Abstract
Damage by iron-mediated Fenton reactions under aerobic or anaerobic conditions to deoxycytidine, deoxycytidine-5′-monophosphate, d-CpC, d-CpCpC, and dCMP residues in DNA resulted in at least 26 distinguishable products. Of these, 24 were identified by high performance liquid chromatography retention times, radiolabeling, UV absorption spectra, chemical synthesis, fast atom bombardment mass spectrometry, high resolution fast atom bombardment mass spectrometry, and/or NMR. The nature of the products was qualitatively similar for each substrate except for d-CpC (and possibly d-CpCpC) under anaerobic conditions for which 5-hydroxy-deoxycytidine was uniquely present and 1-carbamoyl-1-carboxy-4-(2-deoxy-β—erythropentofuranosyl)glycinamide was uniquely absent. Damage to dC, d-CpC, and d-CpCpC but not to dCMP or DNA was largely quenched by ethanol, indicating that iron is strongly associated only with dCMP and DNA. The presence of oxygen had little effect with dC or dCMP but had quantitative and qualitative effects with d-CpC and a significantly quantitative but not a qualitative effect with DNA. NADH could drive the Fenton reaction to cause damage to the dC family <i>in vitro</i>, consistent with a previous proposal that NADH was the reducing agent for the Fenton reaction <i>in vivo</i> (Imlay, J.A., and Linn, S. (1988) <i>Science</i> 240, 1302-1309). Finally, the damage spectrum of the dC family by the Fenton reaction is compared with that by ionizing radiation and chemical mechanisms leading to the formation of the 24 identified products are proposed.
Highlights
Active oxygen species such as superoxide anion radical (O2.), hydrogen peroxide (H2O2), and hydroxyl radical (1⁄7OH) can damage almost all cell components, including DNA, membranes, and proteins [1]
In this study of the deoxycytidine family, we have subjected 2Ј-deoxycytidine, 2Ј-deoxycytidine-5Ј-monophosphate, d-CpC, d-CpCpC, and DNA to iron/H2O2 under various conditions and analyzed the resulting dC modifications. The objectives of this and the accompanying studies were extensively and systematically to study DNA damage without the use of chelators and acid hydrolysis, to identify the major stable degradation products of the dC family after Fenton reactions under a variety of conditions so as to obtain a “fingerprint” of oxidative damages that might be useful for identifying the conditions of Fenton reaction-induced DNA damage from cells subjected to oxidative stress, and to establish the chemical pathways that lead to the formation of the damaged products
Products were identified after enzymatic hydrolysis to the nucleoside level by high performance liquid chromatography (HPLC)1 retention times, radiolabeling, UV absorption spectra, chemical synthesis, fast atom bombardment mass spectrometry by both positive mode (FAB-MSϩ) and negative mode (FAB-MSϪ), high resolution FAB-MS, and nuclear magnetic resonance (NMR)
Summary
Active oxygen species such as superoxide anion radical (O2.), hydrogen peroxide (H2O2), and hydroxyl radical (1⁄7OH) can damage almost all cell components, including DNA, membranes, and proteins [1]. DNA base damages induced by hypoxanthine/xanthine oxidase [4] or iron and H2O2 [5] have been identified and quantitated Because iron chelators such as EDTA and buffers such as potassium phosphate were used in these studies, the chemistry of the Fenton reaction has probably been perturbed [6]. Damage to the bases in DNA and mammalian chromatin by H2O2 and transition metals has been investigated [7,8,9] In these cases chemical hydrolysis procedures were used that might destroy, alter, or form various products [5]. We are able to compare damages by these three different agents
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