Reactive oxygen species as mediators of photoreceptor apoptosis in vitro

Retinitis pigmentosa is a heterogeneous group of retinal degenerations characterized by a progressive loss of photoreceptors through the process of apoptosis. The apoptotic cell death of photoreceptors appears to represent a final common pathway in the pathology of retinitis pigmentosa. Previous studies have reported the ability of antioxidants to ameliorate light-induced retinal degeneration, suggesting a role for oxidative stress in photoreceptor cell death. This study demonstrates an early and sustained increase in intracellular reactive oxygen species accompanied by a rapid depletion of intracellular glutathione in an in vitro model of photoreceptor apoptosis. These early changes in the cellular redox state precede disruption of mitochondrial transmembrane potential, nuclear condensation, DNA nicking, and cell shrinkage, all of which are well-characterized events of apoptotic cell death. The ability of zinc chloride and pyrrolidine dithiocarbamate, two established antioxidants, to inhibit photoreceptor apoptosis through the scavenging of intracellular reactive oxygen species establishes a role for reactive oxygen species as possible mediators of in vitro photoreceptor apoptosis. This study provides a molecular basis for the inhibition of photoreceptor apoptosis by antioxidants.     

Ornithine decarboxylase prevents methotrexate-induced apoptosis by reducing intracellular reactive oxygen species production

Methotrexate (MTX), a folate antagonist, was developed for the treatment of malignancies, and is currently used in rheumatoid arthritis (RA) and other chronic inflammatory disorders. It has been proven in short-term and long-term prospective studies that low doses of MTX (0.75 mg/Kg/week) are effective in controlling the inflammatory manifestations of RA. Low-concentrations of MTX achieve apoptosis and clonal deletion of activated peripheral T cells. One of the mechanisms of the anti-inflammatory and immunosuppressive effects may be the production of reactive oxygen species (ROS). However, the drug resistance of MTX in malignancies remains poorly understood. Ornithine decarboxylase (ODC) plays an important role in diverse biological functions, including cell development, differentiation, transformation, growth and apoptosis. In our previous studies, ODC overexpression was shown to prevent TNFα-induced apoptosis via reducing ROS. Here, we also investigated one mechanism of MTX-induced apoptosis and of drug resistance as to the anti-apoptotic effects of ODC during MTX treatment. We found MTX could induce caspase-dependent apoptosis and promote ROS generation together with disrupting the mitochondrial membrane potential (ΔΨ_m) of HL-60 and Jurkat T cells. Putrescine and ROS scavengers could reduce MTX-induced apoptosis, which leads to the loss of ΔΨ_m, through reducing intracellular ROS. Overexpression of ODC in parental cells had the same effects as putrescine and the ROS scavengers. Moreover, ODC overexpression prevented the decline of Bcl-2 that maintains ΔΨ_m, the cytochrome c release and activations of caspase 9 and 3 following MTX treatment. The results demonstrate that MTX-induced apoptosis is ROS-dependent and occurs along a mitochondria-mediated pathway. Overexpressed ODC cells are resistant to MTX-induced apoptosis by reducing intracellular ROS production.     

The role of reactive oxygen species in apoptosis of the diabetic kidney

Increased levels of reactive oxygen species (ROS) by hyperglycemia can induce apoptosis of renal cells and diabetic nephropathy. The redox balance in the renal cell seems, therefore, of the utmost importance. ROS-mediated apoptosis may be further aggravated by an inadequate cytoprotective response against ROS. When there are insufficient cytoprotective and ROS scavenging molecules, ROS lead to considerable cellular damage and to a point of no return in apoptosis. Induction of cytoprotective proteins may prevent or attenuate apoptosis, renal cell injury, and finally diabetic nephropathy. Here, we discuss some mechanisms of apoptosis and several strategies that have been probed to ameliorate, or to prevent apoptosis in the diabetic kidney.     

Cholesterol secoaldehyde induces apoptosis in J774 macrophages via mitochondrial pathway but not involving reactive oxygen species as mediators

Cholesterol secoaldehyde (3β-hydroxy-5-oxo-5,6-secocholestan-6-al or ChSeco) is an oxysterol known to be formed in reactions of ozone with cholesterol and also when cholesterol-5α-hydroperoxide undergoes Hock cleavage. In view of its widespread occurrence and atherogenic potential, we examined the effects of ChSeco on mouse J774 macrophage viability and events associated with apoptosis. A dose-dependent decrease in cell viability, disruptions in mitochondrial transmembrane potential (64 ± 5.5%; mean ± SD, n = 3), increased levels of cytosolic cytochrome c (8.8 ± 0.84 ng/ml; mean ± SD, n = 3), activation of caspase-3 (ca. 3.6-fold) and caspase-9 (ca.1.8-fold), and increased DNA fragmentation (ca. 5-fold), all indicative of apoptosis, were observed in response to exposure to ChSeco. The apoptotic nature of cell death in macrophages was confirmed by dual staining with acridine orange and ethidium bromide. However, unlike the case with cardiomyoblasts and neuronal cells, the apoptotic process in these immune cells was not mediated by increased levels of reactive oxygen species as indicated by a minimal or no increase in 2′,7′-dichlorofluorescein fluorescence. It is suggested that the apoptotic process is mediated via the mitochondrial pathway and that ChSeco formed in biological environments contributes to the initiation, progression, and culmination of atherosclerotic plaque formation, as these processes are critically dependent on macrophage apoptosis.     

Ornithine decarboxylase prevents tumor necrosis factor alpha-induced apoptosis by decreasing intracellular reactive oxygen species

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF- -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential ( _m) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained _m and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of _m and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt _m or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of _m and apoptosis when cells were treated with TNF- . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained _m and prevented apoptosis, but could not reduce ROS until four hours after TNF- treatment. According to these data, we suggest that TNF- induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF- -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain _m, prevent cytochrome c release and deactivate the caspase cascade pathway.     

Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell-ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.     

Beryllium-stimulated reactive oxygen species and macrophage apoptosis

Beryllium (Be), the etiologic agent of chronic beryllium disease, is a toxic metal that induces apoptosis in human alveolar macrophages. We tested the hypothesis that Be stimulates the formation of reactive oxygen species (ROS) which plays a role in Be-induced macrophage apoptosis. Mouse macrophages were exposed to 100 microM BeSO4 in the absence and presence of the catalytic antioxidant MnTBAP (100 microM). Apoptosis was measured as the percentage of TUNEL+ and caspase-8+ cells. ROS production was measured by flow cytometry using the fluorescence probes, dihydroethidine (DHE) and dichlorofluorescein diacetate (DCFH-DA). Be-exposed macrophages had increased TUNEL+ cells (15+/-1% versus controls 1+/-0.2%, P<0.05) and increased caspase-8+ cells (18.7+/-2% versus controls 1.8+/-0.4%, P<0.05). Be-induced caspase-8 activation, and a 4-fold increase in ROS formation, was ameliorated by exposure to MnTBAP. Hydrogen peroxide (30 microM) exposure potentiated Be-induced caspase-8 activation, and was also attenuated by MnTBAP. Our data are the first to demonstrate that Be stimulates macrophage ROS formation which plays an important role in Be-induced macrophage apoptosis.     

Magnificant role of intracellular reactive oxygen species production and its scavenging encompasses downstream processes

Environmental stresses are often associated with production of certain deleterious chemical entities called reactive oxygen species (ROS), which include hydrogen peroxide (H₂O₂), superoxide radical (O₂^?), hydroxyl radical (OH^?). In plants, ROS are formed by the inevitable leakage of electrons onto O₂ from the electron transport activities of chloroplasts, mitochondria, peroxisomes, vacuole and plasma membranes or as a byproduct of various metabolic pathways. Plants have their own antioxidant defense mechanisms to encounter ROS that is of enzymic and non-enzymic nature. Coordinated activities of these antioxidants regulate ROS detoxification and reduces oxidative load in plants. Though ROS are always regarded to impart negative impact on plants, some reports consider them to be important in regulating key cellular functions; however, such reports in plant are limited. On the other hand, specific ROS function as signaling molecules and activate signal transduction processes in response to various stresses is a matter of investigation.     

Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.     

Regulation of T-cell apoptosis by reactive oxygen species

To ensure that a constant number of T cells are preserved in the peripheral lymphoid organs, the production and proliferation of T cells must be balanced out by their death. Newly generated T cells exit the thymus and are maintained as resting T cells. Transient disruption of homeostasis occurs when naïve T cells undergo antigen-induced expansion, a process involving intracellular signaling events that lead to T cell proliferation, acquisition of effector functions, and, ultimately, either apoptosis or differentiation into long-lived memory cells. The last decision point (death vs. differentiation) is a crucial one: it resets lymphoid homeostasis, promotes protective immunity, and limits autoimmunity. Despite its importance, relatively little is known about the molecular mechanisms involved in this cell fate decision. Although multiple mechanisms are likely involved, recent data suggest an underlying regulatory role for reactive oxygen species in controlling the susceptibility of T cells to apoptosis. This review focuses on recent advances in our understanding of how reactive oxygen species modulate T-cell apoptosis.     

Reactive oxygen species as mediators of cell adhesion

The intracellular production of reactive oxygen species (ROS) has a fundamental importance in both cell proliferation and apoptosis induction. Moreover, many experimental and epidemiological evidence indicate that ROS contribute to the initiation and promotion of carcinogenesis, and that drugs or treatments aimed to reduce the tissue content of ROS can be chemopreventive and curative against cancer. Recently, important observations on the role of ROS as physiological regulators of intracellular signaling cascades activated by growth factors through their tyrosine-kinase receptors have shed new light on the possible mechanisms that can sustain the promoting activity of ROS. The downstream effect of ROS production is the reversible oxidation of proteins. Redox sensitive proteins include protein tyrosine phosphatases (PTPs) as the active-site cysteine is the target of specific oxidation, and this modification can be reversed by intracellular reducing agents. The reversible oxidation of PTPs family member was demonstrated firstly for PTP1B during EGF signaling and then for LMW-PTP and SHP-2 during PDGF stimulation. The inhibition exerted by ROS on tyrosine-phosphatases helps the propagation of RTK signals mediated by protein tyrosine phosphorylation, generally associated with the proliferative stimulus. Our new data are consistent with a model in which ROS take a role in integrin signaling, and in which synergistic activation of Rac-1 by growth factors and adhesion molecules translates in a critical increase of intracellular oxidants up to a threshold level where inhibition of the tyrosine phosphatase LMW-PTP takes place. In seeking for potential molecular mechanisms for oxidative signaling by integrins, we found that transient oxidation/inactivation of LMW-PTP, a known negative regulator of RTK signaling, occurred during fibroblast adhesion to matrix, with a kinetic which paralleled the generation of ROS. Moreover, overexpression of LMW-PTP in NIH-3T3 fibroblasts delayed cell attachment to the substrate. Finally, constitutively high levels of intracellular ROS, as are observed in cells expressing active Rac, would attenuate anchorage dependence for growth, by substituting for integrin signaling in non adherent cells.     

Reactive oxygen species as mediators of cellular senescence

Aging has often been viewed as a random process arising from the accumulation of both genetic and epigenetic changes. Increasingly, the notion that aging is a stochastic process is being supplanted by the concept that maximum lifespan of an organism is tightly regulated. This knowledge has led to a growing overlap between classical signal transduction paradigms and the biology of aging. We review certain specific examples where these seemingly disparate disciplines intersect. In particular, we review the concept that intracellular reactive oxygen species function as signalling molecules and that oxidants play a central role as mediators of cellular senescence.     

Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species

Dihydrocalcein (H₂-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H₂-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H₂-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5 min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H₂-calcein but increased the oxidation of H₂-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H₂-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H₂-DCF. In contrast, DPI inhibited H₂-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H₂-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H₂-calcein, but not H₂-DCF inhibited complex I activity.

These observations indicate that H₂-DCF is an indicator for intracellular ROS, whereas the oxidation of H₂-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.     

Mechanical traumatic injury without circulatory shock causes cardiomyocyte apoptosis: role of reactive nitrogen and reactive oxygen species

Apoptotic cell death plays a critical role in tissue injury and organ dysfunction under a variety of pathological conditions. The present study was designed to determine whether apoptosis may contribute to posttraumatic cardiac dysfunction, and if so, to investigate the mechanisms involved. Male adult mice were subjected to nonlethal traumatic injury, and cardiomyocyte apoptosis, cardiac function, and cardiac production of reactive oxygen/nitrogen species were determined. Modified Noble-Collip drum trauma did not result in circulatory shock, and the 24-h survival rate was 100%. No direct mechanical traumatic injury was observed in the heart immediately after trauma. However, cardiomyocyte apoptosis gradually increased and reached a maximal level 12 h after trauma. Significantly, cardiac dysfunction was observed 24 h after trauma in the isolated perfused heart. This was completely reversed when apoptosis was blocked by administration of a nonselective caspase inhibitor immediately after trauma. In the traumatized hearts, reactive nitrogen species (e.g., nitric oxide) and reactive oxygen species (e.g., superoxide) were both significantly increased, and maximal nitric oxide production preceded maximal apoptosis. Moreover, a highly cytotoxic reactive species, peroxynitrite, was markedly increased in the traumatic heart, and there was a significant positive correlation between cardiac nitrotyrosine content and caspase 3 activity. Our present study demonstrated for the first time that nonlethal traumatic injury caused delayed cell death and that apoptotic cardiomyocyte death contributes to posttrauma organ dysfunction. Antiapoptotic treatments, such as blockade of reactive nitrogen oxygen species generation, may be novel strategies in reducing posttrauma multiple organ failure.     

Mitochondrial reactive oxygen species affect sensitivity to curcumin-induced apoptosis

Curcumin exhibits anticancer activity in vivo and triggers tumor cell apoptosis in vivo and in vitro. Several in vitro studies suggest that curcumin-induced apoptosis is associated with reactive oxygen species (ROS) production and/or oxidative stress in transformed cells. This study compared and contrasted the effects of curcumin on human skin cancer cells and their respiration-deficient (rho0) clones to characterize the prospective oxidative stress signaling responsible for initiating apoptosis. Curcumin promoted a dose-and time-dependent G2/M cell cycle arrest and/or apoptosis in COLO 16 cells. Apoptosis induction in COLO 16 cells was associated with DNA fragmentation, cell shrinkage, the externalization of cell membrane phosphatidylserine, and mitochondrial disruption, which were preceded by an increase in intracellular ROS production. Pharmacologically lowering the mitochondrial bioenergetic capacity, as well as the constitutive ROS levels, in COLO 16 cells suppressed the cytotoxic effects of curcumin. Correspondingly, the rho0 counterparts of COLO 16 cells were markedly resistant to ROS production, mitochondrial disruption, and DNA fragmentation following curcumin exposure. These observations implied that the diminution of mitochondrial ROS production protected cells against the cytotoxic effects of curcumin, and support the notion that mitochondrial respiration and redox tone are pivotal determinants in apoptosis signaling by curcumin in human skin cancer cells.     

Reactive oxygen species as mediators of angiotensin II signaling

Angiotensin II stimulates a plethora of signaling pathways leading to cell growth and contraction. Recent work has shown that reactive oxygen species are involved in transducing many of the effects of angiotensin II, and are in fact produced in response to agonist-receptor binding. Angiotensin II stimulates a NAD(P)H oxidase to produce superoxide and hydrogen peroxide, both of which may act on intracellular growth-related proteins and enzymes to mediate the final physiological response. Of particular importance is hydrogen peroxide, which mediates angiotensin II stimulation of such important intracellular signals as EGF-receptor transactivation, p38 mitogen activated protein kinase, and Akt. Future work will be directed towards identifying other important redox-sensitive signaling pathways and their relationship to the physiology and pathophysiology of the renin-angiotensin system.     

Regulation of intracellular signalling through cysteine oxidation by reactive oxygen species

Reactive oxygen species (ROS) have been regarded as harmful molecules that damage various molecules inside cells by oxidation and are responsible for ageing and various human diseases. However, recent studies have revealed an opposite aspect of ROS that these are actively generated in cells and mediate physiological intracellular signalling as second messengers. Several proteins have been shown to function as effectors for ROS, which are sensitively and reversibly oxidized by ROS. Such ROS-effector proteins commonly possess a highly reactive cysteine (Cys) residue, of which oxidation changes the protein function, thus enabling signal transmission to downstream targets. Among the ROS effectors, protein tyrosine phosphatase (PTP), thioredoxin (TRX) and peroxiredoxin (PRX) family proteins possess special domains/motifs to maintain the reactivity of Cys and utilize them to respond to ROS. Progressively advancing identification of ROS-effector proteins reveals the pleiotropic functions of ROS in physiological and pathological cell biology.