DNA adducts of 2,3-epoxy-4-hydroxynonanal:: Detection of 7-(1′,2′-dihydroxyheptyl)-3H-imidazo[2,1-i]purine and 1,N6-ethenoadenine by gas chromatography negative ion chemical ionization mass spectrometry

2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxidation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyunsaturated fatty acids, EH is mutagenic and tumorigenic and capable of modifying DNA bases forming etheno adducts in vitro. Recent studies showed that etheno adducts are present in tissue DNA of humans and untreated rodents, suggesting a potential endogenous role of EE-I in their formation. A sensitive assay is needed so we can determine whether EH is involved in etheno adduct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative ion chemical ionization/mass spectrometry assay for the analysis of 1,N-6-ethenoadenine (epsilon Ade) and 7-(1',2'-dihydroxyheptyl)-3H-imidazo[2,1-i]-purine (DHH-epsilon Ade) in DNA; both are products from the reaction of adenine with EH. The assay entails the following sequence of steps: (1) addition of [N-15(5)]epsilon Ade and [N-15(5)]DHH-epsilon Ade to DNA as internal standards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid phase extraction (SPE) (4) derivatization by pentafluorobenzylation (PFB), (5) separation of PFB-epsilon Ade and PFB-DHH-epsilon Ade on a Si SPE column, (6) acetonide (ACT:I formation of PFB-DHH-epsilon Ade. and (7) GC/MS analysis with selective ion monitoring (SIM). The limit of detection by on-column injection for PFB-epsilon Ade monitoring of the (M - PFB)(-) ion at m/z 158 was 30 amol and for ACT-PFB-DHH-epsilon Ade monitoring of the (M - PFB)(-) ion at m/z 328 was 0.4 fmol: the detection limits for the entire assay were 6.3 fmol for epsilon Ade anrl 36 fmol for DHH-epsilon Ade. In calf thymus DNA modified with EH at 37 degrees C: for 50 h, both epsilon Ade and DHH-epsilon Ade were detected at high levels by this method, 4.5 +/- 0.7 and 90.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also verified by HPLC fluorescence analysis. indicating that EH extensively; reacts with adenine in DNA, forming etheno adducts. The high sensitivity of the assay suggests that it may be used in the analysis of ethenoadenine adducts in vivo.