Postlabeling Methods for Carcinogen-DNA Adduct Analysis

• Published in: Environmental health perspectives Vol. 62; pp. 57 - 65
• Main Authors: Randerath, Kurt, Randerath, Erika, Agrawal, Hari P., Gupta, Ramesh C., Schurdak, Mark E., Reddy, M. Vijayaraj
• Format: Journal Article
• Language: English
• Published: United States National Institute of Environmental Health Sciences. National Institutes of Health. Department of Health, Education and Welfare 01.10.1985
• Subjects: Adduct Detection and Identification; Adducts; Aflatoxins; Animals; Carcinogenicity; Carcinogens; Carcinogens - metabolism; Chemicals; DNA; DNA - isolation & purification; DNA - metabolism; DNA adducts; Dosage; Kinetics; Liver; Liver - metabolism; Mice; Nucleotides; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Rats; Skin - metabolism; Structure-Activity Relationship
• ISSN: 0091-6765; 1552-9924
• DOI: 10.1289/ehp.856257

Radioactive carcinogens have provided most of our present knowledge about the chemistry of interactions between carcinogens and biological systems. The requirement of radioactive carcinogens has restricted carcinogen-DNA binding studies to chemicals that are readily available in isotopically labeled form, i.e., a minute fraction of all potentially mutagenic or carcinogenic chemicals. To extend the scope of carcinogen-DNA binding studies, an alternative method, which does not require radioactive test chemicals, has been developed. In this approach, radioactivity (32P) is being incorporated into DNA constituents by polynucleotide kinase-catalyzed [32P]phosphate transfer from [γ -32P] ATP after exposure of the DNA in vitro or in vivo to a nonradioactive, covalently binding chemical, and evidence for the alteration of DNA nucleotides is provided by the appearance of extra spots on autoradiograms of thin-layer chromatograms of digests of the chemically modified DNA. Quantitation of adduct levels is accomplished by scintillation counting. The sensitivity of the technique depends on the experimental conditions for32P-labeling and on the chemical structure of the adducts. Greater sensitivity may be achieved if adducts can be separated as a class from the normal nucleotides. This is the case for an estimated 80% of all carcinogens, giving rise to bulky and/or aromatic substituents in DNA. Under the present conditions, one such adduct in 109to 1010normal nucleotides can be detected. A total of approximately 80 compounds has been studied thus far. Binding to DNA of rodent tissues was readily detected by the32P-postlabeling assay for all known carcinogens among these compounds, and adducts were detected in DNA from human placenta of smokers.