Hydroxyl Radical Scavenging Agents Research Articles

Non-destructively incorporating ceria in Nafion membrane as hydroxyl radical scavenging agent for long-term PEMFC application

Aiming at enhancing the long-term operation of proton exchange membrane fuel cell (PEMFC), Nafion as a typical example of perfluorosulfonic acid proton exchange membrane is non-destructively modified by sulfonated ceria (CeO2-DS) nanoparticles via a swelling-filling strategy. CeO2-DS nanoparticles in Nafion matrix can not only act as radical scavenging agent to improve the durability of Nafion, but also assist the re-construction of the efficient proton conduction channel in the composite Nafion membrane. Both oxidative stability and proton conductivity of the Nafion membrane are therefore remarkably improved with the great mechanical and thermal stability of Nafion maintained. PEMFC equipped with the modified Nafion membrane therefore outperforms that with the pristine Nafion membrane in terms of both power output and durability. Specifically, the maximum power density of the PEMFC equipped with modified Nafion after Fenton accelerated degradation test was significantly improved by ca. 25 % compared with that of the pristine Nafion membrane. This work hence provides a facile way to improve the durability of the PEMFC for practical applications.

Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.

An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.

Kinetics and mechanism investigation on the destruction of oxytetracycline by UV-254 nm activation of persulfate

Oxytetracycline (OTC), an important broad-spectrum antibiotic, has been detected extensively in various environmental systems, which may have a detrimental impact on ecosystem and human health through the development of drug resistant bacteria and pathogens. In this study, the degradation of OTC was evaluated by UV-254nm activated persulfate (PS). The observed UV fluence based pseudo first-order rate constant (kobs) was found to be the highest at near neutral pH conditions (pH 5.5–8.5). Presence of various natural water constituents had different effects on OTC degradation, with a significant enhancement in the presence of bicarbonate or Cu2+. Limited elimination of total organic carbon (TOC) and PS was observed during the mineralization of OTC. Transformation byproducts in the presence and absence of hydroxyl radical scavenging agent tert-butanol (t-BuOH) were identified using ultra-high definition accurate-mass quadrupole time-of-flight liquid chromatography/mass spectrometer (LC-QTOF/MS). Potential OTC degradation mechanism was subsequently proposed revealing four different reaction pathways by SO4− reaction including hydroxylation (+16Da), demethylation (−14Da), decarbonylation (−28Da) and dehydration (−18Da). This study suggests that UV-254nm/PS is a promising treatment technology for the control of water pollution caused by emerging contaminants such as OTC.

Interaction of Fibrin with Red Blood Cells: The Role of Iron

Activation of coagulation pathways results in the formation of hemostatic fibrin plugs. Under normal physiologic conditions fibrin clots are gradually, albeit completely, degraded by a fibrinolytic enzyme system to ensure proper wound healing and/or blood vessel patency. Yet in pathological situations, thrombi are not effectively removed, leading to chronic thrombosis. The susceptibility of blood clots to enzymatic degradation depends on the structure and properties of fibrin fibers. Many factors have been suspected as culprits, including red blood cells (RBCs) that become transiently trapped within fibrin mesh. Here, the authors show that there is indeed a specific interaction between RBCs and fibrin-like fibers identified here as dense matted deposits (DMDs) by means of scanning electron microscopy (SEM). It is emphasized that such interactions can be observed in ischemic stroke patients, but not from healthy subjects. However, DMD/RBC aggregates can be induced in normal blood by the additions of trivalent iron ions. The plausible mechanism of the enhanced fibrin–red blood cell interaction is based on the previously described iron-induced generation of hydroxyl radicals. These radicals cause, in turn, non-enzymatic formation of fibrinogen aggregates remarkably resistant to fibrinolysis that are also similar to DMDs described in this paper. In conclusion, this relatively simple SEM analysis may become a convenient tool for diagnosing prothrombotic conditions associated with iron overload. It is suggested that future research on prevention and treatment of ischemic stroke and other thrombosis associated diseases should include testing of iron-chelating and hydroxyl radical-scavenging agents.

Inhibitory effects of edaravone on the production of tumor necrosis factor-α in the isolated heart undergoing ischemia and reperfusion

We evaluated the effects of edaravone, a hydroxyl radical scavenging agent, on the production of tumor necrosis factor-alpha (TNF-alpha) in myocardium, and the release of TNF-alpha and P-selectin from myocardium after ischemia-reperfusion injury in isolated Langendorff-perfused rat hearts. Cardiodynamic function at stable points during perfusion and 5, 15, 30, and 60 min after the initiation of reperfusion was evaluated by left ventricular developed pressure, rate of increase in left ventricular pressure and rate of decrease in ventricular pressure, coronary flow, and heart rate. At 60 min after the initiation of reperfusion, myocardial infarct size was estimated microscopically using triphenyltetrazolium chloride staining, and expression of TNF-alpha in myocardium was detected by Western blot and immunohistochemistry. At the same time points as the measurement of cardiodynamic function, TNF-alpha and the soluble form of P-selectin in coronary effluent were measured by enzyme immunoassay. At all time points during reperfusion, edaravone markedly improved cardiodynamic function and reduced myocardial infarct size in comparison to the control. In myocardium in the control, TNF-alpha was detected in the endothelial cells and other cells bearing some resemblance to interstitial cells and monocyte cells. Edaravone suppressed this cytokine expression in the corresponding sites. P-selectin as well as TNF-alpha was found in the coronary effluent of the control, and edaravone significantly decreased soluble P-selectin levels in comparison to the control (P < 0.01). Edaravone might have protective effects on cardiac function through reduction of infarct size via decrease of production of TNF-alpha in myocardium induced by ischemia-reperfusion injury and through reduction of the release of adhesion molecules such as P-selectin from vascular endothelial cells.

The antioxidant activity of the essential oils of Artemisia afra, Artemisia abyssinica and Juniperus procera.

The essential oils of Artemisia afra Jacq., Artemisia abyssinica Schultz-Bip. and Juniperus procera Hoechst ex Endl. were examined for their potential radical scavenging activities. First a rapid evaluation of antioxidants was made using a TLC screening method. The abilities of the volatile oils to act as nonspecific donors for hydrogen atoms or electrons were checked in the diphenylpicrylhydrazyl assay. Oils from all three species showed positive results and were examined further. The oils of A. afra and J. procera were also effective hydroxyl radical scavenging agents when assessed in the deoxyribose degradation assay, whilst oils from A. abyssinica exhibited a paradoxical effect. In the in vitro assay for non-enzymatic lipid peroxidation in liposomes, the oils of A. afra and J. procera also displayed antioxidant potential. It was not possible to measure the effect of A. abyssinica oil in this system because certain components, e.g. alk-2-enals, interfered with the assay. The compounds that contribute to the radical scavenging activities of A. afra and J. procera were identified and then assessed for their effects in the various test systems. Finally, the qualitative and quantitative compositions of the essential oils were studied by GC-MS.

Evidence for Production of Hydroxyl Radicals by Pentachlorophenol Metabolites and Hydrogen Peroxide: A Metal-Independent Organic Fenton Reaction

The production of hydroxyl radicals by tetrachlorohydroquinone (TCHQ, a major metabolite of the widely used biocide pentachlorophenol) in the presence of H2O2 was studied by salicylate hydroxylation method. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl radicals react with salicylate. We found that TCHQ and H2O2 could produce both 2,3- and 2,5-DHBA when incubated with salicylate. Their production was markedly inhibited by hydroxyl radical scavenging agents dimethyl sulfoxide and ethanol, as well as by tetrachlorosemiquinone radical scavengers desferrioxamine and other hydroxamic acids. In contrast, their production was not affected by the nonhydroxamate iron chelators diethylenetriaminepentaacetic acid (DTPA), bathophenanthroline disulfonic acid, and phytic acid, as well as the copper-specific chelator bathocuprione disulfonic acid. A comparison of product formation and distribution from the reaction of ferrous iron with hydrogen peroxide (the classic Fenton system) strongly suggests that the same hydroxyl radical adducts are formed as in the TCHQ/H2O2 experiments. Taken together, we propose that hydroxyl radicals were produced by TCHQ in the presence of H2O2, probably through a metal-independent organic Fenton reaction.

Quantification of Total Oxidant Scavenging Capacity of Antioxidants for Peroxynitrite, Peroxyl Radicals, and Hydroxyl Radicals

We have extended the application of our previously reported total oxidant scavenging capacity (TOSC) assay (Winstonet al., Free Radical Biol. Med.24, 480–493, 1998) to permit facile quantification of the absorbance capacity of antioxidants toward three potent oxidants, i.e., hydroxyl radicals, peroxyl radicals, and peroxynitrite. Respectively, these oxidants were generated by the iron plus ascorbate-driven Fenton reaction, thermal homolysis of 2,2′-azobis(2-methylpropionamidine) dihydrochloride (ABAP), and 3-morpholinosydnonimineN-ethylcarbamide (SIN-1). Each of these oxidants reacts with α-keto-γ-methiolbutyric acid (KMBA), which is oxidized and yields ethylene. The antioxidant capacity of the compounds tested is quantified from their ability to inhibit ethylene formation relative to a control reaction. Assay conditions were established in which control reactions give comparable yields of ethylene with each of the oxidants studied. Thus, the relative efficiency of various antioxidants could be compared under conditions of quantitatively similar KMBA oxidizing capability by the three oxidants. Reduced glutathione was an efficient scavenger of peroxyl radicals, but scavenged peroxynitrite and hydroxyl radicals relatively poorly. Uric acid, Trolox, and ascorbic acid were comparable scavengers of peroxynitrite and peroxyl radicals. Uric acid and Trolox were approximately an order of magnitude less efficient as scavengers of hydroxyl radicals. The classical hydroxyl radical scavenging agents mannitol, dimethyl sulfoxide, and benzoic acid had much higher TOSC values with hydroxyl than with peroxyl radicals or peroxynitrite. The very different chemical reactivity toward KMBA by the SIN-1- and iron–ascorbate-generated oxidants indicates that hydroxyl radical is not a major oxidant produced by the SIN-1 system. The data show that the TOSC assay is useful and robust in distinguishing the reactivity of various oxidants and the relative capacities of antioxidants to scavenge these oxidants.

Generation of Reactive Oxygen Species during Interaction of Diesel Exhaust Particle Components with NADPH-Cytochrome p450 Reductase and Involvement of the Bioactivation in the DNA Damage

Since the toxicity of diesel exhaust particles (DEP) after intratracheal injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al. Free Rad. Biol. Med. 14: 37–47; 1993), the possibility that superoxide could be enzymatically and continuously generated from diesel exhaust particles (DEP), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extract of DEP with the Triton N-101 treated microsomal preparation of mouse lung whereas the cytosolic fraction was less active, suggesting that DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6.2.4, P450 reductase) rather than DT-diaphorase. When purified P450 reductase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate for P450 reductase because reaction was inhibited by addition of glutathione (GSH) to form those GSH adduct or pretreatment with NaBH 4 to reduce those to the hydroxy compounds although a possibility of nitroarenes as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 μg) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P450 reductase caused DNA scission which was reduced in the presence of superoxide dismutase (SOD), catalase, or hydroxyl radical scavenging agents. Taken together, these results indicate that DEP components, probably quinoid or nitroaromatic structures, that appear to promote DNA damage through the redox cycling based generation of superoxide. Copyright © 1996 Elsevier Science Inc.

Two- and one-electron dependent In vitro reductive metabolism of nitroaromatics by Mytilus edulis, Carcinus maenas and Asterias rubens

The subcellular and tissue distribution of 2- and 1-electron NAD(P)H-dependent nitroreductase activity was investigated in marine invertebrates from three phyla. The metabolites of 2-electron nitroreduction were characterised by reverse phase, isocratic HPLC. One-electron nitroreduction, resulting in the formation of reactive oxygen species, was assayed by the iron/EDTA-mediated oxidation of the hydroxyl radical scavenging agent 2-keto-methiolbutyric acid. NADPH-dependent 2-electron nitroreduction occurred only under anaerobic conditions, with the amine end-product, but not the nitroso- or hydroxylamine intermediates, being detected. Activity was present in cytosolic ( p-nitrobenzoic acid) or both microsomal and cytosolic (1-nitropyrene) fractions of the major digestive tissues of mussel Mytilus edulis (digestive gland) and crab Carcinus maenas (hepatopancreas), but not in the gills of either species. No activity was detectable in the pyloric caeca or stomach region of the starfish Asterias rubens. A wider subcellular distribution was evident for NAD(P)Hdependent 1-electron nitroreduction, with activity towards nitrofurantoin, p-nitrobenzoic acid and 1nitropyrene being present in all subcellular (mitochondrial, microsomal and cytosolic) fractions of the major digestive tissues of the three species. Activity was higher for NADH than NADPH, and highest for the model redox cycling compound nitrofurantoin. NAD(P)H-dependent nitrofurantoin-mediated activity was present in several tissues (digestive, respiratory, gonads, other) of all three species, but was highest in the digestive tissues. The results indicate a widespread potential for nitroaromatic metabolism and toxicity, with oxygen availability being a factor in determining the metabolic fate of nitroaromatic xenobiotics.

Ferritin Stimulation of Hydroxyl Radical Production by Rat Liver Nuclei

Iron mobilized from ferritin has been shown to catalyze production of potent reactive oxygen intermediates. Experiments were carried out to evaluate the ability of ferritin to catalyze nuclear generation of hydroxyl radical in the presence of either NADPH or NADH. In the absence of redox cycling agents, ferritin did not catalyze nuclear oxidation of hydroxyl radical scavenging agents (2-keto-4-thiomethylbutyric acid, dimethylsulfoxide, ethanol) even if EDTA was added to chelate any released iron. The addition of menadione or paraquat resulted in a ferritin-dependent oxidation of chemical scavengers; menadione promoted the catalysis by ferritin with either NADPH or NADH, whereas paraquat was much more reactive with NADPH as the nuclear reductant. The presence of an externally added iron chelator was required for elevated rates of scavenger oxidation, with EDTA and DTPA being more reactive than ATP or citrate and desferrioxamine being inhibitory. The ferritin-catalyzed hydroxyl radical scavenger oxidation was sensitive to superoxide dismutase, catalase, and competitive scavengers. In the absence or presence of ferritin, rates of NADPH- or NADH-dependent H 2O 2 production were low; menadione increased H 2O 2 production with both NADPH and NADH, whereas paraquat was mostly effective with NADPH. Depending on the nature of the added chelating agent (e.g., EDTA, ATP) and the reductant, rates of nuclear production of ·OH in the presence of redox cycling agent plus ferritin were 10 to 70% as high as rates found with redox cycling agent plus ferric-chelate (e.g., ferric-EDTA, ferric-ATP). Since reactive oxygen intermediates such as the hydroxyl radical can alter the structural integrity of the nucleus and interact with DNA, the ability of ferritin to promote nuclear generation of hydroxyl radical may play a role in the toxicity associated with iron as well as redox cycling agents.

Hydroxyl radical-mediated conversion of morphine to morphinone

1. The hydroxyl radical-mediated conversion of morphine to morphinone (MO) was examined as an alternative to the enzymic reaction. 2. Hydroxyl radicals were generated by autoxidation of ascorbate in the presence of iron and EDTA. This system oxidized morphine to MO which was identified by h.p.l.c. and t.l.c. The reaction was dependent on the concentration of added Fe2+ and required the addition of ascorbate when Fe3+ was used. 3. Catalase inhibited production of MO whereas superoxide dismutase (SOD) had no effect. Addition of a large amount of H2O2 to the system resulted in a significant decrease in production of MO. No MO production was initiated by H2O2 itself. The oxidation of morphine was inhibited by typical hydroxyl radical-scavenging agents. These results indicate that morphine undergoes oxidation to MO by hydroxyl radical.

Decreased stability of digestive gland lysosomes from the common musselMytilus edulis L. byin vitro generation of oxygen-free radicals

A consequence of xenobiotic exposure is oxidative stress resulting from one electron metabolism with the concomitant generation of oxygen-derived free radicals. The growing interest in molluscs as sentinels of environmental pollution has prompted studies on the responsiveness of lysosomal membranes from the common marine mussel,Mytilus edulis L. to oxidative stress by the oxyradicalgenerating hypoxanthine/xanthine oxidase reaction. We report that oxyradicals generated external to the lysosomal membrane cause a decrease in the stability of the lysosomal membrane, thereby resulting in leakage of enzymes from this organelle. This leakage can be prevented by catalase but not by hydroxyl radical scavenging agents. Superoxide dismutase appears to be a major enzyme exhibiting latent activity upon attack of lysosomes by oxyradicals but does not afford sufficient protection to prevent enzyme leakage under the conditions studied. Further, non-fluorescent dihydrorhodamine 123 is converted to highly fluorescent products after uptake into the lysosomal compartment of digestive gland cells suggesting that oxyradicals may be produced within the lysosomal compartment in association with the pinocytotic activity of the digestive cells. Thus, a consequence of xenobiotic-mediated proliferation of oxyradicals might be the release of potent hydrolytic enzymes into the cytosol with ensuing enzyme degradation and damage to other organelles.

NADH-dependent generation of reactive oxygen species by microsomes in the presence of iron and redox cycling agents

NADH was found previously to catalyze the reduction of various ferric complexes and to promote the generation of reactive oxygen species by rat liver microsomes. Experiments were conducted to evaluate the ability of NADH to interact with ferric complexes and redox cycling agents to catalyze microsomal generation of potent oxidizing species. In the presence of iron, the addition of menadione increased NADPH- and NADH-dependent oxidation of hydroxyl radical (·OH) scavenging agents; effective iron complexes included ferric-EDTA, -diethylenetriamine pentaacetic acid, -ATP, -citrate, and ferric ammonium sulfate. The stimulation produced by menadione was sensitive to catalase and to competitive ·OH scavengers but not to superoxide dismutase. Paraquat, irrespective of the iron catalyst, did not increase significantly the NADH-dependent oxidation of ·OH scavengers under conditions in which the NADPH-dependent reaction was increased. Menadione promoted H 2O 2 production with either NADH or NADPH; paraquat was stimulatory only with NADPH. Stimulation of H 2O 2 generation appears to play a major role in the increased production of ·OH-like species. Menadione inhibited NADH-dependent microsomal lipid peroxidation, whereas paraquat produced a 2-fold increase. Neither the control nor the paraquat-enhanced rates of lipid peroxidation were sensitive to catalase, superoxide dismutase, or dimethyl sulfoxide. Although the NADPH-dependent microsomal system shows greater reactivity and affinity for interacting with redox cycling agents, the capability of NADH to promote menadione-catalyzed generation of ·OH-like species and H 2O 2 or paraquat-mediated lipid peroxidation may also contribute to the overall toxicity of these agents in biological systems. This may be especially significant under conditions in which the production of NADH is increased, e.g. during ethanol oxidation by the liver.

Inhibition of the oxidation of hydroxyl radical scavenging agents after alkaline phosphatase treatment of rat liver microsomes

Treatment of rat liver microsomes with alkaline phosphatase results in a loss in the FMN but not the FAD flavin prosthetic group of NADPH-cytochrome P-450 reductase (Taniguchi, H. and Pyerin, W. (1987) Biochim. Biophys. Acta 912, 295-307). Experiments were carried out to evaluate the effect of preventing electron transfer from the FADH2 to FMN component of the reductase, and subsequent mixed function oxidase activity, on reduction of ferric chelates, production of H2O2, and the generation of .OH-like species by microsomes. Treatment with alkaline phosphatase was confirmed to decrease NADPH-cytochrome c, but not NADPH-ferricyanide, reductase activity by microsomes and by purified NADPH cytochrome P-450 reductase. The oxidation of hydroxyl radical scavenging agents by microsomes and reductase was decreased by the alkaline phosphatase treatment in accordance with the decline in cytochrome c reductase activity. This decrease in hydroxyl radical production occurred in the presence of various ferric chelate catalysts. Rates of microsomal reduction of the ferric chelates were also inhibited after alkaline phosphatase treatment. Production of H2O2 was decreased in accordance to the fall in cytochrome c reductase activity and .OH production. Rates of H2O2 production appeared to be rate-limiting for the overall generation of .OH as the addition of an external H2O2-generating system stimulated .OH production as well as prevented the decline in .OH production caused by the alkaline phosphatase treatment. These results suggest that both the FAD and FMN flavin prosthetic groups of the reductase contribute towards the reduction of various ferric chelates. However, loss of the FMN component and activities dependent on electron transfer from this prosthetic group result in a decrease in H2O2 production, which appears to be responsible for the decline in the generation of .OH-like species by microsomes after treatment with alkaline phosphatase.

NADPH- and NADH-dependent oxygen radical generation by rat liver nuclei in the presence of redox cycling agents and iron

Redox cycling agents such as paraquat and menadione increase the generation of reactive oxygen species in biological systems. The ability of NADPH and NADH to catalyze the generation of oxygen radicals from the metabolism of these redox cycling agents by rat liver nuclei was determined. The oxidation of hydroxyl radical scavenging agents by the nuclei was increased in the presence of menadione or paraquat, especially with NADPH as the reductant. Paraquat, even at high concentrations, was relatively ineffective with NADH. The highest rates of generation of ·OH-like species occurred with ferric-EDTA as the iron catalyst. Certain ferric complexes such as ferric-ATP, ferric-citrate, or ferric ammonium sulfate, which were ineffective catalysts for ·OH generation in the absence of paraquat or menadione, were reactive in the presence of the redox cycling agents. Oxidation of ·OH scavengers was sensitive to catalase and competitive ·OH-scavenging agents under all conditions. The redox cycling agents increased NADPH-dependent nuclear generation of H 2O 2; stimulation of H 2O 2 production may play a role in the increase in ·OH generation by menadione and paraquat. Menadione inhibited nuclear lipid peroxidation, whereas paraquat and adriamycin were stimulatory. The nuclear lipid peroxidation with either NADPH or NADH plus the redox cycling agents was not sensitive to catalase or ·OH scavengers. These results indicate that the interaction of rat liver nuclei with redox cycling agents and iron leads to the production of potent oxidants which initiate lipid peroxidation or oxidize ·OH scavengers. Although NADPH is more effective, NADH can also participate in catalyzing the production of reactive oxygen intermediates from the interaction of quinone redox cycling agents with nuclei. The ability of redox cycling agents to interact with various ferric complexes to catalyze nuclear generation of potent oxidizing species with either NADPH or NADH as reductants may contribute to the oxidative stress, toxicity, and mutagenicity of these agents in biological systems.

Hydroxyl Radical Production during Oxidative Deposition of Iron in Ferritin

The chemistry of oxidative deposition of iron(III) in ferritin and apoferritin is poorly understood. This study was undertaken to look for radicals formed as the hydrous ferric oxide core is developed from Fe(II) and O2. Radicals were observed indirectly by using the spin-trapping reagent N-tert-butyl-alpha-phenylnitrone (PBN) at room temperature and directly by measuring ESR spectra of frozen solutions at 77 K. In both instances, radical production was inhibited by the hydroxyl radical scavenging agents dimethyl sulfoxide, thiourea, and mannitol and enhanced by the addition of hydrogen peroxide. These findings strongly suggest that hydroxyl radical, produced from the iron-catalyzed Haber-Weiss reaction, is a by-product of core formation in ferritin and is a precursor to the observed radicals. The yield of ESR-observable and spin-trapped radicals is quite low, being at the micromolar level when millimolar concentrations of ferrous ion are employed. Furthermore, radical production appears to be confined to the interior of the ferritin molecule, where cellular components would be protected from the oxygen-derived toxic effects of iron. It is postulated that hydroxyl radical-medicated oxidative damage to the protein, a process that may contribute to the formation of hemosiderin from ferritin, leads to the observed radicals. By serving as a sink for hydroxyl radical, the protein shell may therefore efficiently minimize damage to other biomolecules in the cell.

Chemiluminescence from Acetaldehyde Oxidation by Xanthine Oxidase Involves Generation of and Interactions with Hydroxyl Radicals

The ability of acetaldehyde to generate free radicals is often ascribed to its oxidation by xanthine oxidase, with the subsequent production of reactive oxygen intermediates. Chemiluminescence associated with the oxidation of acetaldehyde by xanthine oxidase was inhibited by superoxide dismutase, catalase, or several hydroxyl radical scavenging agents, and was stimulated by the addition of EDTA or ferric-EDTA. This suggests that the light emission is primarily due to the production of hydroxyl radicals via an iron-catalyzed Haber-Weiss type of reaction. Chemiluminescence with hypoxanthine as substrate for xanthine oxidase was much lower than that found with acetaldehyde, yet rates of hydroxyl radical production were greater with hypoxanthine. Acetaldehyde increased light emission in the presence of hypoxanthine by a greater than additive effect. These results suggest a complex role for acetaldehyde in catalyzing xanthine oxidase-dependent chemiluminescence. It appears that besides being a substrate for xanthine oxidase, acetaldehyde also reacts with the generated hydroxyl radical to produce acetaldehyde radicals, which yield chemiluminescence upon their decay. Further studies will be required to evaluate whether the production of such species contributes to or plays a role in the generation of reactive oxygen intermediates and toxicity associated with acetaldehyde metabolism.

Oxyradical production by hepatopancreas microsomes from the red swamp crayfish, Procambarus clarkii

Microsomes catalyze the one electron reduction of many xenobiotics leading to proliferative production of damaging oxyradicals via redox cycling. The oxidation of 2-keto-4-thiomethylbutyric acid (KMBA) to ethylene was used as a means to study the O 2-/H 2O 2-generating oxidase pathway of hepatopancreas microsomes from the red swamp crayfish, Procambarus clarkii. Ethylene was produced by P. clarkii hepatopancreas microsomes in a chelex-treated incubation medium. The reaction was dependent upon NAD(P)H, O 2 and intact microsomes. NADH supported a 2- to 3-fold greater rate of KMBA oxidation by hepatopancreas microsomes than did NADPH, whereas NADPH supported a higher rate of KMBA oxidation than did NADH using rat liver microsomes. Ethylene production was increased approximately 3-fold in the presence of azide, an inhibitor of catalase, suggesting that H 2O 2 served as the precursor of · OH ∗ ∗ ·OH is taken here to include any 3 electron reduction product of O 2 with the oxidizing power of ·OH, e.g. a ferryl species (Aust and Svingen, 1982) production. Likewise, the addition of exogenous catalase in an azide-free system almost completely eliminated ethylene production. Superoxide dismutase was without any significant effect on KMBA oxidation by P. clarkii microsomes. Ethylene production by P. clarkii hepatopancreas microsomes was suppressed by competing hydroxyl radical scavenging agents, i.e. benzoate, mannitol, ethanol, and dimethyl sulfoxide (DMSO). Ferric ethylenediaminetetracetic acid (EDTA) stimulated NAD(P)H-dependent ethylene production in a dose-related manner, thus ferric was reduced by the system. KMBA oxidation was stimulated by EDTA alone and inhibited by the potent ferric-binding chelate, desferrioxamine suggesting the presence of iron in the microsomal system. Rates of · OH generation as measured by formaldehyde production from DMSO were consistent with rates calculated via ethylene production from KMBA. These data show that hepatopancreas microsomes generate potent oxidants during NAD(P)H-dependent microsomal electron transport. The kinetics of oxidant production appears to reflect differential reactivities of ferric-EDTA with NADH- vs. NADPH-dependent reductase(s) which varies considerably between hepatopancreas and rat liver microsomal systems.

Increased NADPH-dependent chemiluminescence by microsomes after chronic ethanol consumption

The generation of reactive oxygen intermediates by microsomes from ethanol-fed rats and pair-fed controls was determined by assaying for NADPH-dependent chemilumi-nescence. In the absence or presence of added ferric complexes, microsomal light emission was elevated several-fold after chronic ethanol consumption. Iron complexes such as ferric-citrate or ferric-ATP stimulated, while ferric-EDTA, inhibited microsomal chemiluminescence. Freeze-thawing the microsomes to elevate their content of lipid hydroperoxides resulted in large increases in chemiluminescence; under all conditions, the light emission remained several-fold higher with microsomes from the ethanol-fed rats. Chemiluminescence was not sensitive to Superoxide dismutase, catalase, or the hydroxyl radical scavenging agent, dimethyl sulfoxide, but was inhibited by antioxidants and by glutathione. Replacing air with a mixture of 50% nitrogen-50% air or 50% carbon monoxide-50% air had no effect on chemiluminescence by microsomes from the pair-fed controls. However, the chemiluminescent response by microsomes from the ethanol-fed rats was inhibited about 50% by the nitrogen mixture, and was further inhibited (about 75% of values found with 100% air, and 50% of values found with 50% nitrogen-50% air) with the carbon monoxide mixture. The sensitivity to carbon monoxide suggests the possibility that the alcohol-inducible cytochrome P-450 isozyme may contribute, in part, to the elevated light emission produced by microsomes from the ethanol-fed rats. The increase in chemiluminescence by microsomes after chronic ethanol consumption appears to reflect an elevated level of lipid hydroperoxides as well as an increased rate of generation of reactive oxygen species.