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

Abstract

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.

Liquid chromatography-electrospray ionization-mass spectrometry: the future of DNA adduct detection

“…Ham et al showed that N 2 ,N-3 ethenoguanine was formed by direct alkylation following the treatment calf thymus DNA with 13 C-labelled ethyl linoleate under peroxidizing conditions. The authors showed that the level of incorporation of the 13 C label was 86% suggesting that N 2 ,N-3 ethenoguanine was also being formed by an alternative mechanism (169).…”

Accumulation of M1dG DNA adducts after chronic exposure to PCBs, but not from acute exposure to polychlorinated aromatic hydrocarbons

“…Such oxidative DNA lesions are suspected to be increased with non-DNA reactive chemicals, resulting in the induction of mutations followed by tumor formation. While base oxidation and abasic sites are the most frequent insults to DNA, substantial evidence supports that secondary DNA damage produced from byproducts of primary DNA damage or lipid membrane damage may play an important role in mutation [23,24]. Recently, we have developed ultra sensitive and highly specific mass spectrometric methods for the quantitation of M 1 dG adducts.…”

A New LC-MS/MS Method for the Quantification of Endogenous and Vinyl Chloride-Induced 7-(2-Oxoethyl)Guanine in Sprague–Dawley Rats

“…52 Previously, the key LPO intermediates that have been identified for the formation of DNA adducts 19,58-60 are the result of reactions of HNE, ONE and MDA with dG and are associated with DNA adducts formed by VC exposure. 58 …”

“…We hypothesized that the source leading to the formation of 7-OEG was lipid peroxidation and present data to support our hypothesis. The reaction of calf thymus DNA (CtDNA) with [ 13 C 18 ]-ethyl linoleate (EtLa) under peroxidizing conditions resulted in the formation of [ 13 C 2 ]-7-OEG, 52 which could be quantified by LC/MS-MS (Figure 3). In addition to quantitating endogenous and exogenous 7-OEG after [ 13 C 2 ]-VC exposures, the half-life of [ 13 C 2 ]-7-OEG in liver and lung was determined.…”