SHORT COMMUNICATION Enzyme-mediated phosphorylation of polycyclic hydrocarbon metabolites: detection of non-adduct compounds in the 32P-postlabelling assay

2To whom correspondence should be addressed 32P-Postlabelling analysis is a sensitive method of detecting covalent modification of DNA by chemical carcinogens. We demonstrate that tetrol derivatives of the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene (BP) and chrysene become 32P-labelled in the assay in the absence of nucleic acids. The transfer of MP from [7-32P]ATP to the PAH derivatives requires T4 polynucleotide kinase. Phosphorylated dihydrodiols, phenols, triols and parent hydrocarbons were not detected under standard TLC conditions. Labelling of the non-nucleotide substrates was at least 2000-fold less efficient than labelling of a synthetic BP-DNA adduct. Using 75 /*Ci[7-32P]ATP, the detection limit for BP tetrols was 100-200 pg. Labelling of non-adduct substrates is unlikely to interfere with the analysis of DNA isolated from mammalian tissues, but DNA modified by etectrophiks in vitro may, if inadequately purified, give rise to spurious radioactive products. The majority of chemical carcinogens exert their biological effects through the covalent binding of reactive species to cellular DNA to form chemically-stable, altered nucleotides (adducts) (1). Several analytical methods have been developed to detect such adducts in animals and man including immunological (2,3), physico-chemical (4,5) and postlabelling (6—10) techniques. P-Postlabelling analysis has proved to be a highly sensitive method of detecting aromatic DNA adducts and is a technique mat is applicable to the study of DNA adduct formation resulting from exposure to complex mixtures of carcinogens, such as cigarette smoke (11 —13), petrol and diesel exhausts (14,21) and tars (15,16). The ^P-postlabelling assay involves digestion of die isolated DNA using micrococcal nuclease and spleen phosphodiesterase to nucleoside 3'-monophosphates, 5'-phosphorylation using T4 polynucleotide kinase and [7-32P]ATP, resolution of the P