DETECTION AND MEASUREMENT OF HUMAN DNA ADDUCTS

人类 DNA 加合物的检测和测量

• 批准号: 3548869
• 负责人: KURT RANDERATH
• 金额: $ 15.56万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-08-01 至 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3548869
• 关键词: DNA; aging; autoradiography; azo compounds; carbopolycyclic compound; chemical carcinogen; chemical fingerprinting; dyes; environment related neoplasm /cancer; environmental toxicology; formaldehyde; gas poisoning; laboratory mouse; laboratory rat; occupational hazard; polystyrenes; radioassay; radiotracer; thin layer chromatography; tobacco abuse

A 32P-Postlabeling method developed in the applicant's laboratory will be applied to study potential covalent DNA lesions in human white blood cells and tissues exposed to occupational or environmental mixtures of genotoxic compounds. The basic procedure entails the enzymatic digestion of the DNA preparation to be tested for the presence of adducts to deoxyribonucleoside 3'-monophosphates, conversion of these mononucleotides to 5'-32P-labeled deoxyribonucleoside 3',5'-bisphosphates, resolution of the 32P-labeled nucleotides by thin-layer chromatography, and autoradiography. 32P-Labeled adduct fractions are detected as extra spots on X-ray film and are quantitated by scintillation (Cerenkov) counting. Characteristic "fingerprints" are obtained for DNA adducts formed in vivo from authentic carcinogens or mixtures of genotoxic compounds such as cigarette smoke. The 32P-postlabeling assay will be suitable specifically for the analysis of traces of adducts in microgram amounts of human DNA. For the characterization of adducts observed in human DNA, co-chromatography and stability studies will be conducted with adducts obtained from DNA of experimental animals treated with crude environmental/occupational mixtures or authentic carcinogens known to occur in the exposure of interest. Cigarette smoking will be considered as a confounding variable on the basis of the detection of specific smoking-associated DNA adducts. The project will focus on occupational exposures to polycyclic aromatic hydrocarbons, aromatic amines/amides, azo compounds, dyestuffs, formaldehyde, and styrene. In addition, it is proposed to study whether the exposure to emissions of wood-burning stoves and the natural process of aging give rise to adducts in human DNA.

申请人实验室开发的 32P-Postlabeling 方法将 用于研究人类白细胞中潜在的共价 DNA 损伤 以及暴露于职业或环境遗传毒性混合物的组织 化合物。 基本程序包括 DNA 的酶消化 待测试脱氧核糖核苷加合物存在的制剂 3'-单磷酸，将这些单核苷酸转化为 5'-32P 标记 脱氧核糖核苷 3',5'-二磷酸，32P 标记的分辨率 通过薄层色谱法和放射自显影法检测核苷酸。 32P-标记 加合物部分被检测为 X 射线胶片上的额外斑点，并被 通过闪烁（切伦科夫）计数进行定量。 特征 “指纹”是从真实的体内形成的DNA加合物获得的 致癌物或遗传毒性化合物的混合物，例如香烟烟雾。 32P 标记后测定特别适合分析 微克量的人类 DNA 中的加合物痕迹。 为了表征在人类 DNA 中观察到的加合物， 将使用加合物进行共色谱和稳定性研究 取自经粗处理的实验动物的DNA 已知发生的环境/职业混合物或真正的致癌物质 在利益的曝光中。 吸烟将被视为 基于特定检测的混杂变量 与吸烟相关的 DNA 加合物。 该项目将重点关注多环芳烃的职业暴露 碳氢化合物、芳香胺/酰胺、偶氮化合物、染料、 甲醛和苯乙烯。 此外，建议研究是否 暴露于燃木炉的排放物和自然过程 衰老会导致人类 DNA 中产生加合物。