DETECTION AND MEASUREMENT OF HUMAN DNA ADDUCTS

在申请人实验室开发的32p标签后方法将是

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.