DNA DAMAGE AS A MARKER OF EXPOSURE TO BETEL QUID/TOBACCO

DNA 损伤是接触槟榔/烟草的标志

• 批准号: 3548853
• 负责人: HELMUT BARTSCH
• 金额: $ 6.68万
• 依托单位: INTERNATIONAL AGENCY FOR RES ON CANCER
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-03-01 至 1990-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3548853
• 关键词: DNA; biopsy; carcinogens; chemical addition; early diagnosis; epithelium; fluorimetry; gas chromatography mass spectrometry; genetic markers; high performance liquid chromatography; human subject; immunologic techniques; mouth neoplasms; nucleobase; radioimmunoassay; tobacco

The objective of this proposal is to develop, validate and apply methods for measuring DNA damage as biological markers of carcinogen exposure in chewers of betel quid (BQ) with or without tobacco (T). Three approaches will be used: (a) in vitro modification of DNA by BQ/T constituents, (b) detection of DNA damage and toxic effects in cultured human buccal epithelial cells and explants treated as in (a), and (c) measurement of DNA damage in exfoliated buccal epithelial cells and buccal mucosa biopsies from exposed human subjects. For (c) specimens will be collected from about 50 subjects in each of four habit groups: (i) chewers of BQ with T, (ii) chewers of BQ only; (iii) chewers of T only and (iv) subjects with no chewing and smoking habits. The following assays for DNA-damage will be studied: 1) Assays for DNA strand breaks: Fluorimetric analysis of DNA unwinding in buccal epithelial cells exposed in vitro and in vivo to BQ/T. 2) Assays for oxidative DNA base damage: Quantification of thymine glycol, and 8-hydroxyguanine levels by HPLC with electrochemical detection or by GC-MS. 3) Immunoassays for carcinogen-DNA adducts: Detection and estimation of O6- alkyldeoxyguanine residues by RIA using available poly- or monoclonal antibodies (after HPLC clean-up), immuno slot-blot techniques and immunocytological assays. 4) Detection of carcinogen-DNA adducts by 32P-postlabelling: Randerath's method with modification will be employed to detect the in vitro and in vivo modification of DNA by BQ/T constituents. 5) Structural identification of DNA base adducts: They will be identified/confirmed after formation of electron-capturing derivatives by NICI-MS and by GC-ECD comparisons with authentic carcinogen adducts. Immunoassays for relevant adducts will then be developed. This study should provide a tissue dosimeter of carcinogen exposure through the quantitation of several types of DNA adducts/modifications produced by BQ/T in human buccal epithelial cells. As such DNA lesions can now be detected with high sensitivity, their relationship to the duration and frequency of use of BQ/T and to cancer risk can be examined in molecular/epidemiological studies. By comparing DNA-base damage in the study groups i-iv, the major cancer-causing ingredients in BQ/T should be identified, and the relative contributions of tobacco and areca nut-related compounds to betel quid associated cancers should be discerned. The methodology developed should also be applicable to snuff dippers and tobacco chewers in western countries.

该建议的目的是开发，验证和应用 测量DNA损伤作为生物标记的方法 槟榔（BQ）的咀嚼（BQ）中的致癌物暴露 烟草（T）。 将使用三种方法：（a）体外 通过BQ/T成分修饰DNA，（b）检测DNA 培养的人颊上皮细胞的损害和毒性作用 和被视为（a）和（c）DNA的外植体 去角质颊上皮细胞和颊粘膜的损害 暴露于人类受试者的活检。 对于（c）标本将是 从四个习惯群中的每个人中收集了大约50名受试者：（i） bq的咀嚼t，（ii）仅bq的咀嚼； （iii）t 只有（iv）没有咀嚼和吸烟习惯的受试者。 将研究以下DNA破坏的测定法：1）测定法 对于DNA链断裂：DNA的荧光分析 在体外和体内暴露于bq/t的颊上皮细胞中。 2） 氧化DNA碱损伤的测定：定量 HPLC与胸腺乙醇和8-羟基鸟嘌呤水平 电化学检测或通过GC-MS。 3）免疫测定 致癌基因加合物：O6-的检测和估计 RIA使用可用的多聚或 单克隆抗体（HPLC清理后），免疫插槽印象 技术和免疫细胞学测定。 4）检测 32p poStlabelling的致癌物加合物：Randerath's 将采用修饰方法来检测体外 BQ/T成分对DNA的体内修饰。 5） DNA碱加合物的结构鉴定：它们将是 形成电子捕获后被识别/确认 NICI-MS的导数以及与GC-ECD的比较 正宗的致癌加合物。 相关加合物的免疫测定 然后将开发。 这项研究应提供组织 通过定量的致癌剂量计 BQ/T在IN中产生的几种类型的DNA加合物/修饰 人颊上皮细胞。 因为这样的DNA病变现在可以 以高灵敏度检测到它们与持续时间的关系 可以检查BQ/T和癌症风险的使用频率 在分子/流行病学研究中。 通过比较DNA碱 研究小组I-IV的损害，主要由癌症引起 应识别BQ/T中的成分，相对 与烟草和槟榔相关化合物的贡献 槟榔相关的癌症应分辨。 这 开发的方法也应适用于鼻烟 西方国家的烟草咀嚼。