Cytotoxicity and Site-specific DNA Damage Induced by Nitroxyl Anion (NO−) in the Presence of Hydrogen Peroxide

Abstract

Nitroxyl anion (NO ؊ ), the one-electron reduction product of nitric oxide (NO ⅐ ), is formed under various physiological conditions. We have used four different assays (DNA strand breakage, 8-oxo-deoxyguanosine formation in calf thymus DNA, malondialdehyde generation from 2-deoxyribose, and analysis of site-specific DNA damage using 32 P-5-end-labeled DNA fragments of the human p53 tumor suppressor gene and the c-Haras-1 protooncogene) to study the effects of NO ؊ generated from Angeli's salt on DNA damage. It was found that strong oxidants are generated from NO

Induction of Heme Oxygenase 1 by Nitrosative Stress

“…Despite this observation, no increase in apoptosis was detected in cells treated with AS or DEA/NO, indicating that the reduction in cell metabolism by NO could be a reversible process. Although these findings appear to be in contrast with previous reports showing cytotoxicity for NO Ϫ , it needs to be pointed out that in those studies higher concentrations of AS were used (2-5 mM) (63), and exacerbation of cellular damage was observed only when AS was combined with hydrogen peroxide (56). Of interest, and in agreement with results using NO donors (62), we also found that NO Ϫ -mediated HO-1 expression is transient and gradually disappears once the spontaneous generation of NO Ϫ ceases.…”

“…The fate of NO Ϫ within the cellular system is unknown, but this unstable species has been reported to react readily with thiols (41,42), and in the presence of molecular oxygen or other oxidants, it can generate other RNS with specific reactivity toward cellular targets (54,55). In the presence of hydrogen peroxide and transition metals, NO Ϫ but not NO appears to cause loss of cell viability by site-specific DNA damage (56). Recent reports using donors of nitroxyl revealed opposite effects by showing either NO Ϫ -mediated exacerbation of post-ischemic myocardial injury (43) or cytoprotection against neuronal damage (57).…”

Formation of Nitroxyl and Hydroxyl Radical in Solutions of Sodium Trioxodinitrate

“…In the presence of reductants, nitroxides can be readily reduced to the corresponding EPR silent hydroxylamines (32). In support of the latter assumption, the addition of K 3 [Fe(CN) 6 ] to an extract of a reaction solution consisting of 1b, Me 2 SO, and PBN resulted in a pronounced increase of the EPR signal of 4. This effect most likely reflected the oxidation of the EPR silent hydroxylamine 5 to 4 (Scheme 2).…”

“…When the reaction solution was analyzed by HPLC with electrochemical detection, the predominant formation of 5 was observed (Fig. 3); an addition of K 3 [Fe(CN) 6 ] to the analyzed solutions resulted in the interconversion of the HPLC peaks reflecting the elution of 5 and 4, respectively. The identity of compounds 4 and 5 was confirmed by coinjections of authentic HPLC standards as described previously (32,38).…”

“…In cells, the biosynthesis of HNO is believed to proceed via reduction of NO ⅐ by superoxide dismutase (1) and cytochrome c (2), and reduction of S-nitrosoglutathione by low molecular weight and protein thiols (3)(4)(5). It has been suggested that HNO can affect the etiology of various pathophysiological conditions such as inflammation and neurodegenerative diseases, especially when H 2 O 2 and transition metal ions are present (6,7). Similar to NO ⅐ and NO ϩ , HNO is a potent inducer of the antioxidant protein heme oxygenase 1 (8), exhibits vasorelaxant properties (9), and modulates the activity of thiol-containing proteins, such as aldehyde dehydrogenase (10,11) and the N-methyl-D-aspartate receptor (12,13).…”