Detection of PAH:DNA adducts from auto-oxidation using 32P-postlabeling

Abstract

The binding of benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 3-methylcholanthrene, benz[a]anthracene, dibenz[a,c]anthracene and phenanthrene to calf thymus DNA in vitro in the absence of enzymatic or chemical activation was investigated using the 32P-postlabeling assay. Reactions were performed in the dark or under white light in 1 ml of Tris-HCl buffer (pH 7.5), containing 150 mM KCl, 250 μg of DNA and 0.12 nmol–600 nmol of hydrocarbon. Reactions were incubated for 1 h at 37°C and the extent of hydrocarbon:DNA adduct formation was determined. With the exception of phenanthrene, all of the hydrocarbons investigated formed DNA adducts that were easily detected with the 32P-postlabeling assay. The multiplicity and level of hydrocarbon: DNA adducts varied for each hydrocarbon. A dose related increase in adduct formation was observed. Adduct levels ranged from 0.07 to 15.28 adducts per 10 7 nucleotides. Highest adduct levels were detected with 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P). Hydrocarbon: DNA adduct formation was enhanced when reactions were performed under white light. A comparison of the adduct levels formed from auto-oxidation and enzymatic activation suggests that 0.05 and 0.26% of the adducts detected in the enzymatic activation of B[a]P and DMBA, can be attributed to auto-oxidation, respectively. These data demonstrate that in the absence of enzymatic or chemical activation, polycyclic aromatic hydrocarbons can undergo autooxidation in vitro and form hydrocarbon:DNA adducts that are detectable with the 32P-postlabeling assay.