DNA Oxidation as a Source of Endogenous Electrophiles: Formation of Ethenoadenine Adducts in γ-Irradiated DNA

Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 10(8) normal nucleotides from 100 microg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethane-treated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.

Section: Resultsmentioning

confidence: 99%

Quantification of Etheno-DNA Adducts Using Liquid Chromatography, On-Line Sample Processing, and Electrospray Tandem Mass Spectrometry

Doerge

Churchwell

Fang

et al. 2000

“…To test this hypothesis, we used thymidine, as a source of deoxyribose separate from the deoxyguanosine, in 10-fold molar excess of both EtLA and dGuo. Although deoxyadenosine had been used previously as a source of deoxyribose oxidation products ( , ), we chose to use thymidine in these experiments since etheno adducts of dAdo can be formed, but no such adduct for thymidine exists. By using thymidine instead of dAdo, therefore, we wanted to eliminate any competitive reactions for the formation of etheno adducts.…”

Section: Resultsmentioning

confidence: 99%

“…It is possible, therefore, that N 2 ,3-Gua may be formed from the reaction of dGuo with phosphoglycoaldehyde, resulting from sugar oxidation in DNA, or hydroxyacetaldehyde, resulting from sugar oxidation in deoxyguanosine. In fact, Jones and Dedon (32) have demonstrated that Ade is formed from the reaction of dAdo with 2-phosphoglycoaldehyde. They were unable to reproduce, however, the formation of Ade in γ-irradiated DNA, which should form phosphoglycoaldehyde in DNA.…”

Section: Discussionmentioning

confidence: 99%

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

Ham

Ranasinghe

Koç

et al. 2000

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.

“…Our previous study showed that 4-hydroxynonal can be epoxidized to 2,3-epoxy-4-hydrox-ynonanal by biological oxidants and, thus, could contribute to the endogenous formation of etheno DNA adducts (17). Background levels of etheno DNA adducts detected in rodents and human (18) may originate from lipid peroxidation (12) or from oxidation of the sugar backbone of DNA (19). Their levels appear to increase with oxidative stress and are implicated in cancer development (20)(21)(22).…”

Section: Introductionmentioning

confidence: 99%

Analysis of 3,N⁴-Ethenocytosine in DNA and in Human Urine by Isotope Dilution Gas Chromatography/Negative Ion Chemical Ionization/Mass Spectrometry

Chen

Lin

Hong

et al. 2001

The promutagenic etheno DNA adducts have been detected in tissue DNA of rodents and humans from various exogenous and endogenous sources. While other etheno DNA adducts have been detected and quantified by isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry (GC/NICI/MS), similar analysis for 3,N(4)-ethenocytosine (epsilonCyt) has not been available. In this report, a GC/NICI/MS assay was developed for detection and quantification of epsilonCyt in DNA and in human urine samples. The stable isotope of epsilonCyt with 7 mass units higher than the normal epsilonCyt was synthesized and used as internal standard of the assay. The adduct-enriched fraction of DNA hydrolysate was derivatized with pentafluorobenzyl bromide before GC/NICI/MS analysis with selective ion monitoring at [M - 181](-) fragments of pentafluorobenzylated epsilonCyt and its isotope analogue. One femtogram (S/N > 40) of pentafluorobenzylated epsilonCyt was detected when injected on column with selective ion monitoring mode. The limit of quantification for the entire assay was 7.4 fmol of epsilonCyt, which was approximately one thousand times lower than that of the HPLC/fluorescence assay for the nucleoside 3,N(4)-etheno-2'-deoxycytidine in DNA. Analysis of chloroacetaldehyde-treated calf thymus DNA by both GC/NICI/MS and HPLC/fluorescence methods provided similar adduct levels and thus verified the assay. This GC/NICI/MS method was used for analysis of epsilonCyt in two smokers' urine samples and the average level of epsilonCyt was 101 +/- 17 pg/mL/g of creatinine. Thus, quantification of epsilonCyt in DNA and in urine by this highly specific and ultrasensitive isotope dilution GC/NICI/MS assay may facilitate research on the role of epsilonCyt in carcinogenesis and in cancer development.