4-Hydroxy-2-nonenal and Ethyl Linoleate Form N2,3-Ethenoguanine under Peroxidizing Conditions

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Abstract
In these studies, we demonstrate that N2,3-ethenoguanine (N2,3-εGua) 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 °C. The resulting N2,3-εGua 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 N2,3-εGua formed were 825 ± 20 and 1720 ± 50 N2,3-εGua adducts/106 normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increases in the amount of N2,3-εGua compared to controls containing only t-BuOOH. Controls containing t-BuOOH but no lipid resulted in a >1000-fold increase in the level of N2,3-εGua 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 °C for 89 h. The amounts of N2,3-εGua formed in intact ctDNA were 114 ± 32 and 52.9 ± 16.7 N2,3-εGua adducts/106 normal dGuo bases for EtLA and HNE, respectively. These compared to 2.02 ± 0.17 and 2.05 ± 0.47 N2,3-εGua adducts/106 normal dGuo bases in control DNA incubated with t-BuOOH, but no lipid. [13C18]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 13C label into N2,3-εGua 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-εGua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The data also suggest, however, that N2,3-εGua is also formed by an alternative mechanism that involves some other oxidative reaction which remains unclear.

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