Nj. Gorelick et Nl. Reeder, DETECTION OF MULTIPLE POLYCYCLIC AROMATIC HYDROCARBON DNA ADDUCTS BY A HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY P-32 POSTLABELING METHOD, Environmental health perspectives, 99, 1993, pp. 207-211
A P-32-postlabeling procedure for identifying and quantifying hydropho
bic DNA adducts was developed (by modifying the method of Randerath an
d co-workers) in which labeled adducts are separated by high-performan
ce liquid chromatography (HPLC) and quantified by liquid scintillation
counting. This method was first developed for fluoranthene-DNA adduct
s, and methods for optimal detection and quantification of DNA adducts
with diol epoxide metabolites of benzo[a]pyrene (BPDE), chrysene (CHD
E), 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 s
ensitivity to nuclease Pl, mediated dephosphorylation. Thus, multiple
adducts can be detected in one sample as long as the recovery of adduc
ts under the applied conditions has been determined and chromatographi
c 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 rela
ted polycyclic aromatic hydrocarbon diol epoxide adducts cross-react f
or the quantification of adducts.