Effects of Diethyldithiocarbamate on Activating Mechanisms of Neutrophils

The effects of diethyldithiocarbamate on superoxide release by rat neurophils were investigated in a chemiluminescence study. Diethyldithiocarbamate augmented lucigenin-dependent chemiluminescence in a concentration-dependent manner and inhibited luminol-dependent chemiluminescence at concentrations of 0.1-1 μM. In contrast, after the addition of 0.1 mM diethyldithiocarbamate, the chemiluminescence was markedly enhanced. Diethyldithiocarbamate inhibited both the myeloperoxidase activity of neutrophils and the chemiluminescence generated in a cell-free horseradish peroxidase/H₂O₂ and H₂O₂/HOCl system. The increase in lucigenin-dependent chemiluminescence brought about by diethyldithiocarbamate was inhibited by H-7, ML-7, W-7, EGTA and pertussis toxin. These results suggest that diethyldithiocarbamate may stimulate O₂^? production by a guanosine 5′-triphosphate protein-mediated and Ca²⁺–dependent process and that the increase in O₂^? release by neutrophils may be dependent not only on the direct stimulation of the signal transduction pathway but also on the increase in O₂^? by reducing the effect of the hydroperoxide (H₂O₂)-myeloperoxidase system.     

Zinc hydroxide induced respiratory burst in rat neutrophils

The effects of zinc hydroxide on the respiratory burst and phagocytosis by rat neutrophils were examined. Zinc hydroxide induced an increase in oxygen consumption and O₂⁻ production. Electronmicroscopy showed that neutrophils engulfed zinc hydroxide particles by phagocytosis. Pertussis toxin (0.25, 0.5, 1.0 μ/ml) and EGTA (1, 2, 5 mM) inhibited zinc hydroxide-induced O₂⁻ production in a dose-dependent manner. The inhibitors of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide inhibited zinc hydroxide-induced O₂⁻ production with IC₅₀ values ranging between 10 μM and 25 μM. The inhibitory study using an inhibitor of myosin light chain kinase, 1-(5-iodonapthalene-1-sulfonyl)-1H- hexahydro-1,4-diazepine, showed IC₅₀ values ranging from 5 μM to 10 μM. These findings indicate that zinc hydroxide induces respiratory burst and phagocytosis by rat neutrophils.     

Hydrogen sulfide protects endothelial nitric oxide function under conditions of acute oxidative stress in vitro.

The aim of this study was to examine the ability of H₂S, released from NaHS to protect vascular endothelial function under conditions of acute oxidative stress by scavenging superoxide anions (O₂ ^?) and suppressing vascular superoxide anion production. O₂ ^? was generated in Krebs' solution by reacting hypoxanthine with xanthine oxidase (Hx-XO) or with the O₂ ^? generator pyrogallol to model acute oxidative stress in vitro. O₂ ^? generation was measured by lucigenin-enhanced chemiluminescence. Functional responses in mouse aortic rings were assessed using a small vessel myograph. NaHS scavenged O₂ ^? in a concentration-dependent manner. Isolated aortic rings exposed to either Hx-XO or pyrogallol displayed significantly attenuated maximum vasorelaxation responses to the endothelium-dependent vasodilator acetylcholine, and significantly reduced NO bioavailability, which was completely reversed if vessels were pre-incubated with NaHS (100 μM). NADPH-stimulated aortic O₂ ^? production was significantly attenuated by the NADPH oxidase inhibitor diphenyl iodonium. Prior treatment of vessels with NaHS (100 nM–100 μM; 30 min) inhibited NADPH-stimulated aortic O₂ ^? production in a concentration-dependent manner. This effect persisted when NaHS was washed out prior to measuring NADPH-stimulated O₂ ^? production. These data show for the first time that NaHS directly scavenges O₂ ^? and suppresses vascular NADPH oxidase-derived O₂ ^? production in vitro. Furthermore, these properties protect endothelial function and NO bioavailability in an in vitro model of acute oxidative stress. These results suggest that H₂S can elicit vasoprotection by both scavenging O₂ ^? and by reducing vascular NADPH oxidase-derived O₂ ^? production.     

Impact of zinc uptake regulator Zur on the susceptibility and oxidative stress response of Acinetobacter baumannii to antibiotics

Zinc is a highly coveted redox-inactive micronutrient required for the growth and virulence of Acinetobacter baumannii. In this study, the role of the zinc uptake regulator Zur in the susceptibility and oxidative stress response of A. baumannii to antibiotics was evaluated. Inactivation of zur increased the susceptibility of A. baumannii AB5075 to colistin, gentamicin, rifampicin and tigecycline. Furthermore, activities of superoxide dismutase and catalase decreased significantly in the Δzur mutant compared with the parental strain. Colistin, gentamicin, rifampicin and tigecycline raised the superoxide anion radical (·O₂^?) and hydrogen peroxide (H₂O₂) contents of the Δzur mutant compared with the parental strain. In addition, the antibiotics lowered glutathione and concomitantly raised glutathione disulphide levels in the Δzur mutant. All of the antibiotics, except tigecycline, significantly raised the NAD⁺/NADH and ADP/ATP ratios in A. baumannii. We conclude that decreased capability of the Δzur mutant to detoxify reactive oxygen species increased its susceptibility to antibiotics.     

Reactivity of various phenothiazine derivatives with oxygen and oxygen radicals

7,8-Dihydroxychlorpromazine, 7,8-dihydroxyprochlorperazine and 7,8-dihydroxyperphenazine all reacted with O₂ to make hydrogen peroxide (H₂O₂). The rate of reaction between the ortho-dihydroxyphenothiazines and O₂ was increased by superoxide dismutase, an enzyme which catalyzes the dismutation of the superoxide radical (O₂^?) to H₂O₂ and O₂. This indicated the formation of O₂^? during the autoxidation of the ortho-dihydroxyphenothiazines. Several phenothiazines lacking the ortho-dihydroxy groups did not consume O₂ at a detectable rate (did not generate H₂O₂). All ortho-dihydroxyphenothiazines tested also reacted with O₂ in the presence of methional to form ethylene. Ethylene formation was inhibited by catalase and hydroxyl radical (·OH) trapping agents, such as sodium benzoate; this indicated ·OH formation from the ortho-dihydroxyphenothiazines. In addition, several nonhydroxylated phenothiazines and monohydroxylated phenothiazines inhibited ethylene generation from 6-aminodopamine, which also generates ·OH. This inhibition was probably mediated by a reaction between the phenothiazine and ·OH. All of the above reactions (generation of H₂O₂, O₂^? or ·OH or reaction with ·OH) may be responsible for some of the beneficial and/or adverse effects of administered phenothiazines.     

The cGMP Modulator,LY83583 Alters Oxygen Metabolites Differently in Cultured Endothelial Cells and Isolated Neutrophilic Granulocytes

Abstract: The present study was designed to assess the effect of LY83583 on H₂O₂/O₂^- production from endothelial cells and neturophils, as determined by chemiluminiscence generation in vitro. We found that LY83583 increased H₂O₂/O₂^-production from endothelial cells, but inhibited the H₂O₂/O₂^- production from phorbol myristate acetate-stimulated neutrophils. Furthermore, LY83583 consumed NADPH under certain conditions. Since neutrophils generate superoxide anion radicals via an NADPH-oxidase, we suggest that the reduction of chemiluminiscence, seen after addition of LY83583 to phorbol myristate acetate-stimulated neutrophils, is due to increased consumption of NADPH. In endothelial cells, NADPH is required as a co-factor in the generation of nitric oxide, which may interact with superoxide anion. A consumption in NAPDH would therefore be expected to decrease the production of nitric oxide and increase H₂O₂/O₂^- generation. The consumption of NADPH in endothelial cells could also cause reduced scavenger functions of the glutathion system, resulting in a further increase in H₂O₂ release.     

H2O2 production,modification of the glutathione status and methemoglobin formation in red blood cells exposed to diethyldithiocarbamate in vitro

Human red blood cells treated with the CuZn superoxide dismutase inhibitor diethyldithio-carbamate (DDC) undergo metabolic modifications in addition to the superoxide dismutase inhibition: oxidation of the reduced glutathione (GSH) to oxidized glutathione (GSSG), methemoglobin formation, and increased hexose monophosphate shunt activity were observed. The magnitudes of these changes are dependent on the DDC concentration. Under nitrogen, only superoxide dismutase inhibition occurs. After removal of the GSH with N-ethylmaleimide, production of H₂O₂ can be detected by measuring the red cell catalase inhibition in the presence of 3-amino-1,2,4-triazole. H₂O₂ production is not altered by conversion of oxyhemoglobin to methemoglobin by sodium nitrite prior to incubation. GSH oxidation and methemoglobin formation are stopped when DDC is eliminated from the incubation medium after completion of the superoxide dismutase inhibition. These data indicate that methemoglobin formation and modification of the GSH status in red cells treated by DDC are not a direct consequence of the CuZn Superoxide dismutase inhibition but are due rather to a DDC-dependent production of H₂O₂.     

Evidence for ligand-specific conformations of the histamine H2-receptor in human eosinophils and neutrophils

The histamine H₂-receptor (H₂R) couples to G_S-proteins and induces adenylyl cyclase-mediated cAMP accumulation. In human neutrophils and eosinophils, the H₂R reduces chemotactic peptide-stimulated superoxide anion (O₂^?) formation. However, pharmacological characterization of the H₂R in these cells is far from being complete. The aim of this study was to provide a comprehensive profiling of the H₂R in neutrophils and eosinophils. Histamine inhibited O₂^? formation in human neutrophils more effectively than in eosinophils. H₂R agonists mimicked the effects of histamine and H₂R antagonists blocked the effects of histamine. We noticed multiple discrepancies in the potencies and efficacies of H₂R agonists with respect to cAMP accumulation and inhibition of O₂^? formation in both cell types. There were also differences in the antagonist profiles between cAMP accumulation and inhibition of O₂^? formation in neutrophils. Moreover, the pharmacological profile of the recombinant H₂R did not match the H₂R profile in native cells. The H₂R sequence identified in human neutrophils corresponds to the published H₂R sequence, excluding the exclusive expression of a new H₂R isoform as explanation for the differences. Very likely, the differences between ligands are explained by the existence of ligand-specific receptor conformations with unique affinities, potencies and efficacies. Thus, our data provide evidence for the notion that the concept of ligand-specific receptor conformations can be extended from recombinant systems to native cells.     

Manganese promotes increased formation of hydrogen peroxide by activated human macrophages and neutrophils in vitro

Although pro-inflammatory mechanisms have been implicated in the pathogenesis of manganese (Mn²⁺)-related neurological and respiratory disorders, relatively little is known about the potential of this metal to interact pro-oxidatively with human phagocytes. The primary objective of the current study was to investigate the effects of Mn²⁺ as MnCl₂ (0.5–100 µM) on the generation of the reactive oxygen species (ROS), superoxide, hydrogen peroxide (H₂O₂), and hypohalous acids by isolated human blood neutrophils and monocyte-derived macrophages following activation of these cells with the chemotactic tripeptide, FMLP (1 µM), or the phorbol ester, PMA (25?ng/mL). Generation of ROS was measured using the combination of oxygen consumption, lucigenin/luminol-enhanced chemiluminescence, spectrofluorimetric detection of oxidation of 2,7-dichlorodihydrofluorescein, radiometric assessment of myeloperoxidase (MPO)-mediated protein iodination, release of MPO by ELISA, and spectrophotometric measurement of nitrite formation. Treatment of activated neutrophils with either FMLP or PMA resulted in significantly decreased reactivity of superoxide in the setting of increased formation of H₂O₂ and MPO-mediated iodination, with no detectable effects on either oxygen consumption or MPO release. Similar effects of the metal with respect to superoxide reactivity and H₂O₂ formation were observed with activated macrophages, while generation of NO was unaffected. Taken together with the findings of experiments using cell-free ROS-generating systems, these observations are compatible with a mechanism whereby Mn²⁺, by acting as a superoxide dismutase mimetic, increases the formation of H₂O₂ by activated phagocytes. If operative in vivo, this mechanism may contribute to the toxicity of Mn²⁺.     

Paraquat-induced chemiluminescence of microsomal fractions

Paraquat-induced chemiluminescence of microsomal fractions requires electron donors (from DADPH through NADPH-cytochrome P-450 reductase) and oxygen; the latter is a deciding factor to observe this chemiluminescence, in accordance with the known requirements for the redox cycling of the herbicide with formation of O₂^? and derived oxygen species. Paraquat-dependent chemiluminescence was not associated with accumulation of malondialdehyde (thiobarbituric acid reactive material), and addition of the bipyridylium compound to an ongoing lipid peroxidation reaction produced a different time course for this chemiluminescence and inhibited malondialdehyde formation.Accumulation of the paraquat radical under anaerobiosis was not accompanied by chemiluminescence, but subsequent oxygenation of the sample produced a burst of chemiluminescence, the intensity of which was related to the time of incubation under anaerobiosis and paraquat radical accumulated.Superoxide dismutase inhibits this chemiluminescence, thus suggesting the involvement of O₂^?; catalase was without effect.Selective light emission at 634 and 703 nm with a minimum intensity at 668 nm points to the involvement of singlet molecular oxygen in the paraquat-elicited photoemission.At variance with the microsomal fraction, paraquat-infused perfused liver did not show any noticeable chemiluminescence, probably due to the presence of cellular defense mechanisms.     

Lindane-Induced Oxidative Stress. I. Time Course of Changes in Hepatic Microsomal Parameters,Antioxidant Enzymes,Lipid Peroxidative Indices and Morphological Characteristics

1. Lindane (60 mg/kg) administered orally to rats increased liver cytochrome P-450 content and superoxide radical (O^?₂) generation 24 h after treatment, while formation of thiobarbituric acid reactants and NADPH/ADP-supported microsomal chemiluminescence were significantly increased 4 h after treatment.

2. Hepatic superoxide dismutase (SOD) and catalase decreased 6h after lindane treatment and SOD/O^?₂ ratio progressively decreased during 4 to 24 h after lindane treatment.

3. Morphological evidence of hepatic cell injury after lindane treatment was seen at all times studied, and appeared to increase with time.

4. Lindane administration results in time-dependent oxidative stress in liver which involves an early component (4–6 h) related to the reductive metabolism of lindane, and a late component (24 h) associated with the induction of cytochrome P-450; the biochemical changes correlated with the observed morphological lesions.     

An aqueous pomegranate peel extract inhibits neutrophil myeloperoxidase in vitro and attenuates lung inflammation in mice

Punica granatum peel aqueous extract (PGE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. In this study we investigated the effect of PGE on human neutrophil reactive oxygen species (ROS) production in vitro and on LPS-induced lung inflammation in vivo in mice. Neutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H₂O₂ was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethyl benzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. PGE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLF)- or phorbol myristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. PGE had no effect on superoxide anion generation, suggesting that it does not directly inhibit NADPH oxidase activity or activation pathways, or scavenge superoxide anions. PGE did not scavenge H₂O₂ but directly inhibited myeloperoxidase activity in vitro. In vivo studies showed that PGE also attenuated LPS-induced lung inflammation in mice. So this study reveals that PGE inhibits neutrophil MPO activity and attenuates LPS-induced lung inflammation in mice. Inhibition of MPO activity by PGE could explain its anti-inflammatory action.     

Formation of active oxygen species by dithranol, III. Dithranol, active oxygen species and lipid peroxidation in vivo

At pH 7.8 the dithranol anion 1a is oxidized in the dark to yield the bianthrone 3 besides dithranol brown, whilst O₂ is reduced to O₂^·? and H₂O₂. O₂^·? was detected by the reduction of nitroblue-tetrazolium (NBT, 4 ). The formation of the formazan 5 was inhibited by superoxide dismutase (SOD). Exclusion of O₂ and addition of SOD reveal a direct electron transfer from 1a to 4 and to cytochrome c which was not affected in the presence of SOD. — In vivo experiments indicate a low rate of lipid peroxidation which was determined on the basis of ethane exhalation.     

Nicotine- and tar-free cigarette smoke induces cell damage through reactive oxygen species newly generated by PKC-dependent activation of NADPH oxidase

We examined cytotoxic effects of nicotine/tar-free cigarette smoke extract (CSE) on C6 glioma cells. The CSE induced plasma membrane damage (determined by lactate dehydrogenase leakage and propidium iodide uptake) and cell apoptosis {determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] reduction activity and DNA fragmentation}. The cytotoxic activity decayed with a half-life of approximately 2 h at 37°C, and it was abolished by N-acetyl-l-cysteine and reduced glutathione. The membrane damage was prevented by catalase and edaravone (a scavenger of ^?OH) but not by superoxide dismutase, indicating involvement of ^?OH. In contrast, the CSE-induced cell apoptosis was resistant to edaravone and induced by authentic H₂O₂ or O₂^? generated by the xanthine/xanthine oxidase system, indicating involvement of H₂O₂ or O₂^? in cell apoptosis. Diphenyleneiodonium [NADPH oxidase (NOX) inhibitor] and bisindolylmaleimide I [BIS I, protein kinase C (PKC) inhibitor] abolished membrane damage, whereas they partially inhibited apoptosis. These results demonstrate that 1) a stable component(s) in the CSE activates PKC, which stimulates NOX to generate reactive oxygen species (ROS), causing membrane damage and apoptosis; 2) different ROS are responsible for membrane damage and apoptosis; and 3) part of the apoptosis is caused by oxidants independently of PKC and NOX.[Supplementary methods and Figure: available only at http://dx.doi.org/10.1254/jphs.11166FP]     

Effects of superoxide dismutase and catalase on catalysis of 6-hydroxydopamine and 6-aminodopamine autoxidation by iron and ascorbate

The effects of superoxide dismutase and catalase on autoxidation of 6-hydroxydopamine and 6-aminodopamine in several chemical environments were studied. Inhibition by superoxide dismutase of autoxidation of 6-hydroxydopamine to its p-quinone required the presence of metal chelators, EDTA or diethylenetriamine pentaacetic acid (DETAPAC). A “lag” period in 6-hydroxydopamine autoxidation in the presence of superoxide dismutase couid be prevented by carrying out autoxidation in a mixture of 6-hydroxydopamine and its p-quinone, conditions in which adequate levels of the semiquinone are available for reaction with O₂. Catalase potentiated the inhibitory effect of superoxide dismutase in the presence of EDTA but had no effect in the presence of DETAPAC. Superoxide dismutase and catalase had no effect on the initial rate of 6-aminodopamine autoxidation to the p-quinone imine or on later intracyclization and polymer formation. Iron chelated by EDTA functioned as a catalyst in 6-hydroxydopamine autoxidation, making the reaction independent of superoxide as shown by the lack of effect of superoxide dismutase. the presence of iron-EDTA resulted in bleaching of the p-quinone product and the consumption of the H₂O₂ formed during autoxidation. Catalase had no effect on the rate of 6-hydroxydopamine autoxidation but completely prevented bleaching of the p-quinone, probably by preventing formation of hydroxyl radical by a Fenton reaction between iron-EDTA and H₂0₂. Ethanol, which scavenges hydroxyl radical but not superoxide of H₂O₂, similarly prevented bleaching of the p-quinone with no other effects on autoxidation. Iron-EDTA also catalyzed 6-aminodopamine autoxidation with associated consumption of H₂O₂. Superoxide dismutase, catalase and ethanol had no effect on 6-aminodopamine autoxidation in the presence or absence of iron-EDTA, showing the independence of the kinetics of 6-aminodopamine autoxidation and polymerization from its products, superoxide, H₂0₂ and hydroxyl radical. Chelation by DETAPAC prevented the effects of iron on 6-hydroxydopamine and 6-aminodopamine autoxidation. Autoxidation of 6-hydroxydopamine in the presence of ascorbate exhibited a lag phase followed by a linear phase of autoxidation. Superoxide dismutase, catalase and ethanol had no effect on 6-hydroxydopamine or 6-aminodopamine autoxidation in the presence of ascorbate. Autoxidation of 6-hydroxydopamine or 6-aminodopamine in the combined presence of iron-EDTA and ascorbate showed the full effects of both additions, except that 6-hydroxydopamine autoxidation exhibited no lag phase and the iron-catalyzed autoxidation of ascorbate occurred simultaneously with the 6-hydroxydopamine or 6-aminodopamine reactions.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H₂O₂) and leukotriene B₄ (LTB₄) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated.
2. At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H₂O₂ in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml⁻¹)- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 μM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB₄ in the supernatants of OZ- and fMLP-stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ-stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC).
3. NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H₂O₂ when incubated with exogenous H₂O₂ concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB₄ when incubated with exogenous LTB₄.
4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H₂O₂ had no significant effect on LTB₄ production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H₂O₂ to H₂O and O₂, caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB₄ measured in the supernatants of OZ- and fMLP-stimulated neutrophils.
5. H₂O₂ (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB₄ (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml⁻¹).
6. In conclusion, the anti-oxidant drug, NAC, increases LTB₄ production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H₂O₂. LTB₄ undergoes a H₂O₂-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB₄, suggesting that NAC may increase LTB₄ production by blocking the H₂O₂-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.

     

Inhibitory effect of rosiglitazone on the acid-induced intracellular generation of hydrogen peroxide in cultured feline esophageal epithelial cells

Peroxisome proliferator-activated receptor-gamma (PPARγ) agonists have been reported to enhance antioxidant defenses by increasing levels of catalase and copper–zinc superoxide dismutase (Cu/Zn SOD) in oligodendrocyte progenitor cells. In this study, we investigated the effects of the PPARγ agonist, rosiglitazone, on hydrogen peroxide (H₂O₂) generation by acidified medium at pH 5.5 (AM5.5), which is in the pH range of duodenogastric refluxates, in primary cultured feline esophageal epithelial cells (EEC). Successful isolation of EEC was identified by immunocytochemistry. AM5.5- and rosiglitazone-induced cell viabilities were determined using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assays. The NAD(P)H oxidase activity was measured, and expression of catalase or SOD protein by AM5.5 in the absence and presence of rosiglitazone was assessed using western blotting analysis. PPARγ protein and mRNA were constitutively expressed in EEC. In the incubation with rosiglitazone alone, cell viability was shown more than 90% at 0–10 μM for 72 h. After exposure to AM5.5 for 8 h, intracellular H₂O₂ was significantly generated. Treatment with rosiglitazone prior to and during exposure to AM5.5 inhibited the H₂O₂ generation whereas the specific PPARγ antagonist GW9662 offsets the inhibitory action of rosiglitazone. H₂O₂ generation was also prevented by a nonspecific ROS scavenger N-acetylcysteine or an inhibitor of NADPH oxidase diphenyleneiodonium. The enhanced AM5.5-induced NAD(P)H oxidase activity was not suppressed by rosiglitazone. Instead, the pretreatment of rosiglitazone enhanced the protein expression of catalase, Cu/Zn SOD, and Mn SOD, which are endogenous antioxidative enzymes. These findings indicate that rosiglitazone inhibits AM5.5-induced intracellular H₂O₂ production, which occurs via NAD(P)H oxidase activation, by using a PPARγ-dependent pathway, and that the underlying mechanism involves an increase in the expression of catalase and SOD proteins.     

Icaritin inhibits oxidative stress in murine astrocytes by binding to Orai1 to block store-operated calcium channel

Previous study has shown that icaritin (ICT) has meaningful protective effect on cerebral ischemic stroke, and this study aimed to investigate its mechanism from the aspect of protecting astrocytes from oxidative stress. Murine primary astrocytes were pretreated by ICT and exposed to H₂O₂ to induce oxidative stress. The results indicated that ICT inhibited H₂O₂-induced astrocytes apoptosis, decreased Bax and cleaved caspase-3, and increased Bcl-2. In addition, ICT inhibited H₂O₂-induced oxidative stress, increased mitochondrial membrane potential (ΔΨ_m), and maintained mitochondrial morphology. ICT decreased the synthesis of malondialdehyde and increased the activity of glutathione peroxidase, catalase, and superoxide dismutase. Moreover, ICT suppressed the transient and resting intracellular Ca²⁺ overload. Further investigation revealed that ICT could target the combination with Orai1 to block store-operated calcium channel induced by H₂O₂. However, ICT did not enhance the protective effect of RO2959, a selective blocker of Orai1. These results indicate that ICT can play a neuroprotective role against oxidative stress injury by binding to Orai1 to block SOCC.     

The histamine receptor 2 may be a pacemaker for neutrophil activation

There is considerable debate regarding the relative contributions of the histamine (HA) H₁ and H₂ receptors in neutrophil activation. The effects of HA-ligands on human neutrophils were assayed by luminol-dependent chemiluminescence (CL) and analyzed by a two-tailed paired Student's t-test. Histamine alone did not significantly alter the CL generation, but the H₁-antagonist (phenylenediamine, 10^-6 M) inhibited histamine- or FMLP-mediated CL significantly (p < 0.01). An H₂-antagonist, cimetidine (10^-6 M), partially inhibited the FMLP-mediated CL generation with slight stimulation of histamine-mediated CL. H₁-antagonist and H₂-agonist, dB-c-AMP, together produced the same degree of inhibition of FMLP challenge seen with H₁-antagonist. A significant sensitivity of HA to EGTA-, H₁-antagonist- or quinacrine-treatment (p < 0.01) indicated the H₁-receptor mediated activation of phospholipase C (PLC) may require extracellular Ca²⁺. The histamine-mediated CL was also sensitive to inhibitors of protein kinase C (PKC) and tyrosine kinase (TK), indicating a Ca²⁺-mediated phosphorylation. Further, HA-mediated CL generation was partially inhibited by a G-protein blocker, pertussis toxin (PTX). We conclude that histamine receptor 1-mediated neutrophil activation via the PLC pathway may be modulated via H₂-site cascade in a G-protein dependent manner.     

EVALUATION OF UV-INDUCED SUPEROXIDE RADICAL GENERATION POTENTIAL OF SOME COMMON ANTIBIOTICS

Photosensitization reaction of drugs leading to the formation of reactive oxygen species (ROS) cause tissue injury causing damage to various cellular macromolecules. The aim of this study was to determine the superoxide anion (O₂^?) generation potential of commonly used antibiotics so that due precaution could be exercised to minimize their photosensitizing action and oxidative stress potential. The selected antibiotics were examined for their ability to produce (O₂^?) under sunlight and artificial UVA (320–400 nm) and UVB (290–320 nm). Lincomycin, cephalothin and erythromycin generated significant amount of O₂^?. under various irradiation conditions. Superoxide dismutase (SOD), an enzymatic quencher of O₂^? inhibited O₂^? production in all drugs tested. The results suggest that due precaution are necessary to avoid ultraviolet radiation (UVR) after the intake of photoreactive drugs, especially in tropical and sub tropical countries.