DETECTION AND MEASUREMENT OF HUMAN DNA ADDUCTS

申请人实验室开发的32 P-标记后方法将 用于研究人类白色血细胞中潜在的共价DNA损伤 和组织暴露于职业或环境混合物的遗传毒性 化合物. 基本程序需要DNA的酶消化 用于检测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'-32 P标记的 脱氧核糖核苷3 '，5'-二磷酸，32 P标记的 通过薄层色谱法和放射自显影法测定核苷酸。 32 P标记 加合物部分在X射线胶片上被检测为额外的斑点， 通过闪烁（Cerenkov）计数定量。 特性 “指纹”是从体内形成的DNA加合物中获得的， 致癌物或遗传毒性化合物的混合物，如香烟烟雾。 32 P-标记后测定将特别适用于分析 微量的加合物在微克数量的人类DNA。 为了表征在人DNA中观察到的加合物， 将对加合物进行共色谱和稳定性研究 从用粗品处理的实验动物的DNA中获得 已知存在的环境/职业混合物或真实致癌物 利益的曝光。 吸烟将被视为 基于特定检测的混杂变量 吸烟相关的DNA加合物 该项目将侧重于多环芳烃的职业暴露 烃，芳族胺/酰胺，偶氮化合物，染料， 甲醛和苯乙烯。 此外，建议研究是否 暴露于燃烧木材的炉灶的排放物和 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.