Biochemical (postlabelling) methods for analysis of carcinogen-DNA adducts

Radioactive carcinogens have provided most of our present knowledge about the interactions between carcinogens and components of biological systems. The requirement of radioactive carcinogens restricts carcinogen-DNA binding studies to chemicals that are readily available in isotopically labelled 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 incorporated into DNA constituents by polynucleotide kinase-catalysed (32P)-phosphate transfer from (gamma-32P)ATP after exposure of the DNA, in vitro or in vivo, to a nonradioactive, covalently binding chemical; alteration of DNA nucleotides is shown by the appearance of extra spots on autoradiograms from thin-layer chromatograms of digests of the chemically modified DNA. Adduct levels are quantitated by scintillation counting. The sensitivity of the technique depends, to some extent, on the chemical structure of the adducts, in that greater sensitivity is achieved if adducts can be separated, as a class, from the normal nucleotides. An estimated 80% of all carcinogens can be separated in this way, giving rise to bulky and/or aromatic substituents in DNA. Under present conditions, one such adduct in 10(9)-10(10) normal nucleotides can be detected. A total of 41 compounds has been studied, so far. Binding to DNA of rodent liver and skin was readily detected by the 32P-postlabelling assay for all known carcinogens among these compounds, and adducts were detected in DNA from tissues of smokers.