scholarly journals 2′,7′-dichlorofluorescin-based analysis of Fenton chemistry reveals auto-amplification of probe fluorescence and albumin as catalyst for the detection of hydrogen peroxide

2020 ◽  
Vol 477 (24) ◽  
pp. 4689-4710
Author(s):  
Teresa Gonzalez ◽  
Franck Peiretti ◽  
Catherine Defoort ◽  
Patrick Borel ◽  
Roland Govers
Keyword(s):  
Hydrogen Peroxide ◽  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Ferrous Iron ◽  
Fenton Reaction ◽  
Iron Chelation ◽  
Intact Cells ◽  
Free System ◽  
Fenton Chemistry ◽  
In Cells

Fluorophore 2′,7′-dichlorofluorescin (DCF) is the most frequently used probe for measuring oxidative stress in cells, but many aspects of DCF remain to be revealed. Here, DCF was used to study the Fenton reaction in detail, which confirmed that in a cell-free system, the hydroxyl radical was easily measured by DCF, accompanied by the consumption of H2O2 and the conversion of ferrous iron into ferric iron. DCF fluorescence was more specific for hydroxyl radicals than the measurement of thiobarbituric acid (TBA)-reactive 2-deoxy-D-ribose degradation products, which also detected H2O2. As expected, hydroxyl radical-induced DCF fluorescence was inhibited by iron chelation, anti-oxidants, and hydroxyl radical scavengers and enhanced by low concentrations of ascorbate. Remarkably, due to DCF fluorescence auto-amplification, Fenton reaction-induced DCF fluorescence steadily increased in time even when all ferrous iron was oxidized. Surprisingly, the addition of bovine serum albumin rendered DCF sensitive to H2O2 as well. Within cells, DCF appeared not to react directly with H2O2 but indirect via the formation of hydroxyl radicals, since H2O2-induced cellular DCF fluorescence was fully abolished by iron chelation and hydroxyl radical scavenging. Iron chelation in H2O2-stimulated cells in which DCF fluorescence was already increasing did not abrogate further increases in fluorescence, suggesting DCF fluorescence auto-amplification in cells. Collectively, these data demonstrate that DCF is a very useful probe to detect hydroxyl radicals and hydrogen peroxide and to study Fenton chemistry, both in test tubes as well as in intact cells, and that fluorescence auto-amplification is an intrinsic property of DCF.

scholarly journals Superoxide dismutase enhances the formation of hydroxyl radicals in the reaction of 3-hydroxyanthranilic acid with molecular oxygen

1988 ◽  
Vol 251 (3) ◽  
pp. 893-899 ◽  
Author(s):  
H Iwahashi ◽  
T Ishii ◽  
R Sugata ◽  
R Kido
Keyword(s):  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Hydroxyl Radical ◽  
Molecular Oxygen ◽  
Hydroxyl Radicals ◽  
Fenton Reaction ◽  
Potassium Phosphate ◽  
Radical Formation ◽  
Superoxide Anions ◽  
Hydroxyanthranilic Acid

Superoxide dismutase (SOD) enhanced the formation of hydroxyl radicals, which were detected by using the e.s.r. spin-trapping technique, in a reaction mixture containing 3-hydroxyanthranilic acid (or p-aminophenol), Fe3+ ions, EDTA and potassium phosphate buffer, pH 7.4. The hydroxyl-radical formation enhanced by SOD was inhibited by catalase and desferrioxamine, and stimulated by EDTA and diethylenetriaminepenta-acetic acid, suggesting that both hydrogen peroxide and iron ions participate in the reaction. The hydroxyl-radical formation enhanced by SOD may be considered to proceed via the following steps. First, 3-hydroxyanthranilic acid is spontaneously auto-oxidized in a process that requires molecular oxygen and yields superoxide anions and anthranilyl radicals. This reaction seems to be reversible. Secondly, the superoxide anions formed in the first step are dismuted by SOD to generate hydrogen peroxide and molecular oxygen, and hence the equilibrium in the first step is displaced in favour of the formation of superoxide anions. Thirdly, hydroxyl radicals are generated from hydrogen peroxide through the Fenton reaction. In this Fenton reaction Fe2+ ions are available since Fe3+ ions are readily reduced by 3-hydroxyanthranilic acid. The superoxide anions do not seem to participate in the reduction of Fe3+ ions, since superoxide anions are rapidly dismuted by SOD present in the reaction mixture.

Rotting by radicals – the role of cellobiose oxidoreductase?

2003 ◽  
Vol 31 (6) ◽  
pp. 1335-1336 ◽  
Author(s):  
M.G. Mason ◽  
P. Nicholls ◽  
M.T. Wilson
Keyword(s):  
Hydrogen Peroxide ◽  
Hydroxyl Radicals ◽  
Ferrous Iron ◽  
Functional Role ◽  
Fenton Chemistry ◽  
Lignocellulose Biodegradation

Cellobiose oxidoreductase is a flavocytochrome secreted by wood-rotting fungi. The structure and functional role of the enzyme are reviewed, and a mechanism through which the enzyme produces superoxide, ferrous iron and hydrogen peroxide is proposed. The reactions of hydroxyl radicals formed by Fenton chemistry are discussed in the context of lignocellulose biodegradation.

scholarly journals Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

2015 ◽  
Vol 81 (18) ◽  
pp. 6393-6398 ◽  
Author(s):  
Ying Wang ◽  
Anni B. Hougaard ◽  
Wilhelm Paulander ◽  
Leif H. Skibsted ◽  
Hanne Ingmer ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Staphylococcus Aureus ◽  
Free Radicals ◽  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Bacterial Species ◽  
Iron Chelation ◽  
Radical Formation ◽  
Bacterial Cells ◽  
Content Type

ABSTRACTDetection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrrolineN-oxide) to assess free radical formation in the human pathogenStaphylococcus aureustreated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that whenS. aureuswas exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However,S. aureuscells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reducedkatAexpression and catalase activity in the presence of either antibiotic. Therefore, our results show that inS. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.

A Systematic DFT Study of Two Elementary Steps Involving Hydrogen Peroxide and the Hydroxyl Radical in the Fenton Reaction

2018 ◽  
Author(s):  
Danilo Carmona ◽  
David Contreras ◽  
Oscar A. Douglas-Gallardo ◽  
Stefan Vogt-Geisse ◽  
Pablo Jaque ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Hydroxyl Radical ◽  
Ab Initio ◽  
Fenton Reaction ◽  
Basis Set ◽  
Basis Sets ◽  
Dft Methods ◽  
Elementary Steps ◽  
Oxygen Oxygen ◽  
Complete Basis Set

The Fenton reaction plays a central role in many chemical and biological processes and has various applications as e.g. water remediation. The reaction consists of the iron-catalyzed homolytic cleavage of the oxygen-oxygen bond in the hydrogen peroxide molecule and the reduction of the hydroxyl radical. Here, we study these two elementary steps with high-level ab-initio calculations at the complete basis set limit and address the performance of different DFT methods following a specific classification based on the Jacob´s ladder in combination with various Pople's basis sets. Ab-initio calculations at the complete basis set limit are in agreement to experimental reference data and identified a significant contribution of the electron correlation energy to the bond dissociation energy (BDE) of the oxygen-oxygen bond in hydrogen peroxide and the electron affinity (EA) of the hydroxyl radical. The studied DFT methods were able to reproduce the ab-initio reference values, although no functional was particularly better for both reactions. The inclusion of HF exchange in the DFT functionals lead in most cases to larger deviations, which might be related to the poor description of the two reactions by the HF method. Considering the computational cost, DFT methods provide better BDE and EA values than HF and post--HF methods with an almost MP2 or CCSD level of accuracy. However, no systematic general prediction of the error based on the employed functional could be established and no systematic improvement with increasing the size in the Pople's basis set was found, although for BDE values certain systematic basis set dependence was observed. Moreover, the quality of the hydrogen peroxide, hydroxyl radical and hydroxyl anion structures obtained from these functionals was compared to experimental reference data. In general, bond lengths were well reproduced and the error in the angles were between one and two degrees with some systematic trend with the basis sets. From our results we conclude that DFT methods present a computationally less expensive alternative to describe the two elementary steps of the Fenton reaction. However, choice of approximated functionals and basis sets must be carefully done and the provided benchmark allows a systematic validation of the electronic structure method to be employed

scholarly journals Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions

2003 ◽  
Vol 69 (4) ◽  
pp. 2245-2252 ◽  
Author(s):  
J. B. Cross ◽  
R. P. Currier ◽  
D. J. Torraco ◽  
L. A. Vanderberg ◽  
G. L. Wagner ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Ascorbic Acid ◽  
Dissolved Oxygen ◽  
Hydroxyl Radicals ◽  
Fenton Reaction ◽  
Copper Ions ◽  
Transition Metal Ions ◽  
Fenton Reagent ◽  
Bacillus Spores ◽  
Modified Fenton

ABSTRACT An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu2+) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O2 is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.

scholarly journals Involvement of a local Fenton reaction in the reciprocal modulation by O2 of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase gene and the insulin-dependent activation of the glucokinase gene in rat hepatocytes

1998 ◽  
Vol 335 (2) ◽  
pp. 425-432 ◽  
Author(s):  
Thomas KIETZMANN ◽  
Torsten PORWOL ◽  
Karl ZIEROLD ◽  
Kurt JUNGERMANN ◽  
Helmut ACKER
Keyword(s):  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Fenton Reaction ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gene Activation ◽  
Three Dimensional ◽  
Rat Hepatocytes ◽  
Confocal Laser ◽  
Radical Scavenger ◽  
Insulin Dependent

H2O2 mimicked the action of periportal pO2 in the modulation by O2 of the glucagon-dependent activation of the phosphoenolpyruvate carboxykinase (PCK) gene and the insulin-dependent activation of the glucokinase (GK) gene. H2O2 can be converted in the presence of Fe2+ in a Fenton reaction into hydroxyl anions and hydroxyl radicals (•OH). The hydroxyl radicals are highly reactive and might interfere locally with transcription factors. It was the aim of the present study to investigate the role of and to localize such a Fenton reaction. Hepatocytes cultured for 24 h were treated under conditions mimicking periportal or perivenous pO2 with glucagon or insulin plus the iron chelator desferrioxamine (DSF) or the hydroxyl radical scavenger dimethylthiourea (DMTU) to inhibit the Fenton reaction. PCK mRNA was induced by glucagon maximally under conditions of periportal pO2 and half-maximally under venous pO2. GK mRNA was induced by insulin with reciprocal modulation by O2. DSF and DMTU reduced the induction of PCK mRNA to about half-maximal and increased the induction of GK mRNA to maximal under both O2 tensions. Hydroxyl radical formation was maximal under arterial pO2. Perivenous pO2, DSF and DMTU each decreased the formation of •OH to about 70% of control. The Fenton reaction could be localized in a perinuclear space by confocal laser microscopy and three-dimensional reconstruction techniques. In the same compartment, iron could be detected by electron-probe X-ray microanalysis. Thus a local Fenton reaction is involved in the O2 signalling, which modulated the glucagon- and insulin-dependent PCK gene and GK gene activation.

Hydroxyl radical formation from bacteria-assisted Fenton chemistry at neutral pH under environmentally relevant conditions

2016 ◽  
Vol 13 (4) ◽  
pp. 757 ◽  
Author(s):  
Jarod N. Grossman ◽  
Tara F. Kahan
Keyword(s):  
Hydrogen Peroxide ◽  
Hydroxyl Radical ◽  
Natural Waters ◽  
Shewanella Oneidensis ◽  
Radical Formation ◽  
Production Rates ◽  
Fenton Chemistry ◽  
Iron Reducing Bacteria ◽  
Neutral Ph ◽  
Reducing Bacteria

Environmental contextReactions in natural waters such as lakes and streams are thought to be extremely slow in the absence of sunlight (e.g. at night). We demonstrate that in the presence of iron, hydrogen peroxide and certain bacteria (all of which are common in natural waters), certain reactions may occur surprisingly quickly. These findings will help us predict the fate of many compounds, including pollutants, in natural waters at night. AbstractDark Fenton chemistry is an important source of hydroxyl radicals (OH•) in natural waters in the absence of sunlight. Hydroxyl radical production by this process is very slow in many bodies of water, owing to slow reduction and low solubility of FeIII at neutral and near-neutral pH. We have investigated the effects of the iron-reducing bacteria Shewanella oneidensis (SO) on OH• production rates from Fenton chemistry at environmentally relevant hydrogen peroxide (H2O2) and iron concentrations at neutral pH. In the presence of 2.0 × 10–4M H2O2, OH• production rates increased from 1.3 × 10–10 to 2.0 × 10–10Ms–1 in the presence of 7.0 × 106cellsmL–1 SO when iron (at a concentration of 100μM) was in the form of FeII, and from 3.6 × 10–11 to 2.2 × 10–10Ms–1 when iron was in the form of FeIII. This represents rate increases of factors of 1.5 and 6 respectively. We measured OH• production rates at a range of H2O2 concentrations and SO cell densities. Production rates depended linearly on both variables. We also demonstrate that bacteria-assisted Fenton chemistry can result in rapid degradation of aromatic pollutants such as anthracene. Our results suggest that iron-reducing bacteria such as SO may be important contributors to radical formation in dark natural waters.

Formation of Hydroxyl Radicals from Hydrogen Peroxide and Iron Salts by Superoxide- and Ascorbate-Dependent Mechanisms: Relevance to the Pathology of Rheumatoid Disease

1983 ◽  
Vol 64 (6) ◽  
pp. 649-653 ◽  
Author(s):  
D. A. Rowley ◽  
B. Halliwell
Keyword(s):  
Hydrogen Peroxide ◽  
Hydroxyl Radical ◽  
Hydroxyl Radicals ◽  
Oxygen Radicals ◽  
Rheumatoid Disease ◽  
Iron Salts ◽  
Low Concentrations ◽  
Short Period ◽  
Generating System

1. Superoxide and hydrogen peroxide are formed by activated phagocytes and react together in the presence of iron salts to form the hydroxyl radical, which attacks hyaluronic acid. Ascorbic acid also interacts with hydrogen peroxide and iron salts to form hydroxyl radical in a reaction independent of superoxide. Since iron salts, ascorbate and activated phagocytes are present in the rheumatoid joint, experiments were designed to see whether ascorbate-dependent or superoxide-dependent formation of hydroxyl radicals would be more important in vivo. 2. in the present study, addition of ascorbate to a superoxide-generating system at concentrations of 100 μmol/l provoked a superoxide-independent formation of hydroxyl radicals for a short period. Lower concentrations of ascorbate did not do this. It is therefore suggested that the superoxide-dependent reaction is probably more important. 3. It is further suggested that destruction of ascorbate by oxygen radicals formed by activated phagocytes accounts for the previously reported low concentrations of this compound in the serum and synovial fluid of rheumatoid patients.

Fenton chemistry in biology and medicine

2007 ◽  
Vol 79 (12) ◽  
pp. 2325-2338 ◽  
Author(s):  
Josef Prousek
Keyword(s):  
Hydroxyl Radical ◽  
Fenton Reaction ◽  
Brown Rot ◽  
White Rot ◽  
Practical Utilization ◽  
Fenton Chemistry ◽  
Radical Production

Various aspects of the participation of Fenton chemistry in biology and medicine are reviewed. Accumulated evidence shows that both hydroxyl radical and ferryl [Fe(IV)=O]2+ can be formed under a variety of Fenton and Fenton-like reactions. Some examples of metal-independent hydroxyl radical production are included. Extracellular Fenton reaction is illustrated by the white rot and brown rot wood-decaying fungi. The natural and practical utilization of catechol-driven Fenton reaction is also presented.

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