Management of multicellular senescence and oxidative stress

Progressively sophisticated understanding of cellular and molecular processes that contribute to age‐related physical deterioration is being gained from ongoing research into cancer, chronic inflammatory syndromes and other serious disorders that increase with age. Particularly valuable insight has resulted from characterization of how senescent cells affect the tissues in which they form in ways that decrease an organism's overall viability. Increasingly, the underlying pathophysiology of ageing is recognized as a consequence of oxidative damage. This leads to hyperactivity of cell growth pathways, prominently including mTOR (mammalian target of rapamycin), that contribute to a build‐up in cells of toxic aggregates such as progerin (a mutant nuclear cytoskeletal protein), lipofuscin and other cellular debris, triggering formation of senescent cellular phenotypes, which interact destructively with surrounding tissue. Indeed, senescent cell ablation dramatically inhibits physical deterioration in progeroid (age‐accelerated) mice. This review explores ways in which oxidative stress creates ageing‐associated cellular damage and triggers induction of the cell death/survival programs’ apoptosis, necrosis, autophagy and ‘necroapoptophagy’. The concept of ‘necroapoptophagy’ is presented here as a strategy for varying tissue oxidative stress intensity in ways that induce differential activation of death versus survival programs, resulting in enhanced and sustained representation of healthy functional cells. These strategies are discussed in the context of specialized mesenchymal stromal cells with the potential to synergize with telocytes in stabilizing engrafted progenitor cells, thereby extending periods of healthy life. Information and concepts are summarized in a hypothetical approach to suppressing whole‐organism senescence, with methods drawn from emerging understandings of ageing, gained from Cnidarians (jellyfish, corals and anemones) that undergo a unique form of cellular regeneration, potentially conferring open‐ended lifespans.     

Ethylene as a regulator of senescence in tobacco leaf discs

The regulatory role of ethylene in leaf senescence was studied with excised tobacco leaf discs which were allowed to senesce in darkness. Exogenous ethylene, applied during the first 24 hours of senescence, enhanced chlorophyll loss without accelerating the climacteric-like pattern of rise in both ethylene and CO₂, which occurred in the advanced stage of leaf senescence. Rates of both ethylene and CO₂ evolution increased in the ethylene-treated leaf discs, especially during the first 3 days of senescence. The rhizobitoxine analog, aminoethoxy vinyl glycine, markedly inhibited ethylene production and reduced respiration and chlorophyll loss. Pretreatment of leaf discs with Ag⁺ or enrichment of the atmosphere with 5 to 10% CO₂ reduced chlorophyll loss, reduced rate of respiration, and delayed the climacteric-like rise in both ethylene and respiration. Ag⁺ was much more effective than CO₂ in retarding leaf senescence. Despite their senescence-retarding effect, Ag⁺ and CO₂, which are known to block ethylene action, stimulated ethylene production by the leaf discs during the first 3 days of the senescing period; Ag⁺ was more effective than CO₂. The results suggest that although ethylene production decreases prior to the climacteric-like rise during the later stages of senescence, endogenous ethylene plays a considerable role throughout the senescence process, presumably by interacting with other hormones participating in leaf senescence.     

The senescence accelerated mouse (SAMP8) as a model for oxidative stress and Alzheimer's disease

The senescence accelerated mouse (SAMP8) is a spontaneous animal model of overproduction of amyloid precursor protein (APP) and oxidative damage. It develops early memory disturbances and changes in the blood-brain barrier resulting in decreased efflux of amyloid-β protein from the brain. It has a marked increase in oxidative stress in the brain. Pharmacological treatments that reduce oxidative stress improve memory. Treatments that reduce amyloid-β (antisense to APP and antibodies to amyloid-β) not only improve memory but reduce oxidative stress. Early changes in lipid peroxidative damage favor mitochondrial dysfunction as being a trigger for amyloid-β overproduction in this genetically susceptible mouse strain. This sets in motion a cycle where the increased amyloid-beta further damages mitochondria. We suggest that this should be termed the Inflammatory-Amyloid Cycle and may well be similar to the mechanisms responsible for the pathophysiology of Alzheimer's disease. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.     

Pollination-induced floral senescence in orchids: Status of oxidative stress

The orchid flowers may stay fresh in unpollinated state from few weeks to months but show rapid senescence upon pollination. Metabolic changes related to this phenomenon are less well understood in orchid flowers. Presently, two orchid species, Aerides multiflora Roxb. and Rhynchostylis retusa (L.) Bl., varying in their floral life span were evaluated for their postpollination-induced responses, involving the oxidative stress. The unpollinated flowers of A. multiflora stayed fresh for 17 days and attained senescence in 5 days after pollination (DAP), while those of R. retusa. remained fresh for 24 days and showed senescence in 7 DAP. After pollination, wilting began in 2 to 3 days in A. multiflora and 3 to 4 days in R. retusa. There was a higher electrolyte leakage accompanied by a concomitant increase in the levels of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), indicators of oxidative damage in all the organs after pollination while ascorbic acid decreased significantly. The flowers of A. multiflora showed a greater electrolyte leakage, MDA and H₂O₂ contents as compared to those of R. retusa. Ascorbic acid content, on the other hand, was lower in A. multiflora than in R. retusa, suggesting a higher oxidative damage to the floral organs in the former species. An application of triiodobenzoic acid ( an auxin inhibitor; 0.25 mM) and silver nitrate (ethylene inhibitor; 0.25 mM) to pollinated flowers partially prevented the oxidative damage and consequently the senescence, suggesting the involvement of these hormones. AgNO₃ was more effective in delaying senescence.     

The correlation between oxidative stress and leaf senescence during plant development

In plants, besides being the final step leading to the death of the whole organism, senescence has a developmental function involving the coordinated degradation of macromolecules and the mobilization of nutrients out of senescing tissues into developing parts of the plant. Free radicals are thought to play an essential role in senescence, especially those derived from oxygen. Since these molecules are extremely toxic, the levels of the different reactive oxygen species have to be tightly regulated. However, at low concentrations, hydrogen peroxide may also serve as a signalling molecule. Therefore, a coordinated regulation of the free radical scavenging system, which comprises enzymatic components such as catalase, superoxide dismutase and ascorbate peroxidase, and non-enzymatic molecules such as ascorbate and glutathione is essential. The increased radical levels displayed during senescence are not only caused by the elevated production of radicals but also by a loss in antioxidant capacity.     

Death-associated protein 3 regulates cellular senescence through oxidative stress response

Death-associated protein 3 (DAP3) has been originally identified as a positive mediator of apoptosis. It has been revealed recently that the predominant localization of DAP3 to mitochondria implies its functional involvement in mitochondrial metabolism in addition to apoptosis. However, little is known about the molecular basis of these physiological functions of DAP3. Here, we demonstrate that DAP3 is reduced in both replicative and premature senescence induced by oxidative stress, and the DAP3 reduction induced by oxidative stress is observed mostly in a mitochondrial fraction. Using DAP3-specific short hairpin RNA (shRNA) in a clonogenic survival assay, we reveal that reduction of DAP3 induces resistance to oxidative stress and decreases intracellular reactive oxygen species (ROS) production. Furthermore, this strategy allows us to show that loss of DAP3 is involved in the avoidance of replicative senescence in mouse embryonic fibroblasts (MEFs). Thus, our study offers an insight into the potential regulatory function of mitochondrial DAP3 involved in cellular senescence.     

Lactoferrin-protector against oxidative stress and regulator of glycolysis in human erythrocytes

Binding of lactoferrin (Lf) to its membrane receptors requires an electron for the reduction of Fe(3+)LF to Fe(2+)LF. It is possible that glyceraldehyde -3-phosphate dehydrogenase, a glycolytic enzyme part of the erythrocyte membrane, delivers that electron. Then Lf, obtaining an electron from the coenzyme NADH, might stimulate glycolysis, which requires the oxidised state of the coenzyme NAD+. Such possibility is supported by the finding that another extracellular e- acceptor--potassium ferricyanide activates glycolysis by the similar mechanism. Present results show that ferricyanide inhibited the specific 59Fe-lactoferrin binding to its erythrocyte membrane receptors. It may be assumed that ferricyanide competes with lactoferrin for an electron which leads to decrease of the binding of 59Fe-lactoferrin to its receptors. Lactoferrin (50 and 100 nM), similar to ferricyanide, increased the accumulation of lactate (respectively by 25% and 30%). These results support the assumption that ferricyanide and lactoferrin are final acceptors of a common electron transport chain connected with the regulation of glycolysis. We established an antioxidative effect of lactoferrin on erythrocytes, which was expressed as: a) an influence on content and on activity of intracellular antioxidants--namely an enhancement of the content of reduced glutathione; b) a decreased content both of products of lipid peroxidation (thiobarbituric acid reactive substances) and hemolysis under normal conditions and oxidative stress. Lactoferrin is capable to bind metal ions and thus to block their catalytic participation in the oxidative disturbances of the membrane. In most of our experiments there were no metal ions in the incubation mixtures (except those stimulating oxidative stress). Our results showed that Lf limited both the generation of thiobarbituric acid reactive substances and hemolysis in the absence of metal ions in the media, as well as in their presence. These facts suggest that probably the antioxidative property of lactoferrin is glycolysis stimulation, leading to increased formation of ATP, which is necessary to maintain the ion gradient, membrane potential and morphology of the erythrocyte.     

Mouse models of mitochondrial disease, oxidative stress, and senescence.

During the course of normal respiration, reactive oxygen species are produced which are particularly detrimental to mitochondrial function. This is shown by recent studies with a mouse that lacks the mitochondrial form of superoxide dismutase (Sod2). Tissues that are heavily dependent on mitochondrial function such as the brain and heart are most severely affected in the Sod2 mutant mouse. Recent work with a mouse mutant for the heart/muscle specific isoform of the mitochondrial adenine nuclear translocator (Ant1) demonstrates a potential link between mitochondrial oxidative stress and mitochondrial DNA mutations. These mutations can be detected by Long-extension PCR, a method for detecting a wide variety of mutations of the mitochondrial genome. Such mutations have also been observed in the mitochondrial genome with senescence regardless of the mean or maximal lifespan of the organism being studied. Mutations have been detected with age in Caenorhabditis elegans, mice, chimpanzees, and humans. This implies that a causal relationship may exist between mitochondrial reactive oxygen species production, and the senescence specific occurrence of mitochondrial DNA mutations.     

Bcl-2 protects against oxidative stress while inducing premature senescence

Replicative senescence is a cellular response to stress that has been postulated to serve as a tumor suppression mechanism and a contributor to aging. Numerous experimental studies have demonstrated that an apparently identical senescent state can also be prematurely induced in vitro in different cell types following sublethal oxidative stress stimuli. The former suggests a molecular link between cell cycle regulation and cell survival that could involve regulatory proteins such as Bcl-2. There is strong evidence that, in addition to its well-known effects on apoptosis, Bcl-2 is involved in antioxidant protection and regulation of cell cycle progression. The aim of this work was to determine if the protection against oxidative stress mediated by Bcl-2 could prevent or delay oxidative stress-induced senescence. Using a retroviral infection system, Bcl-2 was overexpressed in primary, nonembryonic mice fibroblasts obtained from lungs derived from 2-month-old CD1 mice. Fibroblasts overexpressing Bcl-2 were exposed to 75 microM H2O2 for 2 h to induce SIPS. The rate of proliferation and the increment of senescent cells were then determined. Our results indicate that overexpression of Bcl-2 protected primary fibroblasts against oxidative stress-mediated reduction in cell proliferation, but did not prevent premature senescence.     

The 1-Cys peroxiredoxin, a regulator of seed dormancy, functions as a molecular chaperone under oxidative stress conditions

Peroxiredoxins are antioxidative enzymes that catalyze the reduction of alkyl hydroperoxides to alcohols and hydrogen peroxide to water. 1-Cys peroxiredoxins (1-Cys Prxs) perform important roles during late seed development in plants. To characterize their biochemical functions in plants, a 1Cys-Prx gene was cloned from a Chinese cabbage cDNA library and designated as “C1C-Prx”. Glutamine synthetase (GS) protection and hydrogen peroxide reduction assays indicated that C1C-Prx was functionally active as a peroxidase. Also C1C-Prx prevented the thermal- or chemical-induced aggregation of malate dehydrogenase and insulin. Hydrogen peroxide treatment changed the mobility of C1C-Prx on a two-dimensional gel, which implies overoxidation of the conserved Cys residue. Furthermore, after overoxidation, the chaperone activity of C1C-Prx increased approximately two-fold, but its peroxidase activity decreased to the basal level of the reaction mixture without enzyme. However, according to the structural analysis using far-UV circular dichroism spectra, intrinsic tryptophan fluorescence spectra, and native-PAGE, overoxidation did not lead to a conformational change in C1C-Prx. Therefore, our results suggest that 1-Cys Prxs function not only to relieve mild oxidative stresses but also as molecular chaperones under severe conditions during seed germination and plant development, and that overoxidation controls the switch in function of 1-Cys-Prxs from peroxidases to molecular chaperones.     

Staphylococcus aureus CymR is a new thiol-based oxidation-sensing regulator of stress resistance and oxidative response

As a human pathogen, Staphylococcus aureus must cope with oxidative stress generated by the human immune system. Here, we report that CymR utilizes its sole Cys-25 to sense oxidative stress. Oxidation followed by thiolation of this cysteine residue leads to dissociation of CymR from its cognate promoter DNA. In contrast, the DNA binding of the CymRC25S mutant was insensitive to oxidation and thiolation, suggesting that CymR senses oxidative stress through oxidation of its sole cysteine to form a mixed disulfide with low molecular weight thiols. The determined crystal structures of the reduced and oxidized forms of CymR revealed that Cys-25 is oxidized to Cys-25-SOH in the presence of H(2)O(2). Deletion of cymR reduced the resistance of S. aureus to oxidative stresses, and the resistance was restored by expressing a C25S mutant copy of cymR. In a C25S substitution mutant, the expression of two genes, tcyP and mccB, was constitutively repressed and did not respond to hydrogen peroxide stress, whereas the expression of the genes were highly induced under oxidative stress in a wild-type strain, indicating the critical role of Cys-25 in redox signaling in vivo. Thus, CymR is another master regulator that senses oxidative stress and connects stress responses to virulence regulation in S. aureus.     

Effect of water stress on some oxidative enzymes and senescence in Vigna seedlings

Seedlings of Vigna catjang Endl. were subjected to water stress for 6, S and 10 days by withholding water to investigate the activities of some oxidative enzymes and the pattern of senescence in leaves of 17-day-old seedlings undergoing water stress. Increasing duration of stress produced a proportional increase in the activities of IAA-oxidase, AA-oxidase, peroxidase and glycolate oxidase but decreased catalase activity and the contents of both chlorophyll and protein, hastening senescence. Leaf water potential and relative water content were also lowered with incresing duration of stress. Permeability was increased in leaf tissue undergoing water stress for 8 days. Seed treatment with CaCl₂ (10⁻² and 10⁻¹⁴ M ) for 6 h improved the water status of leaves, decreased tissue permeability, activities of oxidative enzymes, decline of chlorophyll and protein contents and delayed senescence compared to untreated water stressed plants.     

Dityrosine as a product of oxidative stress and fluorescent probe

Summary. Dityrosine can be a natural component of protein structure, a product of environmental stress, or a product of in vitro protein modification. It is both a cross-link and a fluorescent probe that reports structural and functional information on the cross-linked protein molecule. Diverse reactions produce tyrosyl radicals, which in turn may couple to yield dityrosine. Identification and quantitation of dityrosine in protein hydrolysates usually employs reversed phase high pressure liquid chromatography (RP-HPLC) or gas chromatography. RP-HPLC of protein hydrolysates that have been derivatized with dabsyl chloride gives a complete amino acid analysis that includes dityrosine and 3-nitrotyrosine. Calmodulin, which contains a single pair of tyrosyl residues, undergoes both photoactivated and enzyme-catalyzed dityrosine formation. Polarization measurements, employing the intrinsic fluorescence of dityrosine, and catalytic activity determinations show how different patterns of inter- and intramolecular cross-linking affect the interactions of calmodulin with Ca²⁺ and enzymes.     

Magmas functions as a ROS regulator and provides cytoprotection against oxidative stress-mediated damages

Redox imbalance generates multiple cellular damages leading to oxidative stress-mediated pathological conditions such as neurodegenerative diseases and cancer progression. Therefore, maintenance of reactive oxygen species (ROS) homeostasis is most important that involves well-defined antioxidant machinery. In the present study, we have identified for the first time a component of mammalian protein translocation machinery Magmas to perform a critical ROS regulatory function. Magmas overexpression has been reported in highly metabolically active tissues and cancer cells that are prone to oxidative damage. We found that Magmas regulates cellular ROS levels by controlling its production as well as scavenging. Magmas promotes cellular tolerance toward oxidative stress by enhancing antioxidant enzyme activity, thus preventing induction of apoptosis and damage to cellular components. Magmas enhances the activity of electron transport chain (ETC) complexes, causing reduced ROS production. Our results suggest that J-like domain of Magmas is essential for maintenance of redox balance. The function of Magmas as a ROS sensor was found to be independent of its role in protein import. The unique ROS modulatory role of Magmas is highlighted by its ability to increase cell tolerance to oxidative stress even in yeast model organism. The cytoprotective capability of Magmas against oxidative damage makes it an important candidate for future investigation in therapeutics of oxidative stress-related diseases.Reactive oxygen species (ROS) are the chemical species formed by the incomplete reduction of oxygen and includes superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), singlet oxygen and hydroxyl radicals (·OH).^{1, 2} ROS is generated primarily as a by-product of cellular metabolism through leakage of electrons by electron transport chain (ETC) in mitochondria and from other sources such as plasma membrane, peroxisomes and endoplasmic reticulum.^{3, 4} ROS acts as signaling molecule when present at an appropriate level through the covalent modification of specific cysteine residues of redox-sensitive target proteins.⁵ The optimum level of ROS is maintained by equilibrium between its production and scavenging through the involvement of antioxidant system. An alteration in this equilibrium gives rise to oxidative stress, leading to cellular damage that finally precipitates into neurodegenerative disorders, cancer and metabolic disorders such as diabetes.^{6, 7, 8, 9, 10, 11}Therefore, for the maintenance of redox equilibrium, surveillance on generation of ROS is as critical as ROS scavenging. Mitochondria being the primary source of ROS generation by ETC assume the important center for maintenance of ROS levels. The factors that control ROS production by ETC complexes are not well defined. Mitochondria harbors a number of ROS scavenging enzymes such as intermembrane space-associated Cu–Zn superoxide dismutase (Cu-Zn SOD), matrix-localized MnSOD, isoforms of peroxiredoxins and glutaredoxins.¹² Together, these proteins help in maintaining the appropriate cellular ROS level. Maintenance of optimum level of ROS is important for developing tissues and stem cells and in metabolically active tissues where the high-energy demand makes them more vulnerable to oxidative stress. In addition to this, cancer cells having aberrant metabolism tend to have enhanced ROS that helps in tumor progression; however, abnormally high ROS may lead to apoptosis.^{13, 14, 15, 16, 17} Thus, in cancer cells, for their prolonged survival, there exists an evolved mechanism to maintain the balance of redox state through upregulation of many antioxidant enzymes^{9, 10, 11} and a number of signaling molecules modulating the expression level of these enzymes.^{18, 19}In earlier reports, overexpression of ‘Magmas'' was observed in patient samples of prostate cancer, pituitary adenoma, in various developmental stages and in metabolically active human tissues.²⁰ Originally, Magmas was identified as a protein involved in granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling and was found to localize in mitochondria.^{21, 22} Magmas belongs to type IV class of J-proteins and acts as co-chaperone of mitochondrial heat shock protein 70 (mtHsp70). It is an inner membrane-associated protein and an essential component of mitochondrial protein translocation machinery. Magmas inhibits the activity of its J-protein counterpart DnaJC19 in stimulating ATPase activity of mtHsp70 at the transport channel and regulates the import of nuclear-encoded mitochondrial proteins into the matrix.²²Although overexpression of Magmas in energy-demanding tissues and cancer cells is well known, the physiological advantage provided by the enhanced expression of the protein is still enigmatic. In the present study, we have provided compelling evidence favoring an additional function of Magmas as a ‘ROS regulator''. The overexpression of Magmas led to reduction in ROS and increased cellular tolerance to oxidative stress, whereas its downregulation elevated the cellular ROS level and made the cells more susceptible to ROS-mediated apoptosis. To maintain the redox equilibrium and provide cytoprotection against oxidative stress, Magmas controls ROS production by enhancing the ETC complex activity. On the other hand, it increases the activity of two major antioxidant enzymes such as MnSOD and glutathione peroxidase (GPx) to promote ROS scavenging. In summary, we propose a novel essential role of Magmas as a ‘ROS regulatory protein'' in the maintenance of cellular redox homeostasis and imparting cytoprotection under oxidative stress.     

Potential involvement of oxidative stress in cartilage senescence and development of osteoarthritis: oxidative stress induces chondrocyte telomere instability and downregulation of chondrocyte function

Oxidative stress leads to increased risk for osteoarthritis (OA) but the precise mechanism remains unclear. We undertook this study to clarify the impact of oxidative stress on the progression of OA from the viewpoint of oxygen free radical induced genomic instability, including telomere instability and resulting replicative senescence and dysfunction in human chondrocytes. Human chondrocytes and articular cartilage explants were isolated from knee joints of patients undergoing arthroplastic knee surgery for OA. Oxidative damage and antioxidative capacity in OA cartilage were investigated in donor-matched pairs of intact and degenerated regions of tissue isolated from the same cartilage explants. The results were histologically confirmed by immunohistochemistry for nitrotyrosine, which is considered to be a maker of oxidative damage. Under treatment with reactive oxygen species (ROS; 0.1 μmol/l H₂O₂) or an antioxidative agent (ascorbic acid: 100.0 μmol/l), cellular replicative potential, telomere instability and production of glycosaminoglycan (GAG) were assessed in cultured chondrocytes. In tissue cultures of articular cartilage explants, the presence of oxidative damage, chondrocyte telomere length and loss of GAG to the medium were analyzed in the presence or absence of ROS or ascorbic acid. Lower antioxidative capacity and stronger staining of nitrotyrosine were observed in the degenerating regions of OA cartilages as compared with the intact regions from same explants. Immunostaining for nitrotyrosine correlated with the severity of histological changes to OA cartilage, suggesting a correlation between oxidative damage and articular cartilage degeneration. During continuous culture of chondrocytes, telomere length, replicative capacity and GAG production were decreased by treatment with ROS. In contrast, treatment with an antioxidative agent resulted in a tendency to elongate telomere length and replicative lifespan in cultured chondrocytes. In tissue cultures of cartilage explants, nitrotyrosine staining, chondrocyte telomere length and GAG remaining in the cartilage tissue were lower in ROS-treated cartilages than in control groups, whereas the antioxidative agent treated group exhibited a tendency to maintain the chondrocyte telomere length and proteoglycan remaining in the cartilage explants, suggesting that oxidative stress induces chondrocyte telomere instability and catabolic changes in cartilage matrix structure and composition. Our findings clearly show that the presence of oxidative stress induces telomere genomic instability, replicative senescence and dysfunction of chondrocytes in OA cartilage, suggesting that oxidative stress, leading to chondrocyte senescence and cartilage ageing, might be responsible for the development of OA. New efforts to prevent the development and progression of OA may include strategies and interventions aimed at reducing oxidative damage in articular cartilage.     

Peroxiredoxins as biomarkers of oxidative stress

Background

Peroxiredoxins (Prxs) are a class of abundant thiol peroxidases that degrade hydroperoxides to water. Prxs are sensitive to oxidation, and it is hypothesized that they also act as redox sensors. The accumulation of oxidized Prxs may indicate disruption of cellular redox homeostasis.

Scope of review

This review discusses the biochemical properties of the Prxs that make them suitable as endogenous biomarkers of oxidative stress, and describes the methodology available for measuring Prx oxidation in biological systems.

Major conclusions

Two Prx oxidation products accumulate in cells under increased oxidative stress: an intermolecular disulfide and a hyperoxidized form. Methodologies are available for measuring both of these redox states, and oxidation has been reported in cells and tissues under oxidative stress from external or internal sources.

General significance

Monitoring the oxidation state of Prxs provides insight into disturbances of cellular redox homeostasis, and complements the use of exogenous probes of oxidative stress. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.