5-hydroxy-2--deoxycytidine and 5-hydroxy-2--deoxyuridine

The reaction of hydroxyl radicals with 2'-deoxycytidine (dCyd), as well as the decomposition of dCyd radical cations, leads to a complex mixture of oxidation products in aqueous aerated solutions. The oxidation of dCyd gives products with a relatively low oxidation potential that are highly susceptible to further oxidation, including 5-hydroxy-2'-deoxycytidine (5-oh-dCyd) and 5-hydroxy-2'-deoxyuridine (5-oh-dUrd). Previously, we showed that the oxidation of 2'-deoxyuridine (dUrd) involves the formation of dialuric acid and isodialuric acid intermediates, followed by ring contraction to N1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-hydroxyhydantoin (5-oh-dHyd). In this work, we have examined the oxidation of 5-oh-dCyd and 5-oh-dUrd in greater detail. The oxidation of these substrates by Br2 led to a similar profile of intermediate and stable products indicating that the dialuric and isodialuric acid derivatives of dCyd largely undergo deamination before they transform into 5-oh-dHyd. Analysis of the final mixture of oxidation products by HPLC revealed the formation of two novel products. On the basis of NMR and MS, these products were identified as the diastereomers of N1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-hydroxyimidazolidine-2,5-dione (iso-4-oh-dHyd). These products arise from alpha-hydroxy-ketone isomerization of 5-oh-dHyd. The isomerization of 5-oh-dHyd to iso-4-oh-dHyd was reversible, and each diastereomer produced a specific diastereomer of the other structural isomer. The rate of isomerization was accelerated in going from pH 5 to pH 9, whereas all isomers decomposed at higher pH. In contrast, interconversion between each pair of diastereomers was minor. Thus, we conclude that the oxidation of 5-oh-dCyd or 5-oh-dUrd gives a mixture of four isomers of 5-oh-dHyd and iso-4-oh-dHyd as final products. The biological consequences of dCyd oxidation may ultimately depend on the effects of these products.

Topics: Chromatography, High Pressure Liquid; Deoxycytidine; Deoxyuridine; Hydantoins; Hydrogen-Ion Concentration; Indicators and Reagents; Isomerism; Ketones; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Nucleosides; Oxidation-Reduction; Stereoisomerism

Spontaneous oxidative DNA damage occurs as a consequence of aerobic metabolism, lipid peroxidation, immune responses, ionizing radiation, and some chemical oxidants. These processes yield a vast array of oxidized DNA bases and sugars. The existence of significant steady-state levels of oxidized DNA bases in the genome suggests that these lesions are not completely repaired on a biologically relevant time scale and thus may contribute to mutagenesis. In particular, studies have shown that the steady-state levels of 5-hydroxy-2'-deoxycytidine (dC5-OH) and its deamination product, 5-hydroxy-2'-deoxyuridine (dU5-OH), are similar to those found for 7,8-dihydro-8-oxoguanosine, a known highly mutagenic lesion formed by oxidation of guanosine. Structural and biological properties of dC5-OH and dU5-OH have been constrained by the lack of synthetic methodology for oligonucleotides containing these modified bases. A method is described here for the solid-phase synthesis of oligonucleotides containing dC5-OH and dU5-OH. Preparation of each of the required phosphoramidites involved the selective protection of the base 5-hydroxyl group over the deoxyribose 5'- and 3'-hydroxyl groups. The base composition and the incorporation of the adducts into synthetic heptanucleotides were confirmed after purification of the modified oligonucleotides by enzymatic digestion and HPLC analysis. Mass spectrometric analysis of the oligonucleotide products by electrospray MS and GC/MS further confirmed their composition. Most significantly, deamination of the dC5-OH oligomer to a putative dU5-OH product during solid-phase DNA synthesis or oligonucleotide deprotection was not detected by any analytical technique employed.

Topics: Chromatography, High Pressure Liquid; Deoxycytidine; Deoxyuridine; Gas Chromatography-Mass Spectrometry; Mutagenesis; Oligonucleotides; Organophosphorus Compounds

The oxidation of cytosine in DNA by free radicals and other oxidants leads to an assortment of products including pyrimidine ring 5,6-saturated, 5,6-unsaturated, contraction, and fragmentation products. The formation of these products in cellular DNA may explain in part the preponderance of C to T transitions induced spontaneously and by H2O2 or ionizing radiation. Our studies have focused on the biological effects of two major 5,6-unsaturated oxidation products of cytosine: 5- hydroxycytosine and 5-hydroxyuracil. In the present work, we have attempted to study the repair of these two lesions by specifically incorporating them into cellular DNA upon incubation of cells with 5-hydroxy-2'deoxycytidine and 5-hydroxy-2'-deoxyuridine. Incubation of mouse L1210 cells with 250 M 5-hydroxy-2'-deoxycytidine led to the incorporation of this lesion to a level 20 times higher (43 lesions/10(5) cytosines) than base-line levels; however, there was no evidence for its repair following a 15-h chase. In contrast, we did not observe any significant incorporation of 5-hydroxy-2'-deoxyuridine into the DNA of L1210 cells but did observe an unidentified product, presumably an oxidation product. This unidentified pyrimidine was incorporated at a very high level (about 2000 lesions/10(5) cytosine residues) and then partially repaired in chase experiments.

Topics: Animals; Deoxycytidine; Deoxyuridine; DNA; DNA Damage; DNA Repair; HeLa Cells; Humans; Jurkat Cells; Leukemia L1210; Mice; Oxidation-Reduction

5-Hydroxy-2'-deoxycytidine (5-OHdC) and 5-hydroxy-2'-deoxyuridine (5-OHdU) are major products of oxidative DNA damage with mutagenic potential. Until now, no enzymatic activity responsible for their removal has been identified. We report here that both 5-OHdC and 5-OHdU are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase (FPG). 5-OHdU is also a substrate for uracil DNA N-glycosylase. Consistent with their mechanisms of action on previously described substrates, endonuclease III removes 5-OHdC and 5-OHdU via a N-glycosylase/beta-elimination reaction, FPG follows a N-glycosylase/beta,delta-elimination reaction, and uracil N-glycosylase removes 5-OHdU by N-glycosylase action leaving behind an abasic site. Endonuclease III removes both lesions more efficiently than FPG, and both endonuclease III and FPG remove 5-OHdC slightly more efficiently than 5-OHdU. Uracil DNA N-glycosylase removes 5-OHdU more efficiently than the other two enzymes and has no activity on 5-OHdC even when present in great excess. Analysis of crude extracts obtained from wild type and endonuclease III deletion mutants of E. coli correlated well with data obtained with the purified enzymes.

Topics: Base Sequence; Deoxycytidine; Deoxyribonuclease (Pyrimidine Dimer); Deoxyuridine; DNA Glycosylases; DNA Repair; DNA-Formamidopyrimidine Glycosylase; Endodeoxyribonucleases; Escherichia coli; Escherichia coli Proteins; Molecular Sequence Data; N-Glycosyl Hydrolases; Oligodeoxyribonucleotides; Substrate Specificity; Uracil-DNA Glycosidase

Three major oxidation products of 2'-deoxycytidine (dC)--5-hydroxy-2'-deoxycytidine (oh5dC), 5-hydroxy-2'-deoxyuridine (oh5dU), and 5,6-dihydroxy-5,6-dihydro-2'-deoxyuridine (dUg)--were analyzed from enzymatically hydrolyzed DNA with reversed-phase high-performance liquid chromatography coupled to electrochemical detection. oh5dC and oh5dU can be detected with high sensitivity (50 fmol) and selectivity (0-0.2 V) from hydrolyzed DNA. dUg is not electrochemically active but can be measured by dehydrating it into oh5dU. The quantities of oh5dC, dUg, and oh5dU in untreated commercial-grade calf thymus DNA are 10, 10, and 0.75 fmol/micrograms of DNA, respectively. These levels increased substantially when calf thymus DNA was exposed to ionizing radiation, H2O2 alone, H2O2 and combinations of Fe3+ or Cu2+ and ascorbate, near-UV light (365 nm), near-UV light in the presence of menadione, and OsO4, indicating that oh5dC, oh5dU, and dUg are major oxidative DNA damage products. The steady-state levels of these products were determined from freshly extracted rat tissues and ranged from less than 0.5 fmol/micrograms of DNA for oh5dU to about 10 fmol/micrograms of DNA for oh5dC and dUg in liver and kidney and 22 fmol/micrograms of DNA for oh5dC in brain. The levels of oxo8dG were also determined and in general were somewhat lower than the levels of oh5dC. These findings reinforce the link between DNA damage induced by oxidative metabolism and spontaneous mutagenesis leading to cancer and aging.

Topics: Animals; Brain Chemistry; Chromatography, High Pressure Liquid; Deoxycytidine; Deoxyuridine; DNA; DNA Damage; Humans; Kidney; Leukocytes; Liver; Male; Oxidation-Reduction; Rats; Rats, Inbred F344