Configuration of thiols dictates their ability to promote iron-induced reactive oxygen species generation

Abstract

Iron catalyzes the production of reactive oxygen species (ROS) through the Fenton reaction. The modification of this phenomenon in the presence of various thiol compounds that are nominally reducing agents has been studied. Using the synaptosomal/mitochondrial (P2) fraction of rat cerebral cortex as a biological source of reactive oxygen species (ROS) production, we studied the influence of four compounds, glutathione (GSH), cysteine, N-acetyl-cysteine (NAC), and homocysteine on iron-induced ROS production. None of the thiol compounds alone, at the concentrations used, affected the basal rate of ROS production in the P2 fraction. GSH, homocysteine and NAC did not alter Fe-induced ROS generation, while cysteine greatly potentiated ROS formation. Measurement of the rate of ROS production in the presence of varying concentrations of cysteine together with 20 µM ferrous iron revealed a dose-response relationship. The mechanism whereby free cysteine, but not the cysteine-containing peptide GSH, homocysteine or NAC with a blocked amino group, exacerbates the prooxidant properties of ferrous iron probably involves formation of a complex between iron, a sulfhydryl and a free carboxyl residue located at a critical distance from the –SH group. Cysteine-iron interactions may, in part, account for the excessive toxicity of free cysteine in contrast to GSH and NAC.     

Role of mitochondrial thiols of different localization in the generation of reactive oxygen species

The role of thiols of the outer and the inner membranes of mitochondria in the regulation of generation of reactive oxygen species (ROS) has been studied. It was found that N-ethylmaleimide (NEM), which penetrates through the mitochondrial membrane and binds thiols to form thioesters, at concentrations from 20 to 250 μM activates the production of superoxide anion and hydrogen peroxide during the oxidation of the substrates of complexes I and II of the respiratory chain. 5′,5′-Dithiobis-(2-nitrobenzoate) (DTNB), which does not penetrate into mitochondria and binds thiols to form disulfides, weakly activates hydrogen peroxide production during the oxidation of NAD-dependent substrates and inhibits the ROS production upon succinate oxidation. DTNB is particularly effective in inhibiting the menadione-induced formation of ROS. The differences in the ROS formation by these reagents are explained by the fact that they influence different thiol-containing proteins and enzymes. As distinct from NEM, which inhibits complex I of the respiratory chain, DTNB has no effect on the respiratory chain of mitochondria but can bind the SH-groups of NADH-quinone oxidoreductase, which is localized in the outer mitochondrial membrane and participates in the redox cycle of menadione. It was also shown that the ability to inhibit the ADP-stimulated respiration, a feature inherent in both reagents, does not significantly contribute to ROS production.     

Fungal variegatic acid and extracellular polysaccharides promote the site-specific generation of reactive oxygen species

Journal of Industrial Microbiology & Biotechnology - This study aims to clarify the role of variegatic acid (VA) in fungal attack by Serpula lacrymans, and also the generation and scavenging of...     

Neuregulin induces sustained reactive oxygen species generation to mediate neuronal differentiation

Neuregulins (NRGs), which are highly expressed in the nervous system, bind and activate two receptor tyrosine kinases, ErbB-3 and ErbB-4. Recently, we have shown that ErbB-4 receptors expressed in PC12 cells mediate NRG-induced differentiation through the MAPK signaling pathway. Here we demonstrate that NRG induces an increase in the intracellular concentration of reactive oxygen species (ROS). N-acetylcysteine, a ROS scavenger, inhibited NRG-induced activation of Ras and Erk and PC12-ErbB-4 cell differentiation. These results suggest that ROS production is involved in NRG-mediated neuronal differentiation and that ROS can regulate activation of Ras and Erk. Constitutively active Ras enhanced ROS production and dominant negative Ras inhibited NRG-induced ROS production, suggesting, a positive regulatory loop between Ras and ROS. The mitogen, EGF, induced short-term ROS production whereas NRG and NGF, which induce cell differentiation, induced prolonged ROS production. These results strongly suggest that the kinetics of ROS production may determine whether the cells will differentiate or proliferate.     

Regulation of reactive oxygen species generation in cell signaling

Reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H₂O₂) are thought to be byproducts of aerobic respiration with damaging effects on DNA, protein, and lipid. A growing body of evidence indicates, however, that ROS are involved in the maintenance of redox homeostasis and various cellular signaling pathways. ROS are generated from diverse sources including mitochondrial respiratory chain, enzymatic activation of cytochrome p450, and NADPH oxidases further suggesting involvement in a complex array of cellular processes. This review summarizes the production and function of ROS. In particular, how cytosolic and membrane proteins regulate ROS generation for intracellular redox signaling will be detailed.     

Balancing reactive oxygen species generation by rebooting gut microbiota

Reactive oxygen species (ROS; free radical form O₂^•−, superoxide radical; OH^•, hydroxyl radical; ROO^•, peroxyl; RO^•, alkoxyl and non-radical form ¹O₂, singlet oxygen; H₂O₂, hydrogen peroxide) are inevitable companions of aerobic life with crucial role in gut health. But, overwhelming production of ROS can cause serious damage to biomolecules. In this review, we have discussed several sources of ROS production that can be beneficial or dangerous to the human gut. Micro-organisms, organelles and enzymes play crucial role in ROS generation, where NOX1 is the main intestinal enzyme, which produce ROS in the intestine epithelial cells. Previous studies have reported that probiotics play significant role in gut homeostasis by checking the ROS generation, maintaining the antioxidant level, immune system and barrier protection. With current knowledge, we have critically analysed the available literature and presented the outcome in the form of bubble maps to suggest that the probiotics help in controlling the ROS-specific intestinal diseases, such as inflammatory bowel disease (IBD) and colon cancer. Finally, it has been concluded that rebooting of the gut microbiota with probiotics, postbiotics or faecal microbiota transplantation (FMT) can have crucial implications in the structuring of gut communities for the personalized management of the gastrointestinal (GI) diseases.     

Nucleotide receptor signalling and the generation of reactive oxygen species

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology. This work was supported by National Institutes of Health Grants HL56396 and AI50500. The first author was supported by the Hematology Training Program NIH 5 T32 HL07899 at the University of Wisconsin.     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.     

Cobalt-mediated generation of reactive oxygen species and its possible mechanism

Electron spin resonance spin trapping was utilized to investigate free radical generation from cobalt (Co) mediated reactions using 5,5-dimethyl-l-pyrroline (DMPO) as a spin trap. A mixture of Co with water in the presence of DMPO generated 5,5-dimethylpyrroline-(2)-oxy(1) DMPOX, indicating the production of strong oxidants. Addition of superoxide dismutase (SOD) to the mixture produced hydroxyl radical (OH). Catalase eliminated the generation of this radical and metal chelators, such as desferoxamine, diethylenetriaminepentaacetic acid or 1,10-phenanthroline, decreased it. Addition of Fe(II) resulted in a several fold increase in the OH generation. UV and O₂ consumption measurements showed that the reaction of Co with water consumed molecular oxygen and generated Co(II). Since reaction of Co(II) with H₂O₂ did not generate any significant amount of OH radicals, a Co(I) mediated Fenton-like reaction [Co(I) + H₂O₂ → Co(II) + OH + OH⁻] seems responsible for OH generation. H₂O₂ is produced from O₂⁻ via dismutation. O₂⁻ is produced by one-electron reduction of molecular oxygen catalyzed by Co. Chelation of Co(II) by biological chelators, such as glutathione or β-ananyl-3-methyl- -histidine alters, its oxidation–reduction potential and makes Co(II) capable of generating OH via a Co(II)-mediated Fenton-like reaction [Co(II) + H₂O₂ → Co(III) + OH + OH⁻]. Thus, the reaction of Co with water, especially in the presence of biological chelators, glutathione, glycylglycylhistidine and β-ananyl-3-methyl- -histidine, is capable of generating a whole spectrum of reactive oxygen species, which may be responsible for Co-induced cell injury.     

Synchronized generation of reactive oxygen species with the cell cycle

A possible appearance of reactive oxygen species (ROS) with the normal cell cycle was studied to find how ROS are generated in cells in relation to the cell cycle. The production of ROS in relation to the cell cycle was examined by determining the changes in intracellular ROS concentrations at different phases of the cell cycle by culturing BALB 3T3 cells in the presence and absence of aphidicolin. The amounts of intracellular ROS and the cell population at specific phases (S and G2/M) were determined as the fluorescence of dichlorodihydrofluorescein and propidium iodide taken up simultaneously by the cells, respectively, by flow cytometry. Although intracellular ROS remained at the control levels when the cell growth was arrested with aphidicolin at the G1 phase, they increased when the arrest was released to result in the increase of the cell population at the S phase. Furthermore, ROS was shown to disturb/stop the cell cycle by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cell cycle was regulated through oxidative stress by exposure to hydrogen peroxide and glutathione ethyl ester. The cell cycle was prevented more sensitively in metallothionein-null cells than in the wild type cells. Based on the present observations, we proposed for the first time that ROS are generated synchronously with the normal cell cycle, and that they have to be controlled at certain level for normal progress of the cell cycle.     

Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear. In this study, we report that the P2X7 agonists ATP and 3'-O-(4-benzoyl) benzoic ATP (BzATP) stimulate ROS production by RAW 264.7 murine macrophages. These effects are potentiated in lipopolysaccharide-primed cells, demonstrating an important interaction between extracellular nucleotides and microbial products in ROS generation. In terms of nucleotide receptor specificity, RAW 264.7 macrophages that are deficient in P2X7 are greatly reduced in their capacity to generate ROS in response to BzATP treatment (both with and without LPS priming), thus supporting a role for P2X7 in this process. Because MAP kinase activation is key for nucleotide regulation of macrophage function, we also tested the hypothesis that P2X7-mediated MAP kinase activation is dependent on ROS production. We observed that BzATP stimulates MAP kinase (ERK1/ERK2, p38, and JNK1/JNK2) phosphorylation and that the antioxidants N-acetylcysteine and ascorbic acid strongly attenuate BzATP-mediated JNK1/JNK2 and p38 phosphorylation but only slightly reduce BzATP-induced ERK1/ERK2 phosphorylation. These studies reveal that P2X7 can contribute to macrophage ROS production, that this effect is potentiated upon lipopolysaccharide exposure, and that ROS are important participants in the extracellular nucleotide-mediated activation of several MAP kinase systems.     

Effects of glucocorticoids on generation of reactive oxygen species in platelets

Since prednisolone and dexamethasone are known as potent anti-inflammatory agents, the effects of prednisolone and dexamethasone on production of intracellular reactive oxygen species (ROS) were investigated in human platelets. Platelet ROS were measured using the intracellular fluorescent dye dichlorofluorescein diacetate after activation of protein kinase C by phorbol-12-myristate-13-acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). NAD(P)H oxidase activity was measured photometrically. PMA and OAG significantly increased ROS in platelets (P<0.001). Prednisolone or dexamethasone concentration-dependently reduced the PMA-induced ROS production. The PMA-induced ROS increase was significantly reduced in the presence of 10 micromol/l prednisolone to 9+/-1% (n=31; P<0.001) or in the presence of 10 micromol/l dexamethasone to 9+/-1% (n=24; P<0.001). The inhibitory effect of prednisolone or dexamethasone could also be observed in the presence of the glucocorticoid receptor inhibitor, mifepristone (RU486). Administration of testosterone or aldosterone did not significantly reduce PMA-induced ROS increase. Prednisolone had no effect on platelet NAD(P)H oxidase activity. The inhibition of oxidative phosphorylation by sodium azide reduced platelets ROS to 8+/-1% (n=35). It is concluded that glucocorticoids, prednisolone and dexamethasone, directly inhibit production of intracellular ROS. This effect may contribute to the anti-inflammatory actions of these agents.     

Analysis of mitochondrial generation and release of reactive oxygen species.

BACKGROUND: Reactive oxygen species (ROS) are mainly produced in mitochondria and are important contributors to many forms of cell death. ROS also function as second messengers within the cell and may constitute a signaling pathway from mitochondria to the cytoplasm and nucleus. The aim of the present study was to develop a protocol to detect changes in intra- and extramitochondrial releases of ROS, which could be used to analyze the role of mitochondria in cell signaling and cell death. METHODS: Fluorescence-based assays were used to measure (a) total production of ROS, (b) intramitochondrial ROS, (c) extramitochondrial hydrogen peroxide, and (d) superoxide outside inverted (inside-out) submitochondrial particles. ROS generation in the samples was increased or decreased by the addition of different substrates, enzymes, and inhibitors of the electron transport chain. RESULTS: The individual assays used were sensitive to increased (e.g., after addition of antimycin A; increased signal) and decreased (ROS scavenging; decreased signal) levels of ROS. In combination, the assays provided information about mitochondrial ROS generation and release dynamics from small samples of isolated mitochondria. CONCLUSIONS: The combination of fluorescent techniques described is a useful tool to study the role of ROS in cell death and in cellular redox signaling.     

Intracellular generation of reactive oxygen species by contracting skeletal muscle cells

The aim of this work was to examine the intracellular generation of reactive oxygen species in skeletal muscle cells at rest and during and following a period of contractile activity. Intracellular generation of reactive oxygen species was examined directly in skeletal muscle myotubes using 2',7'-dichlorodihydrofluorescein (DCFH) as an intracellular probe. Preliminary experiments confirmed that DCFH located to the myotubes but was readily photoxidizable during repeated intracellular fluorescence measurements and strategies to minimize this were developed. The rate of oxidation of DCFH did not change significantly over 30 min in resting myotubes, but was increased by approximately 4-fold during 10 min of repetitive, electrically stimulated contractile activity. This increased rate was maintained over 10 min following the end of the contraction protocol. DCF fluorescence was distributed evenly throughout the myotube with no evidence of accumulation at any specific intracellular sites or localization to mitochondria. The rise in DCF fluorescence was effectively abolished by treatment of the myotubes with the intracellular superoxide scavenger, Tiron. Thus these data appear to represent the first direct demonstration of a rise in intracellular oxidant activity during contractile activity in skeletal muscle myotubes and indicate that superoxide, generated from intracellular sites, is the ultimate source of oxidant(s) responsible for the DCFH oxidation.     

Mequindox induced cellular DNA damage via generation of reactive oxygen species

Mequindox, a quinoxaline-N-dioxide derivative that possesses antibacterial properties, has been widely used as a feed additive in the stockbreeding industry in China. While recent pharmacological studies have uncovered potential hazardous effects of mequindox, exactly how mequindox induces pathological changes and the cellular responses associated with its consumption remain largely unexplored. In this study, we investigated the cellular responses associated with mequindox treatment. We report here that mequindox inhibits cell proliferation by arresting cells at the G2/M phase of the cell cycle. Interestingly, this mequindox-associated deleterious effect on cell proliferation was observed in human, pig as well as chicken cells, suggesting that mequindox acts on evolutionarily conserved target(s). To further understand the mequindox-host interaction and the mechanism underlying mequindox-induced cell cycle arrest, we measured the cellular content of DNA damage, which is known to perturb cell proliferation and compromise cell survival. Accordingly, using γ-H2AX as a surrogate marker for DNA damage, we found that mequindox treatment induced cellular DNA damage, which paralleled the chemical-induced elevation of reactive oxygen species (ROS) levels. Importantly, expression of the antioxidant enzyme catalase partially alleviated these mequindox-associated effects. Taken together, our results suggest that mequindox cytotoxicity is attributable, in part, to its role as a potent inducer of DNA damage via ROS.     

Rapid upregulation of endothelial P-selectin expression via reactive oxygen species generation

Endothelial cell ICAM-1 upregulation in response to TNF-alpha is mediated in part by reactive oxygen species (ROS) generated by the endothelial membrane-associated NADPH oxidase and occurs maximally after 4 h as the synthesis of new protein is required. However, thrombin-stimulated P-selectin upregulation is bimodal, the first peak occurring within minutes. We hypothesize that this early peak, which results from the release of preformed P-selectin from within Weibel-Palade bodies, is mediated in part by ROS generated from the endothelial membrane-associated xanthine oxidase. We found that this rapid expression of P-selectin on the surface of endothelial cells was accompanied by qualitatively parallel increases in ROS generation. Both P-selectin expression and ROS generation were inhibited, dose dependently, by the exogenous administration of disparate cell-permeable antioxidants and also by the inhibition of either of the known membrane-associated ROS-generating enzymes NADPH oxidase or xanthine oxidase. This rapid, posttranslational cell signaling response, mediated by ROS generated not only by the classical NADPH oxidase but also by xanthine oxidase, may well represent an important physiological trigger of the microvascular inflammatory response.     

Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species

The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.