Manganese complexes of curcumin analogues: evaluation of hydroxyl radical scavenging ability, superoxide dismutase activity and stability towards hydrolysis

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl radical (HO*) scavenging ability. SOD activity was evaluated by both the nitroblue tetrazolium (NBT) reduction assay and electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. CpCpx and AcylCpCpx inhibited the NBT reduction and decreased the DMPO/OOH adduct much greater than corresponding antioxidants or ligands, with IC50 values of 29.9 and 24.7 microM (NBT), and 1.09 and 2.40 mM (EPR), respectively. For EPR, potassium superoxide (KO2) was used as a source of O2- where qualitative results suggested that CpCpx and AcylCpCpx were SOD mimics, which catalyze the conversion of O2- to dioxygen and hydrogen peroxide (H2O2). Additionally, CpCpx and AcylCpCpx exhibited the great inhibition of DMPO/OH adduct formation with an IC50 of 0.57 and 0.37mM, respectively, which were comparable to that of curcumin (IC50 of 0.64 mM), indicating that both Mn complexes are also an effective HO* scavenger. The stability against hydrolysis in water, various buffers and human blood/serum was carried out in vitro. It was found that both Mn complexes were pH and salt concentration dependent, being more stable in basic pH. In the human blood/serum test, CpCpx was more stable against hydrolysis than AcylCpCpx with about 10 and 20% of free Mn2+ releasing, respectively.     

Studies on the reduction of nitroblue tetrazolium chloride mediated through the action of NADH and phenazine methosulphate.

The general features of the reduction of nitroblue tetrazolium chloride (NBT) by NADH and phenazine methosulphate (PMS) have been studied under aerobic and anaerobic conditions. Under aerobic condition the reduction appears to be mediated through the intermediate formation of the superoxide anion radical O2-.; this reaction is strongly inhibited by superoxide dismutase and by a number of O2-. scavengers such as propyl gallate, (+)-catechin, manganous ions, reduced glutathione and benzoquinone. Cupric ions inhibited the overall reaction by reoxidising reduced PMS. Under anaerobic conditions, superoxide dismutase had only a small inhibitory action and, with the exception of cupric ions, the other substances mentioned above were ineffective as inhibitors. The data presented show that the use of NBT to detect the presence of O2-. is fraught with difficulties due to an equally rapid reduction of NBT by NADH and PMS under anaerobic conditions.     

Differences in the effect of arachidonic acid on polymorphonuclear and mononuclear leukocyte function

Incubation of human polymorphonuclear leukocytes with arachidonic acid resulted in a stimulation of the oxidative metabolism of the cells. Upon stimulation with 80 microM arachidonic acid, neutrophils (5 X 10(6) cells/ml) produced superoxide (53 +/- 8 nmol/5 X 10(6) cells per 15 min), generated chemiluminescence (1211 100 +/- 157 000 cpm) and consumed oxygen (20 +/- 1 nmol/10(6) cells per 5 min). The stimulation of the cell metabolism could be reduced 40-60% by prior incubation of the cells with 10 microM indomethacin. Incubating polymorphonuclear leukocytes with arachidonic acid also resulted in a diminished chemotaxis towards an attractant, a decreased uptake of opsonized staphylococci and aggregation of the cells. This may be due to inhibitory products of arachidonic acid metabolism and toxic oxygen species produced during stimulated oxidative metabolism. The effects of arachidonic acid are specific for neutrophils, as mononuclear phagocytes only produced 17 +/- 8 nmol superoxide/5 X 10(6) cells per 15 min and generated 27 000 +/- 15 000 cpm chemiluminescence when stimulated with 80 microM arachidonic acid. When monocytes and neutrophils were stimulated with particles such as opsonized staphylococci, the amount of superoxide produced, oxygen consumed and chemiluminescence generated were similar. The phagocytic activity of the monocytes was also not affected by prior incubation with arachidonic acid. We conclude that in contrast to monocytes, neutrophil metabolism can be stimulated with arachidonic acid and this stimulation resulted in a decreased phagocytic activity of these cells.     

Modulation of superoxide dismutase by electron donors and acceptors

The competition between superoxide dismutase (SOD) and nitroblue tetrazolium (NBT) for O2- radicals in the presence of a number of physiologically active compounds was studied. The Na+ channel blockers, ajmaline, tetracaine, bipuvacaine, lidocaine and etmozine produced an increase in the amount of O2- reacting with SOD. Nitroprusside, ferricyanide, BAY K8644, levomycetin, cGMP, cAMP and GMP acted in the opposite way. All the SOD activtors had in common the property of being electron donors in the reactions with the light-induced free radicals of eosin whereas the SOD inhibitors behaved as electron acceptors. The electron activity of SOD modulators correlated qualitatively with their regulating efficacy.     

Characteristics of Eliciting Effects of Furostanol Glycosides on Cultured Yam Cells

To unravel mechanisms of elicitor action of furostanol glycosides (FGs), the formation of superoxide anion after the addition of FGs to a suspension culture of yam (Dioscorea deltoidea Wall. ex Griseb) cells was studied. The substantial increase in superoxide level, evaluated by nitroblue tetrazolium (NBT) reduction to formazan, was found at the exponential phase of cell growth. The involvement of NADPH oxidase in the superoxide generation was revealed by means of inhibitory analysis. Diphenyliodonium chloride (DPI), the inhibitor of NADPH oxidase, compromised the action of FGs. Meanwhile, the elimination of apoplastic peroxidase did not affect the accumulation of formazan, which suggests the involvement of NADPH oxidase but not peroxidase in the superoxide generation. In addition to NBT-test, the superoxide formation was judged by changes in activity of superoxide dismutase (SOD). Exogenous FGs activated the enzyme due to the increased production of superoxide anion. In this case, DPI decreased SOD activity that conforms to the NADPH oxidase involvement in the superoxide generation. The analysis of antioxidant activity of FGs by inhibition of radicals of 2,2-diphenyl-1-picrylhydrazyl showed that FGs are weak reductants in comparison with ascorbic acid. The results of the work allow for the suggestion that, supposing a weak reducing capacity of FGs, the special feature of their exogenous action on cultured yam cells is the increase in the level of superoxide anion radical mainly produced by NADPH oxidase.     

Superoxide Radical Generation by Amadori Compounds

Several D-sugars were incubated with L-lysine or with L-arginine for 10 days. The resulting compounds are able to reduce nitrobluetetrazolium (NBT). This is prevented by superoxide dismutase (SOD), indicating that the superoxide radical is generated by the resulting Amadori compounds.

The formation of superoxide radical in vivo, as a result of nonenzymatic glycosylation of proteins, may be considered to be a contributory factor to the appearance of chronic complications of diabetes.     

Superoxide Radical Generation by Amadori Compounds

Several D-sugars were incubated with L-lysine or with L-arginine for 10 days. The resulting compounds are able to reduce nitrobluetetrazolium (NBT). This is prevented by superoxide dismutase (SOD), indicating that the superoxide radical is generated by the resulting Amadori compounds.

The formation of superoxide radical in vivo, as a result of nonenzymatic glycosylation of proteins, may be considered to be a contributory factor to the appearance of chronic complications of diabetes.     

牡丹花水提液对氧自由基的清除作用

牡丹花水提液加入O 2和·OH的产生及检测系统中,能显著降低介导的氮兰四唑(NBT)的光化学还原和·OH作用下的水杨酸羟基化作用.这提示牡丹花水提液具有显著的清除氧自由基的活性,红色牡丹花水提液清除氧自由基的效能高于浅色花.     

Re-evaluation of superoxide scavenging capacity of xanthohumol

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3'-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.     

NOC/oFQ PKC-dependent superoxide generation contributes to hypoxic-ischemic impairment of NMDA cerebrovasodilation

This study determined whether nociceptin/orphanin FQ (NOC/oFQ) generates superoxide anion (O(2)(-)) in a protein kinase C (PKC)-dependent manner and whether such production contributes to hypoxic-ischemic (H-I) impairment of N-methyl-D-aspartate (NMDA)-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was an index of O(2)(-) generation. Under non-H-I conditions, topical NOC/oFQ (10(-10) M, concentration present in cerebrospinal fluid after I or H-I) increased SOD-inhibitable NBT reduction from 1 +/- 1 to 20 +/- 3 pmol/mm(2). PKC inhibitors staurosporine and chelerythrine (10(-7) M) blunted NBT reduction (1 +/- 1 to 7 +/- 2 pmol/mm(2) for chelerythrine), whereas the NOC/oFQ receptor antagonist [F/G]NOC/oFQ (1-13)-NH(2) (10(-6) M) blocked NBT reduction. [F/G]NOC/oFQ(1-13)-NH(2) and staurosporine also blunted the NBT reduction observed after I or H-I. NMDA (10(-8), 10(-6) M)-induced pial artery dilation was reversed to vasoconstriction after H-I. The NOC/oFQ antagonist staurosporine and free radical scavengers partially prevented this impaired dilation (sham: 9 +/- 1 and 16 +/- 1; H-I: -5 and -10 +/- 1; H-I staurosporine pretreated: 3 +/- 1 and 6 +/- 1%). These data show that NOC/oFQ increased O(2)(-) production in a PKC-dependent manner and contributed to this production after insult and that NOC/oFQ contributed to impaired NMDA-induced pial artery dilation after H-I, suggesting, therefore, that PKC-dependent O(2)(-) generation by NOC/oFQ links NOC/oFQ release to impaired NMDA dilation after H-I.     

An assay for superoxide dismutase activity in mammalian tissue homogenates

During the course of measuring superoxide dismutase (SOD) activity in rat breast tissue, interferences in the nitroblue tetrazolium (NBT) and cytochrome c assay systems were noted. These interferences inhibit accurate measurement of SOD activity in breast tissues, necessitating the development of a new NBT-based assay that includes compounds capable of inhibiting tissue specific interferences. The most effective compounds were metal chelators that were also electron transport chain inhibitors. Bathocuproine sulfonate (BCS) was the most effective of these compounds. The inclusion of BCS in the NBT assay system was shown to make the accurate measurement of SOD activity in tissues with interferences possible.     

The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte

Superoxide dismutase was found to partially inhibit both chemiluminescence and nitroblue tetrazolium (NBT) reduction from intact human polymorphonuclear leukocytes. This capacity to reduce NBT was lost when the polymorphonuclear leukocytes were sonicated, but could be regained if exogenous NADPH (or NADH) was added to the system. Superoxide dismutase was found to inhibit this NADPH- and NADH-dependent NBT reduction. A mechanism is proposed that relates superoxide anion generation to the univalent reduction of O₂ by the activated NADPH (and NADH) oxidase. The relationship of superoxide anion production to NBT reduction, singlet molecular oxygen generation, and chemiluminescence is discussed.     

Re-evaluation of superoxide scavenging capacity of xanthohumol

Abstract

The chemopreventive chalcone xanthohumol (Xh) has been reported to decrease xanthine oxidase (XOD) catalysed formation of formazan from nitroblue tetrazolium (NBT) and is discussed as a potent scavenger of superoxide. Re-evaluation of the scavenging capacity indicated that Xh disturbed detection of superoxide with NBT, in case of an insufficient NBT/Xh ratio. Xh lacked superoxide scavenging activity in contrast to the Xh-derivative 3′-hydroxy-Xh with catechol substructure, used as positive control. This was shown by the use of sufficient concentration of NBT and other detectors such as hydroxylamine, XTT, cytochrome c and hydroethidine. HPLC analysis of reaction products in a xanthine/XOD/peroxidase system demonstrated beside enhanced inhibition of NBT-formazan by Xh that NBT even prevented oxidation of Xh. p-coumaric acid or ferulic acid could replace Xh in that system, indicating that superoxide detection using NBT is likely jeopardized by interference of phenoxyl-radicals. Furthermore, this study provides evidence that Xh can moderately generate superoxide via auto-oxidation.     

Superoxide-dependent nitroblue tetrazolium reduction and expression of cytochrome b-245 components by human tonsillar B lymphocytes and B cell lines

EBV-transformed B lymphocyte cell lines can generate superoxide, using an electron transport chain homologous, or even identical, to phagocytic NADPH-oxidase. We searched for normal, not virally transformed, B lymphocytes with analogous properties, using tonsils as the source of B cells. Unseparated tonsillar leukocytes contained cells capable of PMA-triggered superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT+ cells) well in excess of phagocytes (18.9 +/- 6.4% NBT+ cells with 1.3 +/- 0.9% granulocytes and 1.9 +/- 2.3% monocytes/macrophages, n = 8). NBT reduction was also inhibited by diphenylene iodonium, a selective inhibitor of phagocytic NADPH-oxidase. Cross-linking of surface Ig was equally effective as PMA in inducing NBT reduction among tonsillar leukocytes. NBT+ cells co-distributed with B cells on Percoll density gradients and were enriched among purified B cells obtained by SRBC rosetting twice and Sephadex G10 adherence (47.8 +/- 15.2% NBT+ cells among 90.5 +/- 5.5% B cells, 4.8 +/- 5.1% T cells, 1.2 +/- 0.77% monocytes/macrophages, and 0.73 +/- 0.6% granulocytes, n = 10). Further, mAb 7D5, directed against an extracellularly located epitope of the small subunit of cytochrome b-245 of phagocytes, stained the majority of tonsillar B cells (85 +/- 9.2% 7D5+ cells and 91.6 +/- 4.04% B cells, n = 3). Superoxide production, staining with 7D5 antibody, and expression of mRNA for the beta chain of cytochrome b-245 were further analyzed in cell lines. The EBV-BLCL F1 and the Burkitt lymphoma P3HR-1 both carried 7D5-detectable cytochrome b-245 Ag and expressed mRNA for the beta chain of the cytochrome b, both in similar amounts. However, only F1, not P3HR-1, was capable of PMA-triggered superoxide production. These data indicate that also normal nontransformed B lymphocytes possess the capacity to generate superoxide by a system apparently similar to phagocytic NADPH-oxidase, provisionally termed "B cell oxidase." Discrepancies observed in certain B cells and lines between expression of cytochrome b components and stimulus-induced superoxide production may be related to an absence or low level of other oxidase components or of the signal transduction mechanism. Conceivably, production of superoxide and derived reactive oxygen species by B cells may have cytotoxic, immunomodulatory, or mutagenic effects on the B cells themselves or on cells in their immediate vicinity.     

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Tempol catalyzes the formation of H(2)O(2) from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H(2)O(2) by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H(2)O(2). Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 +/- 2%), whereas NBT was ineffective (2 +/- 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K(+) channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 +/- 7%. Tempol, but not NBT, increased H(2)O(2) in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 +/- 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 +/- 4%). The vasodilation to tempol was reduced by 68 +/- 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H(2)O(2) and is largely independent of the endothelium and potassium channels.     

Identification of superoxide dismutase isoenzymes in tobacco pollen

We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetra-zolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).     

Effects of Arachidonic Acid on Glutamate and γ-Aminobutyric Acid Uptake in Primary Cultures of Rat Cerebral Cortical Astrocytes and Neurons

The effects of arachidonic acid on glutamate and gamma-aminobutyric acid (GABA) uptake were studied in primary cultures of astrocytes and neurons prepared from rat cerebral cortex. The uptake rates of glutamate and GABA in astrocytic cultures were 10.4 nmol/mg protein/min and 0.125 nmol/mg protein/min, respectively. The uptake rates of glutamate and GABA in neuronal cultures were 3.37 nmol/mg protein/min and 1.53 nmol/mg protein/min. Arachidonic acid inhibited glutamate uptake in both astrocytes and neurons. The inhibitory effect was observed within 10 min of incubation with arachidonic acid and reached approximately 80% within 120 min in both types of culture. The arachidonic acid effect was not only time-dependent, but also dose-related. Arachidonic acid, at concentrations of 0.015 and 0.03 mumol/mg protein, significantly inhibited glutamate uptake in neurons, whereas 20 times higher concentrations were required for astrocytes. The effects of arachidonic acid were not as deleterious on GABA uptake as on glutamate uptake in both astrocytes and neurons. In astrocytes, GABA uptake was not affected by any of the doses of arachidonic acid studied (0.015-0.6 mumol/mg protein). In neuronal cultures, GABA uptake was inhibited, but not to the same degree observed with glutamate uptake. Lower doses of arachidonic acid (0.03 and 0.015 mumol/mg protein) did not affect neuronal GABA uptake. Other polyunsaturated fatty acids, such as docosahexaenoic acid, affected amino acid uptake in a manner similar to arachidonic acid in both astrocytes and neurons. However, saturated fatty acids, such as palmitic acid, exerted no such effect. The significance of the arachidonic acid-induced inhibition of neurotransmitter uptake in cultured brain cells in various pathological states is discussed.     

Identification of superoxide dismutase isoenzymes in tobacco pollen

We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetrazolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).     

Superoxide levels and function of cerebral blood vessels after inhibition of CuZn-SOD

The goal of this study was to examine the role of endogenous copper/zinc (CuZn)-superoxide dismutase (SOD) on superoxide levels and on responses of cerebral blood vessels to stimuli that are mediated by nitric oxide (acetylcholine) and cyclooxygenase-dependent mechanisms (bradykinin and arachidonic acid). Levels of superoxide in the rabbit basilar artery were measured using lucigenin-enhanced chemiluminescence (5 microM lucigenin). Diethyldithiocarbamate (DDC; 10 mM), an inhibitor of CuZn-SOD, increased superoxide levels by approximately 2.4-fold (P < 0.05) from a baseline value of 1.0 +/- 0.2 relative light units x min(-1) x mm(-2) (means +/- SE). The diameter of cerebral arterioles (baseline diameter, 99 +/- 3 microm) was also measured using a closed cranial window in anesthetized rabbits. Topical application of DDC attenuated responses to acetylcholine, bradykinin, and arachidonate, but not nitroprusside. For example, 10 microM arachidonic acid dilated cerebral arterioles by 40 +/- 5 and 2 +/- 2 microm under control conditions and after DDC, respectively (P < 0.05). These inhibitory effects of DDC were reversed by the superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (10 mM). Arachidonate increased superoxide levels in the basilar artery moderately under normal conditions and this increase was greatly augmented in the presence of DDC. These findings suggest that endogenous CuZn-SOD limits superoxide levels under basal conditions and has a marked influence on increases in superoxide in vessels exposed to arachidonic acid. The results also suggest that nitric oxide- and cyclooxygenase-mediated responses in the cerebral microcirculation are dependent on normal activity of CuZn-SOD.     

Effects of antioxidants on surfactant peroxidation by stimulated human polymorphonuclear leukocytes

Production of oxygen radicals by stimulated phagocytes followed by surfactant lipid peroxidation (LPO) and loss of surfactant function have all been implicated in the pathogenesis of acute lung injury. We studied the interactions between natural lung surfactant (Curosurf) and neutrophils in vitro , and compared various antioxidants; (superoxide dismutase (SOD), vitamin E, vitamin C, ebselen and melatonin), or combinations of them in duplicate and triplicate regarding their ability to decrease superoxide production and the peroxidation level of surfactant caused by activated phagocytes. The superoxide production of neutrophils activated by Candida albicans was measured with the nitroblue tetrazolium (NBT) test. The subsequent LPO was estimated as the content of malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE). We found that lung surfactant decreased the superoxide production by activated neutrophils (29.7%) and that Curosurf was peroxidized with elevated MDA/4-HNE values. With supplements of antioxidants (except vitamin C), superoxide radical production and the surfactant LPO level fell in a dose-dependent manner. The protective effect of the antioxidants differed in each test. SOD had a slight effect in both tests. The findings with vitamin E, melatonin and ebselen were similar. The best combination was that of a natural and a synthetic antioxidant (melatonin-ebselen) with a 60% decrease in comparison to the corresponding control. These findings suggest that antioxidants, particularly in combination, prevent LPO of lung surfactant.