Validation/Application of 32P-Postlabeling Analysis for the Detection of DNA Adducts Resulting from Complex Air Pollution Sources Containing PAHs and Nitrated PAHs

Abstract

Two recent versions of the 32P-postlabeling assay (butanol extraction verses nuclease P1) have been employed to enhance the detection of polycyclic aromatic hydrocarbon (PAH)-modified DNA. Previously published studies suggest that the DNA adducts derived from N-substituted aryl compounds are poorly recovered in the nuclease P1 version of the 32P-postlabeling assay. Both versions were employed to ascertain whether the apparent differences in sensitivity could be used to diagnostically select for nitroaromatic-derived DNA adducts formed following in vitro (calf thymus DNA) and in vivo (rodent) exposure to complex air pollution particle extracts. DNA adduct levels and patterns of radioactivity were compared to examine putative DNA adducts resulting from treatment with combustion emission extracts with and without mono- or dinitropyrenes. Following topical application (50 mg) with nitrated PАН containing particle extract (diesel) and complex mixture particle extract without nitrated PAH’s (coke oven and coal soot) multiple DNA adducts were detected along a zone of radioactivity, irrespective of the version of the assay employed. The patterns of radioactivity, however, were characteristically different for each of the complex mixture-modified DNA samples. Nuclease P1-sensitive adducts were not apparent in any of the mouse skin DNA samples examined. In contrast, calf thymus DNA incubated with xanthine oxidase (XO) and nitrated-PAH containing particle extracts, resulted in the formation of DNA adducts detectable only by the butanol extraction version of the 32P-postlabeling assay. These nuclease P1-sensitive adducts were chromatographically similar to mono- and dinitropyrene-modified DNA adducts formed following in vitro incubation of rabbit tracheal epithelial cells (RTE) with 1,6- and 1,8 dinitropyrene (10 μM). Our data suggest that under optimal nitroreducing conditions, the in vitro calf thymus DNA model described in this study can enhance the diagnostic potential of the 32P-postlabeling assay by the identification of nuclease P1-sensitive, N-substituted aryl-derived DNA adducts from the array of PAH-adducts formed following in vivo exposure to a variety of air pollution particle extracts.