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Quantitation of four guanine oxidation products from reaction of DNA with varying doses of peroxynitrite.

ABSTRACT: The oxidation products obtained from the reaction of peroxynitrite (ONOO-) with dG include-among others-8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 2,2-diamino-4[(2-deoxy-beta-d-erythro-pentafuranosyl)amino]-5(2H)-oxazolone (oxazolone), spiroiminodihydantoin, and N1-(beta-d-erythro-pentofuranosyl)-5-guanidinohydantoin (guanidinohydantoin). In the present work, the formation of these products from the treatment of calf thymus DNA with varying amounts of ONOO- was studied quantitatively in vitro. 13C-, 15N-labeled standards were synthesized for the nucleosides of interest, and calf thymus DNA was reacted with ONOO- and digested enzymatically down to the nucleoside level. Specific modifications in the DNA were measured by HPLC separation followed by electrospray ionization tandem mass spectrometric analysis in the selected reaction-monitoring mode. Artifacts of the above four oxidation products, arising from oxidation of dG and/or 8-oxodG during DNA digestion and subsequent workup, were evaluated with 7-15N-dG and/or stable-isotope-labeled 8-oxodG as internal standards. Levels of artifactual 8-oxodG were about 5/10(6) nucleosides. The artifacts of spiroiminodihydantoin and guanidinohydantoin, arising from 8-oxodG, were 3.7% and 0.6% of the measured 8-oxodG values, respectively. No artifacts of oxazolone were detected. 8-OxodG and oxazolone were formed dose-dependently in DNA treated with ONOO-, while the levels of spiroiminodihydantoin and guanidinohydantoin increased significantly at low ONOO- doses, and then dropped off at higher ONOO- doses. The complexity of these dose-response relationships is likely due to the dual role of peroxynitrite as both an oxidant and a nucleophile in competition with water.

SUBMITTER: Yu H

PROVIDER: S-EPMC2526347 | biostudies-literature | 2005 Dec

REPOSITORIES: biostudies-literature

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Quantitation of four guanine oxidation products from reaction of DNA with varying doses of peroxynitrite.

Yu Hongbin H Venkatarangan Lata L Wishnok John S JS Tannenbaum Steven R SR

Chemical research in toxicology 20051201 12

The oxidation products obtained from the reaction of peroxynitrite (ONOO-) with dG include-among others-8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 2,2-diamino-4[(2-deoxy-beta-d-erythro-pentafuranosyl)amino]-5(2H)-oxazolone (oxazolone), spiroiminodihydantoin, and N1-(beta-d-erythro-pentofuranosyl)-5-guanidinohydantoin (guanidinohydantoin). In the present work, the formation of these products from the treatment of calf thymus DNA with varying amounts of ONOO- was studied quantitatively in vitr ...[more]

PMID: 16359175

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Project description:Telomere shortening occurs during oxidative and inflammatory stress with guanine (G) as the major site of damage. In this work, a comprehensive profile of the sites of oxidation and structures of products observed from G-quadruplex and duplex structures of the human telomere sequence was studied in the G-quadruplex folds (hybrid (K(+)), basket (Na(+)), and propeller (K(+) + 50% CH3CN)) resulting from the sequence 5'-(TAGGGT)4T-3' and in an appropriate duplex containing one telomere repeat. Oxidations with four oxidant systems consisting of riboflavin photosensitization, carbonate radical generation, singlet oxygen, and the copper Fenton-like reaction were analyzed under conditions of low product conversion to determine relative reactivity. The one-electron oxidants damaged the 5'-G in G-quadruplexes leading to spiroiminodihydantoin (Sp) and 2,2,4-triamino-2H-oxazol-5-one (Z) as major products as well as 8-oxo-7,8-dihydroguanine (OG) and 5-guanidinohydantoin (Gh) in low relative yields, while oxidation in the duplex context produced damage at the 5'- and middle-Gs of GGG sequences and resulted in Gh being the major product. Addition of the reductant N-acetylcysteine (NAC) to the reaction did not alter the riboflavin-mediated damage sites but decreased Z by 2-fold and increased OG by 5-fold, while not altering the hydantoin ratio. However, NAC completely quenched the CO3(•-) reactions. Singlet oxygen oxidations of the G-quadruplex showed reactivity at all Gs on the exterior faces of G-quartets and furnished the product Sp, while no oxidation was observed in the duplex context under these conditions, and addition of NAC had no effect. Because a long telomere sequence would have higher-order structures of G-quadruplexes, studies were also conducted with 5'-(TAGGGT)8-T-3', and it provided oxidation profiles similar to those of the single G-quadruplex. Lastly, Cu(II)/H2O2-mediated oxidations were found to be indiscriminate in the damage patterns, and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) was found to be a major duplex product, while nearly equal yields of 2Ih and Sp were observed in G-quadruplex contexts. These findings indicate that the nature of the secondary structure of folded DNA greatly alters both the reactivity of G toward oxidative stress as well as the product outcome and suggest that recognition of damage in telomeric sequences by repair enzymes may be profoundly different from that of B-form duplex DNA.

Dataset Information

Quantitation of four guanine oxidation products from reaction of DNA with varying doses of peroxynitrite.

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Quantitation of four guanine oxidation products from reaction of DNA with varying doses of peroxynitrite.

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