4-Hydroxy-2-nonenal and ethyl linoleate form N(2),3-ethenoguanine under peroxidizing conditions.
In these studies, we demonstrate that N(2),3-ethenoguanine (N(2), 3-epsilonGua) 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 degrees C. The resulting N(2), 3-epsilonGua 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-epsilonGua formed were 825 +/- 20 and 1720 +/- 50 N(2), 3-epsilonGua 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-epsilonGua compared to controls containing only t-BuOOH. Controls containing t-BuOOH but no lipid resulted in a >1000-fold increase in the level of N(2),3-epsilonGua 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 degrees C for 89 h. The amounts of N(2),3-epsilonGua formed in intact ctDNA were 114 +/- 32 and 52.9 +/- 16.7 N(2),3-epsilonGua 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-epsilonGua adducts/10(6) normal dGuo bases in control DNA incubated with t-BuOOH, but no lipid. [(13)C(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 (13)C label into N(2),3-epsilonGua 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 (13)C label. These data demonstrate that N(2),3-epsilonGua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. The data also suggest, however, that N(2),3-epsilonGua is also formed by an alternative mechanism that involves some other oxidative reaction which remains unclear.
Ham
A J
AJ
Departments of Pathology and Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
Ranasinghe
A
A
Koc
H
H
Swenberg
J A
JA
D000447:Aldehydes / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D018736:DNA Adducts / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006151:Guanosine / Q000031:analogs & derivatives* / Q000737:chemistry*; D008041:Linoleic Acids / Q000737:chemistry*; D015227:Lipid Peroxidation; D009153:Mutagens / Q000737:chemistry*
D000447:Aldehydes; D018736:DNA Adducts; D008041:Linoleic Acids; D009153:Mutagens; C052524:N(2),3-ethenoguanosine; D006151:Guanosine; C027576:4-hydroxy-2-nonenal; C007678:ethyl linoleate