Separation of 32P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC.
The 32P-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 32P-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 32P-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.(ABSTRACT TRUNCATED AT 250 WORDS)
King
L C
LC
Health Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711.
George
M
M
Gallagher
J E
JE
Lewtas
J
J
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D009574:Nitro Compounds / Q000032:analysis* / Q000378:metabolism; D011083:Polycyclic Compounds / Q000032:analysis* / Q000378:metabolism; D051381:Rats; D013950:Thymus Gland / Q000378:metabolism; D001335:Vehicle Emissions / Q000032:analysis
D018736:DNA Adducts; D009574:Nitro Compounds; D011083:Polycyclic Compounds; D001335:Vehicle Emissions