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

摘要报表 基因毒素与dna的共价结合形成dna加合物被认为是 化学诱导的致癌。细胞和组织中DNA加合物的测量提供了直接的 基因损伤的证据，以及评估暴露对人类健康风险的手段。然而， 病变器官中加合物的水平较低，以及经常可获得的DNA数量有限 使他们的准确测量成为一项极具挑战性的任务。基于微毛细管液体的方法学 本项目开发的层析与纳米电喷雾质谱联用技术 使我们能够实现对加合物的检测和量化能力 在人类暴露中经常遇到使用低微克量DNA的情况。 芳香胺，香烟烟雾，染发剂和其他环境来源中发现的化合物 曾与多种癌症有牵连，包括膀胱癌。4-氨基联苯(4-ABP)是一种重要的 这类化合物中的致癌物和最近的证据已经确定了一种潜在的关系 人膀胱癌P53基因突变热点与4-ABP加合位点的关系一个 将开发高灵敏的分析方法来定量尿路上皮中4-ABP的DNA加合物 参与吸烟/戒烟临床研究的人类志愿者细胞，以评估两者之间的联系 吸烟和致癌风险之间的关系。使用尿液作为生理介质 提供了一种非侵入性的程序，更易于纳入人体研究，以 评估环境化学品引发膀胱癌的风险。抑制病毒形成的药物 芳香胺-DNA加合物将在人类膀胱细胞系中进行测试，从而提供更广泛的 展望它们在诱发膀胱癌中的生物学意义。发展中的 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和 将使我们能够更好地理解在碱基序列和 潜在地根据DNA的特定结构域和选定的基因序列来鉴定加成。 基于差示离子迁移率光谱的其他新技术--质量法 光谱分析(DMS-MS)以及高效液相色谱-质谱仪和微核磁共振的结合，将引入新的 强大的工具，以促进和改善最先进的DNA加合物的分析。我们期待着 这项工作的结果将有助于更明确地建立DNA加合物参与的程度 研究人类癌症的病因，并界定膀胱癌和吸烟之间的关系。

Summary Statement 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.