Analysis of benzo(a)pyrene:DNA adducts formed in cells in culture by immobilized boronate chromatography.
A chromatographic procedure using boronic acid residues linked to a cellulose support [(N-(N'-[m-(dihydroxyboryl)-phenyl]succinamyl)amino]ethyl cellulose), used by Sawicki et al. (Cancer Res., 43: 3212-3218, 1983) for analysis of 7,12-dimethylbenz(a)anthracene:DNA adducts, was modified to allow the analysis of benzo(a)pyrene (BaP):DNA adducts formed in cells in culture. Adducts resulting from reaction of 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) contain cis-vicinal hydroxyl groups that complex with the boronic acid residues; adducts resulting from 7 beta, 8 alpha-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (syn-BaPDE) do not. A mixture of [3H]-syn-BaPDE:deoxyguanosine (dGuo) adduct and [14C]-anti-BaPDE:dGuo adduct was completely resolved on a column of boronate:cellulose. Early-passage cultures of Sencar mouse, Syrian hamster, and Wistar rat embryo cells and a culture of a human hepatoma cell line (Hep G2) were exposed to [3H]BaP, and the BaP:DNA adducts were resolved by boronate chromatography and high-performance liquid chromatography. The Hep G2 cells and mouse embryo cells contained two major adducts, a (+)-anti-BaPDE:dGuo adduct and a syn-BaPDE:dGuo adduct. Boronate chromatography permitted the resolution of an additional minor syn-BaPDE:deoxyribonucleoside adduct in the mouse embryo cells. The hamster and rat embryo cells contained a number of major BaP-DNA adducts that were resolved by boronate chromatography followed by high-performance liquid chromatography. The rat embryo cells contained three syn-BaPDE:deoxyribonucleoside adducts and approximately equal amounts of two adducts tentatively identified as dGuo adducts of the (+) and (-) enantiomers of anti-BaPDE. The boronate chromatography-high-performance liquid chromatography procedure improves the separation of the BaP:DNA adducts formed in biological systems and facilitates the identification of the BaP metabolite(s) responsible for the formation of these adducts.
D000818:Animals; D001564:Benzo(a)pyrene; D001580:Benzopyrenes / Q000302:isolation & purification / Q000378:metabolism*; D001897:Boronic Acids; D002273:Carcinogens / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004622:Embryo, Mammalian; D006801:Humans; D008113:Liver Neoplasms; D008647:Mesocricetus; D051379:Mice; D008815:Mice, Inbred Strains; D051381:Rats; D011919:Rats, Inbred Strains
D001580:Benzopyrenes; D001897:Boronic Acids; D002273:Carcinogens; D001564:Benzo(a)pyrene; D004247:DNA