THE GENERATION OF HYDROXYL RADICALS IN BIOLOGIC SYSTEMS: TOXICOLOGICAL ASPECTS

Abstract— The formation of hydroxyl radicals in vitro was studied through their reaction with 2-keto-4-thiomethylbutyric acid to form ethylene gas. The autoxidation reaction of 6-aminodopamine served as a model source of hydroxyl radicals. Ethylene production was suppressed by catalase and by superoxide dismutase, indicating that both hydrogen peroxide and superoxide were involved in the reaction. Hydroxyl radical scavengers (thiourea > benzoate > ethanol) suppressed ethylene production in good agreement with their respective rate constants for reaction with hydroxyl radicals. Urea served as a negative control. Several substituted thiourea derivatives also suppressed ethylene production to a similar degree as thiourea itself. Biologic studies centered on several cytotoxic agents whose mechanisms of action are thought to involve hydroxyl radicals. These agents included alloxan, which destroys the beta cells of the pancreas, and 6-hydroxy- and 6-aminodopamine, which destroy sympathetic nerves. Damage to tissues in vivo was blocked to varying degrees by pretreatment of animals with hydroxyl radical scavengers such as ethanol or the thiourea derivatives. In addition, hydroxyl radical scavengers blocked the action of 5,7-dihydroxytryptamine, a neurotoxin whose effects on noradrenaline neurons were previously shown to be blocked by inhibitors of monoamine oxidase. The data indicate that these cell toxins produce their damaging actions on specific target cells through the intracellular generation of hydroxyl radicals.     

Electrochemical Probe of Hydroxylation of 4-Nitro-phenol by Cytochrome C with Hydrogen Peroxide

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Hydrophobic behavior,ROS production,and heme degradation of hemoglobin upon interaction with <Emphasis Type="Italic">n</Emphasis>-alkyl sulfates

The oxygen-containing free radical species form upon interaction of amphiphilic substances such as sodium dodecyl sulfate and hemoglobin. Under these conditions, hemoglobin is converted to methemoglobin and simultaneously results in heme degradation. Since heme is located in a hydrophobic moiety of hemoglobin, we hypothesized that other hydrophobic substances or amphiphilic xenobiotics can dispose hemoglobin to oxidative stress. Here this hypothesis was tested by investigating heme degradation of hemoglobin during interaction with n-alkyl sulfates. This was accomplished using UV–Vis and fluorescence spectroscopy, chemometric analysis, and chemiluminescence methods. We determined whether a relationship exists between the alkyl tail length (surfactant hydrophobicity) of n-alkyl sulfate homologues, reactive oxygen species (ROS) production, and heme degradation pattern of hemoglobin. We also proposed a mechanism for these types of interactions and induction of heme degradation. Our results indicated that hemoglobin structural–functional changes including globin denaturation are the key factors in starting the heme degradation process, and heme degradation product patterns were dependent on each alkyl sulfate. However, the number of fluorescent components (heme degradation products) was independent of the alkyl sulfate type. The reason for this phenomenon was the mechanism of reaction in which the amount of hydrogen peroxide was changed with each homologue, but the mechanism of degradation remained the same. Thus, an increase in hydrophobic chain length of the surfactants correlated with the enhanced ROS production and heme degradation of hemoglobin.     

Reaction of Thiourea Dioxide and Hydrogen Peroxide with Coumarin

The system thiourea dioxide–hydrogen peroxide is found to effectively hydroxylate coumarin in aqueous solutions. The hydroxyl radical reacting with coumarin is shown to result from decomposition of the complex thiourea dioxide–H₂O₂. The obtained data are compared to the results of investigation of the reactions occurring in the system thiourea dioxide–H₂O₂ with terephthalic acid.     

Quantitation of the Reactive Oxygen Species Generated by the UVA Irradiation of Ascorbic Acid-Glycated Lens Proteins

The oxidation products of ascorbic acid rapidly glycate proteins and produce protein-bound, advanced glycation endproducts. These endproducts can absorb UVA light and cause the photolytic oxidation of proteins (Ortwerth, Linetsky and Olesen, Photochem. Photobiol . 62, 454–463, 1995), which is mediated by the formation of reactive oxygen species. A dialyzed preparation of calf lens proteins, which had been incubated for 4 weeks with 20 mM ascorbic acid in air, was irradiated for 1 h with 200 mW/ cm² of absorbed UVA light (λ > 338 nm), and the concentration of individual oxygen free radicals was measured. Superoxide anion attained a level of 76 μ M as determined by the superoxide dismutase (SOD)-depen-dent increase in hydrogen peroxide formation and of 52 μ M by the SOD-inhibitable reduction of cytochrome c. Hydrogen peroxide formation increased linearly to 81 μM after 1 h. Neither superoxide anion nor hydrogen peroxide, however, could account for the UVA photolysis of Trp and His seen in this system.
Singlet oxygen levels approached 1.0 mM as measured by the oxidation of histidine, which was consistent with singlet oxygen measurements by the bleaching of N,N- dimethyl-4-nitrosoaniline. High concentrations of sodium azide, a known singlet oxygen quencher, inhibited the photolytic destruction of both His and Trp. Little or no protein damage could be ascribed to hydroxyl radical based upon quenching experiments with added mannitol. Therefore, superoxide anion and H₂O₂ were generated by the UVA irradiation of ascorbate advanced glycation endproducts, however, the major reactive oxygen species formed was singlet oxygen.     

Radical-induced aromatic substitution between benzoyl peroxide and benzenediols in the gas phase characterized by mass spectrometry

A radical-induced aromatic substitution mechanism for the reaction between benzoyl peroxide and benzenediols in the gas phase was characterized by mass spectrometry. The benzoyloxy radical produced from the homolysis of benzoyl peroxide associates at its carbonyl group with the phenolic hydroxyl group. The pairing tendency of the unpaired electron on the oxygen of the radical induces electron transfer along the hydrogen bond, which results in the rupture of the O? H bond of the phenol and aromatic substitution at the ortho position of the benzoyloxy radical. Supporting evidence for the mechanism was obtained by isotope labelling.     

胶束增敏催化荧光法测定血红蛋白

基于血红蛋白（Ｈｂ）的过氧化物酶活性,催化过氧化氢氧化对甲基酚的反应体系,发现表面活性剂ＳＤＳ对该体系反应速率有明显的增强效应;从而建立了新的高灵敏测定Ｈｂ的催化荧光分析法。在１．５×１０－３ｍｏｌ／Ｌ　ＳＤＳ存在下,测定Ｈｂ的线性范围１×１０－９～８×１０－８ｍｏｌ／Ｌ,检出限为３．４×１０－１０ｍｏｌ／Ｌ,用于尿中Ｈｂ的测定,结果满意。     

Direct characterization of hydrogen peroxide bleached thermomechanical pulp using spectroscopic methods

The effects of thermomechanical pulp (TMP) bleaching with hydrogen peroxide under acidic and alkaline conditions were studied using different spectroscopic analytical methods. The results of hydroxyl radical determination in bleaching solutions, analyses of carbonyl and carboxyl groups contents in the pulp, and the cellulose fiber surface analysis by X-ray photoelectron spectroscopy (XPS) elucidate the chemistry of the hydrogen peroxide treatment. Diffuse reflectance laser flash photolysis (DRLFP) method showed the differences in the photochemical behavior that reflect the changes of the chromophoric system after the preliminary peroxide bleaching stage under acidic conditions. Fourier transform infrared (FTIR) spectroscopy confirmed the non-delignifying character of the bleaching process. Suppression of carbonyl and formation of carboxyl groups in the case of the two-stage peroxide bleaching performed in the presence of catalysts and stabilizers was also confirmed. FT-Raman studies showed the removal of coniferaldehyde groups after treatment under acidic and alkaline conditions.     

OXYGEN RADICALS MEDIATE CELL INACTIVATION BY ACRIDINE DYES, FLUORESCEIN, and LUCIFER YELLOW CH

Acridine dyes, fluorescein and lucifer yellow CH are fluorescent photosensitizers used experimentally to selectively stain and photodynamically destroy eukaryotic cells and subcellular structures. We have determined that the mechanism of light- and oxygen-dependent inactivation of E. coli by these dyes involves oxygen radicals and hydrogen peroxide. All of the dyes oxidized NAD(P)H+ under illumination. Superoxide (O2), detected as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c, was a major product of the dye sensitized photooxidation. Cationic acridine dyes penetrated the membranes of E. coli and were photoreduced intracellularly. Reduced dyes diffused back into the medium and mediated the reduction of extracellular ferricytochrome c. The anionic dyes fluorescein and lucifer yellow CH were unable to mediate extracellular cytochrome c reduction, indicating that these dyes were impermeable to the E. coli membrane. Acridine dyes, when illuminated, inhibited the growth of E. coli in a rich medium, and induced the synthesis of SOD. Fluorescein and lucifer yellow CH did not inhibit growth or induce SOD synthesis because they were unable to enter the cells. Superoxide (O2) and hydrogen peroxide (H2O2), generated by the enzyme xanthine oxidase were toxic to E. coli B. Inactivation by xanthine oxidase was partially inhibited by exogenous SOD and completely inhibited by exogenous catalase or SOD plus catalase. Similarly, exogenous SOD plus catalase protected against inactivation by acridines and fluorescein-NADH or lucifer yellow CH-NADH mixtures. Prior induction of superoxide dismutase and catalase in E. coli B significantly protected cells against a subsequent challenge by illuminated acridine dyes. SOD and catalases preinduction combined with additions of exogenous SOD and catalase completely protected E. coli B against photodynamic inactivation by acridine yellow. The hydroxyl radical scavengers, dimethyl sulfoxide, sodium benzoate and thiourea, protected E. coli B against photodynamic inactivation by acridine orange. The results implicate O2, H2O2, and the hydroxyl radical (OH) as underlying molecular agents of the phototoxicity mediated by acridine orange, acridine yellow, fluorescein and lucifer yellow CH.     

Layer-by-layer enzyme/polyelectrolyte films as a functional protective barrier in oxidizing media

The influence of a catalase (Cat) layer located at different depths in the layer-by-layer hemoglobin/polystyrene sulfonate films with an (Hb/PSS)(20)(-)(x)/(Cat/PSS)/(Hb/PSS)(x) (x = 0-20) architecture on kinetics of hemoglobin degradation under treatment with hydrogen peroxide solutions of different concentrations and features of H(2)O(2) decay in surrounding solutions has been studied. While assembled on the top of the multilayers, the catalase layer shows the highest activity in hydrogen peroxide decomposition. Hemoglobin in such films retains its nativity for a longer period of time. The effect of catalase layers is compared with that of protamine, horseradish peroxidase, and inactivated catalase. Positioning an active layer with catalytic properties as an outer layer is the best protection strategy for layer-by-layer assembled films in aggressive media.     

Oxidation of vanadium(III) by hydrogen peroxide and the oxomonoperoxo vanadium(V) ion in acidic aqueous solutions: a kinetics and simulation study

The reaction between vanadium(III) and hydrogen peroxide in aqueous acidic solutions was investigated. The rate law shows first-order dependences on both vanadium(III) and hydrogen peroxide concentrations, with a rate constant, defined in terms of -d[H(2)O(2)]/dt, of 2.06 +/- 0.03 L mol(-)(1) s(-)(1) at 25 degrees C; the rate is independent of hydrogen ion concentration. The varying reaction stoichiometry, the appreciable evolution of dioxygen, the oxidation of 2-PrOH to acetone, and the inhibition of acetone formation by the hydroxyl radical scavengers, dimethyl sulfoxide and sodium benzoate, point to a Fenton mechanism as the predominant pathway in the reaction. Methyltrioxorhenium(VII) does not appear to catalyze this reaction. A second-order rate constant for the oxidation of V(3+) by OV(O(2))(+) was determined to be 11.3 +/- 0.3 L mol(-)(1) s(-)(1) at 25 degrees C. An overall reaction scheme consisting of over 20 reactions, in agreement with the experimental results and literature reports, was established by kinetic simulation studies.     

SINGLET OXYGEN INVOLVEMENT IN THE INACTIVATION OF CULTURED HUMAN FIBROBLASTS BY UVA (334 nm, 365 nm) AND NEAR-VISIBLE (405 nm) RADIATIONS

The UVA (320-380 nm) radiation inactivation of mammalian cells is dependent upon the presence of oxygen. In order to examine the intermediates involved, we have irradiated cells in the presence of chemical probes which are able to modify the activity of various oxygen species. We have also examined the possibility that UVA inactivates cultured human fibroblasts via generation of intracellular hydrogen peroxide. An iron scavenger (desferrioxamine) and a hydroxyl radical scavenger (dimethylsulfoxide) protect the cells against hydrogen peroxide. Diethyldithiocarbamate (a superoxide dismutase inhibitor) and aminotriazole (a catalase inhibitor) sensitize the cells to this oxidizing agent. These data support previous reports that hydrogen peroxide inactivates as a result of the iron-catalyzed generation of hydroxyl radical. None of these agents significantly alter the fluence-dependent inactivation of cell populations by radiation at 365 nm. In contrast, the cells are sensitized to radiation at 334, 365 and 405 nm in the presence of deuterium (an enhancer of singlet oxygen lifetime) and are protected against radiation at 365 nm by sodium azide (a quencher of singlet oxygen). These results are consistent with the conclusion that the generation of singlet oxygen, but not hydrogen peroxide or hydroxyl radical, plays an important role in the inactivation of cultured human cells by UVA and near-visible radiations.     

Theoretical study on the oxidation mechanism of thiourea by hydrogen peroxide with water and hydroxyl assistance

This article presents a theoretical study on the oxidation reaction of thiourea by hydrogen peroxide in water or alkaline solutions using density functional and ab initio theories. This work also focuses on the analysis of the thermodynamic and kinetic properties of the predicted oxidation mechanism of thiourea using density functional and ab initio theories. The calculated results show that the activation energies, activation enthalpies, and activation Gibbs free energies of the reaction decreased and the releasable reaction energies, enthalpies and Gibbs free energies increased with the cooperation of water or hydroxyl anion. We conclude that the oxidation reaction of thiourea by hydrogen peroxide in water or alkaline solutions was easier and more completed than that in the gas state. The calculated results are consistent with the experiments.     

Reaction of hydrogen peroxide with low molecular weight iron complexes

Fe(II) complexed with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) reacts with hydrogen peroxide in neutral aqueous solution at room temperature to yield reactive species which are not scavenged by t-butanol, under conditions where >90% of hydroxyl radical would be scavenged. Further, the ratio of the rate constants for the reaction of the reactive species with Fe(II)CDTA and H₂O₂ is 6.2, in contrast to a ratio of 200 which would result if the species were the hydroxyl radical. Thus, it is concluded that the reactive species produced is not the hydroxyl radical, but an iron-oxo species such as the ferryl ion. The reactive species is formed in an apparent first order reaction, when either hydrogen peroxide or Fe(II)CDTA is in kinetic excess. The bimolecular reaction rate constant is (1.26 ± 0.19) × 10³ M^-1 s^-1. In experiments where H₂O₂ was in kinetic excess, a chain decomposition of H₂O₂ was observed in which the initially produced iron-oxo intermediate exhibits hydroperoxidase activity.     

Peroxide Damage of Hemoglobin by the Fenton System

The extent of hemoglobin peroxidation under the action of mixtures of ferrous salts and hydrogen peroxide has been investigated. The rate of accumulation of carbonyl-containing products of the protein fragmentation, detected as 2,4-dinitrophenylhydrazones, is independent of the pH of the reaction medium and proportional to the concentrations of hydrogen peroxide, ferrous ion, and hemoglobin. A chain radical mechanism of the peroxide fragmentation of the polypeptide chains of hemoglobin has been proposed, involving reactions of the alkoxyl radical of hemoglobin with ferrous ion and of the carbon-centered radical of the protein with dissolved oxygen as rate-limiting steps. Chain termination is therewith effected through cross recombination of the above radicals.     

Degradation of a model naphthenic acid, cyclohexanoic acid, by vacuum UV (172 nm) and UV (254 nm)/H2O2

The mechanism of hydroxyl radical initiated degradation of a typical oil sands process water (OSPW) alicyclic carboxylic acid was studied using cyclohexanoic acid (CHA) as a model compound. By use of vacuum ultraviolet irradiation (VUV, 172 nm) and ultraviolet irradiation in the presence of hydrogen peroxide UV(254 nm)/H(2)O(2), it was established that CHA undergoes degradation through a peroxyl radical. In both processes the decay of the peroxyl radical leads predominantly to the formation of 4-oxo-CHA, and minor amounts of hydroxy-CHA (detected only in UV/H(2)O(2)). In UV/H(2)O(2), additional 4-oxo-CHA may also have been formed by direct reaction of the oxyl radical with H(2)O(2). The oxyl radical can be formed during decay of the peroxyl-CHA radical or reaction of hydroxy-CHA with hydroxyl radical. Oxo- and hydroxy-CHA further degraded to various dihydroxy-CHAs. Scission of the cyclohexane ring was also observed, on the basis of the observation of acyclic byproducts including heptadioic acid and various short-chain carboxylic acids. Overall, the hydroxyl radical induced degradation of CHA proceeded through several steps, involving more than one hydroxyl radical reaction, thus efficiency of the UV/H(2)O(2) reaction will depend on the rate of generation of hydroxyl radical throughout the process. In real applications to OSPW, concentrations of H(2)O(2) will need to be carefully optimized and the environmental fate and effects of the various degradation products of naphthenic acids considered.     

Chemical ionization mass spectrometry of benzoyl peroxide: A radical aromatic substitution resulting in biphenylcarboxylic acid in the gas phase

A radical aromatic substitution resulting in biphenylcarboxylic acid is inferred for the decomposition of benzoyl peroxide from the chemical ionization and collision-induced dissociation mass spectra. The thermolysis of benzoyl peroxide gives rise to a benzoyloxy radical, which undergoes rapid decarboxylation and hydrogen abstraction leading to phenyl radical and benzoic acid, respectively. Attack of the resulting phenyl radical on the benzoic acid results in biphenylcarboxylic acid. On the other hand, the phenyl radical abstracts a hydrogen atom to yield benzene, which is then subjected to the attack of a benzoyloxy radical, affording phenyl benzoate. This substitution reaction rather than the recombination of benzoyloxy and phenyl radicals is found to be responsible for the formation of phenyl benzoate under the present conditions.     

Fe(II)-EDTA chelate-induced aromatic hydroxylation of terephthalate as a new method for the evaluation of hydroxyl radical-scavenging ability.

The investigation of Fe(II)-EDTA chelate-induced aromatic hydroxylation of terephthalate in pH 7.4 phosphate buffer solution and a new method for the evaluation of hydroxyl radical-scavenging ability are reported. The method is based on attack of the hydroxyl radical on the terephthalate to produce highly fluorescent 2-hydroxyterephthalate, which is detected fluorimetrically. The formation of hydroxyl radical is believed to be the result of the reduction of molecular oxygen by Fe(II)-EDTA to form superoxide radical, which in turn dismutates to hydrogen peroxide, and then Fe(II)-EDTA catalyzes the decomposition of hydrogen peroxide to produce hydroxyl radical. The mechanism of the generation of hydroxyl radical in the proposed system was confirmed. This study established a simple and inexpensive method for the evaluation of the scavenging ability of some compounds on hydroxyl radicals.     

Experimental studies on the chemiluminescence reaction mechanism of carbonate/bicarbonate and hydrogen peroxide in the presence of cobalt(II)

Chemiluminescence (CL) phenomena of carbonates or bicarbonates of potassium, sodium, or ammonium with hydrogen peroxide in the presence of cobalt sulfate were reported. After cobalt(II) solution was injected into the mixture of carbonate/bicarbonate and hydrogen peroxide, a CL signal was given out briefly. The CL conditions of these systems were optimized. The CL reaction mechanisms were studied experimentally by examining the spectrum emitted by the CL system and the effect of various free radical scavengers on CL emission intensity. The results showed that the maximal emission wavelengths of the CO32--H2O2-Co2+ and HCO3--H2O2-Co2+ systems were 440 and 490 nm, respectively. As a result, a radical scavenger of ascorbic acid, thiourea, and superoxide dismutase exhibited different effects on these CL systems. The different CL mechanisms involving the carbon dioxide dimer and the oxygen dimer were revealed, respectively.     

Aliphatic peptidyl hydroperoxides as a source of secondary oxidation in hydroxyl radical protein footprinting

Hydroxyl radical footprinting is a technique for studying protein structure and binding that entails oxidizing a protein system of interest with diffusing hydroxyl radicals, and then measuring the amount of oxidation of each amino acid. One important issue in hydroxyl radical footprinting is limiting amino acid oxidation by secondary oxidants to prevent uncontrolled oxidation, which can cause amino acids to appear more solvent accessible than they really are. Previous work suggested that hydrogen peroxide was the major secondary oxidant of concern in hydroxyl radical footprinting experiments; however, even after elimination of all hydrogen peroxide, some secondary oxidation was still detected. Evidence is presented for the formation of peptidyl hydroperoxides as the most abundant product upon oxidation of aliphatic amino acids. Both reverse phase liquid chromatography and catalase treatment were shown to be ineffective at eliminating peptidyl hydroperoxides. The ability of these peptidyl hydroperoxides to directly oxidize methionine is demonstrated, suggesting the value of methionine amide as an in situ protectant. Hydroxyl radical footprinting protocols require the use of an organic sulfide or similar peroxide scavenger in addition to removal of hydrogen peroxide to successfully eradicate all secondary oxidizing species and prevent uncontrolled oxidation of sulfur-containing residues.