Detection of PAH:DNA adducts from auto-oxidation using 32P-postlabeling.
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 micrograms of DNA and 0.12 nmol-600 nmol of hydrocarbon. Reactions were incubated for 1 h at 37 degrees 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 auto-oxidation in vitro and form hydrocarbon:DNA adducts that are detectable with the 32P-postlabeling assay.
Bryla
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Rutgers, State University of New Jersey College of Pharmacy, Piscataway 08855-0789.
Weyand
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D015127:9,10-Dimethyl-1,2-benzanthracene / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D001551:Benz(a)Anthracenes / Q000032:analysis / Q000378:metabolism; D001564:Benzo(a)pyrene / Q000032:analysis / Q000378:metabolism; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D018736:DNA Adducts; D066298:In Vitro Techniques; D010084:Oxidation-Reduction; D010761:Phosphorus Radioisotopes; D011083:Polycyclic Compounds / Q000032:analysis* / Q000378:metabolism
C059444:7,12-dimethylbenz(a)anthracene-DNA adduct; D001551:Benz(a)Anthracenes; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; D011083:Polycyclic Compounds; C056615:benz(a)anthracene-DNA adduct; C056612:benzo(a)pyrene-DNA adduct; C006710:benzotriphenylene; D001564:Benzo(a)pyrene; D015127:9,10-Dimethyl-1,2-benzanthracene; D004247:DNA