Chromate-mediated free radical generation from cysteine,penicillamine, hydrogen peroxide,and lipid hydroperoxides

The Cr(VI)-mediated free radical generation from cystein, penicillamine, hydrogen peroxide, and model lipid hydroperoxides was investigated utilizing the electron spin resonance (ESR) spin trapping technique. Incubation of Cr(VI) with cysteine (Cys) generated cysteinyl radical. Radical yield depended on the relative concentrations of Cr(VI) and Cys. The radical generation became detectable at a cysteine: Cr(VI) ration of about 5, reached its highest level at a ratio of 30, and declined thereafter. Cr(VI) or Cys alone did not generate a detectable amount of free radicals. Similar results were obtained with penicillamine. Incubation of Cr(VI), Cys or penicillamine adn H₂O₂ led to hydroxyl (·OH) radical generation, which was verified by quantitative competition experiments utilizing ethanol. The mechanism for ·OH radical generation is considered to be a Cr(VI)-mediated Fenton-like reaction. When model lipid hydroperoxides such as t-butylhydroperoxide and cumene hydroperoxide were used in place of H₂O₂, hydroperoxide-derived free radicals were produced. Since thiols, such as Cys, exist in cellular systems at relatively high concentrations, Cr(VI)-mediated free radical generation in the presence of thiols may participate in the mechanisms of Cr(VI)-induced toxicity and carcinogenesis.     

Lipid peroxidation during the oxidation of haemoproteins by hydroperoxides. Relation to electronically excited state formation

The formation of electronically excited states during hydroperoxide metabolism is analysed in terms of recombination reactions involving secondary peroxyl radicals and scission of the O? O bond of peroxides by haemoproteins, mainly myoglobin. Both processes may be sequentially interrelated, for the cleavage of H₂O₂ by metmyoglobin leads to the formation of a strong oxidizing equivalent with the capability to promote peroxidation of polyunsaturated fatty acids. The decomposition of lipid hydroperoxides by ferryl-hydroxo complexes, as that formed during the oxidation of metmyoglobin by H₂O₂, is a source of peroxyl radicals, the recombination of which proceeds with elimination of a conjugated triplet carbonyl or singlet oxygen.     

Isotope dilution mass spectrometry for the quantification of sulfane sulfurs

Sulfane sulfurs are one type of important reactive sulfur species. These molecules have unique reactivity that allows them to attach reversibly to other sulfur atoms and exhibit regulatory effects in diverse biological systems. Recent studies have suggested that sulfane sulfurs are involved in signal transduction processes regulated by hydrogen sulfide (H₂S). Accurate and reliable measurements of sulfane sulfurs in biological samples are thus needed to reveal their production and mechanisms of actions. Herein we report a convenient and accurate method for the determination of sulfane sulfur concentrations. The method employs a triphenylphosphine derivative (P2) to capture sulfane sulfurs as a stable phosphine sulfide product, PS2. The concentration of PS2 was then determined by isotope dilution mass spectrometry, using a ¹³C₃-labeled phosphine sulfide, PS1, as the internal standard. The specificity and efficiency of the method were proven by model reactions. It was also applied to the measurement of sulfane sulfurs in mouse tissues including brain, kidney, lung, liver, heart, spleen, and blood.     

Singlet oxygen generation from lophine hydroperoxides.

Singlet oxygen was detected from the reaction of lophine hydroperoxides 2 in the presence of 1,3-diphenyl-isobenzofuran as a singlet oxygen detector. In order to examine the substituent effect on the formation of (1)O(2), 2-(p-substituted phenyl) lophine peroxides 2a-c were tested. It was found that an electron-attracting group contributed to the efficient formation of (1)O(2) (80% for a NO(2) group), while an electron-donating group enhanced the efficiency of chemiluminescence.     

Chemiluminescent assay of lipid hydroperoxides

The addition of luminol plus a catalyst such as peroxidase or a heme prosthetic group to a solution containing a small quantity of lipid hydroperoxides results in a flash of chemiluminescence, the intensity of which is a function of the hydroperoxide concentrations. Various protocols for lipid hydroperoxide assays have been described and we have studied conditions to increase their sensitivity and specificity. Plasma lipid hydroperoxide determinations require an extraction, since compounds present in plasma interfere with light emission. Moreover, the sensitivity of the assay is by the presence of hydrogen peroxide in the medium, which causes high background values. Catalase does not act on lipid hydroperoxides and can be used to eliminate hydrogen peroxide from the reaction medium. The determination requires a blank tube in which hydroperoxides are destroyed by incubating the sample with haematin plus ascorbate. The increase in the chemiluminescence of the assay tube caused by the presence of lipid hydroperoxides is then compared to the value obtained for an internal standard.     

Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

H2O2-Mediated Damage to Lysosomal Membranes of J-774 Cells

The effects of hydrogen peroxide on cell viability and, in particular, on lysosomal integrity were investigated in a model system of cultured, established, macrophage-like J-774 cells. The cells were found to rapidly degrade added hydrogen peroxide, withstanding concentrations 250μM without cell death; however, all tested concentrations (100-500/μM) substantially decreased cellular ATP to approximately the same degree. Concentrations of hydrogen peroxide 500/μM resulted in a pronounced and rapid decrease in cell viability preceded by the loss of lysosomal integrity, as judged by the relocalization of acridine orange, a lysosomotropic weak base, in pre-labelled cells. Hydrogen peroxide-induced relocalization of acridine orange and cell death were either enhanced or much prevented, according to if the cells were initially allowed to endocytose ferric iron or the specific iron-chelator deferoxamine, respectively. Depletion of ATP, however, was not associated with the loss of lysosomal integrity and viability regardless of iron or deferoxamine pretreatment. Pre-exposure to E-64, an inhibitor of lysosomal thiol proteases, resulted in the reduction of both lysosomal membrane damage and cell death. The results are interpreted as indicating (i) generation of hydroxyl radicals within the secondary lysosomal compartment due to the occurrence of reactive ferrous iron, leading to (ii) peroxidative alterations of the lysosomal membrane resulting in (iii) loss of lysosomal membrane integrity with dissipation of the proton gradient and leakage of lysosomal contents, including hydrolytic enzymes, into the cell sap. The partial protection by E-64 may result from hydroxyl radical scavening by accumulated non-degraded autophagocytosed lysosomal material, and/or decreased availability of reactive redox-cycling iron due to decreased enzymatic digestion of autophagocytosed iron-containing metalloproteins. Moreover, our results show that the normal lysosomal content of iron, capable of redox cycling, of the cell line under study is enough to induce oxidative damage leading to loss of lysosomal integrity. It is suggested that lysosomal damage may be an important cause of cell degeneration under conditions of increased intra- or extracellular hydrogen peroxide-formation.     

Boronate probes for the detection of hydrogen peroxide release from human spermatozoa

Human spermatozoa are compromised by production of reactive oxygen species (ROS), and detection of ROS in spermatozoa is important for the diagnosis of male infertility. The probes 2′,7′-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE), and MitoSOX red (MSR) are commonly used for detecting ROS by flow cytometry; however, these probes lack sensitivity to hydrogen peroxide (H₂O₂), which is particularly damaging to mammalian sperm cells. This study reports the synthesis and use of three aryl boronate probes, peroxyfluor-1 (PF1), carboxyperoxyfluor-1, and a novel probe, 2-(2-ethoxyethoxy)ethoxyperoxyfluor-1 (EEPF1), in human spermatozoa. PF1 and EEPF1 were effective at detecting H₂O₂ and peroxynitrite (ONOO⁻) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. EEPF1 was more effective at detection of ROS in spermatozoa than DCFH, DHE, or MSR; furthermore it distinguished poorly motile sperm as shown by greater ROS production. EEPF1 should therefore have a significant role in the diagnosis of oxidative stress in male infertility, cryopreservation, age, lifestyle, and exposure to environmental toxicants.     

Multi-isotopic analysis reveals individual mobility and diet at the Early Iron Age monumental tumulus of Magdalenenberg, Germany

For the Early Iron Age western Hallstatt culture, which includes the site of Magdalenenberg in southwest Germany, it has been proposed that people were mobile and maintained far reaching social and trading networks throughout Europe. We tested this hypothesis by analyzing multiple isotopes (strontium, oxygen, sulfur, carbon, and nitrogen) of the preserved skeletons from the Magdalenenberg elite cemetery to determine diets and to look for evidence of mobility. The analysis of carbon, nitrogen, and sulfur isotope ratios in collagen of humans (n = 50) and associated domestic fauna (n = 10) indicates a terrestrial-based diet. There was a heterogeneous range of isotope values in both strontium (0.70725 to 0.71923, n = 76) and oxygen (13.4‰ to 18.5‰, n = 78) measured in tooth enamel. Although many of the individuals had values consistent with being from Hallstatt culture sites within southwest Germany, some individuals likely originated from further afield. Possible areas include the Alps of Switzerland and Austria or even locations in Italy. Our study strongly supports the assumption of far reaching social and economic networks in the western Hallstatt culture.     

Nitrosylmyoglobin as antioxidant--kinetics and proposed mechanism for reduction of hydroperoxides

Nitrosylmyoglobin (MbFe^IINO), which is believed to have a protective role during ischemia and reperfusion injury, was oxidized by tert-butyl hydroperoxide (t-BuOOH), and by hydrogen peroxide (H₂O₂) to the nitrite anion and metmyoglobin (MbFe^III). Further characterization of the reaction of MbFe^IINO with excess of t-BuOOH was investigated with respect to reaction stoichiometry, temperature and pH dependence. It was found that the reaction between MbFe^IINO with excess of t-BuOOH followed a simple stoichiometry and had moderate pH and temperature dependence with the activation parameters ΔH^‡ = 57.4 ± 1.4 kJ mol^- 1 and ΔS^‡ = - 112.0 ± 5.1 J mol^- 1 K^- 1, which is consistent with an associative reaction mechanism. Moreover, t-BuOOH-induced oxidation of MbFe^IINO did not result in any detectable formation of the hypervalent myoglobin (Mb) species, i.e. perferrylmyoglobin, (^√MbFe^IV = O) or ferrylmyoglobin (MbFe^IV = O), and hereby differed from H₂O₂-induced oxidation of MbFe^IINO, which results in the formation of MbFe^IV = O. Based on the obtained results and on published data, different mechanisms for the reaction of the MbFe^IINO with t-BuOOH and H₂O₂ are proposed.     

Effect of low-frequency alternating magnetic fields on the rate of biochemical reactions proceeding with formation of reactive oxygen species

It is (theoretically) shown by an example of the reaction of a radical with an oxygen molecule that the alternating component of a combined weak magnetic field affects the rate constants of chemical reactions. The mechanism of transduction of a weak magnetic perturbation from the primary receptor of the field to experimentally observed biological effects is followed. It is stated that the external magnetic field alters the initial population of energy levels. The magnitude of these changes depends on the field parameters. The exposure to an alternating field with proper parameters can substantially increase the concentration of reactive oxygen species in biological systems. By controlling their concentration by means of weak magnetic field, it is possible to affect the key links of metabolism.     

In vivo thiyl free radical formation from hemoglobin following administration of hydroperoxides

Although free radical formation due to the reaction between red blood cells and organic hydroperoxides in vitro has been well documented, the analogous in vivo ESR spectroscopic evidence for free radical formation has yet to be reported. We successfully employed ESR to detect the formation of the 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)/hemoglobin thiyl free radical adduct in the blood of rats dosed with DMPO and tert-butyl hydroperoxide, cumene hydroperoxide, ethyl hydrogen peroxide, 2-butanone hydroperoxide, 15(S)-hydroperoxy-5,8,11,13-eicosatetraenoic acid, or hydrogen peroxide. We found that pretreating the rats with either buthionine sulfoximine or diethylmaleate prior to dosing with tert-butyl hydroperoxide decreased the concentration of nonprotein thiols within the red blood cells and significantly enhanced the DMPO/hemoglobin thiyl radical adduct concentration. Finally, we found that pretreating rats with the glutathione reductase inhibitor 1,3-bis(2-chloroethyl)-1-nitrosourea prior to dosing with tert-butyl hydroperoxide enhanced the DMPO/hemoglobin thiyl radical adduct concentration and induced the greatest decrease in nonprotein thiol concentration within the red blood cells.     

Bicarbonate-catalyzed hydrogen peroxide oxidation of cysteine and related thiols

The effect of bicarbonate on the rates of the H₂O₂ oxidation of cysteine, gluthathione, and N-acetylcysteine to the corresponding disulfides was investigated. The relative oxidation rates at pH 8 for the different thiols are inversely related to the pK_a values of the thiol groups, and the reactive nucleophiles are identified as the thiolate anions or their kinetic equivalents. The second-order rate constants at 25 °C for the reaction of the thiolate anions with hydrogen peroxide are 17 ± 2 M⁻¹ s⁻¹ for all three substrates. In the presence of bicarbonate (>25 mM), the observed rate of thiolate oxidation is increased by a factor of two or more, and the catalysis is proposed to be associated with the formation of peroxymonocarbonate from the equilibrium reaction of hydrogen peroxide with bicarbonate (via CO₂). The calculated second-order rate constants for the direct reaction of the three thiolate anions with peroxymonocarbonate fall within the range of 900-2000 M⁻¹ s⁻¹. Further oxidation of disulfides by peroxymonocarbonate results in the formation of thiosulfonate and sulfonate products. These results strongly suggest that peroxymonocarbonate should be considered as a reactive oxygen species in aerobic metabolism with relevance in thiol oxidations.     

Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method

Methods were developed for the separation and measurement of lipid and protein hydroperoxides, which can be used for biological materials. Lipids were extracted with methanol:chloroform and their hydroperoxides measured in solutions of methanol and chloroform containing 110mM perchloric acid, xylenol orange, and ferrous iron. Proteins were isolated by precipitation with 0.2M perchloric acid. The precipitates were redissolved in 6M guanidine hydrochloride and washed with chloroform, and the hydroperoxides were measured in the presence of perchloric acid, xylenol orange, and ferrous iron. Optimum conditions for hydroperoxide measurements were established and the assays were applied to oxidized human blood serum and to cultured cells.     

Detection of hydrogen peroxide by lactoperoxidase-mediated dityrosine formation

The aim of this work was to study the dityrosine-forming activity of lactoperoxidase (LPO) and its potential application for measuring hydrogen peroxide (H₂O₂). It was observed that LPO was able to form dityrosine at low H₂O₂ concentrations. Since dityrosine concentration could be measured in a simple fluorimetric reaction, this activity of the enzyme was utilized for the measurement of H₂O₂ production in different systems. These experiments successfully measured the activity of NADPH oxidase 4 (Nox4) by this method. It was concluded that LPO-mediated dityrosine formation offers a simple way for H₂O₂ measurement.     

The effects of superoxide dismutase on H2O2 formation

Numerous reports of the effects of overproduction of SODs have been explained on the basis of increased H2O2 production by the catalyzed dismutation of O2-. In this review we consider the effects of increasing [SOD] on H2O2 formation and question this explanation.     

Hydrogen Peroxide Dependent Oxidative Degradation of DNA by Copper Epinephrine

The hydrogen peroxide dependent oxidation of the epinephrinecopper complex to adrenochrome is mediated by free copper ions. The oxidation is enhanced by chloride ions and by the presence of serum albumin. The reaction is not inhibited by SOD or by hydroxyl radical scavengers.

The 2:1 epinephrine or dopamine:Cu(II) complexes are able to bind to DNA and to catalyze its oxidative destruction in the presence of hydrogen peroxide. The DNA-epinephrine-Cu(II) terenary complex has characteristic spectral properties. It has the capacity to catalyze the reduction of oxygen or H₂O₂ and it preserves the capacity over a wide range of comp1ex:DNA ratios. The rate of DNA cleavage is proportional to the rate of epinephrine oxidation and the rate determining step of the reaction Seems to be the reduction of free Cu(II) ions. The ability to form redox active stable DNA ternary complexes, suggests that under specific physiological conditions, when “free” copper ions are available. catecholamina may induce oxidative degradation of DNA and other biological macromolecules.     

Analysis of retinal and 3-dehydroretinal in the retina by high-pressure liquid chromatography

A sensitive analytical method was developed in order to study the rhodopsin-porphyropsin system in the eye. Oximes of 11-cis-retinal, all-trans-retinal, 11-cis-3-dehydroretinal, and all-trans-3-dehydroretinal were determined quantitatively by high-pressure liquid chromatography. This method was applied to the analysis of retinal and 3-dehydroretinal in the retinas of bullfrog and goldfish. The results agreed with those obtained from the bleaching kinetics of visual pigment extracted with detergent. A reliable result is obtained if the tissue contains more than 5 pmol of retinal (or 3-dehydroretinal). The chromophore composition could be determined in the eye of a small freshwater prawn, Palaemon pancidence, using 50 pmol of 11-cis-retinal and no 3-dehydroretinal.     

Luminol-enhanced chemiluminescence after reaction of hydroperoxides with opsonized zymosan

Luminol-enhanced chemiluminescence (LEC) is very sensitive in detecting free radicals but relatively insensitive for hydroperoxides (hydrogen peroxide, tert-butyl hydroperoxide). However, in the presence of opsonized zymosan (often used for stimulation of phagocytic cells) hydroperoxides also induce LEC, suggesting that free radicals are produced under these conditions. Therefore careful interpretation with respect to the nature of the reactive species is necessary when LEC is used for characterization of zymosan-induced phagocytosis. We studied the properties of zymosan-induced LEC under different test conditions and with various inhibitors. Typical radical scavengers, e.g. nordihydroguaiaretic acid and superoxide dismutase, are strong inhibitors, indicating the importance of the superoxide anion. This system is useful for drug testing with respect to antioxidative or radical scavenging activity.     

Trimetazidine protects low-density lipoproteins from oxidation and cultured cells exposed to H(2)O(2) from DNA damage

Trimetazidine is a well-established anti-ischemic drug, which has been used for long time in the treatment of pathological conditions related with the generation of reactive oxygen species. However, although extensively studied, its molecular mode of action remains largely unknown. In the present study, the ability of trimetazidine to protect low-density lipoproteins (LDL) from oxidation and cultured cells from H₂O₂-induced DNA damage was investigated. Trimetazidine, tested at concentrations 0.02 to 2.20 mM, was shown to offer significant protection to LDL exposed to three different oxidizing systems, namely copper, Fe/ascorbate, and met-myoglobin/H₂O₂. The oxidizability of LDL was estimated by measuring, (i) the lag period, (ii) the maximal rate of conjugated diene formation, (iii) the total amount of conjugated dienes formed, (iv) the electrophoretic migration of LDL protein in agarose gels (REM), and (v) the inactivation of the enzyme PAF-acetylhydrolase present in LDL. In addition, the presence of trimetazidine decreased considerably the DNA damage in H₂O₂-exposed Jurkat cells in culture. H₂O₂ was continuously generated by the action of glucose oxidase at a rate of 11.8 ± 1.5 μM per min (60 ng enzyme per 100 μl), and DNA damage was assessed by the single cell gel electrophoresis assay (also called comet assay). The protection offered by trimetazidine in this system (about 30% at best) was transient, indicating modification of this agent during its action. These results indicate that trimetazidine can modulate the action of oxidizing agents in different systems. Although its mode of action is not clarified, the possibility that it acts as a lipid barrier permeable transition metal chelator is considered.