DNA adducts of 2,3-epoxy-4-hydroxynonanal: Detection of 7-(1 ',2 '-dihydroxyheptyl)-3H-imidazo[2,1-i]purine and 1,N-6-ethenoadenine by gas chromatography negative ion chemical ionization mass spectrometry

2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxid ation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyu nsaturated fatty acids, EH is mutagenic and tumorigenic and capable of modi fying 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 sensit ive assay is needed so we can determine whether EH is involved in etheno ad duct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative i on chemical ionization/mass spectrometry assay for the analysis of 1,N-6-et henoadenine (epsilon Ade) and 7-(1',2'-dihydroxyheptyl)-3H-imidazo[2,1-i]-p urine (DHH-epsilon Ade) in DNA; both are products from the reaction of aden ine with EH. The assay entails the following sequence of steps: (1) additio n of [N-15(5)]epsilon Ade and [N-15(5)]DHH-epsilon Ade to DNA as internal s tandards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid ph ase 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 analy sis with selective ion monitoring (SIM). The limit of detection by on-colum n injection for PFB-epsilon Ade monitoring of the (M - PFB)(-) ion at m/z 1 58 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 w ere 6.3 fmol for epsilon Ade anrl 36 fmol for DHH-epsilon Ade. In calf thym us 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 9 0.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also ver ified by HPLC fluorescence analysis. indicating that EH extensively; reacts with adenine in DNA, forming etheno adducts. The high sensitivity of the a ssay suggests that it may be used in the analysis of ethenoadenine adducts in vivo.