Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Hypoxia induces production of nitric oxide and reactive oxygen species in glomus cells of rat carotid body

The carotid body is an arterial chemoreceptor organ that senses arterial pO(2) and pH. Previous studies have indicated that both reactive oxygen species (ROS) and nitric oxide (NO) are important potential mediators that may be involved in the response of the carotid body to hypoxia. However, whether their production by the chemosensitive elements of the carotid body is indeed oxygen-dependent is currently unclear. Thus, we have investigated their production under normoxic (20% O(2)) and hypoxic (1% O(2)) conditions in slice preparations of the rat carotid body by using fluorescent indicators and confocal microscopy. NO-synthesizing enzymes were identified by immunohistochemistry and histochemistry, and the subcellular localization of the NO-sensitive indicator diaminofluorescein was determined by a photoconversion technique and electron microscopy. Glomus cells of the carotid body responded to hypoxia by increases in both ROS and NO production. The hypoxia-induced increase in NO generation required (to a large extent, but not completely) extracellular calcium. Glomus cells were immunoreactive to endothelial NO synthase but not to the neuronal or inducible isoforms. Ultrastructurally, the NO-sensitive indicator was observed in mitochondrial membranes after exposure to hypoxia. The data show that glomus cells respond to exposure to hypoxia by the enhanced production of both ROS and NO. NO production by glomus cells is probably mediated by endothelial NO synthase, which is activated by calcium influx. The presence of NO indicator in mitochondria suggests the hypoxic regulation of mitochondrial function via NO in glomus cells.     

Chromium-induced excretion of urinary lipid metabolites,DNA damage,nitric oxide production,and generation of reactive oxygen species in Sprague-Dawley rats

Chromium and its salts induce cytotoxicity and mutagenesis, and vitamin E has been reported to attenuate chromate-induced cytotoxicity. These observations suggest that chromium produces reactive oxygen species which may mediate many of the untoward effects of chromium. We have therefore examined and compared the effects of Cr(III) (chromium chloride hexahydrate) and Cr(VI) (sodium dichromate) following single oral doses (0.50 ld₅₀) on the production of reactive oxygen species by peritoneal macrophages, and hepatic mitochondria and microsomes in rats. The effects of Cr(III) and Cr(VI) on hepatic mitochondrial and microsomal lipid peroxidation and enhanced excretion of urinary lipid metabolites as well as the incidence of hepatic nuclear DNA damage and nitric oxide (NO) production were also examined. Increases in lipid peroxidation of 1.8- and 2.2-fold occurred in hepatic mitochondria and microsomes, respectively, 48 hr after the oral administration of 25 mg Cr(VI)/kg, while increases of 1.2- and 1.4-fold, respectively, were observed after 895 mg Cr(III)/kg. The urinary excretion of malondialdehyde (MDA), formaldehyde (FA), acetaldehyde (ACT) and acetone (ACON) were determined at 0–96 hr after Cr administration. Between 48 and 72 hr post-treatment, maximal excretion of the four urinary lipid metabolites was observed with increases of 1.5- to 5.4-fold in Cr(VI) treated rats. Peritoneal macrophages from Cr(VI) treated animals 48 hr after treatment resulted in 1.4- and 3.6-fold increases in chemiluminescence and iodonitrotetrazolium reduction, indicating enhanced production of Superoxide anion, while macrophages from Cr(III) treated animals showed negligible increases. Increases in DNA single strand breaks of 1.7-fold and 1.5-fold were observed following administration of Cr(VI) and Cr(III), respectively, at 48 hr post-treatment. Enhanced production of NO by peritoneal exudate cells (primarily macrophages) was monitored following Cr(VI) administration at both 24 and 48 hr post-treatment with enhanced production of NO being observed at both timepoints. The results indicate that both Cr(VI) and Cr(III) induce an oxidative stress at equitoxic doses, while Cr(VI) induces greater oxidative stress in rats as compared with Cr(III) treated animals.     

New insights into reactive oxygen species and nitric oxide signalling under low oxygen in plants

Plants produce reactive oxygen species (ROS) when exposed to low oxygen (O₂). Much experimental evidence has demonstrated the existence of an oxidative burst when there is an O₂ shortage. This originates at various subcellular sites. The activation of NADPH oxidase(s), in complex with other proteins, is responsible for ROS production at the plasma membrane. Another source of low O₂‐dependent ROS is the mitochondrial electron transport chain, which misfunctions when low O₂ limits its activity. Arabidopsis mutants impaired in proteins playing a role in ROS production display an intolerant phenotype to anoxia and submergence, suggesting a role in acclimation to stress. In rice, the presence of the submergence 1A (SUB1A) gene for submergence tolerance is associated with a higher capacity to scavenge ROS. Additionally, the destabilization of group VII ethylene responsive factors, which are involved in the direct O₂ sensing mechanism, requires nitric oxide (NO). All this evidence suggests the existence of a ROS and NO – low O₂ mechanism interplay which likely includes sensing, anaerobic metabolism and acclimation to stress. In this review, we summarize the most recent findings on this topic, formulating hypotheses on the basis of the latest advances.     

Mitochondrial production of reactive oxygen species

Numerous biochemical studies are aimed at elucidating the sources and mechanisms of formation of reactive oxygen species (ROS) because they are involved in cellular, organ-, and tissue-specific physiology. Mitochondria along with other cellular organelles of eukaryotes contribute significantly to ROS formation and utilization. This review is a critical account of the mitochondrial ROS production and methods for their registration. The physiological and pathophysiological significance of the mitochondrially produced ROS are discussed.     

A key enzyme for flavin synthesis is required for nitric oxide and reactive oxygen species production in disease resistance

Nitric oxide (NO) and reactive oxygen species (ROS) play key roles in plant immunity. However, the regulatory mechanisms of the production of these radicals are not fully understood. Hypersensitive response (HR) cell death requires the simultaneous and balanced production of NO and ROS. In this study we indentified NbRibA encoding a bifunctional enzyme, guanosine triphosphate cyclohydrolase II/3,4‐dihydroxy‐2‐butanone‐4‐phosphate synthase, which participates in the biosynthesis of flavin, by screening genes related to mitogen‐activated protein kinase‐mediated cell death, using virus‐induced gene silencing. Levels of endogenous riboflavin and its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are important prosthetic groups for several enzymes participating in redox reactions, decreased in NbRibA‐silenced Nicotiana benthamiana. Silencing NbRibA compromised not only HR cell death, but also the NO and ROS production induced by INF1 elicitin and a constitutively active form of NbMEK2 (NbMEK2^DD), and also induced high susceptibility to oomycete Phytophthora infestans and ascomycete Colletotrichum orbiculare. Compromised radical production and HR cell death induced by INF1 in NbRibA‐silenced leaves were rescued by adding riboflavin, FMN or FAD. These results indicate that flavin biosynthesis participates in regulating NO and ROS production, and HR cell death.     

Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages

Immunosenescence is an age-associated dysregulation of the immune function, which contributes to increased susceptibility to disease in the elderly. Alveolar macrophages (AM) are known phagocytes that generate reactive oxygen species (ROS) and nitric oxide (NO), essential mediators for host defence. We studied phagocytosis, ROS and NO production in AM obtained from young, adult and senescent rats (1-2, 9-12 and 18-24 months old, respectively) after exposure to lipopolysaccharide (LPS, 0.1-10 microg mL(-1)), 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microg mL(-1)) or LPS + TPA in culture. Phagocytosis was significantly lower in control AM from adult rats than in AM from young animals. Nevertheless, AM from adult animals pretreated with LPS exhibited higher phagocytic capacity than AM from younger animals. ROS was identified by the NBT test at single cell level and quantified by automated image analysis. When TPA was added to all three populations, AM from adult and senescent animals responded more than AM from young animals. All LPS-stimulated AM produce more NO than controls. However, NO production increased three-, four- and two-fold in young, adult and senescent animals, respectively. Our results demonstrate that AM from young, adult and senescent animals display differential responsiveness to inflammatory mediators. Therefore, aging processes markedly affect AM metabolic functions and may further compromise the lung immune defence response, increasing adverse long-term health effects.     

Mitochondrial fission in endothelial cells after simulated ischemia/reperfusion: role of nitric oxide and reactive oxygen species

Ischemia (I)/reperfusion (RP)-induced endothelial cell (EC) injury is thought to be due to mitochondrial reactive oxygen species (mtROS) production. MtROS have been implicated in mitochondrial fission. We determined whether cultured EC exposure to simulated I/RP causes morphological changes in the mitochondrial network and the mechanisms behind those changes. Because shear stress results in nitric oxide (NO)-mediated endothelial mtROS generation, we simulated I/RP as hypoxia (H) followed by oxygenated flow over the ECs (shear stress of 10dyn/cm(2)). By exposing ECs to shear stress, H, H/reoxygenation (RO), or simulated I/RP and employing MitoTracker staining, we assessed the differential effects of changes in mechanical forces and/or O(2) levels on the mitochondrial network. Static or sheared ECs maintained their mitochondrial network. H- or H/RO-exposed ECs underwent changes, but mitochondrial fission was significantly less compared to that in ECs exposed to I/RP. I/RP-induced fission was partially inhibited by antioxidants, a NO synthase inhibitor, or an inhibitor of the fission protein dynamin-related protein 1 (Drp1) and was accompanied by Drp1 oligomerization and phosphorylation (Ser616). Hence, shear-induced NO, ROS (including mtROS), and Drp1 activation are responsible for mitochondrial fission in I/RP-exposed ECs, and excessive fission may be an underlying cause of EC dysfunction in postischemic hearts.     

Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA), one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study, lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of varying concentrations of L-DOPA (1 microM-1 mM). The results indicated a slight augmentation of NO(2)(-) production at low concentrations of L-DOPA (<100 microM) and complete inhibition of NO(2)(-) at higher concentrations (500 microM, 1 mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein expression. Total reactive oxygen species (ROS) were detected using 2', 7'-dichlorofluorescein diacetate fluorescence dye (2', 7'-DCFC) and there was an increase of intensity with the increasing concentrations of L-DOPA. Furthermore, large amounts of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were generated from the autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase (SOD); therefore, there was an accumulation of O(2)(-), tantamount to elevation in 2'7'-DCFC intensity. Simultaneous accumulation of O(2)(-) and NO(2)(-) would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data obtained confirmed that the observed effects on iNOS were not due to the activation of the D(1) or beta1 adrenergic receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the increase of O(2)(-) production in the stimulated glioma cells in vitro.     

Plant peroxisomes,reactive oxygen metabolism and nitric oxide

In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. Like mitochondria and chloroplasts, peroxisomes also produce superoxide radicals (O2*-) and there are, at least, two sites of superoxide generation: one in the organelle matrix, the generating system being xanthine oxidase, and another site in the peroxisomal membranes dependent on NAD(P)H. In peroxisomal membranes, three integral polypeptides (PMPs) with molecular masses of 18, 29, and 32 kDa have been shown to generate O2*- radicals. Besides catalase, several antioxidative systems have been demonstrated in plant peroxisomes, including different superoxide dismutases, the four enzymes of the ascorbate-glutathione cycle plus ascorbate and glutathione, and three NADP-dependent dehydrogenases. A CuZn-SOD and two Mn-SODs have been purified and characterized from different types of plant peroxisomes. The presence of the enzyme nitric oxide synthase (NOS) and its reaction product, nitric oxide (NO*), has been recently demonstrated in plant peroxisomes. Different experimental evidence has suggested that peroxisomes have a ROS-mediated cellular function in leaf senescence and in stress situations induced by xenobiotics and heavy metals. Peroxisomes could also have a role in plant cells as a source of signal molecules like NO*, O2*- radicals, H2O2, and possibly S-nitrosoglutathione (GSNO). It seems reasonable to think that a signal molecule-producing function similar to that postulated for plant peroxisomes could also be performed by human, animal and yeast peroxisomes, where research on oxy radicals, antioxidants and nitric oxide is less advanced than in plant peroxisomes.     

Rehydration of the lichen Ramalina lacera results in production of reactive oxygen species and nitric oxide and a decrease in antioxidants

Lichens are slow-growing associations of fungi and unicellular green algae or cyanobacteria. They are poikilohydric organisms whose lifestyle in many cases consists of alternating periods of desiccation, with low metabolic activity, and hydration, which induces increase in their metabolism. Lichens have apparently adapted to such extreme transitions between desiccation and rehydration, but the mechanisms that govern these adaptations are still poorly understood. In this study, the effect of rehydration on the production of reactive oxygen species and nitric oxide as well as low-molecular-weight antioxidants was investigated with the lichen Ramalina lacera. Rehydration of R. lacera resulted in the initiation of and a rapid increase in photosynthetic activity. Recovery of photosynthesis was accompanied by bursts of intracellular production of reactive oxygen species and nitric oxide. Laser-scanning confocal microscopy using dichlorofluorescein fluorescence revealed that formation of reactive oxygen species following rehydration was associated with both symbiotic partners of the lichen. The rate and extent of reactive oxygen species production were similar in the light and in the dark, suggesting a minor contribution of photosynthesis. Diaminofluorescein fluorescence, indicating nitric oxide formation, was detected only in fungal hyphae. Activities associated with rehydration did not have a deleterious effect on membrane integrity as assessed by measurement of electrolyte leakage, but water-soluble low-molecular-weight antioxidants decreased significantly.     

Reactive oxygen and nitrogen species regulate inducible nitric oxide synthase function shifting the balance of nitric oxide and superoxide production

Inducible NOS (iNOS) is induced in diseases associated with inflammation and oxidative stress, and questions remain regarding its regulation. We demonstrate that reactive oxygen/nitrogen species (ROS/RNS) dose-dependently regulate iNOS function. Tetrahydrobiopterin (BH₄)-replete iNOS was exposed to increasing concentrations of ROS/RNS and activity was measured with and without subsequent BH₄ addition. Peroxynitrite (ONOO⁻) produced the greatest change in NO generation rate, ∼95% decrease, and BH₄ only partially restored this loss of activity. Superoxide () greatly decreased NO generation, however, BH₄ addition restored this activity. Hydroxyl radical (OH) mildly decreases NO generation in a BH₄-dependent manner. iNOS was resistant to H₂O₂ with only slightly decreased NO generation with up to millimolar concentrations. In contrast to the inhibition of NO generation, ROS enhanced production from iNOS, while ONOO⁻ had the opposite effect. Thus, ROS promote reversible iNOS uncoupling, while ONOO⁻ induces irreversible enzyme inactivation and decreases both NO and production.     

Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells

The important role of plant peroxisomes in a variety of metabolic reactions such as photorespiration, fatty acid beta-oxidation, the glyoxylate cycle and generation-degradation of hydrogen peroxide is well known. In recent years, the presence of a novel group of enzymes, mainly involved in the metabolism of oxygen free-radicals, has been shown in peroxisomes. In addition to hydrogen peroxide, peroxisomes can generate superoxide-radicals and nitric oxide, which are known cellular messengers with a variety of physiological roles in intra- and inter-cellular communication. Nitric oxide and hydrogen peroxide can permeate the peroxisomal membrane and superoxide radicals can be produced on the cytosolic side of the membrane. The signal molecule-generating capacity of peroxisomes can have important implications for cellular metabolism in plants, particularly under biotic and abiotic stress.     

Ventilatory muscle activation and inflammation: cytokines, reactive oxygen species, and nitric oxide.

Strenuous diaphragmatic contractions that are induced by inspiratory resistive breathing initiate an inflammatory response that involves the elevation of pro- and anti-inflammatory cytokines within the diaphragm, which may then spill into the circulation. The production of reactive oxygen species within working respiratory muscles increases in response to these strenuous diaphragmatic contractions. At the same time, diaphragmatic nitric oxide (NO) production declines significantly, despite a time-dependent increase in NO synthase isoform protein expression. The increase in adhesion molecule expression and infiltration of granulocytes and macrophages that follows may contribute to the contraction-induced diaphragm injury. Enhanced generation of reactive oxygen species, oxidative stress augmentation, reduced NO production, and glycogen depletion are potential stimuli for the cytokine induction that is secondary to strenuous diaphragmatic contractions. This production of cytokines within the diaphragm may contribute to the diaphragmatic muscle fiber injury that occurs with strenuous contractions or to the expected repair process. TNF-alpha is a cytokine that compromises diaphragmatic contractility and may contribute to muscle wasting. IL-6 is a cytokine that may have beneficial systemic effects by mobilizing glucose from the liver and free fatty acids from the adipose tissue and providing them to the strenuously working respiratory muscles. Thus cytokine upregulation within the working diaphragm may be adaptive and maladaptive.     

Parameters of reactive oxygen species and nitric oxide metabolism in people with high arterial blood pressure

An exchange of active forms of oxygen and nitric oxide in normal conditions and under the development of oxidative stress in humans with high of arterial blood pressure was studied. The activity of NO-synthase was estimated in the human thrombocytes. The nitric oxide formations were determined by the quantity level of its final metabolites--nitrites and nitrates. The peroxynitrite formations were determined by the quantity level of 3-nitrotyrosine. An analysis of the investigation results has shown the increase of processes of oxidative stress, violation of nitric oxide formation in humans with high arterial blood pressure. Application of ascorbic acid allows to reduce the level of free radicals and to increase the formation of nitric oxide, but does not result in statistically reliable changes of the parameters describing formation of peroxynitrite and products of peroxide oxidation of lipids in humans with high arterial blood pressure. Application of ascorbic acid does not result in changes of researched parameters in the control group.     

Cytosolic NADP(+)-dependent isocitrate dehydrogenase protects macrophages from LPS-induced nitric oxide and reactive oxygen species

Macrophages activated by microbial lipopolysaccharides (LPS) produce bursts of nitric oxide and reactive oxygen species (ROS). Redox protection systems are essential for the survival of the macrophages since the nitric oxide and ROS can be toxic to them as well as to pathogens. Using suppression subtractive hybridization (SSH) we found that cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDPc) is strongly upregulated by nitric oxide in macrophages. The levels of IDPc mRNA and of the corresponding enzymatic activity were markedly increased by treatment of RAW264.7 cells or peritoneal macrophages with LPS or SNAP (a nitric oxide donor). Over-expression of IDPc reduced intracellular peroxide levels and enhanced the survival of H2O2- and SNAP-treated RAW264.7 macrophages. IDPc is known to generate NADPH, a cellular reducing agent, via oxidative decarboxylation of isocitrate. The expression of enzymes implicated in redox protection, superoxide dismutase (SOD) and catalase, was relatively unaffected by LPS and SNAP. We propose that the induction of IDPc is one of the main self-protection mechanisms of macrophages against LPS-induced oxidative stress.     

Genome damage and reactive oxygen species production in the progenies of irradiated CHO-K1 cells

A series of experiments to study the delayed effects of gamma-radiation exposure in different generations of the progenies of the Chinese hamster ovary CHO-K1 irradiated cells has been conducted. It has been shown that in the progenies of the cells irradiated with a dose of 1 Gy, the following effects are observed: in the 9-27 cell generations - increase in the genome damage, intracellular production of reactive oxygen species, apoptotic cells percentage and cell sensitivity to additional exposure (irradiation at a dose of 10 Gy); in the 30-42 cell generations - decrease of the studied parameters up to control values and increased resistance to additional exposure. It is assumed that the decrease of the studied parameters up to the control values in the 30-42 postirradiation generations of cells is caused by elimination of damaged cells or transition of genomic instability into a hidden (latent) condition.     

Genome damage and reactive oxygen species production in the progenies of irradiated CHO-K1 cells

A series of experiments to study the delayed effects of γ-radiation exposure in different generations of the progenies of irradiated cells of the ovary of the Chinese hamster CHO-K1 is conducted. It is shown that, in the progenies of the cells irradiated at a dose of 1 Gy, the following effects are observed: in cell generations 9–27, an increase in the genome damage, intracellular production of reactive oxygen species, percentage of apoptotic cells, and cell sensitivity to additional exposure (irradiation at a dose of 10 Gy); in cell generations 30–42, a decrease in these parameters to control values and increased resistance to additional exposure. It is assumed that the decrease in these parameters to the control values in postradiation cell generations 30–42 is caused by elimination of damaged cells or transition of genomic instability into a hidden (latent) condition.     

Iron induces protection and necrosis in cultured cardiomyocytes: Role of reactive oxygen species and nitric oxide

We investigate here the role of reactive oxygen species and nitric oxide in iron-induced cardiomyocyte hypertrophy or cell death. Cultured rat cardiomyocytes incubated with 20 μM iron (added as FeCl₃–Na nitrilotriacetate, Fe–NTA) displayed hypertrophy features that included increased protein synthesis and cell size, plus realignment of F-actin filaments along with sarcomeres and activation of the atrial natriuretic factor gene promoter. Incubation with higher Fe–NTA concentrations (100 μM) produced cardiomyocyte death by necrosis. Incubation for 24 h with Fe–NTA (20–40 μM) or the nitric oxide donor Δ-nonoate increased iNOS mRNA but decreased iNOS protein levels; under these conditions, iron stimulated the activity and the dimerization of iNOS. Fe–NTA (20 μM) promoted short- and long-term generation of reactive oxygen species, whereas preincubation with l-arginine suppressed this response. Preincubation with 20 μM Fe–NTA also attenuated the necrotic cell death triggered by 100 μM Fe–NTA, suggesting that these preincubation conditions have cardioprotective effects. Inhibition of iNOS activity with 1400 W enhanced iron-induced ROS generation and prevented both iron-dependent cardiomyocyte hypertrophy and cardioprotection. In conclusion, we propose that Fe–NTA (20 μM) stimulates iNOS activity and that the enhanced NO production, by promoting hypertrophy and enhancing survival mechanisms through ROS reduction, is beneficial to cardiomyocytes. At higher concentrations, however, iron triggers cardiomyocyte death by necrosis.