Detection and quantification of 1,N(6)-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 (epsilonAde) has previously been detected in tissue DNA of untreated rodents and humans by an immunoaffinity/(32)P-postlabeling method. This study reports detection and quantification of the endogenous epsilonAde 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, 1474], the level of epsilonAde 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 epsilondAdo. With this LC/MS assay, epsilondAdo was detected at the level of 2.5 adducts per 10(6) dAdo nucleosides in the same human placental DNA. The stable isotopes of epsilonAde and epsilondAdo 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 epsilonAde 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 epsilonAde level in human placental DNA freshly isolated in the presence of an antioxidant was similar to that in DNA from the commercial source. Since epsilonAde is a potential mutagenic lesion, analysis of epsilonAde by the specific and sensitive GC/NICI/MS method may provide a useful biomarker in cancer risk assessment.
Chen
H J
HJ
Department of Chemistry, National Chung Cheng University, 160 San-Hsing, Ming-Hsiung, Chia-Yi 62142, Taiwan, and Division of Carcinogenesis and Molecular Epidemiology, American Health Foundation, Valhalla, New York 10595, USA.
Chiang
L C
LC
Tseng
M C
MC
Zhang
L L
LL
Ni
J
J
Chung
F L
FL
D000225:Adenine / Q000031:analogs & derivatives* / Q000032:analysis; D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004247:DNA / Q000737:chemistry*; D018736:DNA Adducts / Q000032:analysis*; D005260:Female; D005453:Fluorescence; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D009153:Mutagens / Q000032:analysis*; D010920:Placenta / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018736:DNA Adducts; D009153:Mutagens; C004981:1,N(6)-ethenoadenine; D004247:DNA; D000225:Adenine