Hypoxanthine And Xanthine Oxidase Research Articles

Oxidative Stress Type Influences the Properties of Antioxidants Containing Polyphenols in RINm5F Beta Cells.

The in vitro methods currently used to screen bioactive compounds focus on the use of a single model of oxidative stress. However, this simplistic view may lead to conflicting results. The aim of this study was to evaluate the antioxidant properties of two natural extracts (a mix of red wine polyphenols (RWPs) and epigallocatechin gallate (EGCG)) with three models of oxidative stress induced with hydrogen peroxide (H2O2), a mixture of hypoxanthine and xanthine oxidase (HX/XO), or streptozotocin (STZ) in RINm5F beta cells. We employed multiple approaches to validate their potential as therapeutic treatment options, including cell viability, reactive oxygen species production, and antioxidant enzymes expression. All three oxidative stresses induced a decrease in cell viability and an increase in apoptosis, whereas the level of ROS production was variable depending on the type of stress. The highest level of ROS was found for the HX/XO-induced stress, an increase that was reflected by higher expression antioxidant enzymes. Further, both antioxidant compounds presented beneficial effects during oxidative stress, but EGCG appeared to be a more efficient antioxidant. These data indicate that the efficiency of natural antioxidants is dependent on both the nature of the compound and the type of oxidative stress generated.

Calcium influx-dependent differential actions of superoxide and hydrogen peroxide on microvessel permeability

Our previous study demonstrated that reactive oxygen species (ROS) released from activated blood cells contribute significantly to the increased microvessel permeability during inflammation. This study aims to define the individual roles of hydrogen peroxide (H(2)O(2)) and superoxide in ROS-induced increases in permeability and endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (L(p)). Endothelial [Ca(2+)](i) was measured in fura-2 AM-loaded microvessels. Perfusing microvessels with superoxide generated by hypoxanthine and xanthine oxidase (HX/XO) induced immediate and transient increases in L(p). The mean peak value, which occurred within 5 min of HX/XO exposure, was 4.3 +/- 0.6 times that of the control. In contrast, the perfusion of H(2)O(2) (100 and 500 microM) caused no immediate increases in L(p). A significant L(p) increase, 3.6 +/- 0.6 times the control value, occurred 30 min after the perfusion of H(2)O(2) at 500 microM. The perfusion of H(2)O(2) at 100 or 500 microM for 1 h increased L(p) to 6.6 +/- 0.9 and 11.3 +/- 3.6 times the control value, respectively. The increased endothelial [Ca(2+)](i) in HX/XO or H(2)O(2) perfused vessels was correlated with the time course of the increases in L(p). Inhibiting Ca(2+) influx by LaCl(3) prevented the permeability increase induced by HX/XO or H(2)O(2). These results demonstrated differential actions of superoxide and H(2)O(2) on microvessel permeability and endothelial [Ca(2+)](i). Superoxide-induced permeability increases were immediate and transient, whereas H(2)O(2)-induced permeability increases were progressive, demonstrating concentration and time dependence. Ca(2+) influx plays an essential role in both superoxide and H(2)O(2)-induced permeability increases.

Role of the Nitro Functionality in the DNA Binding of 3-Nitro-10-methylbenzothiazolo[3,2-a]quinolinium Chloride

Interest in DNA binding drugs has increased in recent years due to their importance in the treatment of genome-related diseases, like cancer. A new family of water-soluble DNA binding compounds, the benzothiazolo[3,2- a]quinolinium chlorides (BQCls), is studied here as potential candidates for chemical treatment of solid tumor cells that may encounter low-oxygen environments, a condition known as hypoxia. These compounds are good DNA intercalators; however, no studies have been made of these compounds under hypoxic conditions. This work demonstrates the importance of the nitro-functionality in the DNA binding of 3-nitro-10-methylbenzothiazolo[3,2- a]quinolinium chloride (NBQ-91), which possesses nitro-functionality, and 10-methylbenzothiazolo[3,2- a]quinolinium chloride (BQ-106), which does not. Both NBQ-91 and BQ-106 have similar noncovalent binding affinity toward DNA. Dialysis experiments show that NBQ-91 binds DNA under N2-saturated conditions with increasing concentrations of reducing agent, presumably due to reduction of the nitro-functionality. Conversely, because of the lack of nitro-functionality, the presence of a reducing agent had no effect on BQ-106 binding to DNA under both aerobic and N2-saturated conditions. Clonogenic assays were performed to determine the quinolinium chloride cytotoxicities under both aerobic (95% air and 5% CO2) and hypoxic (80% N2 and 20% CO2) conditions. The calculated ratios of cellular toxicity under aerobic to hypoxic conditions caused by the same concentration of test agent (CTR values) show greater levels of cell death under hypoxia than under aerobic conditions for mitomycin C (MC) (CTR = 0.7 at 1 microM) and NBQ-91 (CTR = 0.4 at 200 microM) than for BQ-106 (CTR = 1.0 at 200 microM), which agreed with the previously reported data for MC and confirmed the importance of nitro-functionality for reactivity under hypoxic conditions. There was no correlation between noncovalent binding affinity constants and their cytotoxicity under hypoxic conditions. Adduct formation between the NBQ-91 and 2'-dG was also assessed by reacting 2'-dG or DNA with NBQ-91 and BQ-106 under N2-saturated conditions in the presence of hypoxanthine and xanthine oxidase (HX/XO). DNA covalent adduct formation was analyzed by two techniques: LC-ESI-MS and Sephadex size exclusion chromatography. LC-ESI-MS results clearly indicate the formation of a prominent molecular ion at masses of 266.0 and 530.58 Da, corresponding to the [M + H](+2) and [M](+) molecular ions of the monitored 2'-dG-NBQ-91 adduct. Results from the Sephadex size exclusion chromatography support these findings because the NBQ-91 elution percentage increases in the presence of HX/XO due to the reduction of the nitro-functionality, which results in covalent binding to DNA. This study reports evidence of the DNA binding capacity of this bioreductive drug. The preferential N2-saturated over aerobic conditions for DNA binding makes NBQ-91 a potential bioreductive compound for hypoxic cell killing.

Modification of oligosaccharides by reactive oxygen species decreases sialyl lewis x-mediated cell adhesion

Modification of cell surface oligosaccharides by reactive oxygen species (ROS) and the biological effect of such modifications on cell adhesion were investigated. Treatment of HL60, a human promyelocyte leukemia cell line, with ROS, generated by a combination of hypoxanthine and xanthine oxidase (HX/XO), decreased the sialic acid content on the cell surface, as indicated by a flow cytometric analysis involving sialic acid-specific lectins, and a concomitant increase of free sialic acid was observed in the supernatant. A cell adhesion assay showed that the HX/XO treatment of HL60 cells decreases their capability of binding to human umbilical vein endothelial cells (HUVEC), probably because of an impairment of the interaction involving E-selectin, whereas the decrease in the binding was canceled by the addition of superoxide dismutase (SOD) and catalase. In fact, cell surface sialyl lewis x (sLe x), but not lewis x (Le x), was decreased by HX/XO treatment. Thus, it is more likely that the impaired interaction is based on diminished levels of the selectin ligand. Cleavage of sialic acid by ROS was further verified by the degradation of 4MU-Neu5Ac by HX/XO in the presence of hydrogen peroxide and iron ion. These results indicate that glycosidic linkage of sialic acid is a potential target for superoxide and other related ROS. It is well known that ROS cause cellular damages such as lipid peroxidation and protein oxidation, but, as suggested by the findings reported in the literature, ROS may also regulate cell adhesion via the structural alteration of sialylated oligosaccharides on the cell surface.

Resveratrol, a red wine constituent polyphenol, prevents superoxide-dependent inflammatory responses induced by ischemia/reperfusion, platelet-activating factor, or oxidants.

Moderate consumption of red wine has been shown to exert cardioprotection against ischemia/reperfusion. Because oxidant-dependent leukocyte infiltration plays a critical role in ischemia/reperfusion-induced tissue injury, we hypothesized that resveratrol, a red wine constituent polyphenol would attenuate postischemic leukocyte recruitment and subsequent endothelial dysfunction. Intravital microscopic approaches were used to quantify leukocyte/endothelial cell interactions and venular protein leakage in rat mesenteries exposed to either 20 min ischemia and 60 min reperfusion (I/R), oxidants generated by the reaction of hypoxanthine and xanthine oxidase (HX/XO), platelet-activating factor (PAF), or leukotriene B4 (LTB4). I/R or HX/HX produced marked increases in the number of adherent (LA) and emigrated (LE) leukocytes, which were associated with significant increases in venular albumin leakage (VAL). Intravenous administration of resveratrol or superoxide dismutase (SOD) attenuated these increases in LA, LE, and VAL. Superfusion of the mesentery with PAF or LTB4 also markedly increased LA, LE, and VAL. While resveratrol attenuated the proinflammatory effects of PAF, LTB4-induced changes were not affected by resveratrol. Resveratrol prevents leukocyte recruitment and endothelial barrier disruption induced by a number of superoxide-dependent proinflammatory stimuli, including I/R, HX/XO, or PAF. These salutary effects appear to be related to the antioxidant properties of resveratrol and contribute to the cardioprotective actions associated with consumption of red wine.

Actions of reactive oxygen species on AH/type 2 myenteric neurons in guinea pig distal colon.

With conventional intracellular recording methods, we investigated the mechanism of actions of reactive oxygen species (ROS) derived from hypoxanthine and xanthine oxidase (HX/XO) reactions on AH/type 2 myenteric neurons in the guinea pig distal colon. Of the 54 neurons to which HX/XO was applied, 32 neurons showed a transient membrane hyperpolarization(s) followed by a long-lasting membrane depolarization. Two additional groups of 10 myenteric neurons exhibited only a membrane hyperpolarization(s) or a late-onset membrane depolarization, respectively, and the remaining two neurons did not show any response to HX/XO. Analysis of changes of the input resistance induced by HX/XO indicated that suppression and augmentation of the conductance of Ca(2+)-dependent K(+) channels are the ionic mechanisms underlying the membrane hyperpolarization and depolarization, respectively. The effects of HX/XO on myenteric neurons were mimicked by application of caffeine or H(2)O(2). The results suggest that OH(.), but neither H(2)O(2) nor O(2)(.-), is responsible for HX/XO-induced responses. The intracellular Ca(2+) store may be the acting site of ROS in colonic AH/type 2 neurons.

Apoptosis of Myelin-Reactive T Cells Induced by Reactive Oxygen and Nitrogen Intermediatesin Vitro

Apoptosis is a major mechanism of T cell elimination during ontogeny and tolerance induction as well as in autoimmunity. To assess the possible involvement of reactive oxygen and nitrogen intermediates (ROI and NO•) in T cell apoptosis during autoimmune demyelination we investigated the effects of H2O2and NO•in vitroon activated autoreactive CD4+T cell lines capable of transferring experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN). For detection and quantitation of apoptotic cells, DNA fragmentation was assessed byin situtailing with fluorescein-ddUTP and subsequent flow cytometric analysis. H2O2applied directly to the cell cultures for 6 to 18 hr at concentrations of 10 to 300 μMand ROI released by combination of hypoxanthine and xanthine oxidase (HX/XO) caused apoptosis in a dose-dependent manner in 13–33% of T cells of neuritogenic and encephalitogenic T cell lines. Apoptosis induction could be suppressed by the H2O2-neutralizing enzyme catalase. NO•released by the penicillamine derivative SNAP induced apoptosis to a similar extent as ROI. Maximum values were 38% in an encephalitogenic Vβ8.2-T cell receptor-bearing T cell line and 26% in a neuritogenic T cell line. T cell lines with specificity to ovalbumin revealed slightly lower susceptibility to apoptosis induction by all three kinds of trigger, which is, however, most probably not due to the different antigen specificity, but rather a result of fewerin vitrorestimulation cycles of these cells. In neuritogenic cells high-dose (100 units/ml) exogenous interleukin-2 (IL-2) prevents H2O2-induced apoptosis. In conclusion, macrophage-derived reactive oxygen and nitrogen intermediates have the potency to limit inflammatory demyelination by elimination of autoreactive and bystander T cells via apoptotic cell death, and IL-2 is a rescue factor.

Molecular mechanisms involved in superoxide-induced leukocyte-endothelial cell interactions in vivo

Intravital microscopy was used to monitor leukocyte adherence, flux, rolling velocity, and number of rolling leukocytes (flux/velocity) in venules 25-40 microns in diameter. The superoxide-generating system, hypoxanthine and xanthine oxidase (HX/XO), was infused into the mesenteric circulation in untreated animals or in animals pretreated with either catalase (a hydrogen peroxide scavenger), WEB-2086 [a platelet-activating factor (PAF) receptor antagonist], or monoclonal antibodies directed against adhesion molecules CD18 (CL26) or P-selectin (PB1.3). HX/XO infusion caused a decrease in leukocyte rolling velocity and an increase in the number of rolling and adherent leukocytes. WEB-2086 prevented the increase in leukocyte adhesion and markedly increased leukocyte rolling velocity. PB1.3 abolished the HX/XO-associated rise in the flux of rolling leukocytes and proportionally decreased the number of adherent leukocytes. CL26 abolished HX/XO-induced leukocyte adhesion and also reduced the number of rolling leukocytes. In conclusion, P-selectin mediates the increased leukocyte flux induced by superoxide, whereas PAF and CD18 modulate leukocyte adhesion. PAF also reduces leukocyte rolling velocity, possibly as a result of CD18, but not P-selectin.

Nitric oxide prevents leukocyte adherence: role of superoxide

The objective of this study was to determine whether the antiadhesive effects of NO for leukocytes are related to its ability to scavenge superoxide in vivo. Intravital microscopy was used to monitor leukocyte adherence and flux as well as velocity and number of rolling leukocytes in 25- to 40-microns venules. The superoxide-generating system, hypoxanthine and xanthine oxidase (HX-XO), was infused into the mesenteric circulation in untreated animals and in animals pretreated with either superoxide dismutase (SOD) or the NO donor, SIN 1. In another series of studies, the mesenteric preparation was superfused with either platelet-activating factor (PAF) or leukotriene B4 (LTB4) followed by the administration of either SIN 1 or SOD. HX-XO infusion caused a significant increase in the number of rolling and adherent leukocytes (responses that were entirely inhibited by SOD or SIN 1). SOD and SIN 1 both attenuated the PAF-induced but not the LTB4-induced leukocyte adherence. The observation that both SOD and SIN 1 inhibit leukocyte adhesion only under conditions associated with superoxide formation (HX-XO and PAF, but not LTB4) strongly suggests that the antiadhesion properties of NO are related to its ability to inactivate the superoxide anion.

Free radical oxidation products in plasma and synovial fluid of horses with synovial inflammation

Free radical oxidation products, namely conjugated dienes, ultraviolet fluorescence (excitation 325 nm, emission 395 nm) and visible fluorescence (excitation 360 nm, emission 460 nm) were measured in equine synovial fluid exposed to free radicals in vitro and in the plasma and synovial fluids of horses with synovial effusions. The synovial effusions were induced by intra-articularly administered carrageenin (0.3 ml, 1%), which rarely resulted in clinical lameness. The free radicals were generated in vitro by mixtures of iron and ethylene diamine tetra acetate (Fe/EDTA) or mixtures of hypoxanthine and xanthine oxidase (HX/XO). The conjugated diene concentrations and intensity of ultraviolet fluorescence were negligible in plasma and synovial fluid specimens. No increase resulted from incubation of synovial fluids with either a free radical generating system or as a result of the induced inflammation. The intensity of visible fluorescence did not increase in specimens incubated with Fe/EDTA. However, the intensity of visible fluorescence increased in specimens incubated with HX/XO, in synovial effusions induced by carrageenin, in plasma and in synovial fluids aspirated from saline injected controls. The results indicate that the intensity of visible fluorescence of equine synovial fluid increases after exposure to free radicals and during synovitis in the horse, suggesting a possible role for free radicals in the pathogenesis of equine inflammatory joint disease.

Effect of Palosein (superoxide dismutase) and catalase upon oxygen derived free radical induced degradation of equine synovial fluid

The effect of oxygen derived free radicals (ODFR) upon the specific viscosity of equine synovial fluid was studied. ODFR were generated either by a mixture of ferrous iron and EDTA (Fe/EDTA) or by a mixture of hypoxanthine and xanthine oxidase (HX/XO). Incubation of the synovial fluid with both free radical generating systems decreased its specific viscosity. When the synovial fluid was incubated with Fe/EDTA the specific viscosity of the synovial fluid was reduced rapidly. By 2 mins, it was 53 +/- 3 per cent of the original specific viscosity and by 30 mins it was reduced to 39 +/- 5 per cent. In the HX/XO system, the specific viscosity was 75 +/- 4 per cent of the original specific viscosity at 10 mins and by 50 mins it was reduced to 55 +/- 3 per cent. Palosein (superoxide dismutase) was an effective inhibitor of the free radical induced reduction of the viscosity of the synovial fluid when the free radicals were generated with HX/XO but not with Fe/EDTA. Catalase was moderately effective as an inhibitor of reduction in specific viscosity of the synovial fluid when the free radicals were generated by either system. Only minor synergy resulted when mixtures of Palosein and catalase were tested for inhibition of Fe/EDTA induced reduction in the specific viscosity of equine synovial fluid. The results indicate that Palosein may protect equine synovial fluid from the effects of the superoxide radical (O2-) but not from the hydroxyl radical (OH.).(ABSTRACT TRUNCATED AT 250 WORDS)