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⁺.     

The modulation by arylamines of the in vitro formation of superoxide anion radicals and hydrogen peroxide by rat liver microsomes

A concentration-dependent increase in the generation of the superoxide anion radical (O-2), was observed during the incubation of benzidine (0.1-5 mM), but not of the structurally related compounds 4-aminobiphenyl, 2-aminobiphenyl or 4-fluoro-4'-aminobiphenyl, with NADPH-supplemented rat liver microsomes. This increase was partially inhibited by carbon monoxide and catalase but unaffected by 4-aminobiphenyl, a substrate of the cytochrome P-450 system. Microsomes from benzo(a)pyrene-treated, but not microsomes from phenobarbitone-pretreated rats, were responsible for a larger benzidine-dependent effect compared to microsomes from control animals. In contrast to the above observations, benzidine decreased the formation of hydrogen peroxide by NADPH-supplemented microsomal preparations from untreated rats. These results indicate that free radicals of oxygen are generated during the metabolism of some arylamines.     

Release of intermediate reactive hydrogen peroxide by macrophage cells activated by natural products

By determining the hydrogen peroxide (H2O2) released in cultures of peritoneal macrophage cells from Swiss mice, we evaluated the action of 27 vegetable compounds (pristimerin, tingenone, jatrophone, palustric acid, lupeol, cladrastin, ocoteine, boldine, tomatine, yohimbine, reserpine, escopoletin, esculine, plumericin, diosgenin, deoxyschizandrin, p-arbutin, mangiferin, and others) using a 2 mg/ml solution of each compound (100 microg/well). Macrophages are cells responsible for the development of the immunological response reaction, liberating more than one hundred compounds into the extracellular environment. Among these are the various cytokines and the intermediate compounds of nitrogen (NO) and oxygen (H2O2). This coordinated sequence of biochemical reactions is known as the "oxidative burst." When we compared the results with those obtained with zymosan (an important stimulator of H2O2) we observed that the compounds showing the highest activity were substances 2 (tingenone), 16 (reserpine) and 20. Other substances such as compounds 1, 4, 5, 6, 8, 12, 13, 14, 15, 17, 19, 23, 24, 26, and 27 also showed a certain activity, but with less intensity than the aforementioned ones. Compounds 3, 7, 9, 10, 11, 18, 21, 22 and 25 presented no activity. These results suggest that natural products (mainly tingenone and reserpine and others) with different chemical structures are strong immunological modulators. However, further tests are needed to determine the 'oxidative burst' in future studies.     

Manganese dioxide induces alveolar macrophage chemotaxis for neutrophils in vitro

Workers exposed to airborne manganese dioxide (MnO2) may develop pneumonia resistant to chemotherapy. Previous animal experiments have demonstrated that exposure to airborne MnO2 induces an invasion of neutrophils into the airways 4-12 h after exposure. Experiments were undertaken to further study the role of alveolar macrophage (AM) in the development of this response. Guinea pig lung lavage fluid was centrifuged and washed, and cell suspension incubated on one side of a leukocyte migration chamber. MnO2 (2.5 mg/ml) was incubated with the cells for different time periods. Peritoneal neutrophils from guinea pigs were isolated and incubated on the other side of the chamber. Observations of the free lung cells demonstrated that phagocytosis of the MnO2 particles commenced immediately and was virtually completed within 30 min. Cells from control animals caused small directional migration across the filter. Cells which had phagocytosed MnO2 particles showed a significantly higher degree of neutrophil migration. The data suggest that pneumonia, in terms of neutrophil invasion into the lungs after MnO2 exposure, is associated with an activation of AM with subsequent release of neutrophil recruiting factor.     

Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H₂O₂ (200 μM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 μM. Furthermore, in the H₂O₂-treated group, agarose gel electrophoresis displayed a “DNA ladder”. Whereas in the 10 μM crocin-pretreated group, cells remained intact and no “DNA ladder” was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 μM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H₂O₂, which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).     

Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H₂O₂ (200 μM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 μM. Furthermore, in the H₂O₂-treated group, agarose gel electrophoresis displayed a “DNA ladder”. Whereas in the 10 μM crocin-pretreated group, cells remained intact and no “DNA ladder” was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 μM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H₂O₂, which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).     

Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H(2)O(2) (200 microM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 microM. Furthermore, in the H(2)O(2)-treated group, agarose gel electrophoresis displayed a "DNA ladder". Whereas in the 10 microM crocin-pretreated group, cells remained intact and no "DNA ladder" was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 muM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H(2)O(2), which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).     

Hydroxylation of salicylate by activated neutrophils

Salicylates are metabolized in vivo to hydroxylated compounds, including 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid (gentisic acid). The present study hypothesized that activated neutrophils represent one pathway for salicylate hydroxylation. Human neutrophils were incubated in medium containing 10 mM salicylate and stimulated with phorbol myristate acetate (PMA) for 1 hr. The cell-free supernatant fractions were analyzed by HPLC. Neutrophils (1 x 10(6) cells) produced 55 +/- 11 ng of gentisic acid. Neutrophils also produced smaller quantities of 2,3-dihydroxybenzoic acid. Antioxidant inhibitor experiments indicated that superoxide dismutase (SOD), heme protein inhibitors, and glutathione blocked gentisic acid formation, whereas catalase, mannitol, and deferoxamine failed to inhibit. Experiments with the reagent hypochlorous acid (HOCl) and the model myeloperoxidase (MPO) enzyme system did not support a role for the MPO pathway in gentisic acid formation. These findings demonstrate that activated neutrophils can hydroxylate salicylate by an unknown pathway. This pathway may contribute to the increased recovery of hydroxylated salicylates in patients with inflammatory disorders.     

Estimation of lipid peroxidation induced by hydrogen peroxide in cultured human lymphocytes

Malondialdehyde (MDA) is used for the estimation of damage by reactive oxygen species. MDA is a major reactive aldehyde resulting from the peroxidation of biological membranes. The most common method used to assess MDA production is the thiobarbituric acid (TBARS) assay. However, the value of this method is curbed by low specificity and has been criticized for its use in human studies. In the present study we have used an alternative method for the estimation of MDA production i.e. reaction of MDA with a chromogenic agent 1-methyl-2-phenylindole at 45°C. The paper describes the method of preparing standards for the estimation of MDA (lipid peroxidation) after the treatment with an oxidative stress inducing agent hydrogen peroxide (H(2)O(2)). In the present study, the treatments of 1, 5, 10, 20, 50, 100, 150 and 200 μM of H(2)O(2) induced significant increase in lipid peroxidation as compared to the untreated ones. The results suggest that the present method can be used to measure the lipid peroxidation in cultured human peripheral blood lymphocytes and is specific for MDA estimation.     

Pseudoginsenoside-F11 ameliorates ischemic neuron injury by regulating the polarization of neutrophils and macrophages in vitro

Pseudoginsenoside-F11 (PF11), an ocotillol-type saponin, has neuroprotective effects on permanent and transient cerebral ischemia in rats by alleviating autophagic/lysosomal defects and repressing calcium overload, respectively. Ischemic stroke triggers peripheral innate immune cells, mainly neutrophils and macrophages, to infiltrate the damaged brain. The polarization of neutrophils and macrophages after cerebral ischemia is essential for post-stroke damage/recovery. However, it remains elusive whether PF11 ameliorates ischemic neuron injury by regulating the polarization of neutrophils and macrophages. The present study demonstrated for the first time that conditioned media from ischemic neurons induced neutrophils and macrophages to polarize into N1 and M1 phenotypes, respectively. Furthermore, PF11 (30, 100 μM) inhibited the induction of N1 neutrophils by conditioned media from oxygen glucose deprivation/re-oxygenation (OGD/R)-induced ischemic neurons and promoted the polarization of neutrophils to N2 phenotypes. In addition, PF11 (100 μM) attenuated the exacerbation of N1 neutrophils and facilitated the protection of N2 neutrophils on OGD/R-induced neuronal damage. Similarly, PF11 (100 μM) inhibited the induction of M1 macrophages by conditioned media from ischemic neurons and facilitated the polarization of macrophages to M2 phenotypes. What's more, PF11 (100 μM) attenuated the aggravation of M1 macrophages and promoted the protection of M2 macrophages on OGD/R-induced primary neuron injury. In summary, the present study indicates that PF11 ameliorates ischemic neuron damage by regulating neutrophils and macrophages polarization, suggesting that neutrophils and macrophages may be promising targets for the treatment of cerebral ischemia.     

Eplerenone promotes alternative activation in human monocyte-derived macrophages

BackgroundIn this study, we have analyzed the response of human monocyte-derived macrophages to mineralocorticoid axis modulators.MethodsHuman monocyte-derived macrophages were incubated with aldosterone alone, eplerenone alone, and the combination of aldosterone and eplerenone. The analyzed variables were nitric oxide and reactive oxygen species production, and the gene and protein expression of inducible nitric oxide synthase, arginase I, and mannose receptor.ResultsWe showed that aldosterone promotes a classic inflammatory response in macrophages, whereas its antagonist, eplerenone, attenuates aldosterone-induced activity.ConclusionEplerenone did not quantitatively weaken the response of macrophages to aldosterone but instead qualitatively changed their behavior.     

白藜芦醇苷体外对过氧化氢导致小鼠肝细胞损伤的保护作用

目的　观察中药虎杖的活性成分白藜芦醇苷对过氧化氢 (H2 O2 )所致肝细胞损伤的影响。方法　用邻苯三酚自氧化法测过氧化物歧化酶 ,用硫代巴比妥酸 (TBA)法测丙二醛 (MDA)含量 ,用改良Hafeman方法测还原谷胱甘肽含量 ,用 5 ,5’ 二巯基 2 ,2’ 二硝基苯甲酸 (DTNB)法测谷胱甘肽过氧化物酶活性。谷丙转氨酶 (ALT)、一氧化氮 (NO)和一氧化氮合酶 (NOS)采用测试药盒测定。结果　白藜芦醇苷系列浓度 (0 0 5 ,0 1,0 5 ,1,2 ,4mmol·L-1)作用肝细胞后 ,能显著降低H2 O2 引起的NO和MDA水平升高 ,抑制NOS活性 ,升高SOD和GSH px活性 ,减少GSH消耗 ,明显减少了H2 O2 导致的肝细胞悬液中ALT浓度增高。结论　白藜芦醇苷在一定浓度范围内对H2 O2 所致的小鼠肝细胞氧化损伤具有保护作用。     

Immunotherapeutic effects of mangiferin mediated by the inhibition of oxidative stress to activated lymphocytes, neutrophils and macrophages

The effect of mangiferin, a naturally occurring xanthone glucoside on cyclophosphamide-induced immunotoxicity and its mode of action in the immune system were investigated. To induce immunotoxicity, adult male Wistar rats were injected weekly with cyclophosphamide intraperitoneally at 100 mg/kg bodyweight. Mangiferin was injected intraperitoneally at 10 and 20 mg/kg daily for 14 days. Levamisole (3 mg/kg, i.p., daily for 14 days), a known immunostimulant that acts in immunosuppressive conditions was used as a standard drug. The effect of mangiferin on the primary immune response to ovalbumin (200 microg/rat, s.c.) was assessed at weekly intervals by measuring the serum ovalbumin-specific IgM levels. The organ weights and cellularity of spleen, thymus and bone marrow, haematology, T and B cell-dependent mitogen stimulation of splenocytes were assessed for the cellular response. Oxidative changes in lymphocytes, neutrophils and macrophages were measured at the end of the study. As well, the in vitro effect of mangiferin on cytotoxicity caused by H2O2 in primary lymphocytes was studied. The decrease in the lymphoid organ weights, cellular responses and antigen-specific IgM levels by cyclophosphamide treatment were significantly increased by repeated intraperitoneal administration of mangiferin. The enhanced lipid peroxidation and decreased catalase and superoxide dismutase activities found in lymphocytes, polymorphonuclear cells (PMN) and macrophages from cyclophosphamide treated rats were significantly ameliorated in mangiferin treated groups. The tissue injury caused by cyclophosphamide treatment was significantly suppressed by mangiferin as shown by the decrease in serum creatine phosphokinase (CPK) activity. In vitro experiments showed that pretreatment of lymphocytes with mangiferin protected from the toxicity induced by H2O2, further confirming the in vivo findings. From this study, it is evident that mangiferin exhibits an immunoprotective role mediated through the inhibition of reactive intermediate-induced oxidative stress in lymphocytes, neutrophils and macrophages.     

N-Acetylbenzidine-DNA adduct formation by phorbol 12-myristate-stimulated human polymorphonuclear neutrophils

N'-(3'-Monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is the major adduct in exfoliated urothelial cells and in peripheral white blood cells of workers exposed to benzidine. This study was designed to assess the metabolic pathways leading to dGp-ABZ formation in human peripheral white blood cells. [(3)H]-N-Acetylbenzidine (ABZ) transformation was assessed using myeloperoxidase (MPO), hypochlorous acid (HOCl), and human peripheral white blood cells in the absence and presence of DNA or dGp. MPO metabolism required H(2)O(2), but not NaCl. While transformation by HOCl was completely inhibited by 10 mM taurine, the level of metabolism of ABZ by MPO was only reduced 56%. Transformation by either MPO or HOCl was inhibited by 100 mM DMPO, 1 mM glutathione, and 1 mM ascorbic acid. Glutathione formed a new product with MPO, but not with HOCl. Previously identified oxidation products of ABZ, N'-hydroxy-N-acetylbenzidine or 4'-nitro-4-acetylaminobiphenyl, were not detected. With DNA or dGp present, a new product was observed that corresponded to synthetic dGp-ABZ in its HPLC elution profile, in nuclease P(1) hydrolysis to dG-ABZ, and in (32)P-postlabeling analysis. The HOCl-derived adduct was identified by electrospray ionization mass spectrometry, with collision-activated dissociation, as dGp-ABZ. Metabolism of [(3)H]ABZ by peripheral blood cells was stimulated about 3-fold with 30 ng/mL beta-phorbol 12-myristate 13-acetate (PMA). Using (32)P-postlabeling, dGp-ABZ was detected only in the presence of PMA and its level was increased more than 300-fold if either 0.7 mg/mL DNA or dGp was present. Indomethacin (0.1 mM) did not alter adduct formation. With dGp, dGp-ABZ formation could be detected with as little as 0.12 x 10(6) neutrophils. Using specific chromatographic and enzymatic techniques, neutrophil-derived dGp-ABZ was identical to the synthetic standard. Thus, these results are consistent with human polymorphonuclear neutrophils forming dGp-ABZ by a peroxidatic mechanism involving MPO.     

Antioxidant effect of hydroxytyrosol, a polyphenol from olive oil: scavenging of hydrogen peroxide but not superoxide anion produced by human neutrophils

Hydroxytyrosol (HT) (also known as dihydroxyphenylethanol (DPE)) is a polyphenol extracted from virgin olive oil. HT is known to exert an antioxidant effect but the mechanism of action and the identity of the reactive oxygen molecule(s) targeted are not known. In this study, we show that HT inhibits luminol-amplified chemiluminescence of human neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA) and opsonized zymosan. This effect was dose-dependent and occurred immediately after the addition of HT. However, HT had no effect on lucigenin-amplified chemiluminescence, suggesting that it does not inhibit NADPH oxidase activation or scavenge superoxide anions. Furthermore, HT inhibited H(2)O(2)-dependent-dichlorofuoroscein (DCFH) fluorescence of activated neutrophils, as measured by flow cytometry. Finally, HT inhibited luminol-amplified chemiluminescence in a cell-free system consisting of H(2)O(2) and HRPO. These results suggest that HT, a polyphenol derived from olive oil, could exert its antioxidant effect by scavenging hydrogen peroxide but not superoxide anion released during the respiratory burst.     

Effect of epinephrine on glucose metabolism and hydrogen peroxide content in incubated rat macrophages.

The effects of epinephrine on glucose metabolism and hydrogen peroxide content were examined in incubated rat macrophages. An increase in the activities of hexokinase and citrate synthase and a reduction in that of glucose-6-phosphate dehydrogenase was found in resident, inflammatory and activated macrophages incubated for 1 hr in the presence of epinephrine. Glucose utilization by incubated resident, inflammatory and activated macrophages was augmented markedly by the addition of epinephrine, whereas lactate formation was depressed. Under the same conditions, there was a 2.6-fold increment of hydrogen peroxide content and of [U-14C]glucose decarboxylation in activated macrophages incubated for 40 min. Similar results were obtained when pyruvate and oxoglutarate was substituted for glucose. These findings suggest that epinephrine may increase hydrogen peroxide production in activated macrophages possibly through a mitochondrial mechanism other than the pentose-phosphate pathway. Between 40 and 90 min of incubation, the content of hydrogen peroxide decreased markedly, and there was no detectable glucose utilization in the presence of epinephrine. These observations are consistent with the idea that this catecholamine stimulates both hydrogen peroxide production and metabolism, the first process being dependent on mitochondrial fuels.     

Induction of 8-hydroxy-2'-deoxyguanosine by cobalt(II) and hydrogen peroxide in vitro

In order to test the effects of cobalt on oxidative DNA damage, we measured the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the presence of cobalt in calf thymus DNA and in DNA of human diploid fibroblasts. Treatment of calf thymus DNA with Co(II) at 500 microM hydrogen peroxide resulted in a dose-dependent increase in 8-OHdG, which culminated at 25 microM Co(II) (62.6 modified/10(5) dG) and declined at higher Co(II) concentrations [9.6 modified/10(5) dG at 250 microM Co(II)]. Incubation of calf thymus DNA with Co(II) alone did not cause an increase in 8-OHdG. Treatment of calf thymus DNA with H2O2, (0.25-2 mM) caused only a slight generation of 8-OHdG (2.7/10(5) dG, at 2 mM H2O2). Exposure of human diploid fibroblasts to Co(II) at 5-250 microM did not result in an increase in 8-OHdG in a dose-dependent manner, although treated cells showed significantly higher 8-OHdG levels than untreated controls (2.026 +/- 0.695 vs. 1.395 +/- 0.433 8-OHdG/10(5) dG) at all concentrations. Our data indicate that in the presence of H2O2, Co(II) stimulates the in vitro formation of 8-OHdG.     

Hydrogen peroxide modulation of the respiratory burst of human neutrophils.

Addition of micromolar concentrations of hydrogen peroxide (H2O2) to human neutrophils resulted in a dose-dependent luminol-enhanced chemiluminescent response. Pretreatment of neutrophils with micromolar concentrations of H2O2 altered their response to the surface acting stimulants serum-treated zymosan (STZ) and formyl-methionyl-leucyl-phenylalanine (fMLP), but not to the intracellular stimulant phorbol myristate acetate (PMA). The alterations were partially reversible by catalase, but exacerbated by superoxide dismutase. These results suggest a modulatory role for H2O2 in the respiratory burst of neutrophils.