On the spin trapping and ESR detection of oxygen-derived radicals generated inside cells.

Abstract

Recently several attempts to identify oxygenderived radicals in whole cells by spin trapping and electron spin resonance have been reported by using 5,5-dimethyl-1-pyrroline-N-oxide as the spin trap. In the present study, the feasibility of this method is examined. Chinese hamster V79 cells and human erythrocytes served as the test systems, while OH radicals were generated by y radiolysis. Several spin traps were used to scavenge the radicals and a distinction between exo-and endocellular ESR observable species was achieved using tri(oxalato) chromiate(I) as a line broadening agent. To distinguish between exo-and endocellular sites of radical formation, we studied the effects of high molecular weight scavengers (polyethylene glycols), which do not enter the cell. Various possible obstacles associated with trapping and detecting the radicals inside the cells were examined. The results indicate that the primary radicals react with the spin traps. However, these spin adducts decayed within the cells.Cellularly induced decay of 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl radical presented the major difficulty in detecting the endogenous radicals, and potential experimental approaches to overcome this difficulty are discussed.Oxygen-derived radicals in various biological systems under physiological conditions have been identified by using spintrapping and electron spin resonance (ESR) spectrometry mainly in solutions or in in vitro systems containing subcellular components as with tissue homogenates (1)(2)(3) Chemicals. a-Phenyl-N-tert-butylnitrone (PBN), a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a-(4-N-methylpyridinium-Ntert-butylnitrone (PyBN), and the stable nitroxide 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were purchased from Aldrich. Polyethylene glycols (PEG) Abbreviations: PBN, a-phenyl-N-tert-butylnitrone; 4-POBN, a-(4-pyridyl-1-oxide)-N-tert-butylnitrone; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; PyBN, a-(4-N-methylpyridinium)-N-tert-butylnitrone; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; Me2SO, dimethyl sulfoxide.

Oxoammonium cation intermediate in the nitroxide-catalyzed dismutation of superoxide.

“…It is known that nitroxides, formed from the addition of unstable radicals to a variety of spin traps, are destroyed by recombination with other radical species (17). In cells, the spin adduct nitroxides have short lifetimes and are rapidly metabolized to species other than the hydroxylamines (16).…”

“…Whereas the metabolic fate of spin adduct nitroxides is not clearly understood, oxidative degradation has been suggested (16,17).…”

“…The cellular and in vivo pharmacology of stable nitroxides and persistent spin adduct nitroxides has been investigated in detail (8-10). Oneelectron reduction of stable nitroxides to the corresponding hydroxylamine is the primary metabolic pathway (11-15).Whereas the metabolic fate of spin adduct nitroxides is not clearly understood, oxidative degradation has been suggested (16,17).Previous studies have identified a stable five-membered nitroxide, 2-ethyl-2,5,5-trimethyl-3-oxazolidine-1-oxyl (OX-ANO), as a cell-permeable, nontoxic, catalyst of superoxide (°O2) dismutation (18). Subsequent cellular studies established this and other stable nitroxides to be protective agents for cells subjected to oxidative stress induced by agents such as -Os, H202, organic hydroperoxides, and ionizing radiation (19,20).…”

Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps

“…the spin adduct) that accumulates and can be detected by ESR. Some drawbacks such as the poor spin trapping rate, the short lifetime of the adducts, and their instability in biological media limit the application of this method [5][6][7] .…”

The first 1H and 14N ENDOR spectra of an oxygen-centered radical adduct of DMPO-type nitrones

“…The sulfur analogues of alkoxyl radicals (thiyls) are also trapped at fast rate constants (-lo8 M-I s-' ) (13). spin trapping studies have suggested that oxygen-centered radicals may be important intermediates in radiolysis (14) and sonolysis (15) reactions, ozone-related chemistry (16,17), xanthinelxanthine oxidase systems (18), carcinogenesis (19), antitumour drug redox interactions (20)(21)(22), cigarette smoke (23), druglskin phototoxicity (24,25), neutrophil action (26a-c), peroxidation reactions (27)(28)(29)(30)(31), as well as myocardial ischemia/reperfusion injury (32a-c).…”