Prooxidant capacity of phenolic acids defines antioxidant potential

Phenolic acids derived from vegetables, fruits and beverages are considered abundant sources of natural antioxidants consumed in the human diet. In addition to having well-known antioxidant activity, phenolic acids also exhibit pro-oxidant activity under selected conditions. We hypothesized that the availability of extracellular H₂O₂ derived from phenolic acid autoxidation will diffuse across cell membranes to participate as a messenger molecule to activate intracellular redox signaling in response to oxidative stress. We report on the relative activity of structurally different phenolic acids to generate specific changes in the extracellular - intracellular H₂O₂ flux that induces intracellular redox signaling corresponding to a function to reduce intracellular oxidative stress. HyPer-3 methodology was used to measure increases in intracellular H₂O₂ in differentiated Caco-2 intestinal cells in response to phenolic acid autoxidation and changes in extracellular H₂O₂ production. The potential for different phenolic acids to autoxidize and generate H₂O₂ was dependent on the structure and concentration of phenolic acid. Activation of nuclear factor erythroid 2-related factor (Nrf2) cell signaling was enhanced (p < 0.05) by phenolic acid induced H₂O₂ production, and mitigated when present along with catalase (p < 0.05), or, alternatively by blocking aquaporin 3 (AQP3) function (p < 0.05) using DFP00173 as the AQP3 inhibitor. The relative capacity of phenolic acids to generate H₂O₂ via autoxidation was structure specific and corresponded to the level of Nrf2 cell signaling in differentiated Caco-2 epithelial cells. The Nrf2-Keap1 response paralleled the extent of reduced oxidative stress observed in differentiated Caco-2 cells determined by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.     

Protection of nicotinic acid against oxidative stress-induced cell death in hepatocytes contributes to its beneficial effect on alcohol-induced liver injury in mice

Oxidative stress plays a pathological role in the development of alcoholic liver disease. In this study, we investigated the effects of nicotinic acid (NA) supplementation on H₂O₂-induced cell death in hepatocytes and alcohol-induced liver injury in mice. Hepatocytes were exposed to H₂O₂ (0–0.4 mM) for 16 h after a 2-h pretreatment with NA (0–100 μM). Cell viability, intracellular glutathione and total NAD contents were determined. In animal experiments, male C57BL/6 mice were exposed to Lieber-De Carli liquid diet [+/? ethanol with/without NA supplementation (0.5%, w/v) for 4 weeks]. Nicotinic acid phosphoribosyltransferase (NaPRT) is the first enzyme participated in the NA metabolism, converting NA to nicotinic acid mononucleotide (NaMN). In NaPRT-expressing Hep3B cells, H₂O₂-induced cell death was attenuated by NA, whereas in NaPRT-lost HepG2 cells, only NaMN conferred protective effect, suggesting that NA metabolism is required for its protective action against H₂O_2. In Hep3B cells, NA supplementation prevented H₂O₂-inudced declines in intracellular total NAD and GSH/GSSG ratios. Further mechanistic investigations revealed that conservation of Akt activity contributed to NA's protective effect against H₂O₂-inudced cell death. In alcohol-fed mice, NA supplementation attenuated liver injury induced by chronic alcohol exposure, which was associated with alleviated hepatic lipid peroxidation and increased liver GSH concentrations. In conclusion, our findings indicate that exogenous NA supplementation may be an ideal choice for the treatment of liver diseases that involve oxidative stress.     

Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

p38 MAPK and Ca2+ contribute to hydrogen peroxide-induced increase of permeability in vascular endothelial cells but ERK does not

To examine the involvement of p38 mitogen-activated protein kinase (p38 MAPK) and extra-cellular signal-regulated kinase (ERK) in the oxidative stress-induced increase of permeability in endothelial cells, the effects of a p38 MAPK inhibitor (SB203580) and ERK inhibitor (PD90859) on the H₂O₂-induced increase of permeability in bovine pulmonary artery endothelial cells (BPAEC) were investigated using a two-compartment system partitioned by a semi-permeable filter. H₂O₂ at 1 mM caused an increase of the permeation rate of fluorescein isothiocyanate (FITC)-labeled dextran 40 through BPAEC monolayers. SB203580 inhibited the H₂O₂-induced increase of permeability but PD98059 did not, though activation (phosphorylation) of both p38 MAPK and ERK was observed in H₂O₂-treated cells in Western blot analysis. An H₂O₂-induced increase of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) was also observed and an intracellular Ca²⁺ chelator (BAPTA-AM) significantly inhibited the H₂O₂-induced increase of permeability. However, it showed no inhibitory effects on the H₂O₂-induced phosphorylation of p38 MAPK and ERK. The H₂O₂-induced increase of [Ca²⁺]_i was not influenced by SB203580 and PD98059. These results indicate that the activation of p38 MAPK and the increase of [Ca²⁺]_i are essential for the H₂O₂-induced increase of endothelial permeability and that ERK is not.     

MAPK signaling in H9c2 cardiomyoblasts exposed to cholesterol secoaldehyde – Role of hydrogen peroxide

3β-Hydroxy-5,6-secocholestan-6-al (cholesterol secoaldehyde or ChSeco), an oxysterol known to be formed in ozone- and singlet oxygen-mediated oxidations of cholesterol, has been detected in the atherosclerotic plaque and in the brain of patients suffering from Alzheimer’s disease and Lewy body dementia. Previously, we have shown that, in H9c2 cardiomyoblasts, ChSeco induces oxidative stress followed by apoptosis involving both intrinsic and extrinsic signaling pathways. In the present study, we investigated the nature of reactive oxygen species (ROS) and its associated redox signaling in H9c2 cells upon treatment with ChSeco. Both catalase and deferoxamine, which lowered intracellular ROS, were found to alleviate the ChSeco-induced cytotoxicity. ChSeco-treated H9c2 cells showed a significant decrease in the intracellular catalase activity, suggesting the involvement of H₂O₂ in the associated cytotoxicity. Additionally, in ChSeco-exposed cells, there was a marked increase in lipid peroxidation and pre-treatment with SB 203580 (p38 MAPK inhibitor) and MEK1/2 inhibitor (ERK1/2 and JNK inhibitor) rendered protection against the cytotoxicity. An early increase in the expression of p-SAPK/JNK or delayed p38 MAPK did not alter ATF-2 but decreased c-Jun expression in these cells. Overall, these findings are consistent with MAPK signaling resulting from increased cellular H₂O₂ in ChSeco-induced cytotoxicity in cardiomyoblasts.     

Neuroprotective effect of polysaccharide separated from Perilla frutescens Britton var. acuta Kudo against H2O2-induced oxidative stress in HT22 hippocampus cells

This study was carried out to evaluate the neuroprotective activity of polysaccharide extracts isolated from Perilla frutescens (PEPF) in H₂O₂-treated HT22 hippocampus cells. The PEPF treatment was found to increase the anti-oxidant activities of HT22 hippocampus cells. PEPF treatment resulted in a significant protection of HT22 hippocampus cells against H₂O₂-induced neurotoxicity, this protection ultimately occurred through an inhibition of ROS-mediated intracellular Ca²⁺ levels leading to MAPKs and NF-κB, as well as the accumulation of PI3K/AKT and Nrf2-mediated HO-1/NQO1 pathways. Furthermore, PEPF not only decreased the expression of Bax, cytochrome c, and cleaved caspases-3, -8, and -9, but also increased the expression of PARP and Bcl-2 in the H₂O₂-treated HT22 hippocampus cells, which overall contributed to the neuroprotective action. PEPF retains its mitochondrial membrane potential and reduces the elevated levels of sub-G1 phase and apoptotic morphological features induced by H₂O₂. It also reduces the malondialdehyde levels and enhances the intracellular SOD activity.     

Protective Effect of Selenoprotein X Against Oxidative Stress-Induced Cell Apoptosis in Human Hepatocyte (LO2) Cells via the p38 Pathway

Oxidative stress, as mediated by ROS (reactive oxygen species), is a significant factor in initiating the cells damaged by affecting cellular macromolecules and impairing their biological functions; SelX, a selenoprotein also known as MsrB1 belonging to the methionine sulfoxide reductase (Msr) family, is the redox repairing enzyme and involved in redox-related functions. In order to more precisely analyze the relationship between oxidative stress, cell oxidative damage, and SelX, we stably overexpressed porcine Selx full-length cDNA in human normal hepatocyte (LO2) cells. Cell viability, cell apoptosis rate, intracellular ROS, and the expression levels of mRNA or protein of apoptosis-related genes under H₂O₂-induced oxidative stress were detected. We found that overexpression of SelX can prevent the oxidative damage caused by H₂O₂ and propose that the main mechanism underlying the protective effects of SelX is the inhibition of LO2 cell apoptosis. The results revealed that overexpressed SelX reduced the H₂O₂-induced intracellular ROS generation, inhibited the H₂O₂-induced upregulation of Bax and downregulation of Bcl-2, and increased the mRNA and protein ratio of Bcl-2/Bax. Furthermore, it inhibited H₂O₂-induced p38 MAPK phosphorylation. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and that its protective effect is partly via the p38 pathway by acting as a ROS scavenger.     

Extinction of cells of cyanobacterium Anabaena circinalis in the presence of humic acid under illumination

Laboratory experiments targeting the effect of humic acid (HA) on the cell lysis of cyanobacterium Anabaena circinalis have been performed. Light irradiation was found to be an important factor for the cell lysis phenomenon, whereas intracellular hydrogen peroxide (H₂O₂) might be a chemical factor for the process. An exogenous H₂O₂ concentration of 1.0 mg l⁻¹ was determined as the threshold for cell survival. Our results indicated that HA or its possible product(s) of photochemical reaction can induce damage to intracellular catalase under artificial illumination, which leads intracellular H₂O₂ to be accumulated to an abnormally high concentration, eventually resulting in cell death. Moreover, H₂O₂ released into the culture from dead cells can damage other cells, which in turn brings about the population extinction.     

Effects of SOD/catalase mimetic platinum nanoparticles on radiation-induced apoptosis in human lymphoma U937 cells

Since polyacrylic acid capped platinum nano-particles (nano-Pts) are known to have a unique ability to quench superoxide (O₂ ^?) and hydrogen peroxide (H₂O₂), the anti-oxidant activity of nano-Pts against apoptosis induced by x-irradiation in human lymphoma U937 cells was investigated. DNA fragmentation assay, Annexin V-FITC/PI by flow cytometry and Giemsa staining revealed a significant decrease in apoptosis induced by 10 Gy, when cells were pre-treated with nano-Pts in a dose-dependent manner. Pre-treatment with nano-Pts significantly decreased radiation-induced reactive oxygen species (ROS) production, Fas expression and loss of mitochondrial membrane potential as determined by flow-cytometry. Furthermore, western blot analysis also showed that the expression of cleaved caspase-3, Bid and cytosolic cytochrome-c were significantly reduced in nano-Pts pretreated cells. Due to the catalase mimetic activity of nano-Pts, these results indicate that pre-treatment of U937 cells with nano-Pts significantly protect radiation-induced apoptosis by inhibiting intracellular ROS (mainly H₂O₂), which plays a key role in the induction of apoptosis, because of no practical observation of intracellular O₂ ^? formation.     

Phosphorylation is not essential for protection of L929 cells by Hsp25 against H2O2 -mediated disruption actin cytoskeleton, a protection which appears related to the redox change mediated by Hsp25

Small stress proteins protect against the cytotoxicity mediated by oxidative stress. The relationship between Hsp25 expression and the integrity of the actin network was studied in H₂O₂-treated murine L929 fibrosarcoma cells overexpressing endogenous wild-type (wt-) or non-phosphorylatable mutant (mt-) Hsp25. We show here that both proteins prevented actin network disruption induced by a 1 h treatment with 400 μM H₂O₂. In contrast, SB203580, a p38 MAPkinase inhibitor which suppresses Hsp25 phosphorylation, abolished the protective activity conferred by both wt- and mt-Hsp25. Hence, phosphorylation does not appear essential for Hsp25 protective activity against H₂O₂-induced actin disruption, and SB203580-sensitive events other than Hsp25 phosphorylation may be important for actin network regulation. Since, in L929 cells, wt- or mt-Hsp25 expression modulates intracellular glutathione levels, analyses were performed which revealed a direct correlation between glutathione and the integrity of the actin network. Moreover, laser scanning confocal immunofluorescences revealed that only a small fraction of wt- or mt-Hsp25 colocalized with actin microfilaments. Taken together, our results suggest that, in L929 cells, the protection against actin network disruption is probably a consequence of the redox change mediated by Hsp25 rather than a direct effect of this stress protein towards actin.     

Puerarin inhibits the retinal pericyte apoptosis induced by advanced glycation end products in vitro and in vivo by inhibiting NADPH oxidase-related oxidative stress

Activation of NAD(P)H oxidase has been reported to produce superoxide (O₂^??) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use an NAD(P)H oxidase product, NAD⁺ or NADP⁺, to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate, which mediates intracellular Ca^2 + signaling. This study was designed to test a hypothesis that the CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O₂^?? in mouse coronary arterial myocytes (CAMs). By fluorescence microscopic imaging, we simultaneously monitored extra- and intracellular O₂^?? production in wild-type (CD38^+/+) and CD38 knockout (CD38^?/?) CAMs in response to oxotremorine (OXO), a muscarinic type 1 receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O₂^?? production in CAMs. Consistently, the OXO-induced intracellular O₂^?? production was markedly inhibited by CD38 shRNA or the CD38 inhibitor nicotinamide in CD38^+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O₂^?? production, whereas Nox1 siRNA attenuated both intracellular and extracellular O₂^?? production in CD38^+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca^2 + and O₂^?? production in CD38^?/? CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that the CD38/cADPR pathway is an important controller of Nox4-mediated intracellular O₂^?? production and that CD38-dependent intracellular O₂^?? production is augmented in an autocrine manner by CD38-independent Nox1-derived extracellular O₂^?? production in CAMs.     

Depletion of reactive oxygen species induced by chlorogenic acid triggers apoptosis-like death in Escherichia coli

Chlorogenic acid (CGA) is a phenolic compound with various health-promoting properties, including antioxidant effects and a wide range of antibacterial activities. However, the antibacterial mechanism remains unclear. We investigated the underlying mode of action of CGA against Escherichia coli, which shows bacterial apoptosis-like death. Cells treated with CGA showed apoptotic features such as membrane depolarisation, caspase-like protein expression, increased intracellular Ca²⁺ levels, phosphatidylserine externalisation, and DNA fragmentation. In contrast to common bacterial apoptosis-like death, which is caused by reactive oxygen species (ROS) accumulation, CGA depleted intracellular ROS. Because ROS are important intracellular signalling molecules, and ROS depletion may affect bacterial intracellular signalling pathways, leading to cell death. To determine whether deficiencies in intracellular ROS cause apoptosis-like death, the cells were treated with H₂O₂ after CGA treatment. H₂O₂ restored depleted intracellular ROS levels to similar levels as in untreated cells, and cell viability was increased compared to CGA-treated cells. Moreover, apoptotic features were attenuated in H₂O₂ post-treated cells. These results demonstrate that CGA induces bacterial apoptosis in E. coli and intracellular ROS depletion is a core regulator in the progression of bacterial apoptosis-like death.     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.