Novel approach to measure DNA adducts in humans

描述(申请人提供)：这项申请的广泛、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害的化学标记，以评估致癌物质的风险。由于缺乏明确识别和定量体内DNA加合物的分析方法，以及无法轻松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。我们推测，由于高效液相色谱/电喷雾电离-质谱仪(LC/ESI-MS)在灵敏度方面的最新进展，可以在暴露于烟草烟雾和饮食相关致癌物的个体口腔脱落细胞中鉴定和定量DNA加合物。要成功地利用口腔黏膜细胞作为可靠的组织来测量DNA加合物，需要优化分析MS方法，并建立分离人口腔黏膜细胞DNA的标准化方法。为了解决这一假设，其具体目标是：1)建立超痕量LC/ESI-MS方法来测量烟草烟雾中发现的疑似人类致癌物的DNA加合物，包括：4-甲基亚硝氨基-1-(3-吡啶)-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；以及烤肉中形成的丰富的杂环芳胺(HAAs)致癌物质；2)与分离口腔细胞相比，细胞刷洗和漱口水洗涤技术将被用于分离口腔细胞，从而最大限度地回收人类DNA，并将细菌DNA污染降至最低。口腔细胞DNA分离方法的标准化将使对遗传毒素暴露和DNA损伤的可靠评估成为可能；以及3)招募志愿者，包括吸烟者、非吸烟者和肉食者，并用LC/ESI-MS探测口腔细胞DNA加合物。加合物的形成将与接触烟草的唾液尼古丁水平和饮食中的哈斯酸相关。这项研究的结果有望表明，口腔细胞DNA加合物的LC/ESI-MS定量分析可用于评估遗传毒素造成的损害，并通过验证脱落细胞样本来减少风险评估程序的侵袭性。这项研究对公众健康的影响是开发了可用于生物监测和风险评估的新分析工具，这些工具检查对遗传毒物的暴露，评估基因-环境相互作用和癌症的决定因素，或化学预防效果。

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.