(32)P-Postlabeling Analysis of DNA Adducts of Styrene 7,8-Oxide at the O^6-Position of Guanine

A(32) P-postlabeling method was established for the quantitative characterization of 2ʹdeoxyguanosyl O^6-adducts of styrene 7,8-oxide in DNA. The two regioisomeric adducts, O^6-(2hydroxyl-l-phenylethyl)-2ʹ-deoxyguanosine Sʹ-phosphate ((alpha)-isomer) and O^6-(2-hydroxyl-2phenylethyl)-2ʹ-deoxyguanosine 3ʹ-phosphate ((beta)-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 PI (NPI) treatment. Within 30 min, 73 and 94% of the (alpha)- and (beta)-isomers were digested. Adducts could not be extracted into butanol, and micropreparative chromatography on reversedphase 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 NPI treatment. The phosphorylation efficiency with polynucleotide kinase was 5 and 15% for the (alpha)- and (beta)-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 (mu)g of DNA isolated from a control rat liver showed limits of detection of three and two adducts per 10^7 nucleotides for the (alpha)- and (beta)-isomers, respectively. The applicability of the newly developed method was demonstrated by the DNA analysis of styrene-exposed rats.