32P-Postlabeling Analysis of DNA Adducts of Styrene 7,8-Oxide at the O6-Position of Guanine

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Abstract
A 32P-postlabeling method was established for the quantitative characterization of 2‘-deoxyguanosyl O6-adducts of styrene 7,8-oxide in DNA. The two regioisomeric adducts, O6-(2-hydroxyl-1-phenylethyl)-2‘-deoxyguanosine 3‘-phosphate (α-isomer) and O6-(2-hydroxyl-2-phenylethyl)-2‘-deoxyguanosine 3‘-phosphate (β-isomer), were synthesized and used for optimizing and quantifying the various analytical steps. The adducts were stable at pH 7 and 10, but not at pH 4. The adducts were sensitive to dephosphorylation during the standard nuclease P1 (NP1) treatment. Within 30 min, 73 and 94% of the α- and β-isomers were digested. Adducts could not be extracted into butanol, and micropreparative chromatography on reversed-phase thin layers resulted in a loss of adducts at low levels. Therefore, further methods of enrichment had to be investigated. Micropreparative reversed-phase HPLC chromatography on a C18 column resulted in a many thousand-fold purification from the normal nucleotides. Further enrichment was achieved with a mild NP1 treatment. The phosphorylation efficiency with polynucleotide kinase was 5 and 15% for the α- and β-isomers, respectively. Adduct analysis was performed with reversed-phase TLC followed by contact transfer of the origin to a polyethyleneimine-cellulose sheet and two-dimensional development. Addition of various amounts of adduct standard to the hydrolysate of 30 μg of DNA isolated from a control rat liver showed limits of detection of three and two adducts per 107 nucleotides for the α- and β-isomers, respectively. The applicability of the newly developed method was demonstrated by the DNA analysis of styrene-exposed rats.