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的共价结合形成DNA加合物被认为是化学诱导致癌的第一步。对细胞和组织中DNA加合物的测量提供了遗传损害的直接证据，并提供了一种评估暴露对人类健康风险的手段。然而，加合物在受影响的器官中的水平很低，而且经常可以获得的DNA数量有限，这使得准确测量加合物成为一项极具挑战性的任务。在该项目中开发的基于微毛细管液相色谱与纳米电喷雾质谱仪相结合的方法使我们能够使用低微克DNA在类似于人类经常接触的水平上实现加合物的检测和定量。芳香胺、香烟烟雾中发现的化合物、染发剂和其他环境来源都与包括膀胱癌在内的各种癌症有关。4-氨基联苯(4-ABP)是这类化合物中的一种主要致癌物质，最近的证据表明，人类膀胱癌中P53基因的突变热点与4-ABP的加合位点之间存在潜在的关系。为了评估吸烟和致癌风险之间的联系，将开发一种高灵敏的分析方案，以定量测定参与吸烟/戒烟临床研究的人类志愿者尿路上皮细胞中4-ABP的DNA加合物。使用尿液作为生理媒介提供了一种非侵入性的程序，更容易被纳入人体研究，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类膀胱细胞系中进行测试，从而为它们在诱发膀胱癌方面的生物学意义提供更广阔的前景。发展表征寡核苷酸加合物的方法学将扩大这些临床研究的广度，并将使我们能够更好地了解碱基序列背景下加合物的形成，并潜在地根据DNA的特定结构域和选定的基因序列识别加合物。本文提出的基于差示离子迁移率光谱-质谱仪(DMS-MS)的其他新技术，以及高效液相色谱与MS和微核磁共振的结合，将引入新的强大工具来促进和改善DNA加合物分析的最新技术水平。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，并确定膀胱癌与吸烟之间的关系。与公共健康相关：环境中存在的化学物质对DNA的破坏可能是致癌的第一步。这项建议涉及开发灵敏和新颖的分析方法，以在分子水平上检测和量化这一事件。这项研究调查了香烟烟雾中存在的致癌物质，它们在膀胱癌发病中的作用，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.