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5‘-(2-Phosphoryl-1,4-dioxobutane) as a Product of 5‘-Oxidation of Deoxyribose in DNA: Elimination as trans-1,4-Dioxo-2-butene and Approaches to Analysis
Oxidation of deoxyribose in DNA leads to the formation of a spectrum of electrophilic products unique to each position in the sugar. For example, chemical reactions following abstraction of the C5‘-hydrogen atom partition to form either a nucleoside 5‘-aldehyde residue attached to the 5‘-end of the...
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Published in: | Chemical research in toxicology 2004-11, Vol.17 (11), p.1406-1413 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Oxidation of deoxyribose in DNA leads to the formation of a spectrum of electrophilic products unique to each position in the sugar. For example, chemical reactions following abstraction of the C5‘-hydrogen atom partition to form either a nucleoside 5‘-aldehyde residue attached to the 5‘-end of the DNA strand or a 5‘-formyl phosphate residue attached to the 3‘-end of the DNA strand that is accompanied by a four-carbon fragment on the 5‘-end. We now present two approaches that both identify the latter fragment as 5‘-(2-phosphoryl-1,4-dioxobutane) and provide a means to quantify the formation of this residue by different oxidizing agents. The first approach involves oxidation of DNA followed by reaction with O-benzylhydroxylamine to form stable dioxime derivatives of the putative 5‘-(2-phosphoryl-1,4-dioxobutane) residues. The β-elimination product of this dioxime proved to be the expected trans-1,4-dioxo-2-butene, as judged by gas chromatographic and mass spectrometric (GC/MS) comparison to authentic dioximes of cis- and trans-1,4-dioxo-2-butene, which revealed a unique pattern of three signals for each isomer, and by X-ray crystallography. Using a benzylhydroxylamine dioxime derivative of [2H4]-labeled cis-1,4-dioxo-2-butene as an internal standard, the dose−response for the formation of 5‘-(2-phosphoryl-1,4-dioxobutane) was determined to be linear for γ-radiation, with ∼6 lesions per 106 nt per Gy, and nonlinear for Fe2+-EDTA. A comparison of 5‘-(2-phosphoryl-1,4-dioxobutane) formation to total deoxyribose oxidation suggests that γ-radiation produces ∼0.04 lesions per deoxyribose oxidation event. As a positive control for 5‘-oxidation of deoxyribose, the enediyne calicheamicin was observed to produce 5‘-(2-phosphoryl-1,4-dioxobutane) at the rate of ∼9 lesions per 106 nt per μM. A second approach to identifying and quantifying the sugar residue involved derivatization with hydrazine and β-elimination to form pyridazine followed by quantification of the pyridazine by GC/MS. Using this approach, it was observed that the enediyne, neocarzinostatin, produced a linear dose−response for pyridazine formation, as expected given the ability of this oxidant to cause 1‘-, 4‘-, and 5‘-oxidation of deoxyribose in DNA. The antitumor antibiotic, bleomycin, on the other hand, produced pyridazine at a 10-fold lower rate, which is consistent with 4‘-chemistry as the predominant mode of deoxyribose oxidation by this agent. These results provide novel insights into the chemis |
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ISSN: | 0893-228X 1520-5010 |
DOI: | 10.1021/tx049818e |