32P-Postlabeling analysis of DNA adducts of styrene 7,8-oxide at the O6-position of guanine.
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 (alpha-isomer) and O6-(2-hydroxyl-2-phenylethyl)-2'-deoxyguanosine 3'-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 P1 (NP1) 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 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 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 microg 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.
Otteneder
M
M
Department of Toxicology, University of Würzburg, Versbacher Strasse 9, D-97078 Würzburg, Germany.
Eder
E
E
Lutz
W K
WK
D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002273:Carcinogens / Q000737:chemistry* / Q000633:toxicity; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D004852:Epoxy Compounds / Q000737:chemistry* / Q000633:toxicity; D005260:Female; D006147:Guanine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling; D008099:Liver / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D010761:Phosphorus Radioisotopes; D051381:Rats; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet
D002273:Carcinogens; D018736:DNA Adducts; D004852:Epoxy Compounds; D010761:Phosphorus Radioisotopes; D006147:Guanine; D000255:Adenosine Triphosphate; C013690:styrene oxide; D015719:Single-Strand Specific DNA and RNA Endonucleases