4-hydroperoxy-2-nonenal, a novel precursor to the formation of unsubstituted etheno adducts. Free

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Increased lipid peroxidation of polyunsaturated fatty acids is thought to occur during redox cycling of polycyclic aromatic hydrocarbon catechols that are formed by sequential action of specific cytochromes P-450 and aldo-keto reductases. Transition metal ions or vitamin C induce the decomposition of lipid hydroperoxides to the α,β-unsaturated aldehyde, which react with DNA bases to form DNA-adducts [1]. 4-Oxo-2-nonenal (4-ONE), 4-hydroxy-2-nonenal (4-HNE), 4,5-epoxy-2(E)-decenal (4,5-EDE) and 4-hydroperoxy-2-nonenal (4-HPNE) were identified as breakdown products from homolytic decomposition of ω-6 lipid hydroperoxide, 13-hydroperoxyoctadecadienoic acid (13-HpODE). The heptanone-etheno-2′-deoxyguanosine (dGuo), 2′-deoxyadenosine (dAdo) and 2′-deoxycytidine (dCyd) adducts are formed in high yield from the reaction of DNA and 4-ONE, a major product of 13-HpODE decomposition. 4,5-EDE forms unsubstituted etheno adducts, a mutagenic lesion observed in human tissue DNA samples. 4-HPNE was shown to undergo transition metal ion- or vitamin C-mediated breakdown to 4-ONE and 4-HNE, suggesting that 4-HPNE can form heptanone-etheno- and propano- adducts with DNA 4-HPNE, 4-ONE, 4-HNE (25 mM) and 4-HNE (25 mM) with 70% t-butyl hydroperoxide (BTH) were allowed to decompose respectively in the presence of dGuo (5 mM) in Chelex-treated phosphate buffer (pH 7.4) at 37°C for 24h or at 60°C for 48h. Reaction products were analyzed by LC/APCI/MS. Mass spectrometry was conducted with a Finnigan LCQ ion trap mass spectrometer equipped with an APCI source. LC/MS analysis of the reaction between 4-HPNE and dGuo at 37°C for 24h revealed sixteen adducts. The mass spectrum of the most abundant adduct (35.8 min) was identical to that for heptanone-etheno-dGuo (B). Four isomers of heptanone-ethano-dGuo were also observed at 30.4 (A_1a), 30.8 (A_1b), 32.7 (A₂) and 33.9 (A₃) min. When the reaction was conducted at 60°C for 48h, all of heptanone-ethano-dGuo adducts were converted to heptanone-etheno-dGuo, which was consistent with the reaction of 4-ONE with dGuo. The dGuo adduct that eluted at 13.2 min had an MH⁺ at m/z 292 and a BH₂⁺ ion at m/z 176. These LC/MS characteristics were identical to those for 1,N²-etheno-dGuo (G₁). The later eluting adducts (G₂ –G₇) had identical LC/MS characteristics to those formed by the reaction of 4-HNE and dGuo in the presence of BTH. Propano adducts (H₁ –H₄) were not detected in the reaction mixture after 24h at 37°C and appeared as minor adducts when the reaction mixture was heated at 60°C for 48h. When 4-HPNE was allowed to decompose in the presence of vitamin C and dGuo, the same adduct profile was observed. These results confirmed that 4-HPNE is a novel precursor to the formation of unsubstituted etheno-adducts as well as heptanone-etheno- and propano-adducts. 1. Lee SH, Oe T, Blair IA. Science 2001; 292: 2083-86. Suported by NIH grants RO1 CA 91016 and PO1 CA 92537