Oxygen radical formation induced by gossypol in rat liver microsomes and human sperm

Gossypol, a polyphenolic compound found in cotton plants, has many potential uses, including use as a male antifertility drug and spermicide. Gossypol affects a variety of cell processes and many of these effects may be explained by a common underlying mechanism. Here we report that gossypol promotes the formation of oxygen radicals when incubated with rat liver microsomes and human sperm suggesting that oxygen radical production may be the underlying basis of its biological activity.     

Superoxide-dependent reduction of nitroxides by thiols

It was found that superoxide can reduce certain nitroxide free radicals to their corresponding hydroxylamines in the presence of most sulfhydryl-containing compounds. The stoichiometry of the reaction was found to be three nitroxides reduced per superoxide. Evidence is presented indicating that superoxide directly reacts with a nitroxide to yield a N-hydroxy-N-hydroperoxyl compound. This product rapidly decomposes, giving a hydroxylamine and an oxidized sulfhydryl compound, which is postulated to be a sulfenyl hydroperoxide. It is proposed that this sulfenyl hydroperoxide reduces two additional nitroxyl free radicals to account for the unusual stoichiometry.     

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Background

Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant with a short half-life and a low steady-state concentration in biological systems; it promotes nitroxidative damage.

Scope of review

We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable steady–state levels in living systems.

Major conclusions

The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only < 1% of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5 μM s^− 1 and its steady-state concentration at ~ 1 nM.

General significance

The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Early specific free radical-related cytotoxicity of gas phase cigarette smoke and its paradoxical temporary inhibition by tar: An electron paramagnetic resonance study with the spin trap DEPMPO

Electron paramagnetic resonance (EPR) spin trapping studies demonstrated aqueous tar particulate matter (TPM) and gas phase cigarette smoke (GPCS) to behave as different sources of free radicals in cigarette smoke (CS) but their cytotoxic implications have been only assessed in CS due to its relevance to the natural smoking process. Using a sensitive spin trapping detection with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), this study compared the respective roles of CS- and GPCS-derived free radicals on smoke-induced cytotoxicity and lipid peroxidation of filtered and unfiltered, machine-smoked experimental and reference cigarettes yielding a wide range of TPM yields. In buffer bubbled with CS the DEPMPO/superoxide spin adduct was the major detected nitroxide. Use of appropriate control experiments with nitric oxide radical (NO*) or carbonyl sulfide, and a computer analysis of spin adduct diastereoisomery showed that the hydroxyl radical (HO*) adduct of DEPMPO seen in GPCS-bubbled was rather related to metal-catalyzed nucleophilic synthesis than to direct HO* trapping. Unexpectedly a protective effect of TPM on murine 3T3 fibroblasts was observed in early (<3h) free radical-, GPCS-induced cell death, and carbon filtering decreased free radical formation, toxicity and lipid peroxidation in three cell lines (including human epithelial lung cells) challenged with GPCS. These results highlight an acute, free radical-dependent, harmful mechanism specific to the GPCS phase, possibly involving NO* chemistry, whose physical or chemical control may be of great interest with the aim of reducing the toxicity of smoke.     

Detection and characterisation of radicals in biological materials using EPR methodology

Background

Electron paramagnetic resonance (EPR) spectroscopy (also known as electron spin resonance, ESR, spectroscopy) is widely considered to be the “gold standard” for the detection and characterisation of radicals in biological systems.

Scope of review

The article reviews the major positive and negative aspects of EPR spectroscopy and discusses how this technique and associated methodologies can be used to maximise useful information, and minimise artefacts, when used in biological studies. Consideration is given to the direct detection of radicals (at both ambient and low temperature), the use of spin trapping and spin scavenging (e.g. reaction with hydroxylamines), the detection of nitric oxide and the detection and quantification of some transition metal ions (particularly iron and copper) and their environment.

Major conclusions

When used with care this technique can provide a wealth of valuable information on the presence of radicals and some transition metal ions in biological systems. It can provide definitive information on the identity of the species present and also information on their concentration, structure, mobility and interactions. It is however a technique that has major limitations and the user needs to understand the various pitfalls and shortcoming of the method to avoid making errors.

General significance

EPR remains the most definitive method of identifying radicals in complex systems and is also a valuable method of examining radical kinetics, concentrations and structure. This article is part of a Special Issue entitled Current methods to study reactive oxygen species — pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals () generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normal-physiological serum concentrations of IS (0.1-10 μM) resulted in decreased formation of EMPO-superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal-physiological conditions.