4-Hydroxy-2-nonenal and Ethyl Linoleate Form N2,3-Ethenoguanine under Peroxidizing Conditions
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- 作者：Amy-Joan L. Ham ; Asoka Ranasinghe ; Hasan Koc ; and James A. Swenberg
- 刊名：Chemical Research in Toxicology
- 出版年：2000
- 出版时间：December 2000
- 年：2000
- 卷：13
- 期：12
- 页码：1243 - 1250
- 全文大小：100K
- 年卷期：v.13,no.12(December 2000)
- ISSN：1520-5010
文摘
In these studies, we demonstrate that N2,3-ethenoguanine (N2,3-
Gua) is formed from lipidperoxidation 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 hat 50 
C. The resulting N2,3-Gua was characterized by liquid chromatography/electrospraymass 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-Gua formed were 825 ± 20 and 1720 ± 50 N2,3-Gua adducts/106normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increasesin the amount of N2,3-Gua compared to controls containing only t-BuOOH. Controls containingt-BuOOH but no lipid resulted in a >1000-fold increase in the level of N2,3-Gua over dGuothat was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reactedwith calf thymus DNA at 37 C for 89 h. The amounts of N2,3-Gua formed in intact ctDNAwere 114 ± 32 and 52.9 ± 16.7 N2,3-Gua adducts/106 normal dGuo bases for EtLA and HNE,respectively. These compared to 2.02 ± 0.17 and 2.05 ± 0.47 N2,3-Gua adducts/106 normaldGuo bases in control DNA incubated with t-BuOOH, but no lipid. [13C18]EtLA was reactedwith dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. Thesereactions resulted in a 89-93% level of incorporation of the 13C label into N2,3-Gua whenEtLA 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 ctDNAresulted in an 86% level of incorporation of the 13C label. These data demonstrate that N2,3-Gua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. Thedata also suggest, however, that N2,3-Gua is also formed by an alternative mechanism thatinvolves some other oxidative reaction which remains unclear.
Gua) is formed from lipidperoxidation 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 hat 50 C. The resulting N2,3-Gua was characterized by liquid chromatography/electrospraymass 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-Gua formed were 825 ± 20 and 1720 ± 50 N2,3-Gua adducts/106normal dGuo bases for EtLA and HNE, respectively, corresponding to 38- and 82-fold increasesin the amount of N2,3-Gua compared to controls containing only t-BuOOH. Controls containingt-BuOOH but no lipid resulted in a >1000-fold increase in the level of N2,3-Gua over dGuothat was not subjected to incubation. EtLA and HNE, in the presence of t-BuOOH, were reactedwith calf thymus DNA at 37 C for 89 h. The amounts of N2,3-Gua formed in intact ctDNAwere 114 ± 32 and 52.9 ± 16.7 N2,3-Gua adducts/106 normal dGuo bases for EtLA and HNE,respectively. These compared to 2.02 ± 0.17 and 2.05 ± 0.47 N2,3-Gua adducts/106 normaldGuo bases in control DNA incubated with t-BuOOH, but no lipid. [13C18]EtLA was reactedwith dGuo to determine the extent of direct alkylation by lipid peroxidation byproducts. Thesereactions resulted in a 89-93% level of incorporation of the 13C label into N2,3-Gua whenEtLA 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 ctDNAresulted in an 86% level of incorporation of the 13C label. These data demonstrate that N2,3-Gua is formed from EtLA and HNE under peroxidizing conditions by direct alkylation. Thedata also suggest, however, that N2,3-Gua is also formed by an alternative mechanism thatinvolves some other oxidative reaction which remains unclear.