Xanthine-xanthine Oxidase System Research Articles

Possible mechanism of antioxidant action of dinitrosyl iron complexes

The antioxidant effect of dinitrosyl iron complexes (DNICs) was studied in various model systems. DNICs with glutathione ligands effectively inhibited Cu2+-induced peroxidation of low density lipoproteins (LDL). The antioxidant effect of DNICs with phosphate ligands and free reduced glutathione (GSH) was less pronounced. In addition, DNICs with glutathione suppressed the formation of reactive oxygen species during co-oxidation of lecithin liposomes and glucose. Free radical oxidation in this system was induced with a lipophilic azo initiator and evaluated by luminol-dependent chemiluminescence. NO sharply stimulated chemiluminescence during co-oxidation of glucose and liposomes, thus suggesting the formation of potent oxidants under these conditions. Glutathione DNICs scavenge the superoxide radical anion generated in the xanthine-xanthine oxidase system. Superoxide production was assessed by lucigenin-dependent chemiluminescence and electron paramagnetic resonance (EPR) spectroscopy. Chemiluminescence revealed the dose-dependent character of antiradical effect of glutathione DNICs; moreover, these complexes turned out to be more efficient than GSH. EPR spectra of the adducts of the DEPMPO spin trap with free radicals suggest that the interaction of glutathione DNICs and superoxide does not result in the formation of the thiyl radical of glutathione. Here we propose a mechanism of the antioxidant action of glutathione DNICs, suggesting that unstable intermediate complexes are formed upon their interaction with superoxide or lipid radicals. Further, as a result of intramolecular rearrangement, these intermediates decompose without the free radical as the by-products.

184 Effects of cyanidin supplementation on invitro maturation of pig oocytes

Oxidative stress can have a negative effect on oocyte maturation during invitro production of pig embryos. Imbalance of reactive oxygen species and antioxidant levels can affect the progression of oocyte maturation up to the point of fertilization. Antioxidants are effective in maintaining more ideal reactive oxygen species levels, which help to protect oocytes from potential harmful effects of oxidative stress. Berries from the elder plant (Sambucus sp.) contain high levels of a broad spectrum of antioxidants. One of these antioxidants, cyanidin, when supplemented to maturation medium at 100μM concentrations, reduces reactive oxygen species formation and improves IVF and early embryonic development in pigs. However, changes in the enzyme mechanisms of action during oocyte maturation due to cyanidin supplementation are unknown. Therefore, the objective of this study was to characterise the intracellular oocyte enzyme mechanisms between oocytes supplemented with 100μM cyanidin during 40 to 44h of maturation (n=600) and oocytes without supplementation of cyanidin during maturation (n=558). At the end of maturation, oocytes were evaluated for either glutathione peroxidase (n=300), catalase (n=564), or superoxide dismutase (n=294) activities. Glutathione peroxidase activity was determined by following the rate of NADPH oxidation, catalase activity was determined by following the rate of hydrogen peroxide decomposition, and superoxide dismutase activity was determined by following the reduction rate of cytochrome c, utilising the xanthine-xanthine oxidase system. Data were analysed using ANOVA and Tukey's test. There were no significant differences between oocytes matured with 100μM cyanidin and those that were not when comparing glutathione peroxidase and superoxide dismutase activities. Supplementation of 100μM cyanidin to maturation medium increased (P<0.05) catalase activity in oocytes (0.78±0.15 units/oocyte) compared with no cyanidin supplementation (0.14±0.11 units/oocyte). These results indicate that supplementing 100μM cyanidin to the maturation medium of pig oocytes could reduce the negative effects of oxidative stress by increasing intracellular catalase activity during oocyte maturation.

H2O2 Induces Association of RCA with the Thylakoid Membrane to Enhance Resistance of Oryza meyeriana to Xanthomonas oryzae pv. oryzae.

Oryza meyeriana is a wild species of rice with high resistance to Xanthomonas oryzae pv. oryzae (Xoo), but the detailed resistance mechanism is unclear. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activase (RCA) is an important enzyme that regulates photosynthesis by activating Rubisco. We have previously reported that Xoo infection induced the relocation of RCA from the chloroplast stroma to the thylakoid membrane in O. meyeriana, but the underlying regulating mechanism and physiological significance of this association remains unknown. In this study, “H2O2 burst” with rapid and large increase in the amount of H2O2 was found to be induced by Xoo invasion in the leaves of O. meyeriana. 3, 3-diaminobenzidine (DAB) and oxidative 2, 7-Dichlorodi-hydrofluorescein diacetate (H2DCFDA) staining experiments both showed that H2O2 was generated in the chloroplast of O. meyeriana, and that this H2O2 generation as well as Xoo resistance of the wild rice were dramatically dependent on light. H2O2, methyl viologen with light, and the xanthine-xanthine oxidase system all induced RCA to associate with the thylakoid membrane in vitro, which showed that H2O2 could induce the relocation of RCA. In vitro experiments also showed that H2O2 induced changes in both the RCA and thylakoid membrane that were required for them to associate and that this association only occurred in O. meyeriana and not in the susceptible cultivated rice. These results suggest that the association of RCA with the thylakoid membrane helps to protect the thylakoid membrane against oxidative damage from H2O2. Therefore, in addition to its universal function of activating Rubisco, RCA appears to play a novel role in the resistance of O. meyeriana to Xoo.

Oxidation of the plastoquinone pool in chloroplast thylakoid membranes by superoxide anion radicals.

The plastoquinone (PQ)-pool in chloroplast thylakoid membranes is a key electron carrier in the photosynthetic electron transport chain (PETC), and its redox state plays an essential role in the control of plant metabolism. Oxygen reduction in thylakoid membranes producessuperoxide anion radicals ( ), which may react with the PQ-pool. Here, using isolated thylakoids, we show for the first time the oxidation of the PQ-pool by . The xanthine-xanthine oxidase system was used to supply externally to the thylakoid membrane and the redox state of the PQ-pool was monitored by tracking chlorophyll a fluorescence. We propose that,invivo,the reaction of produced in Photosystem I with reduced PQ (plastohydroquinone) creates hydrogen peroxide, which serves as a messenger that signals the redox state of the PETC.

Oxidation of Antioxidant Species with Superoxide Radicals Produced By the Xanthine-Xanthine Oxidase System

Free radicals have become very important in recent years due to the damage they cause to biological molecules. The superoxide radical (O2•-) is the primary species of reactive oxygen species (ROS) that plays a major role in physiological processes. This radical normally is produced physiologically at such a rate that the human body is able to catabolize it. However, when the production of O2•- radicals exceeds the capacity of the human body, physiological imbalances become harmful. In this work the xanthine oxidase enzyme is proposed to catalyze the oxidation of xanthine and take advantage of the production of O2•- radicals. The O2•- formed in the enzymatic reaction are reacted with different antioxidant compounds (caffeic acid, gallic acid, ascorbic acid) and the oxidized species are quantified by cyclic voltammetry in an electrochemical cell thermostatted at 30 °C in a medium buffered with phosphate buffer 0.1 mol L− 1 pH 7.0. A carbon paste electrode was the working electrode, Ag/AgCl the reference and platinum the counter electrode. All antioxidants have an oxidation process between 200 and 600 mV, as well as a reduction process between −200 and 200 mV. The enzyme and the substrate do not present redox processes in the work potentials. However, uric acid has an oxidation process around 400 mV, but does not present reduction processes.

Lignans with NADPH Oxidase 2 (NOX2)-inhibitory Activity from the Fruits ofSchisandra chinensis

An isoform of NADPH oxidase (NOX), NOX2 is a superoxide-generating enzyme involved in diverse pathophysiological events. Although its potential as a therapeutic target has been validated, there is no clinically available inhibitor. Herein, NOX2-inhibitory activity was screened with the constituents isolated from Schisandra chinensis, which has been reported to have antioxidant and reactive oxygen species (ROS)-scavenging effects. Among the partitions prepared from crude methanolic extract, a chloroform-soluble partition showed the highest NOX2-inhibitory activity in PLB-985 cell-based NOX2 assay. A total of twenty nine compounds (1 - 29) were identified from the chloroform fraction, including two first isolated compounds; dimethyl-malate (25) and 2- (2-hydroxyacetyl) furan (27) from this plants. Of these constituents, two compounds (gomisin T, and pregomisin) exhibited an NOX2-inhibitory effect with the IC50 of 9.4 ± 3.6, and 62.9 ± 11.3 μM, respectively. They are confirmed not to be nonspecific superoxide scavengers in a counter assay using a xanthine-xanthine oxidase system. These findings suggest the potential application of gomisin T (6) and other constituents of S. chinensis to inhibit NOX2.

Supplementation of sperm media with zinc, D-aspartate and co-enzyme Q10 protects bull sperm against exogenous oxidative stress and improves their ability to support embryo development.

High levels of reactive oxygen species in the semen of infertile patients or spontaneously generated during in vitro sperm handling may impair sperm quality, fertilization and embryo developmental competence. We recently reported that zinc, d-aspartate and co-enzyme Q10, contained in the dietary supplement Genadis® (Merck Serono), have protective effects on human and bull sperm motility, lipid peroxidation and DNA fragmentation in vitro; furthermore, in bovine, treated spermatozoa had an improved ability to support embryo development. However, only a few studies have investigated the protective role of antioxidants during in vitro sperm handling in the presence of an exogenous oxidative stress. Herein, to simulate such conditions in an animal model, we induced exogenous oxidative stress on spermatozoa through the xanthine-xanthine oxidase system and investigated its effects on sperm function and subsequent embryo developmental competence in the presence of zinc, d-Asp and CoQ10 protection. The main results showed that exogenous oxidative stress decreased sperm motility, increased sperm DNA fragmentation, and reduced fertilization and blastocyst rates and quality. Pre-treatment with zinc, d-aspartate and co-enzyme Q10 before exogenous oxidative stress was able to prevent these effects. Supplementation of sperm culture media with zinc, d-aspartate and co-enzyme Q10 could protect sperm from oxidative stress damage during in vitro handling in assisted reproductive technologies.

Chemical composition, antioxidative and enzyme inhibition activities of chickweed herb (Stelaria media L., Vill.) ethanolic and aqueous extracts

The aim of the study was an attempt of justification of chickweed’s herb (Stelaria media L., Vill.) traditional use as anti-inflammatory agent in the treatment of skin diseases. In the study a phytochemical characterization and potential anti-inflammatory effect of the aqueous and ethanolic extracts of chickweed herb were investigated in vitro, by determining their antioxidant and enzyme inhibition activities. Moreover, the effect on reactive oxygen species (ROS) generation by human skin fibroblasts and keratinocytes after UV irradiation was determined. The main components of both extracts were apigenin glycosides. The content of flavonoids in the aqueous extract was 25.6mg/g of liophilized extract and in the ethanolic extract 63.9mg/g of liophilized extract. The ethanolic extract showed stronger scavenging activity against three of the tested radicals. The determined SC50 (the concentration required to inhibit radical formation by 50%) average values for hydrogen peroxide (H2O2), nitric oxide (NO) and peroxynitrite (ONOO−) were 132.83, 16.5, 11.9μg/mL, respectively. On the other hand, in xanthine-xanthine oxidase system activity of aqueous extract was more potent, with SC50 average value 62.75μg/mL, than activity of ethanolic extract. Scavenging of DPPH, and hyaluronidase and lipoxidase activity inhibition of both the extracts was weak, on the contrary to the strong inhibition of xanthine oxidase activity. Subsequently, the influence of the extracts on the human skin cells was examined. The study showed that the aqueous extract decreased intracellular ROS production by fibroblasts in a concentration-dependent manner. In the case of keratinocytes the aqueous extract also reduced intracellular ROS production, but not in a concentration-dependent manner. On the basis of this study it can be concluded that the possible anti-inflammatory activity on the skin of the extracts obtained from chickweed herb is due to their antioxidant activity, but not via an influence on activity of pro-inflammatory enzymes like hyaluronidase or lipoxygenase.

Sensitive electrochemical measurement of hydroxyl radical generation induced by the xanthine–xanthine oxidase system

A sensitive electrochemical measurement system for hydroxyl radical (OH) was developed using enzyme-catalyzed signal amplification. In the presence of 2,6-xylenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ–GDH) as a catalyst, the amperometric signal of the trapping adduct 2,6-dimethylhydroquinone (DMHQ) produced by the hydroxylation of 2,6-xylenol was able to be amplified and detected sensitively. The limit of detection (signal/noise [S/N]=3) for DMHQ was 1nM. There was no significant interference from urate and other oxidizable compounds in the reaction mixture at the applied potential of 0V versus Ag/AgCl. This method was employed to observe the OH generation induced by the xanthine–xanthine oxidase (XO) system. The reaction rates of the DMHQ production induced from the xanthine–XO system in the presence and absence of various Fe(III) complexes and proteins were compared. Those with a free coordination site on the Fe atom effectively enhanced the OH generation.

Chlorovirus PBCV-1 encodes an active copper-zinc superoxide dismutase.

Superoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Chlorovirus PBCV-1 encodes a 187-amino-acid protein that resembles a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc (named cvSOD). cvSOD has an internal Met that results in a 165-amino-acid protein (named tcvSOD). Both cvSOD and tcvSOD recombinant proteins inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution. tcvSOD was chosen for further characterization because it was easier to produce. Recombinant tcvSOD also inhibited a riboflavin photochemical reduction system in a polyacrylamide gel assay, which was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A k(cat)/K(m) value for cvSOD was determined by stop-flow spectrophotometry as 1.28 × 10(8) M(-1) s(-1), suggesting that cvSOD-catalyzed O2 (-) dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene, and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection, and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described to occur in 3 other families of large DNA viruses, poxviruses, baculoviruses, and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms. Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this article, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.

Protection of common carp (Cyprinus carpio L.) spermatozoa motility under oxidative stress by antioxidants and seminal plasma.

The protective influence of seminal plasma and the antioxidants catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) on quality parameters, oxidative stress indices, and antioxidant activity was studied in common carp (Cyprinus carpio) spermatozoa exposed to the xanthine-xanthine oxidase (X-XO) system. Fish spermatozoa were incubated for 5 and 20 min at 4 °C with X-XO concentrations of 1 mM X-0.1 U/mL, 0.6 mM X-0.05 U/mL, 0.3 mM X-0.025 U/mL, and 0.1 mM X-0.0125 U/mL. A dose-dependent reduction in spermatozoa motility and velocity was observed at concentrations of 0.1 mM X-0.0125 U/mL to 1 mM X-0.1 U/mL XO. Increase in spermatozoa motility parameters was recorded following treatment with antioxidants and seminal plasma. The level of the oxidative stress indices lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was significantly reduced after addition of CAT, SOD, or GTH along with seminal plasma. Significant differences in SOD, glutathione reductase, and glutathione peroxidase activity were seen in spermatozoa incubated with, compared to that without, seminal plasma at all studied X-XO concentrations. The data demonstrated that CAT, SOD, or GTH in combination with SP can reduce reactive oxygen species stress in fish spermatozoa and improve spermatozoa quality.

English

In this study, the antioxidant properties of volatile oils obtained from the earth parts of the Artemisia taurica Willd. and Salvia kronenburgii Rech. Fil. plants and their effects on xanthine oxidase enzyme were studied. The chemical contents of each volatile oil were determined by applying gas chromatograpghy-mass spectrometry (GC-MS) analysis. The total phenol and flavonoid amounts of each volatile oil and the total antioxidant capacities were determined and it was observed that there was a positive correlation among these values. It was found out that the volatile oils were effective on inhibiting the reactive oxygen kinds and that they scavenged the superoxide radical made up with xanthine-xanthine oxidase system and the hydroxyl radical made up with Fe+3 ascorbate EDTA-H2O2 system. Moreover, it was observed that both volatile oil samples reduced the 2,2-diphenyl-1-picrylhydrazyl (DPPH), a determined independent. It was also found out that A. taurica and S. kronenburgii volatile oils inhibited the xanthine oxidase enzyme and that Artemisia volatile oil was more effective on inhibiting this enzyme than Salvia volatile oil. When the inhibition kinetics were studied, it was observed that the inhibition kinds was successively competitive inhibition for A. taurica and uncompetitive inhibition for S. kronenburgii.   Key words: Artemisia taurica Willd,  Salvia kronenburgii Rech.Fil, antioxidant activity, free radical, xanthine oxidase.

Synthesis and Electrochemical Evaluation of Some Organic Molecules as an Antioxidant Agents

A series of new benzamides were synthesized. The chemicals structures were confirmed by elemental analyses 1H NMR and 13C studies. The antioxidant activity of the synthesized compounds was evaluated by square wave voltammetry. A new approach, for antioxidant capacity determination was proposed. It is based on the using of the xanthine-xanthine oxidase system coupled with H2O2 electrochemical sensor.

Potential of a 70 kDa IL‐10‐like factor in synovial fluid from rheumatoid arthritis patients to augment superoxide generation by human neutrophils

To elucidate the role of polymorphonuclear leukocytes (PMNs) in joint destruction during the inflammatory process in rheumatoid arthritis (RA) as related to superoxide generation. Superoxide generation by human peripheral PMNs was measured by using a water-soluble formazan dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt, under PMN stimulation with N-formylmethionyl-leucyl-phenylalanine (fMLP) and cytochalasin B. Factors in synovial fluids (SF) from RA patients that may augment PMN superoxide generation were characterized via high-performance liquid chromatography and isoelectric focusing. The formazan dye allowed measurement of superoxide generated in the xanthine-xanthine oxidase system and by PMNs stimulated by cytochalasin B and fMLP in the presence of the intermediate electron transporter phenazine methosulfate. By using chromatography and electrophoresis, an RA-SF protein with an apparent molecular size of 70kDa and an isoelectric point of 8.3 was isolated and was demonstrated to increase superoxide generation by PMNs. The factor was heat-labile and susceptible to protease treatment. This enhancing activity of the factor was absorbed by human PMNs and was somewhat immunoadsorbed with a specific monoclonal antibody against interleukin (IL)-10. The 70-kDa protein factor in RA-SF increased superoxide generation by human PMNs, which suggests the possibility of its being related to IL-10. This factor may have a pathological role in RA joint destruction caused by PMNs and coinciding with rheumatoid inflammation, which suggests that PMNs, via superoxide generation, play an important role in RA joint destruction. IL-10 therefore likely has biological activity toward PMNs during synovial inflammatory chain reactions in RA.

Oxidative stress in sickle cell disease

In the 18th century, Priestley, Scheele and Lavoisier discovered oxygen and reported its critical role and toxic effects in living organisms. In the last century, several studies highlighted the importance of biological oxidation for energy production by aerobic organisms, in defense and the elimination of drugs. Oxidation is mediated by oxidants and free radicals, generically called reactive oxygen species (ROS), are formed as a byproduct of the oxygen metabolism. Antioxidant enzymatic and non-enzymatic molecules play a crucial role in maintaining the balance of ROS; an imbalance may lead to attack on all the components of the cell, including proteins, lipids and DNA. Collectively, oxidative stress is described as an imbalance between oxidants/free radicals and antioxidants(1-3). Recently, several reports have suggested that oxidative stress is a complex mechanism rather than a simple imbalance between the production and elimination of ROS. Oxidants and free radicals are continuously produced in living organisms with endogenous and external sources such as oxygen and nitric oxide [reactive nitrogen species (RNS)]. An increase in the normal redox state of a cell causes toxic effects that may lead to cell and tissue damage. Furthermore, a decrease in free radicals may be harmful, due to their critical role in microbial defense, cell proliferation, apoptosis, migration, inflammatory gene expression and vascular matrix regulation. In addition, free radicals are increasingly recognized as vital messengers in cellular signal transduction in several organisms(3-5). Sickle cell anemia is an inherited blood disorder affecting approximately 5% of the world's population. This disease results from a mutation in the beta globin chain inducing the substitution of Val for Glu at position 6, shifting the isoelectric point of the protein(6). This single mutation induces the production of hemoglobin S (Hb S), which is abnormal and insoluble. Sickle cell disease promotes harmful pathological effects that includes sickling of erythrocytes, vaso-occlusion and ischemia-reperfusion injury. Increasing evidence points towards an oxidative stress response responsible for increased pathophysiology of secondary dysfunctions in sickle cell patients(7,8). Several molecular mechanisms have been proposed to contribute towards a high oxidative burden in sickle cell patients. Some of the mechanisms that disturb the redox state include, the excessive levels of free hemoglobin that catalyze the Fenton reaction(9),the recurrent ischemia-reperfusion injury promoting the activation of the xanthine-xanthine oxidase system(10) and higher autoxidation of Hb S generating superoxide anion radicals and hence hydrogen peroxide(11). Furthermore, a chronic proinflammatory response in sickle cell patients induced by constant recruitment of neutrophils and monocytes has been shown to play an important role in causing complications(12,13). ROS and RNS are not only potential markers of sickle cell disease severity but are also important targets for antioxidant therapies(14,15). Several reports have indicated lower levels of carotenoids, flavonoids, vitamins C and E and zinc (structural component of superoxide dismutase) in sickle cell anemia patients(14). Nevertheless, no measurable parameters in clinical studies have shown to ameliorate sickle cell disease in patients that received antioxidant supplementation(16). In contrast, the treatment of erythrocytes from sickle cell anemia patients with the flavonoid quercetin has been shown to provide protection against hemoglobin oxidation and other cellular modifications promoted by peroxides(17). Henneberg et al.(18) in this issue of the Revista Brasileira e Hematologia e Hemoterapia demonstrate the use of an unspecific probe (2'7'-dichlorfluorescein-diacetate) to qualitatively assess the intracellular redox state of erythrocytes from sickle cell anemia patients. The authors describe the effect of the flavonols quercetin and rutin to reduce intracellular oxidation promoted by peroxide formation in the cells by their established method. Moreover, an additional antioxidant effect was observed in erythrocytes of patients treated with hydroxyurea. Accordingly, further studies are necessary to understand the mechanistic aspects of free radicals and oxidants in sickle cell disease to improve therapies and find better diagnostic tools. The promising results by Henneberg et al.(18) in monitoring the redox state should encourage the investigation of potential biomolecules and antioxidant therapy for sickle cell treatment in combination with drugs that specifically target ROS/RNS production.

Antioxidant and Antiradical Properties of Esculin, and its Effect in A Model of Epirubicin-Induced Bone Marrow Toxicity

To evaluate the effect of esculin, a plant coumarin glucoside, on free radicals and against epirubicin-induced toxicity on bone marrow cells. Antioxidant activity was assessed by a luminol-dependent chemiluminescence method or NBT test in a xanthine-xanthine oxidase system, and two iron-dependent lipid peroxidation systems. In vivo experiments were carried out in epirubicin-treated mice, alone or in a combination with esculin. Genotoxicity of the anthracycline drug was assessed by cytogenetic analysis and an autoradiographic assay. Esculin inactivated superoxide anion radicals in both systems we used. It exerted SOD-mimetic effect and reduced the level of superoxide radicals generated in a xanthine-xanthine oxidase system by 30%. Esculin also showed an antioxidant effect in a model of Fe2+-induced lipid peroxidation. Cytogenetic analysis showed that epirubicin had a marked influence on the structure of metaphase chromosomes of normal bone marrow cells. Inclusion of esculin in the treatment protocol failed to ameliorate the epirubicin-induced antiproliferative effects and genotoxicity in bone marrow cells. In this study the ability of the coumarin glucoside esculin to scavenge superoxide radicals and to decrease Fe-induced lipid peroxidation was documented. However, despite the registered antioxidant effects the tested compound failed to exert cytoprotection in models of anthracycline-induced genotoxicity in bone marrow cells. The results of this study warrant for more precise further evaluation of esculin, employing different test systems and end-points and a wider range of doses to more precisely appraise its potential role as a chemoprotective/resque agent.

Oxidative Stress in Zebrafish (Danio rerio) Sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.

Copper complexes with nitronylnitroxyl radicals as lipoxygenase inhibitors with antioxidant activity

The effects of the complexes Cu(LRH)2(NO3)2 (R = H, Me) with the nitronylnitroxyl radicals LRH on the lipoxygenase (LOX-1) activity upon peroxidation of linoleic acid were studied. The complexes under study are reversible enzyme inhibitors, competitive and non-competitive inhibition type being observed for the complex with the methyl-substituted and unsubstituted ligands, respectively. The results of molecular docking showed that the complexes under study are incorporated into the hydrophobic pocket of the LOX-1 active site and the change in the type of inhibition can be explained by introduction of additional methyl group into the imidazole fragment of the ligand, which provides a slightly more favorable interaction of the inhibitor with the enzyme and results in the non-competitive mechanism of inhibition. The starting diamagnetic compounds (LRH2), nitronylnitroxyl radicals (LRH), and CuII complexes exhibit high activity in the nonenzymatic peroxidation of linoleic acid, as well as in generation of the superoxide radical anion O2·− in the xanthine-xanthine oxidase system.

1,2-Di-O-α-linolenoyl-3-O-β-galactosyl-sn-glycerol as a Superoxide Generation Inhibitor fromPerilla frutescensvar.crispa

Using a superoxide (O(2)(-)) generation assay system with differentiated HL-60 cells, 1,2-di-O-α-linolenoyl-3-O-β-galactosyl-sn-glycerol (DLGG) was identified as an O(2)(-) generation inhibitor from Perilla frutescens var. crispa (a local variety, kida-chirimen shiso). DLGG suppressed the O(2)(-) level in a dose-dependent manner with an IC(50) value of 21 µM, comparable to those of rosmarinic acid (RoA, IC(50) = 29 µM) and caffeic acid (CA, IC(50) = 30 µM). While RoA and CA also dose-dependently inhibited O(2)(-) generation in a xanthine-xanthine oxidase system, DLGG had no effect in the same system. Thus DLGG appeared to decrease the O(2)(-) level in the HL-60 assay system by mechanisms different from those of RoA and CA, which appeared to act as O(2)(-) scavengers.

Combined Effects of Manganese, Iron, Copper, and Dopamine on Oxidative DNA Damage

Manganese (Mn) damages the central nervous system and causes Parkinson's disease (PD)-like syndrome called manganism. Abnormal accumulation of Mn and iron (Fe) is observed in the basal nuclei of patients with PD. Dopamine induces oxidative DNA damage in the presence of Fe or copper (Cu) in vitro. Therefore, an Mn-induced neural disorder may involve combined actions of Mn, Fe/Cu, and catecholamines. Here we investigated the combined effects of Mn(II) on the DNA damage induced by dopamine and either Fe(III) or Cu(II). Mn(II) enhanced the formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) induced by dopamine and Fe(III)/Cu(II), whereas Mn(II) did not induce 8-oxodG formation in the absence of Fe(III)/Cu(II). Mn(II) accelerated hydrogen peroxide (H2O2) production in the dopamine-Fe(III)/Cu(II) system. Furthermore, Mn(II) decreased superoxide anion radicals (O2•-) generated in the xanthine-xanthine oxidase system. These results indicate that Mn(II)-mediated enhancement of DNA damage is caused by facilitated conversion of O2•- to H2O2. In addition, Mn(II) accelerated the formation of aminochrome, an oxidized dopamine product, in the presence of Cu(II). This reaction may have involved Mn(III) accompanied by H2O2 production from O2•-, suggesting a mechanism for Mn-induced oxidative stress on dopaminergic neurons through the combined actions of Mn, Fe, Cu, and dopamine.