SEPARATION OF P-32 POSTLABELED DNA-ADDUCTS OF POLYCYCLIC AROMATIC-HYDROCARBONS AND NITRATED POLYCYCLIC AROMATIC-HYDROCARBONS BY HPLC

The P-32-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO2-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a] anthracene. The NO2-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO2-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. A second NO2-PAH HPLC gradient system was developed to separate NO2-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO2-PAH-DNA adducts were compared using both adduct enhancement versions of the P-32-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO2-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the P-32-postlabeled DNA adduct standards (PAHs and NO2-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO2-PAH standards used in this study. HPLC analyses of the NO2-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts. HPLC analyses of a nuclease P1 digestion of these xanthine oxidase-derived NO2-PAH-DNA adducts resulted in a significant reduction and in some samples complete loss of adducts when compared to the HPLC profiles of the butanol-extracted samples, suggesting that these DNA adducts are derived from N-substituted aryl compounds. Rat liver S9-mediated metabolism of the described NO2-PAH standards did not produce any measurable DNA adducts using the described methodology. The results of this study demonstrate the potential of the P-32-postlabeling assay coupled to HPLC for the separation of both PAH and NO2-PAH-DNA adducts in complex environmental mixtures.