Enzyme-mediated phosphorylation of polycyclic hydrocarbon metabolites: detection of non-adduct compounds in the 32P-postlabelling assay.
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 32P from [gamma-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 microCi[gamma-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 electrophiles in vitro may, if inadequately purified, give rise to spurious radioactive products.
Masento
M S
MS
Chester Beatty Laboratory, Institute of Cancer Research, London, UK.
Hewer
A
A
Grover
P L
PL
Phillips
D H
DH
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D001564:Benzo(a)pyrene / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D002911:Chrysenes / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D010616:Phenanthrenes / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011865:Radioisotope Dilution Technique
D002911:Chrysenes; D010616:Phenanthrenes; D010761:Phosphorus Radioisotopes; C031180:chrysene; D001564:Benzo(a)pyrene; D000255:Adenosine Triphosphate; D004247:DNA