Figures & data
Fig. 1. LC-MS/MS analysis of 7-(2-oxo-hexyl)-εdG (OOE-dG) and 2-alkenal-derived 7-(2-oxo-alkyl)-εdG adducts.
Note: (A) LC-MS/MS chromatograms of dG adduct (m/z 390.2 → 274.2) in the reaction of dG with oxidized AA, 2-octenal, and OOE. Chemical structure of OOE-dG and the MS/MS fragmentation are also illustrated. (B) Each 2-alkenal (crotonaldehyde, 2-pentenal, 2-hexenal, 2-heptenal, 2-octenal, 2-nonenal, 20 mM) was incubated with dG (2 mM) at 37 °C for 3 days. Chromatograms at the MRM transitions for possible 4-oxo-2-alkenal-derived adducts are shown.
Fig. 2. Effects of transition metals on the formation of OOE-dG.
Note: (A) 2-Octenal (10 mM) was incubated with dG (1 mM) in the presence or absence of CuSO4 (10 μM) or FeSO4 (0.1 mM)/EDTA (0.1 mM) at 37 °C for 3 days The amount of OOE-dG was calculated based on the authentic standard (n = 3). (B) LC-MS/MS chromatograms of enzymatic hydrolysates of native calf thymus DNA (ctDNA), monitoring OOE-dG (m/z 390.1 → 274.1) and internal standard (m/z 395.1 → 279.1). (C) The amounts of OOE-dG in ctDNA treated with 2-octenal (10 mM) in the presence or absence of CuSO4 (10 μM) or FeSO4 (0.1 mM)/EDTA (0.1 mM) were determined using stable-isotope dilution assay (n = 3).
Fig. 3. LC-MS/MS analysis of semicarbazone derivatives of 2-octenal and OOE.
Note: (A) Oxidized AA and authentic 2-octenal were derivatized with semicarbazide and then analyzed by LC-MS/MS. (B) 2-Octenal was incubated in phosphate buffer at 37 °C for 24 h in the presence or absence of CuSO4 (10 μM) or FeSO4 (0.1 mM)/EDTA (0.1 mM), derivatized with semicarbazide and then analyzed by LC-MS/MS. Fragmentation patterns for the derivatives of 2-octenal and OOE are also illustrated.
Fig. 4. Proposed scheme for the formation of OOE-dG through the autoxidation of 2-octenal in the reaction of oxidized AA and dG.
Supplemental material