P-32-postlabeling analysis of DNA adducts of styrene 7,8-oxide at the O-6-position of guanine

A P-32-postlabeling method was established for the quantitative characteriz ation of 2'-deoxyguanosyl O-6-adducts of styrene 7,8-oxide in DNA. The two regioisomeric adducts, O-6-(2-hydroxyl-1-phenylethyl)-2'-deoxyguanosin 3'-p hosphate (alpha-isomer) and O-6-(2-hydroxyl-2-phenylethyl)-2'-deoxyguanosin e 3'-phosphate (beta-isomer), were synthesized and used for optimizing and quantifying the various analytical steps. The adducts were stable at pH 7 a nd 10, but not at pH 4. The adducts were sensitive to dephosphorylation dur ing the standard nuclease P1 (NP1) treatment. Within 30 min, 73 and 94% of the alpha- and beta-isomers were digested. Adducts could not be extracted i nto butanol, and micropreparative chromatography on reversed-phase thin lay ers 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 C-18 column resulted in a many thousand-fold purificat ion from the normal nucleotides. Further enrichment was achieved with a mil d NP1 treatment. The phosphorylation efficiency with polynucleotide kinase was 5 and 15% for the alpha- and beta-isomers, respectively. Adduct analysi s was performed with reversed-phase TLC followed by contact transfer of the origin to a polyethyleneimine-cellulose sheet and two-dimensional developm ent. Addition of various amounts of adduct standard to the hydrolysate of 3 0 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-isom ers, respectively. The applicability of the newly developed method was demo nstrated by the DNA analysis of styrene-exposed rats.