Controllable Release and High‐Efficiency Collection of Hydrogen Peroxide: Application on the Quantitative Investigation of Biomolecule Oxidation Induced by Reactive Oxygen Species

Hydrogen peroxide and hydroxyl radical, both important members of the reactive oxygen species (ROS) family, can cause serious oxidative damages in biological systems. In order to proclaim and prevent oxidation stress, researches on the biomolecule oxidation induced by H₂O₂ or OH^. are in crucial need. However, due to the high reactivity of ROS, traditional methods are difficult to achieve the in situ quantitative investigations on those reactions involving ROS. In this work, using scanning electrochemical microscopy technique (SECM) in a tip generation‐substrate collection mode (TG‐SC), the controllable release and the high‐efficiency collection of electrogenerated H₂O₂ were achieved. Compared to ex situ fluorescence method, SECM improved the collection efficiency approximately two times larger. Based on it, SECM combined with surface plasmon resonance (SPR) was employed to in situ monitor the protein oxidation (taking Cu₁₂⁺? MT as a model) induced by H₂O₂. OH^., which was generated from the interaction between H₂O₂ and Cu₁₂⁺? MT, can attack the peptide chain and induced the unrepairable protein oxidation damage. The whole process was quantitatively characterized by SPR, and the linear relationship between SPR dip shift and the amounts of released H₂O₂ was successfully built. Our work proves that the combined SECM‐SPR technique can realize the in situ quantitative determinations of the biomolecule oxidation induced by ROS, which affords an avenue for further elucidation on the mechanisms of oxidation stress in organisms.     

Determination of Cobalt(II)-Hydrogen Peroxide-Induced DNA Oxidative Damage and Preventive Antioxidant Activity by CUPRAC Colorimetry

Abstract

The classical Fenton system composed of Fe(II) and H₂O₂ uses harsh oxidative conditions and cannot realistically simulate physiological oxidations which are less severe. Here, reactive oxygen species (ROS) were generated with a combination of CoSO₄ and H₂O₂ to provide milder conditions. DNA was used as a biologically meaningful probe for monitoring the oxidative conversion. Oxidative hazard on DNA was accomplished in ammonia/ammonium chloride buffer at 37?°C, and the Fenton reaction was stopped with trichloroacetic acid (TCA). A suitable aliquot of this solution was added to cupric ion reducing antioxidant capacity (CUPRAC) reaction mixture, and the absorbance at 450?nm was recorded. The oxidized species derived from DNA were CUPRAC-reactive while intact DNA was not. The protective effects of antioxidants (AOxs), known to have radical scavenging effects, were tested; green tea and a synthetic fetal bovine serum (FBS) were also successfully used as real ROS scavengers. Although the classical iron-based Fenton procedure applied in ethanol medium generated CUPRAC-responsive products, the proposed system was perfectly ethanol-tolerant, enabling the CUPRAC measurement of DNA oxidation products against an unaffected reagent blank. The protective effects of phenolic antioxidants, perfectly solubilized in ethanol, could also be measured.     

Analysis of oxidation process of cholecystokinin octapeptide with reactive oxygen species by high‐performance liquid chromatography and subsequent electrospray ionization mass spectrometry

The C‐terminal octapeptide of cholecystokinin (CCK8) includes some easily oxidizable amino acids. The oxidation of CCK8 by reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH^?) was investigated using reversed‐phase high performance liquid chromatography (RP‐HPLC) and subsequent electrospray ionization mass spectrometry. The mechanism of oxidation of CCK8 in the H₂O₂ system differed from that of CCK8 in the Fenton system, in which OH^? are produced. In the H₂O₂ system, ²⁸Met and ³¹Met were oxidized to methionine sulfoxide, and no further oxidation or degradation/hydrolysis occurred. On the other hand, in the Fenton system, ²⁸Met and ³¹Met residues were oxidized to methionine sulfone via the formation of methionine sulfoxide. In addition, the oxidized product was observed at the Trp residue but not at the Tyr residue, and small peptide fragments from CCK8 were observed in the Fenton system. From these results, it was concluded that ²⁸Met and ³¹Met residues of CCK8 are susceptible to oxidation by ROS. Copyright © 2009 John Wiley & Sons, Ltd.     

Reduction mechanisms of different oxidizing agents at diamond surfaces

“Electroless” oxidation, at room temperature, of boron-doped diamond (BDD) films with oxidizing agents as Ce⁴⁺, MnO₄^?, H₂O₂ or S₂O₈^2? is an efficient way to transform hydrogen terminations (C-H) into oxygen ones (C-O). To investigate the oxidation mechanism of diamond surfaces through these open current potential (OCP) processes, we study in the present work the reduction mechanisms of the different oxidizing agents at BDD surfaces. Current-voltage measurements were performed using a rotating disk electrode of diamond immersed in a solution containing one of the species. Two different mechanisms were evidenced: an electrochemical for Ce⁴⁺ and MnO₄^? and a chemical one based on the production of radicals under light exposure for H₂O₂ and S₂O₈^2?.     

Enhanced anodic Ru(bpy)3(2+) electrogenerated chemiluminescence by polyphenols

Anodic Ru(bpy)₃²⁺ electrogenerated chemiluminescence (ECL) can be enhanced by polyphenols in alkaline solution. Spin trapping-electron spin resonance (ESR) experiments verified that reactive oxygen species (ROS) were generated during the electrolysis of Ru(bpy)₃²⁺ in alkaline solution, and oxidation of quercetin enhanced Ru(bpy)₃²⁺ ECL at anodic potential by producing additional ROS. This ECL enhancement can be used to analyze real sample and evaluate antioxidant activity of polyphenols.     

Colorimetric determination of radical scavenging activity of antioxidants using Fe3O4 magnetic nanoparticles

We report the first use of iron oxide magnetic nanoparticles (Fe₃O₄ MNPs) as a novel, alternative, simple and reliable agents for colorimetric measurement of radical scavenging activity of the antioxidants. In the presence of H₂O₂ and the peroxidase colorimetric substrate, Fe₃O₄ MNPs catalyzed the oxidation of colorless peroxidase substrate to form colorimetric products via the generation of hydroxyl radicals. After adding antioxidants, the catalytic activity of Fe₃O₄ MNPs was inhibited due to scavenging of hydroxyl radicals by the antioxidants, producing less colorimetric products resulting in the reduction of color intensity. Two model antioxidant standards including gallic acid (GA) and epigallocatechin gallate (EGCG) were successfully evaluated for their hydroxyl radical scavenging activity using the developed assay. The performance of the developed method was validated against traditional antioxidant assays for 9 tea samples. Using the Spearman rank correlation coefficient method, the antioxidant activity of tea samples obtained from the Fe₃O₄ MNP assay correlated well with the traditional assays at the 95% confidence level. Furthermore, the developed assay was successfully carried out on a paper-based device to provide for high throughput analysis with low amounts of reagents and sample consumption and low analysis cost for screening of radical scavenging activity of the antioxidants. The results from the analysis of antioxidant activity in tea samples obtained from the Fe₃O₄ MNP paper-based assay were not significantly different to those obtained from the developed Fe₃O₄ MNP spectrophotometric assay indicating that the developed assay was also applicable in a low-cost analysis platform.     

Electrochemical investigation of flavonolignans and study of their interactions with DNA in the presence of Cu(II)

Flavonolignans, silybin and its derivatives (2,3-dehydrosilybin, 7-O-methylsilybin, 20-O-methylsilybin) and isosilybin were studied using ex situ (adsorptive transfer, AdT) cyclic and square wave voltammetry (SWV). The two oxidation steps were described for flavonolignans at potentials E_p1 + 0.5 V and E_p2 + 0.85 V depending on experimental conditions. An additional oxidation peak at E_p3 + 0.35 V was observed only for 2,3-dehydrosilybin. The anodic currents of flavonolignans are related to their electron transfer processes (oxidation of hydroxyl groups), which was supported by density functional theory (DFT) and B3P86 theory level. Our electrochemical results confirmed that 2,3-dehydrosilybin is a relatively strong antioxidant, which is strictly associated with oxidation at E_p3. The oxidation processes and antioxidant parameters of flavonolignans can be affected by transition metal complexation via hydroxyl groups. We found that silybin and 2,3-dehydrosilybin are able to chelate transition metals, especially Cu²⁺. The formation of silybin/Cu complexes was studied by AdT SWV and the observation was also confirmed using fluorescence spectroscopy. The electrochemical investigation of DNA interactions and damage caused in the presence of silybin/Cu complex and hydrogen peroxide is described. We present evidence that flavonolignans are involved not only in antioxidant abilities but also in the prooxidation effects under in vitro conditions.     

An immunomodulator from Tinospora cordifolia with antioxidant activity in cell-free systems

Several plant products are known to exhibit immense medicinal value against human diseases. Our earlier studies showed that dry stem crude extract (DSCE) ofTinospora cordifolia contained a polyclonal B cell mitogen, G1-4A. DSCE as well as G1-4A also enhanced immune response in mice. In order to explore the possibility of using G1-4A/PPI (partially purified immunomodulator) to modulate radiation induced immunosuppression, the antioxidant effect of PPI from this plant was examined against reactive oxygen and nitrogen species (ROS/RNS), generated by photosensitization/peroxynitrite. Levels of lipid peroxidation products, superoxide dismutase (SOD) and catalase in liver/spleen homogenate from mouse were monitored. Photosensitization induced significant increase in thiobarbituric acid reactive substances (TBARS) in liver. The activities of SOD and catalase were reduced considerably. PPI, present during photosensitisation, prevented lipid peroxidation and restored the activities of both the enzymes. Likewise, oxidative damage induced by peroxynitrite was inhibited by PPI. The degradation of proteins due to photosensitization as assessed by SDS-PAGE was effectively reduced by simultaneous treatment with PPI during photosensitization. Selective inhibitors of ROS like mannitol, SOD, sodium azide and antioxidants, GSH and vitamin C brought about significant inhibition of formation of TBARS suggesting possible involvement of O₂ ^•,^•OH and¹O₂. Photosensitization in deuterated buffer enhanced formation of TBARS thus indicating generation of¹O₂. Thus, the action of PPI may be against oxidative damage through Type I and II photosensitization mechanisms. Therefore, the immunomodulator fromTinospora cordifolia may also be beneficial as an antioxidant.     

Magnesium Corrosion Triggered Spontaneous Generation of H2O2 on Oxidized Titanium for Promoting Angiogenesis

Although the use of reactive oxygen species (ROS) has been extensively studied, current systems employ external stimuli such as light or electrical energy to produce ROS, which limits their practical usage. In this report, biocompatible metals were used to construct a novel electrochemical system that can spontaneously generate H₂O₂ without any external light or voltage. The corrosion of Mg transfers electrons to Au‐decorated oxidized Ti in an energetically favorable process, and the spontaneous generation of H₂O₂ in an oxygen reduction reaction was revealed to occur at titanium by combined spectroscopic and electrochemical analyses. The controlled release of H₂O₂ noticeably enhanced in vitro angiogenesis even in the absence of growth factors. Finally, a new titanium implant prototype was developed by Mg incorporation, and its potential for promoting angiogenesis was demonstrated.     

An Experimental and Mechanistic Investigation of the Complexities Arising During Inhibition of the Briggs?Rauscher Reaction by Antioxidants

A method to determine the relative antioxidant capacity of radical scavengers based on the inhibition of the oscillations of the Briggs? Rauscher (BR) oscillating reaction was previously reported. A semiquantitative mechanistic interpretation of the inhibitory effects required two steps to obtain simulated inhibition times in very good agreement with the experimental ones. The first step is inhibitory, involving H‐atom transfer from antioxidant to the HOO^. radical; the second step is a first‐order degradation of the antioxidant to unspecified products. Since the degradation may be due to oxidation and/or iodination of the antioxidant, we studied the kinetics of the subsystems IO (H⁺)+antioxidant and I₂(H⁺)+antioxidant. We used 2,5‐ and 2,6‐dihydroxybenzoic acids, caffeic acid (=3‐(3,4‐dihydroxyphenyl)prop‐2‐enoic acid), ferulic acid (=3‐(4‐hydroxy‐3‐methoxyphenyl)prop‐2‐enoic acid), pyrocatechol (=benzene‐1,2‐diol), and hydroquinone (=benzene‐1,4‐diol) as antioxidants. Spectra in the wavelength range 500–250 nm were repeated at given time intervals to follow the peaks of the iodine and oxidation products, which were mainly quinones. For the iodination of the above diphenols (=benzenediol derivatives) the substitution and/or addition reactions with I₂ or HOI were found to be relatively slow compared to oxidation by IO . Approximate rate constants for oxidation were obtained on the basis of a reasonable kinetic model by using a suitable numerical integration program. Although these complexities can arise also in the completely inhibited BR oscillator, we believe that the inhibitory effects are due to the HOO^. scavenging action by diphenols or by quinones since HOO^. radicals are also potential reducing agents. We propose two steps that could maintain a small reservoir of diphenol, while both quinone and diphenol deplete HOO^. radicals. In short, the complexities do not affect the method for monitoring the relative activity of antioxidants based on the BR oscillating reaction. The effects of temperature on the inverse of the oscillatory time in the BR‐uninhibited system, on the inverse of inhibition times, and on the time length of the resumed oscillations for four antioxidants were also investigated. Apparent average activation energies were obtained.     

Cellular Antioxidant Properties of Ischnoderma Resinosum Polysaccharide

A predominant polysaccharide isolated from Ischnoderma resinosum underwent evaluation for its capacity to scavenge free radicals and its potential antioxidant properties at a cellular-oriented level. This proved that Ischnoderma resinosum polysaccharide (IRP) remarkably curtailed AAPH-induced erythrocyte hemolysis through the inhibition of the generation of ROS (p < 0.05). Rather, it caused the restoration of intracellular antioxidant enzyme (SOD, GSH-Px, and CAT) activities at an acceptable pace and the silencing of intracellular MDA formation, as well as the rescaling of LDH leakage. Furthermore, a model of oxidative stress in HepG2 cells was established by adopting 400 μM of hydrogen peroxide, which suggested that IRP manifests promising antioxidant activity. Notably, after the intervention of IRP in the H₂O₂-induced HepG2 cells, there was a statistical elevation in cell survivability (p < 0.05). IRP diminished the morphological alterations in the nucleus and decreased the secretion of ROS (p < 0.05), with a dose-dependent abrogation of apoptosis (p < 0.05). Consequently, IRP, which was isolated and purified, was able to scavenge free radicals and possessed favorable antioxidant activity that could dampen the occurrence of oxidative stimulation and effectively alleviate the AAPH-induced erythrocyte hemolysis and H₂O₂-induced oxidative damage in HepG2 cells. This provides a basis and theoretical reference for the development and utilization of IRP as a natural antioxidant, with emphasis on the exploitation of environmentally friendly and cost-effective antioxidants.     

The NSAIDs Indomethacin and Diflunisal as Scavengers of Photogenerated Reactive Oxygen Species

Diflunisal (DFNS) and Indomethacin (IMTC) are two profusely employed NSAIDs that provide anti‐inflammatory and analgesic effects in humans. The scavenging of reactive oxygen species (ROS) by both NSAIDs was systematically studied in pH 7 aqueous solution. The ROS O₂(¹Δ_g), O₂^?? and H₂O₂, generated by visible light irradiation of Riboflavin (Rf) in the presence of DFNS and IMTC, are deactivated by the NSAIDs. The ROS scavenging action by both NSAIDs constitutes an interesting result and adds one more positive aspect to the beneficial actions attributed to these drugs. Nevertheless it should be taken into account that several NSAIDs, in particular IMTC, have been connected to the pathogenesis of gastric mucosal lesions, which in some cases includes ROS generating‐ability. DFNS quenches ROS in a dominant physical fashion. It constitutes an excellent protective‐antioxidant provided that is practically not destroyed/oxidized after the ROS scavenging action. IMTC, being also an efficient interceptor of ROS, belong to the so‐called group of sacrificial‐ROS quenchers: It is easily degraded by the oxidative species in the scavenging action. Although this property is negative in the context of prolonged ROS elimination, exhibits a promissory aspect for the degradation of pharmaceutical contaminants, such as NSAIDs, in waste waters.     

Plant-mediated fabrication of cobalt oxide nanoparticles and evaluation of their catalytic effects on electro-oxidation of formaldehyde

Cobalt oxide nanoparticles (Co₃O₄-NPs) were synthesized by aqueous extract of Artemisia vulgaris plant at 50°C. The biosynthesized extract mediated Co₃O₄-NPs were characterized through different methods containing FT-IR (Fourier transform infrared spectroscopy), FESEM (Field emission scanning electron microscopy), EDS (Energy-dispersive x-ray spectroscopy), and XRD (x-ray diffraction). Co₃O₄-NPs as an efficient and practical catalyst was employed for the electrochemical oxidation of formaldehyde; which was an irreversible charge transfer controlled by mass transfer. The coefficients of electron transfer and diffusion were 0.87 and 0.122 cm²/s for formaldehyde oxidation on Co₃O₄ electrode, respectively. The formation of the CoOOH species during potential sweeping appears to be involved in the catalytic activity of the Co₃O₄ toward formaldehyde oxidation.     

Electrochemical Study on DNA Damage Based on the Direct Oxidation of 8‐Hydroxydeoxyguanosine at an Electrochemically Modified Glassy Carbon Electrode

The study of DNA damage induced by Fenton reaction (Fe²⁺/H₂O₂) in vitro was performed based on the direct electrochemical oxidation of 8‐hydroxydeoxyguanosine (8‐OH‐dG), the biomarker of DNA oxidative damage, at an electrochemically modified glassy carbon electrode (GCE). The effects of antioxidants, such as ascorbic acid, and hydroxyl‐radical scavenger (mannitol) on the DNA damage were also investigated. 8‐OH‐dG, the oxidation product of guanine residues in DNA, has shown significantly oxidative peak on the electrochemically modified GCE. The oxidative peak current of 8‐OH‐dG was linear with the damaged DNA concentration in the range of 10–200 mg/L. The experimental results demonstrate that ascorbic acid has ambivalent effect on DNA oxidative stress. It can promote DNA oxidative damage when ascorbic acid concentration is below 1.5 mM and protect DNA from damage in the range of 1.5–2.5 mM. As a hydroxyl‐radical scavenger, mannitol inhibits significantly DNA oxidative damage. The influence of Fe²⁺, as reactant, and EDTA as iron chelator in the system were also studied. The proposed electrochemical method can be used for the estimation of DNA oxidative damage from new point of view.     

Cytoprotective effect of selenium polysaccharide from Pleurotus ostreatus against H2O2-induced oxidative stress and apoptosis in PC12 cells

One main fraction of Selenium-enriched Pleurotus ostreatus (P. ostreatus) polysaccharide (Se-POP-1) was extracted and purified by DEAE-52 and sephadex G-100. Se-POP-1 was an approximate homogenous polysaccharide with an average molecular weight of 1.62 × 10⁴ Da, and mainly composed of glucose, mannose and galactose, with molar ratio of 5.30:1.55:2.14. The absorption peaks at 941 cm^?1 and 1048 cm^?1 in FT-IR analysis were ascribed as C-O-Se and Se=O bonds. Pr-treatment of Se-POP-1 (400 μg/mL) increased the cell survival of H₂O₂-stimulated PC12 cells and inhibited intrinsic apoptosis and oxidative stress in H₂O₂-stimulated PC12 cells via limiting DNA degradation and decreasing the reactive oxygen species (ROS) generation. In addition, up-regulation of anti-apoptotic protein Bcl-2, down- regulation of pro-apoptotic protein Bax, cleaved caspase 3, and cytochrome c were also observed. Se-POP-1 presented an obvious effect to alleviate oxidative damage and apoptosis in PC12 cells induced by H₂O₂. Therefore, Se-POP-1 possessed potent antioxidant and biological activities with the ability to prevent oxidation via scavenging ROS and free radicals in cells. It could be developed as organic selenium dietary supplement and functional food.     

Electrocatalytic Response of a Glassy-Carbon Electrode Modified with a Polyvinylpyridine Film with Electrodeposited Palladium in the Oxidation of Oxalic Acid

The electrochemical properties of a glassy-carbon electrode modified with a polyvinylpyridine film with electrodeposited palladium were studied. Conditions were selected for preparing a composite film on a glassy-carbon surface. It was found that palladium particles deposited on the polyvinylpyridine film exhibited electrocatalytic activity in the oxidation of H₂C₂O₄. Compared to an unmodified electrode, the oxidation potential of oxalic acid decreased and the current of its oxidation multiply increased. The catalytic current of oxalic acid oxidation was a linear function of its concentration in the range from 1 × 10^–2 to 1 × 10^–6 M.     

Voltammetric Study of Some Synthetic Antioxidants on Platinum Microelectrodes in Acetic Acid Medium

The electrochemical oxidation of the antioxidants tert‐butylhydroquinone (BHQ), tert‐butyl‐4‐hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) has been investigated by voltammetry at platinum microelectrodes in glacial acetic acid containing sodium perchlorate or sodium acetate as a background electrolyte. BHQ, a compound with para‐diphenyl groups, was typically found to have lower oxidation potential than isolated phenols BHA and BHT. Particular attention has been devoted to the mechanism of the electrode processes. Linear scan voltammograms recorded under steady‐state conditions showed well‐defined oxidation waves of the compounds studied even at relatively high concentrations (2 mM), due to a low proclivity for adsorption of the oxidation products on the electrode surface in acetic acid. The optimal conditions for the voltammetric determination of BHQ, BHA and BHT have also been studied. The results reveal that sensitive response for all the oxidants studied can be obtained in solution containing 0.1 M NaClO₄ and 0.1 M CH₃COONa by using the differential pulse voltammetry and square‐wave voltammetry. Peak currents were found to vary linearly with the antioxidant concentration over the range 3×10^?5?1×10^?3 M.     

Simultaneous Electroluminescence

A proof‐of‐concept for the production of simultaneous electroluminescence in a single electrochemical cell in a basic medium and using H₂O₂ as a co reactant is obtained: at the anode by means of the direct oxidation of luminol to yield an excited emitting species, and in the catholyte by an indirect mediated process involving the initial reduction of ClO₂^? to produce ClO^?, which then reacts with luminol (also to produce an excited emitting species). Emission spectra and possible mechanistic pathways are discussed.     

Electroactivity of Superoxide Anion in Aqueous Phosphate Buffers Analyzed with Platinized Microelectrodes

The reactivity of platinized ultramicroelectrodes (Pt-black UMEs) towards superoxide anion O₂^.−, an unstable Reactive Oxygen Species (ROS), and its relatives, H₂O₂ and O₂, was studied. Voltammetric studies in PBS demonstrate that Pt-black UMEs provide: i) a well-resolved reversible redox signature for O₂^.− detected in both alkaline and physiological buffers (pH 12 and 7.4); ii) irreversible oxidation and reduction waves for H₂O₂ at pH 7.4. The oxygen reduction reaction (ORR) at Pt-black surfaces solely yields H₂O₂ (2 electrons/2 H⁺) at physiological pH. Consequently, Pt-black UMEs allow to sense different ROS including superoxide anion for future biomedical or physico-chemical investigations.