Novel approach to measure DNA adducts in humans

测量人类 DNA 加合物的新方法

• 批准号: 7142350
• 负责人: Robert J. Turesky
• 金额: $ 19.19万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142350
• 关键词: DNA damage; adduct; benzopyrenes; biphenyl compounds; cancer risk; chemical carcinogen; clinical research; electrospray ionization mass spectrometry; environmental exposure; human subject; liquid chromatography; meat; mouthwash; mutagens; nitrosamines; nucleic acid quantitation /detection; nutrition related neoplasm /cancer; nutrition related tag; oral mucosa; pyridoindole; smoking; technology /technique development; tissue /cell culture; tobacco; tobacco abuse

DESCRIPTION (provided by applicant): The broad, long-term objective of this application is to establish chemical markers of internal exposure and genetic damage through biomonitoring DNA adducts to assess the risk of carcinogens. Human risk assessment has been severely impeded by the paucity of analytical methods that unambiguously identify and quantitate DNA adducts in vivo, and by the inability to easily obtain relevant tissues to assess cancer development. We hypothesize that the recent advances in sensitivity of liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) instrumentation allow for the identification and quantification of DNA adducts in exfoliated buccal cells of individuals exposed to tobacco smoke and diet-related carcinogens. The successful use of buccal cells as a reliable tissue to measure DNA adducts requires optimization of the analytical MS methods and the establishment of standardized methods to isolate human buccal cell DNA. To address this hypothesis, the specific aims are: 1) To establish ultra-trace LC/ESI-MS methods to measure DNA adducts of suspected human carcinogens found in tobacco smoke that include: 4-methylnitrosamino-1-(3-pyridyl)-1-butanone, benzo(a)pyrene, 2-amino-a-carboline, 2-amino-3-methyl-a-carboline, 4-aminobiphenyl; and abundant heterocyclic aromatic amine (HAAs) carcinogens formed in grilled meats; 2) Cytobrush and mouthwash rinsing techniques will be compared to isolate buccal cells that maximize the recovery of human DNA and minimize contaminating bacterial DNA. The standardization of the buccal cell DNA isolation method will permit reliable assessment of genotoxin exposure and DNA damage; and 3) Recruit volunteers that include: smokers, non-smokers, and meat-eaters and probe for buccal cell DNA adducts by LC/ESI-MS. Adduct formation will be correlated to salivary nicotine levels for tobacco exposure and HAAs in the diet. The results of this research are expected to show that quantitative LC/ESI-MS analysis of buccal cell DNA adducts may be used to assess damage posed by genotoxins, and reduce the invasiveness of risk assessment procedures by validation of exfoliated cell specimens. The impact of this research on public health is the development of new analytical tools that may be used for biomonitoring and risk assessment, which examine exposure to genotoxicants, assess gene-environment interactions and determinants of cancer, or chemopreventive efficacy.

描述（由申请人提供）：本申请的广泛、长期目标是通过生物监测DNA加合物来建立内部暴露和遗传损伤的化学标志物，以评估致癌物的风险。由于缺乏明确鉴定和定量体内DNA加合物的分析方法，以及无法容易地获得相关组织来评估癌症发展，人类风险评估受到严重阻碍。我们假设，液相色谱/电喷雾电离质谱（LC/ESI-MS）仪器的灵敏度的最新进展允许识别和量化的个体暴露于烟草烟雾和饮食相关的致癌物脱落的口腔细胞中的DNA加合物。成功使用颊细胞作为一个可靠的组织来测量DNA加合物，需要优化的分析MS方法和建立标准化的方法来分离人颊细胞DNA。为了解决这一假设，具体的目标是：1）建立超痕量LC/ESI-MS方法来测量烟草烟雾中发现的可疑人类致癌物的DNA加合物，包括：4-甲基亚硝胺-1-（3-吡啶基）-1-丁酮、苯并（a）芘、2-氨基-a-咔啉、2-氨基-3-甲基-a-咔啉、4-氨基联苯;以及在烤肉中形成的大量杂环芳香胺（哈斯）致癌物; 2）将比较细胞刷和漱口水冲洗技术以分离口腔细胞，其最大化人DNA的回收并最小化污染的细菌DNA。口腔细胞DNA分离方法的标准化将允许可靠地评估遗传毒素暴露和DNA损伤;和3）招募志愿者，包括：吸烟者、非吸烟者和肉食者，并通过LC/ESI-MS探测口腔细胞DNA加合物。加合物形成将与烟草暴露的唾液尼古丁水平和饮食中的哈斯相关。本研究的结果预计将表明，定量LC/ESI-MS分析颊细胞DNA加合物可用于评估遗传毒素造成的损害，并通过脱落细胞标本的验证减少风险评估程序的侵入性。这项研究对公共卫生的影响是开发新的分析工具，可用于生物监测和风险评估，检查暴露于遗传毒物，评估基因-环境相互作用和癌症的决定因素，或化学预防功效。