The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.     

The importance of sphingolipids and reactive oxygen species in cardiovascular development

The heart is the first organ in the embryo to form. Its structural and functional complexity is the result of a thorough developmental program, where sphingolipids play an important role in cardiogenesis, heart maturation, angiogenesis, the regulation of vascular tone and vessel permeability. Sphingolipids are necessary for signal transduction and membrane microdomain formation. In addition, recent evidence suggests that sphingolipid metabolism is directly interconnected to the modulation of oxidative stress. However, cardiovascular development is highly sensitive to excessive reactive species production, and disturbances in sphingolipid metabolism can lead to abnormal development and cardiac disease. Therefore, in this review, we address the molecular link between sphingolipids and oxidative stress, connecting these pathways to cardiovascular development and cardiovascular disease.     

Role of reactive oxygen species (ROS) in apoptosis induction

Reactive oxygen species (ROS) and mitochondria play an important role in apoptosis induction under both physiologic and pathologic conditions. Interestingly, mitochondria are both source and target of ROS. Cytochrome c release from mitochondria, that triggers caspase activation, appears to be largely mediated by direct or indirect ROS action. On the other hand, ROS have also anti-apoptotic effects. This review focuses on the role of ROS in the regulation of apoptosis, especially in inflammatory cells.     

Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

Critical roles of reactive oxygen species in mitochondrial permeability transition in mediating evodiamine-induced human melanoma A375-S2 cell apoptosis

Previous studies have shown that evodiamine could trigger apoptosis in human malignant melanoma A375-S2 cells within 24 h. To further investigate the biochemical basis of this activity, the roles of reactive oxygen species (ROS) and mitochondrial permeability transition (MPT) were evaluated. Exposure to evodiamine led to a rapid increase in intracellular ROS followed by an onset of mitochondrial depolarization. ROS scavenger rescued the ΔΨm dissipation and cell death induced by evodiamine, whilst MPT inhibitor blocked the second-time ROS formation as well as cell death. Expressions of key proteins in Fas- and mitochondria-mediated pathways were furthermore examined. Both pathways were activated and regulated by ROS and MPT and were converged to a final common pathway involving the activation of caspase-3. These data suggested that a phenomenon termed ROS-induced ROS release (RIRR) was involved in evodiamine-treated A375-S2 cells and greatly contributed to the apoptotic process through both extrinsic and intrinsic pathways.     

BRCA1 down-regulates cellular levels of reactive oxygen species

Previous studies have shown that the breast cancer suppressor BRCA1 stimulates antioxidant gene expression and protects cells against oxidative stress. To further examine this important function, we tested whether BRCA1 could modulate intracellular levels of reactive oxygen species (ROS). Wild-type BRCA1 (but not a cancer-associated mutant) significantly reduced ROS levels, determined by DCF fluorescence assays by flow cytometry and confocal microscopy. The BRCA1 and REF1 pathways for reduction of ROS levels appear to exhibit cross-talk. BRCA1 also reduced the levels of protein nitration and H₂O₂-induced oxidative damage to DNA. Thus, BRCA1 may protect cellular macromolecules by reducing intracellular ROS levels.     

Seed birth to death: dual functions of reactive oxygen species in seed physiology

Background Reactive oxygen species (ROS) are considered to be detrimental to seed viability. However, recent studies have demonstrated that ROS have key roles in seed germination particularly in the release of seed dormancy and embryogenesis, as well as in protection from pathogens.Scope This review considers the functions of ROS in seed physiology. ROS are present in all cells and at all phases of the seed life cycle. ROS accumulation is important in breaking seed dormancy, and stimulating seed germination and protection from pathogens. However, excessive ROS accumulation can be detrimental. Therefore, knowledge of the mechanisms by which ROS influence seed physiology will provide insights that may not only allow the development of seed quality markers but also help us understand how dormancy can be broken in several recalcitrant species.Conclusions Reactive oxygen species have a dual role in seed physiology. Understanding the relative importance of beneficial and detrimental effects of ROS provides great scope for the improvement and maintenance of seed vigour and quality, factors that may ultimately increase crop yields.     

Cellular reactive oxygen species inhibitory constituents of Hypericum thasium Griseb

The phytochemical investigation of the ethyl acetate extract of Hypericum thasium has led to the characterization of four benzophenone derivatives 1-4, a known benzophenone 5 and four known flavonoids, quercetin (6), quercitrin (7), isoquercetin (8), and 3, 8′′-biapigenin (9). Lucigenin- and luminal-based chemiluminescence assays were employed to monitor the inhibitory activity of these compounds towards the production of reactive oxygen species (ROS) by human polymorphoneutrophils (PMNs). The assay results showed that benzophenones 1 and 3 are extracellular inhibitors of ROS production, while flavonoids 6, 8, and 9 can modulate intracellular ROS production.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Involvement of reactive oxygen species in Microcystin-LR-induced cytogenotoxicity

Microcystin-LR (MCLR) is a potent hepatotoxin. Oxidative stress is thought to be implicated in the cytotoxicity of MCLR, but the mechanisms by which MCLR produces reactive oxygen species (ROS) are still unclear. This study investigated the role and possible sources of ROS generation in MCLR-induced cytogenotoxicity in HepG2, a human hepatoma cell line. MCLR increased DNA strand breaks, 8-hydroxydeoxiguanosine formation, lipid peroxidation, as well as LDH release, all of which were inhibited by ROS scavengers. ROS scavengers partly suppressed MCLR-induced cytotoxicity determined by the MTT assay. MCLR induced the generation of ROS, as confirmed by confocal microscopy with 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid, and upregulated the expression of CYP2E1 mRNA. In addition, CYP2E1 inhibitors chlormethiazole and diallyl sulphide inhibited both ROS generation and cytotoxicity induced by MCLR. The results suggest that ROS contribute to MCLR-induced cytogenotoxicity. CYP2E1 might be a potential source responsible for ROS generation by MCLR.     

Tuberous sclerosis complex 2 loss-of-function mutation regulates reactive oxygen species production through Rac1 activation

The products of the TSC1 (hamartin) and TCS2 (tuberin) tumor suppressor genes negatively regulate cell growth by inhibiting mTOR signaling. Recent research has led to the postulation that tuberin and/or hamartin are involved in tumor migration, presumably through Rho activation. Here we show that LEF-8 cells, which contain a Y1571 missense mutation in tuberin, express higher Rac1 activity than tuberin negative and positive cells. We also provide evidence of obvious lamellipodia formation in LEF-8 cells. Since the production of TSC2^Y1571H cannot form a hetero-complex with hamartin, we further analyzed another mutant, TSC2^R611Q, which also lacks the ability to form a complex with hamartin. Introducing both forms of mutated TSC2 into COS-1 cells increased Rac1 activity as well as cell motility. We also found these two mutants interacted with Rac1. We further demonstrated that the introduction of mutated TSC2 into COS-1 cells can generate higher reactive oxygen species (ROS). These results indicate that loss-of-function mutated tuberin can activate Rac1 and thereby increase ROS production.     

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.     

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.     

Novel Nox inhibitor of oxLDL-induced reactive oxygen species formation in human endothelial cells

In this study, we investigated effects of a novel NAD(P)H oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2870) on oxidized low-density lipoprotein (oxLDL)-mediated reactive oxygen species (ROS) formation in human endothelial cells. Primary cultures of human umbilical vein endothelial cells were cultured to confluence and ROS formation was induced with 50microg/ml oxLDL for 2h. ROS formation was detected by chemiluminescence (CL) using the Diogenes reagent. OxLDL induced ROS formation in human endothelial cells (171+/-12%; n=10, P<0.05 vs. control). This augmented ROS formation in response to oxLDL was completely inhibited by the Nox inhibitor VAS2870 (101+/-9%; n=7, P<0.05 vs. oxLDL). Similar results were obtained with superoxide dismutase (91+/-7%; n=7, P<0.05 vs. oxLDL). However, the Nox4 mRNA expression level was neither changed by oxLDL nor VAS2870. We conclude that VAS2870 could provide a novel strategy to inhibit the augmented endothelial superoxide anion formation in response to cardiovascular risk factors.     

Involvement of Ca in globular adiponectin-induced reactive oxygen species

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca²⁺ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca²⁺ ([Ca²⁺]_i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca²⁺]_i occurred 30 min after gAd treatment, after which [Ca²⁺]_i increased continually and exceeded the initial level. The mitochondrial Ca²⁺ ([Ca²⁺]_m) detected by Rhod-2 fluorescence started to increase at 6 h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca²⁺]_i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca²⁺]_m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity.     

Plant responses to water stress: Role of reactive oxygen species

Terrestrial plants most often encounter drought stress because of erratic rainfall which has become compounded due to present climatic changes.Responses of plants to water stress may be assigned as either injurious change or tolerance index. One of the primary and cardinal changes in response to drought stress is the generation of reactive oxygen species (ROS), which is being considered as the cause of cellular damage. However, recently a signaling role of such ROS in triggering the ROS scavenging system that may confer protection or tolerance against stress is emerging. Such scavenging system consists of antioxidant enzymes like SOD, catalase and peroxidases, and antioxidant compounds like ascorbate, reduced glutathione; a balance between ROS generation and scavenging ultimately determines the oxidative load. As revealed in case of defence against pathogen, signaling via ROS is initiated by NADPH oxidase-catalyzed superoxide generation in the apoplastic space (cell wall) followed by conversion to hydrogen peroxide by the activity of cell wall-localized SOD. Wall peroxidase may also play role in ROS generation for signaling. Hydrogen peroxide may use Ca2+ and MAPK pathway as downstream signaling cascade. Plant hormones associated with stress responses like ABA and ethylene play their role possibly via a cross talk with ROS towards stress tolerance, thus projecting a dual role of ROS under drought stress.