4-hydroxy-2-nonenal and ethyl linoleate form N2,3-ethenoguanine under peroxidizing conditions

In these studies, we demonstrate that N-2,3-ethenoguanine (N-2,3-epsilon Gua) is formed from lipid peroxidation as well as other oxidative reactions. Ethyl Linoleate (EtLA) or 4-hydroxy-2-nonenal (HNE) was reacted with dGuo in the presence of tert-butyl hydroperoxide (t-BuOOH) for 72 h at 50 degreesC. The resulting N-2,3-(epsilon)Gua was characterized by liquid chromatography/electrospray mass spectroscopy and by gas chromatography/high-resolution mass spectral (GC/HRMS) analysis of its pentafluorobenzyl derivative following immunoaffinity chromatography purification. The amounts of N-2,3-epsilon Gua formed were 825 +/- 20 and 1720 +/- 50 N-2,3-epsilon Gua adducts/10(6) normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increases in the amount of N-2,3-epsilon Gua compared to controls containing only t-BuOOH. Controls containing t-BuOOH but no lipid resulted in >1000-fold increase in the level of N-2,3-epsilon Gua over dGuo that was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reacted with calf thymus DNA at 37 degreesC for 89 h. The amounts of N-2,3-epsilon Gua formed in intact ctDNA were 114 +/- 32 and 52.9 +/- 16.7 N-2,3-epsilon Gua adducts/10(6) normal dGuo bases for EtLA and HNE, respectively. These compared to 2.02 +/- 0.17 and 2.05 +/- 0.47 N-2,3-epsilon Gua adducts/10(6) normal dGuo bases in control DNA incubated with t-BuOOH, but no lipid. [C-13(18)]EtLA was reacted with dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. These reactions resulted in a 89-93% level of incorporation of the C-13 label into N-2,3-epsilon Gua when EtLA and dGuo were in equimolar concentrations, when EtLA was in 10-fold molar excess, and when deoxyribose (thymidine) was in 10-fold molar excess. Similar reactions with ctDNA resulted in an 86% level of incorporation of the 13C label. These data demonstrate that N2,3-epsilon Gua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The data also suggest, however, that N-2,3-epsilon Gua is also formed by an alternative mechanism that involves some other oxidative reaction which remains unclear.