32P-postlabelling/HPLC assay reveals an enantioselective adduct formation in N7 guanine residues in vivo after 1,3-butadiene inhalation exposure

We have established a protocol that allows qualitative and quantitative determination of butadiene monoepoxide-DNA adducts formed as a result of inhalation exposure to 1,3-butadiene. We observed that in this particular case in vivo samples required extensive sample purification to facilitate a low background. Sample preparation included a solid phase extraction carried out with a strong anion exchange column and one-dimensional ion exchange TLC. The ultimate analysis is based on reverse phase HPLC with on-line radioactivity and UV detectors. The qualitative identification and quantitation is based on characterized markers, which are used as external and internal standards. Modified 3'-dGMP markers were used to control labelling efficiency, which varies, and modified 5'-dGMP markers were used as an optical standard to qualitatively assign the products and to determine recovery of the sample preparation. Using this method we were able to demonstrate, for the first time, specific enantio- and regioisomeric adduct formation at the N7 position of guanine residues in liver DNA of rats inhalation-exposed to 1,3-butadiene. The major adduct formed was the C-2 isomer derived from the R enantiomer of butadiene monoepoxide, contributing 47% of all adducts formed at the N7 position of guanine. The relative proportions of the remaining three other adducts detected were 22 (R C-1), 18 (S C-2) and 14% (S C-1) respectively. Inhalation exposure to 200 p.p.m. for 5 days resulted in an alkylation level of 7.2 fmol/10 microg DNA or 2.4 adducts/10(-7) normal nucleotides.