Detection and quantification of 1,N6-ethenoadenine in human placental DNA by mass spectrometry

Exocyclic DNA adducts have been reported to derive from various exogenous as well as endogenous sources, such as lipid peroxidation. Among them, 1,N-6-ethenoadenine (epsilon Ade) has previously been detected in tissue DNA of untreated rodents and humans by an immunoaffinity/P-32-postlabeling method. This study reports detection and quantification of the endogenous epsilon Ade adduct in the same human placental DNA by three independent assays, namely, GC/MS, LC/MS, and HPLC/fluorescence. Using a recently reported gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS) method [Chen, H. -J. C., et al. (1998) Chem. Res. Toxicol. 11, 1.474], the level of epsilon Ade in human placental DNA from a commercial source was found to be 2.3 adducts per 10(6) Ade bases. To confirm these findings, a liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) method was developed for epsilon dAdo. With this LC/MS assay, epsilon dAdo was detected at the level of 2.5 adducts per 10(6) dAdo nucleosides in the same human placental DNA. The stable isotopes of epsilon Ade and epsilon dAdo were added as internal standards in both GC/MS and LC/ESI/MS/MS assays, respectively, and thus provided high specificity, reproducibility, and accurate quantification. The relatively high levels of epsilon Ade in this human placental DNA detected by mass spectrometry were further verified by HPLC/fluorescence analysis. The GC/MS method was validated by the HPLC/fluorescence assay using calf thymus DNA treated with chloroacetaldehyde or by the LC/MS method with 2,3-epoxy-4-hydroxynonanal-modified calf thymus DNA. The epsilon Ade level, in human placental DNA freshly isolated in the presence of an antioxidant was similar to that in DNA from the commercial source. Since epsilon Ade is a potential mutagenic lesion, analysis of epsilon Ade by the specific and sensitive GC/NICI/MS method may provide a useful biomarker in cancer risk assessment.