视角。开发表征寡核苷酸加合物的方法将扩大这些临床研究的广度，并使我们能够更好地了解碱基序列背景下的加合物形成，并有可能根据 DNA 的特定域和选定的基因序列识别加合物。这里提出的基于微分离子迁移谱-质谱 (DMS-MS) 以及 HPLC 与 MS 和 microNMR 耦合的其他新技术将引入新的强大工具，以促进和改进 DNA 加合物分析的最新技术。我们期望这项工作的结果将有助于更明确地确定 DNA 加合物在人类癌症病因学中的参与程度，并确定膀胱癌和香烟烟雾之间的关系。公共健康相关性：环境中存在的化学物质对 DNA 的损害可能是致癌的第一步。该提案致力于开发灵敏且新颖的分析方法，以在分子水平上检测和量化该事件。该研究调查了香烟烟雾中存在的致癌物质、它们在膀胱癌发生中的作用以及防止 DNA 损伤的方法。

DESCRIPTION (provided by applicant): The covalent binding of genotoxins to DNA to form DNA adducts is considered to be a first step in chemically induced carcinogenesis. Measurement of DNA adducts in cells and tissues provides direct evidence of genetic damage and a means for assessing human health risk from exposure. However, the low levels at which adducts occur in affected organs and the limited amounts of DNA frequently available make their accurate measurement a highly challenging task. Methodology based on microcapillary liquid chromatography in combination with nano-electrospray mass spectrometry developed in this program has enabled us to achieve detection and quantification capabilities of adducts at levels similar to those frequently encountered in human exposure using low microgram quantities of DNA. Aromatic amines, compounds found in cigarette smoke, hair dyes and other environmental sources have been implicated in a variety of cancers including bladder cancer. 4-Aminobiphenyl (4-ABP), is a major carcinogen in this class of compounds and recent evidence has identified a potential relationship between mutational hot spots of the p53 gene in human bladder cancer and adduction sites of 4-ABP. A highly sensitive analytical protocol will be developed to quantify the DNA adducts of 4-ABP in urothelial cells from human volunteers involved in a smoke / cessation clinical study in order to assess the link between cigarette smoking and the risk for carcinogenesis. Use of urine as the physiological medium provides a non-invasive procedure which is more amenable for incorporation into human studies to assess the risk for bladder cancer from environmental chemicals. Agents which inhibit the formation of aromatic amine-DNA adducts will be tested in a human bladder cell line, thereby providing a broader perspective of their biological significance in their induction of bladder cancer. Development of methodology to characterize oligonucleotide adducts will expand the breadth of these clinical studies and will allow us to gain a better understanding of adduct formation in the context of base sequence and potentially identify the adduction in terms of specific domains of DNA and selected gene sequences. Additional novel technologies proposed here based on differential ion mobility spectrometry - mass spectrometry (DMS - MS) and the coupling of HPLC with MS and microNMR, will introduce new powerful tools to facilitate and improve the state of the art in the analysis of DNA adducts. We expect that the results of this work will help establish more definitively the degree of involvement of DNA adducts in the etiology of human cancer and define the relationship between bladder cancer and cigarette smoke. PUBLIC HEALTH RELEVANCE: Damage of DNA from chemicals present in the environment is potentially the first step to carcinogenesis. This proposal addresses the development of sensitive and novel analytical methodology to detect and quantify this event at the molecular level. The study investigates carcinogens present in cigarette smoke, their role in the initiation of bladder cancer and approaches to prevent the infliction of DNA damage.