Separation and characterization of post-labeled DNA adducts of stereoisomers of benzo[a]pyrene-7,8-diol-9,10-epoxide by immobilized boronate chromatography and HPLC analysis

• Published in: Carcinogenesis (New York) Vol. 15; no. 5; p. 907
• Main Authors: Lau, H H, Baird, W M
• Format: Journal Article
• Language: English
• Published: England 01-05-1994
• Subjects: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide - isolation & purification; 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide - metabolism; 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide - toxicity; Animals; Benzo(a)pyrene - isolation & purification; Boron Compounds; Cells, Cultured; Chromatography, High Pressure Liquid - methods; Cricetinae; DNA - drug effects; DNA - isolation & purification; DNA - metabolism; DNA Adducts; Embryo, Mammalian; Mesocricetus; Stereoisomerism; Sulfur Radioisotopes
• ISSN: 0143-3334
• DOI: 10.1093/carcin/15.5.907

The carcinogenic polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BaP) is enzymatically activated in cells to an ultimate carcinogenic metabolite, benzo[a]pyrene-7,8-dihydrodiol-9,10- epoxide (BaPDE), which reacts with DNA to form covalent adducts involved in the initiation of cancer. Previously, a post-labeling procedure that uses adenosine-5'-O-(3'-[35S]-thiotriphosphate) was developed to facilitate adduct analysis by HPLC. The much greater carcinogenic potency of (+)-anti-BaPDE makes it essential to be able to separate and identify the adducts formed by all four BaPDE enantiomers in DNA of cells exposed to BaP. Reversed-phase HPLC (RPHPLC) resolved the major (+)-anti-BaPDE-N2-deoxyguanosine [(+)-anti-BaPDE-N2-dG] adduct from the (+)-syn-BaPDE-N2-dG adduct. However, anti-BaPDE-N2-dG adducts formed by (+)- and (-)-anti-BaPDE were not resolved. By using ion-pair RPHPLC (IP-RPHPLC) with tetrabutylammonium phosphate, the [35S]post-labeled (-)-anti-BaPDE-N2-dG adduct eluted 3 min prior to the [35S]labeled (+)-anti-BaPDE-N2-dG adduct. In contrast, the major syn-BaPDE-N2-dG adducts were resolved better by RPHPLC than by IP-RPHPLC. The difference in conditions required for optimal separation of anti- and syn-BaPDE-DNA adducts necessitated the development of an immobilized boronate chromatography technique for the separation of anti- from syn-BaPDE-DNA adducts prior to analytical HPLC analysis. At 4 degrees C and with elution buffers containing high salt concentrations, the [35S]post-labeled anti-BaPDE-DNA adducts were selectively retained by a boronate column whereas the [35S]labeled syn-BaPDE-DNA adducts were not. Analysis of the multiple BaP-DNA adducts formed in BaP-treated hamster embryo cells by these techniques gave results comparable to those obtained by other methods. The major BaP-DNA adducts were anti-BaPDE-N2-dG, 14% from (-)- and 86% from (+)-anti-BaPDE. The ability of these techniques to detect low levels of PAH-DNA adducts because of the high specific radioactivity of 35S and to separate the DNA adducts formed by stereoisomeric PAH diol epoxides adducts by boronate by stereoisomeric PAH diol epoxides adducts by boronate chromatography and HPLC will facilitate studies of the role of individual PAH-DNA adducts in the induction of biological effects such as toxicity and carcinogenesis.