DETECTION AND MEASUREMENT OF HUMAN DNA ADDUCTS

在申请人的实验室开发的32P后标记方法将是 用于研究人类白细胞中潜在的共价DNA损伤 以及暴露在职业或环境中的遗传毒性混合物中的组织 化合物。基本程序需要对DNA进行酶消化 用于检测是否存在脱氧核糖核苷加合物的制剂 3‘-单磷酸，这些单核苷酸转化为5’-32P标记 脱氧核糖核苷3‘，5’-二磷酸，~(32)P标记的拆分 用薄层层析法和放射自显影技术测定核苷酸。32P标记 加合物部分在X射线胶片上被检测为额外的斑点，并被 通过闪烁(切伦科夫)计数进行定量。特点 从真品中获得体内DNA加合物的“指纹” 致癌物或遗传毒性化合物的混合物，如香烟烟雾。 ~(32)P-后标记分析将特别适用于分析 微克量的人类DNA中的加合物的痕迹。 为了表征在人类DNA中观察到的加合物， 共层析和稳定性研究将与加合物一起进行 从实验动物的DNA中提取粗品 已知的环境/职业混合物或正宗致癌物质 在利益的曝光上。吸烟将被视为一种 混杂变量在检测特异性的基础上 与吸烟有关的DNA加合物。 该项目将侧重于职业接触多环芳烃。 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，还建议研究是否 暴露在燃烧木柴的炉子的排放物和自然过程中 衰老会导致人类DNA中的加合物。

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.