DETECTION OF MULTIPLE POLYCYCLIC AROMATIC HYDROCARBON DNA ADDUCTS BY A HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY P-32 POSTLABELING METHOD

A P-32-postlabeling procedure for identifying and quantifying hydrophobic DNA adducts was developed (by modifying the method of Randerath and co-workers) in which labeled adducts are separated by high-performance liquid chromatography (HPLC) and quantified by liquid scintillation counting. This method was first developed for fluoranthene-DNA adducts, and methods for optimal detection and quantification of DNA adducts with diol epoxide metabolites of benzo[a]pyrene (BPDE), chrysene (CHDE), and benz[a]anthracene (BADE) have now been established. Analytical conditions slightly different from those adopted for fluoranthene-DNA adducts are required for accurate quantification of BPDE-, CHDE-, and BADE-DNA adducts. In particular, HPLC analysis requires generation of nucleotide 5'-[P-32]monophosphate adducts by treatment with nuclease Pl, and polycyclic aromatic hydrocarbon adducts demonstrate variable sensitivity to nuclease Pl, mediated dephosphorylation. Thus, multiple adducts can be detected in one sample as long as the recovery of adducts under the applied conditions has been determined and chromatographic separation of labeled adducts is achieved. A battery of postlabeling assays can thus make it possible to detect optimally multiple adducts in one DNA sample. Results from these studies indicate that the HPLC-P-32-postlabeling assay is complementary to immunoassays in which related polycyclic aromatic hydrocarbon diol epoxide adducts cross-react for the quantification of adducts.