Steady-state Levels Of Reactive Oxygen Species Research Articles

Role of Oxidative Stress in Heart Failure: Insights from Gene Transfer Studies.

Under physiological circumstances, there is an exquisite balance between reactive oxygen species (ROS) production and ROS degradation, resulting in low steady-state ROS levels. ROS participate in normal cellular function and in cellular homeostasis. Oxidative stress is the state of a transient or a persistent increase of steady-state ROS levels leading to disturbed signaling pathways and oxidative modification of cellular constituents. It is a key pathophysiological player in pathological hypertrophy, pathological remodeling, and the development and progression of heart failure. The heart is the metabolically most active organ and is characterized by the highest content of mitochondria of any tissue. Mitochondria are the main source of ROS in the myocardium. The causal role of oxidative stress in heart failure is highlighted by gene transfer studies of three primary antioxidant enzymes, thioredoxin, and heme oxygenase-1, and is further supported by gene therapy studies directed at correcting oxidative stress linked to metabolic risk factors. Moreover, gene transfer studies have demonstrated that redox-sensitive microRNAs constitute potential therapeutic targets for the treatment of heart failure. In conclusion, gene therapy studies have provided strong corroborative evidence for a key role of oxidative stress in pathological remodeling and in the development of heart failure.

Highlighting reactive oxygen species as multitaskers in root development.

SummaryReactive oxygen species (ROS) are naturally produced by several redox reactions during plant regular metabolism such as photosynthesis and respiration. Due to their chemical properties and high reactivity, ROS were initially described as detrimental for cells during oxidative stress. However, they have been further recognized as key players in numerous developmental and physiological processes throughout the plant life cycle. Recent studies report the important role of ROS as growth regulators during plant root developmental processes such as in meristem maintenance, in root elongation, and in lateral root, root hair, endodermis, and vascular tissue differentiation. All involve multifaceted interplays between steady-state levels of ROS with transcriptional regulators, phytohormones, and nutrients. In this review, we attempt to summarize recent findings about how ROS are involved in multiple stages of plant root development during cell proliferation, elongation, and differentiation.

Talinum paniculatum: a plant with antifungal potential mitigates fluconazole-induced oxidative damage-mediated growth inhibition of Candida albicans

Aims: This study investigated the bioactivity of the crude leaf extract (CLE) and fractions hexane (HX) and ethyl acetate (EtOAc) from Talinum paniculatum alone and in association with fluconazole (FLC) against reference strain and clinical isolates of FLC-resistant Candida albicans. Furthermore, the antioxidant capability, chemical composition of this plant, and the effect’s underlying mechanisms were evaluated. Methods: The antifungal activity was evaluated using checkerboard assay to establish the minimum inhibitory (MIC) and minimum microbicidal concentrations (MMC). During FLC and plant products challenges, the reactive oxygen species (ROS) generation (hydroxyl radicals [HO•]) were detected in C. albicans cells using the membrane-permeable fluorescent probes APF and HPF. High-performance liquid chromatography (HPLC) profile, quantitative analysis of antioxidant compounds, and free radical scavenging activity (DPPH assay) tests were performed. Results: The CLE and fractions presented outstanding antifungal activity and selectivity against C. albicans cells but had no synergistic effects with FLC. The MIC values for CLE and its fractions against C. albicans reference strain were in the order of HX (31.25 μg ml–1) < EtOAc (62.5 μg ml–1) < CLE (500 μg ml–1), and against FLC-resistant C. albicans HX (125 μg ml–1) = EtOAc < CLE (500 μg ml–1). CLE and its fractions had more potent antifungal activities than FLC against the clinical isolates. Moreover, fungicidal effects for these plant products were demonstrated against FLC-resistant C. albicans, which further confirmed an antifungal potential. Conversely, during association, plant products were shown to cause an increase in FLC MIC anywhere from 2- to 16-fold. FLC exposure led to an increase in the steady-state levels of ROS (HO•) in C. albicans cells. Next, we found that the increases in FLC MICs were owing to action of antioxidants containing-CLE and its fractions in preventing FLC-induced ROS-mediated growth inhibition of C. albicans. Conclusion: T. paniculatum can be a source of bioactive compounds with antifungal potential. However, because of the common use of its edible leaf, caution is advised during therapy with FLC (since it can decrease FLC susceptibility).

Resveratrol Enhances Apoptotic and Oxidant Effects of Paclitaxel through TRPM2 Channel Activation in DBTRG Glioblastoma Cells.

Numerous studies have reported a strong association between increased production of reactive oxygen species (ROS) and the pathobiology of several diseases, and cancer in particular. Therefore, manipulation of cellular oxidative stress levels represents an important therapeutic target. Recently, resveratrol (RESV), a naturally occurring phytochemical, has been shown to sensitize several cell lines to the anticancer effects of other chemotherapeutic agents, including paclitaxel (PAX). However, the molecular mechanisms of action of RESV through oxidative sensitive TRPM2 channel activation remain unclear. The aim of this study was to evaluate the effect of combination therapy of RESV and PAX on activation of TRPM2 in DBTRG glioblastoma cells. DBTRG cells were divided into four treatment groups: control, RESV (50 μM), PAX (50 μM), and PAX + RESV for 24 hours. Our data shows that markers for apoptosis, mitochondrial membrane depolarization and mitochondrial function, intracellular steady-state ROS levels, caspase 3 activity, TRPM2 current density, and Ca2+ florescence intensity were significantly increased in DBTRG cells following treatment with PAX and RESV, respectively, although cell viability was also decreased by these treatments. These biochemical markers were further increased to favor the anticancer effects of PAX in DBTRG cells in combination with RESV. The PAX and RESV-mediated increase in current density and Ca2+ florescence intensity was decreased with a TRPM2 blocker. This suggests that for this combination therapy to have a substantial effect on apoptosis and cell viability, the TRPM2 channel must be stimulated.

A mongolian pine specific endoplasmic reticulum localized CALMODULIN-LIKE calcium binding protein enhances arabidopsis growth

Stress-adapted wild plants are natural sources of novel genes for molecular breeding. Here, we conducted a transcriptional analysis of Pinus sylvestris var. mongolica Litv, an evergreen pine in northeastern China, to identify a novel CALMODULIN-LIKE protein-encoding gene, PsCML1, no significant homologs found in other plant species. PsCML1 encodes a protein predicted to have a single trans-membrane domain at its N-terminal. Four EF-hand motifs (calcium [Ca]-binding structures) are located at its C-terminal and showed Ca2+-specific affinity in isothermal titration calorimetric analysis. Transient expression of PsCML1 in Nicotiana benthamiana showed that the PsCML1 localizes to the endoplasmic reticulum (ER). Heterologous expression of PsCML1 in Arabidopsis significantly promoted seedling growth, and increased resistance to stress from NaCl and Ca2+ deficiency. The roots of the transgenic seedlings had higher contents of cellulose and pectin, but less hemicellulose than those of the wild type (WT). The biosynthesis of cell wall components is linked with protein glycosylation in the ER and reactive oxygen species (ROS) homeostasis. No significant difference was found in the extent of protein glycosylation between the transgenic and WT plants. However, the transgenic roots had higher steady-state levels of ROS, NADPH oxidase activity, and endo-membrane dynamics than those of the WT. A working model was proposed to delineate the interaction among Ca2+, ROS homeostasis, and cell wall loosening-dependent cell division.

Reactive oxygen species, abiotic stress and stress combination.

Reactive oxygen species (ROS) play a key role in the acclimation process of plants to abiotic stress. They primarily function as signal transduction molecules that regulate different pathways during plant acclimation to stress, but are also toxic byproducts of stress metabolism. Because each subcellular compartment in plants contains its own set of ROS-producing and ROS-scavenging pathways, the steady-state level of ROS, as well as the redox state of each compartment, is different at any given time giving rise to a distinct signature of ROS levels at the different compartments of the cell. Here we review recent studies on the role of ROS in abiotic stress in plants, and propose that different abiotic stresses, such as drought, heat, salinity and high light, result in different ROS signatures that determine the specificity of the acclimation response and help tailor it to the exact stress the plant encounters. We further address the role of ROS in the acclimation of plants to stress combination as well as the role of ROS in mediating rapid systemic signaling during abiotic stress. We conclude that as long as cells maintain high enough energy reserves to detoxify ROS, ROS is beneficial to plants during abiotic stress enabling them to adjust their metabolism and mount a proper acclimation response.

Contaminant-induced oxidative stress in fish: a mechanistic approach.

The presence of reactive oxygen species (ROS) in living organisms was described more than 60 years ago and virtually immediately it was suggested that ROS were involved in various pathological processes and aging. The state when ROS generation exceeds elimination leading to an increased steady-state ROS level has been called "oxidative stress." Although ROS association with many pathological states in animals is well established, the question of ROS responsibility for the development of these states is still open. Fish represent the largest group of vertebrates and they inhabit a broad range of ecosystems where they are subjected to many different aquatic contaminants. In many cases, the deleterious effects of contaminants have been connected to induction of oxidative stress. Therefore, deciphering of molecular mechanisms leading to such contaminant effects and organisms' response may let prevent or minimize deleterious impacts of oxidative stress. This review describes general aspects of ROS homeostasis, in particular highlighting its basic aspects, modification of cellular constituents, operation of defense systems and ROS-based signaling with an emphasis on fish systems. A brief introduction to oxidative stress theory is accompanied by the description of a recently developed classification system for oxidative stress based on its intensity and time course. Specific information on contaminant-induced oxidative stress in fish is covered in sections devoted to such pollutants as metal ions (particularly iron, copper, chromium, mercury, arsenic, nickel, etc.), pesticides (insecticides, herbicides, and fungicides) and oil with accompanying pollutants. In the last section, certain problems and perspectives in studies of oxidative stress in fish are described.

Antioxidant inhibition of steady-state reactive oxygen species and cell growth in neuroblastoma

Reactive oxygen species (ROS) contribute to adult tumorigenesis; however, their roles in pediatric solid tumors are unknown. Here, we sought to define the steady-state ROS levels in neuroblastoma and to examine whether aggressive cellular behavior, which may predict treatment failure, is regulated by ROS. Neuroblastoma sections were assessed for 4-hydroxynonenal (4-HNE), a marker of intracellular lipid peroxidation and a byproduct of increased levels of ROS. Human neuroblastoma cell lines, MYCN-amplified BE(2)-C and MYCN-nonamplified SK-N-SH, were examined in our study. Superoxide and hydroperoxide oxidation products were detected by staining for dihydroethidium (DHE) and 5, 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate (CDCFH2), using the oxidation-insensitive analog CDCF as a negative control. Cells were treated with N-acetylcysteine (NAC; 10mmol/L) daily for 5days and analyzed. Greater expression of 4-HNE was observed in undifferentiated tumor sections as compared with the more differentiated tumors. Interestingly, increased levels of ROS were detected in MYCN-amplified BE(2)-C cells. Moreover, gastrin-releasing peptide receptor-induced ROS production stimulated upregulation of the hypoxia inducible factor (HIF)-1α/vascular endothelial growth factor (VEGF) pathway and an increase in cell growth. Antioxidant NAC decreased HIF-1α/VEGF expression and inhibited BE(2)-C cell growth. We report a novel observation that shifting the redox balance toward greater ROS levels results in a more aggressive neuroblastoma phenotype. Our data suggest that ROS play a critical role in refractory neuroblastoma.

Oxidative stress shapes breast cancer phenotype through chronic activation of ATM-dependent signaling.

Reactive oxygen species (ROS) are thought to be among the initiating insults that drive carcinogenesis; however, beyond the mutagenic properties of ROS, it is unclear how reactive oxygen species and response to redox imbalance may shape cancer phenotype. We have previously observed that basal activity of the powerfully oncogenic transcription factor NF-κB in cultured breast cancer and other tumor cell lines is dependent upon the DNA damage-responsive kinase ATM. Here we show that, in MDA-MB-231 and HeLa cells, basal ATM-dependent NF-κB activation occurs through a canonical DNA damage-responsive signaling pathway as knockdown of two proteins involved in this signaling pathway, ERC1 and TAB1, results in loss of NF-κB basal activity. We further show that knockdown of ATM in MDA-MB-231, a breast cancer line with a pronounced mesenchymal phenotype, results in the reversion of these cells to an epithelial morphology and gene expression pattern. Culture of MDA-MB-231 and HeLa cells on the antioxidant N-acetyl cysteine (NAC) blunted NF-κB transcriptional activity, and long-term culture on low doses of NAC resulted in coordinate reductions in steady-state ROS levels, acquisition of an epithelial morphology, as well as upregulation of epithelial and downregulation of mesenchymal marker gene expression. Moreover, these reversible effects are attributable, at least in part, to downregulation of ATM-dependent NF-κB signaling in MDA-MB-231 cells as RNAi-mediated knockdown of the NF-κB subunit RelA or its upstream activator TG2 produced similar alterations in phenotype. We conclude that chronic activation of ATM in response to persistent ROS insult triggers continual activation of the oncogenic NF-κB transcriptional complex that, in turn, promotes aggressive breast cancer phenotype.

A New Player in Environmentally Induced Oxidative Stress: Polychlorinated Biphenyl Congener, 3,3′-Dichlorobiphenyl (PCB11)

Recent analysis of air samples from Chicago and Lake Michigan areas observed a ubiquitous airborne polychlorinated biphenyl (PCB) congener, 3,3'-dichlorobiphenyl (PCB11). Our analysis of serum samples also revealed the existence of hydroxylated metabolites of PCB11 in human blood. Because PCBs and PCB metabolites have been suggested to induce oxidative stress, this study sought to determine whether environmental exposure to PCB11 and its 4-hydroxyl metabolite could induce alterations in steady-state levels of reactive oxygen species (ROS) and cytotoxicity in immortalized human prostate epithelial cells (RWPE-1). This study also examines if antioxidants could protect the cells from PCB11-induced cytotoxicity. Exponentially growing RWPE-1 cells were exposed to PCB11 and its metabolite, 3,3'-dichlorobiphenyl-4-ol (4-OH-PCB11), as well as an airborne PCB mixture resembling the Chicago ambient air congener profile, every day for 5 days. Results showed that 4-OH-PCB11 could significantly induce cell growth suppression and decrease the viability and plating efficiency of RWPE-1 cells. 4-OH-PCB11 also significantly increased steady-state levels of intracellular superoxide, O₂•⁻), as well as hydroperoxides. Finally, treatment with the combination of polyethylene glycol-conjugated CuZn superoxide dismutase and catalase added 1h after 4-OH-PCB11 exposures, significantly protected RWPE-1 cells from PCB toxicity. The results strongly support the hypothesis that exposure to a hydroxylated metabolite of PCB11 can inhibit cell proliferation and cause cytotoxicity by increasing steady-state levels of ROS. Furthermore, antioxidant treatments following PCBs exposure could significantly mitigate the PCB-induced cytotoxicity in exponentially growing human prostate epithelial cells.

Stress Granules Inhibit Apoptosis by Reducing Reactive Oxygen Species Production

Cells can undergo two alternative fates following exposure to environmental stress: they either induce apoptosis or inhibit apoptosis and then repair the stress-induced alterations. These processes minimize cell loss and prevent the survival of cells with aberrant DNA and protein alterations. These two alternative fates are partly controlled by stress granules (SGs). While arsenite, hypoxia, and heat shock induce the formation of SGs that inhibit apoptosis, X-ray irradiation and genotoxic drugs do not induce SGs, and they are more prone to trigger apoptosis. However, it is unclear precisely how SGs control apoptosis. This study found that SGs suppress the elevation of reactive oxygen species (ROS), and this suppression is essential for inhibiting ROS-dependent apoptosis. This antioxidant activity of SGs is controlled by two SG components, GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and ubiquitin-specific protease 10 (USP10). G3BP1 elevates the steady-state ROS level by inhibiting the antioxidant activity of USP10. However, following exposure to arsenite, G3BP1 and USP10 induce the formation of SGs, which uncovers the antioxidant activity of USP10. We also found that the antioxidant activity of USP10 requires the protein kinase activity of ataxia telangiectasia mutated (ATM). This work reveals that SGs are critical redox regulators that control cell fate under stress conditions.

Reactive oxygen species and their role in plant defence and cell wall metabolism

Harnessing the toxic properties of reactive oxygen species (ROS) to fight off invading pathogens can be considered a major evolutionary success story. All aerobic organisms have evolved the ability to regulate the levels of these toxic intermediates, whereas some have evolved elaborate signalling pathways to dramatically increase the levels of ROS and use them as weapons in mounting a defence response, a process commonly referred to as the oxidative burst. The balance between steady state levels of ROS and the exponential increase in these levels during the oxidative burst has begun to shed light on complex signalling networks mediated by these molecules. Here, we discuss the different sources of ROS that are present in plant cells and review their role in the oxidative burst. We further describe two well-studied ROS generating systems, the NADPH oxidase and apoplastic peroxidase proteins, and their role as the primary producers of ROS during pathogen invasion. We then discuss what is known about the metabolic and proteomic fluxes that occur in plant cells during the oxidative burst and after pathogen recognition, and try to highlight underlying biochemical processes that may provide more insight on the complex regulation of ROS in plants.

Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g=4.3. This form of iron is referred to as "free" iron. EPR signals at g=4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S=5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g=4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.

Antiproliferative mechanisms of action of the flavin dehydrogenase inhibitors diphenylene iodonium and di-2-thienyliodonium based on molecular profiling of the NCI-60 human tumor cell panel

Flavoprotein-dependent reactive oxygen species (ROS) play a critical role in cytokine-mediated signal transduction in normal tissues and tumor cells. The flavoenzyme inhibitors diphenylene iodonium (DPI) and di-2-thienyliodonium (DTI) have been used to inhibit membrane-bound, flavoprotein-containing NADPH oxidases, including epithelial and leukocyte NADPH oxidases (Nox1-5 and Duox 1 and 2). Recent evidence suggests that DPI can decrease tumor cell proliferation; however, the molecular mechanisms involved remain poorly defined. To explore the mechanisms underlying DPI- and DTI-related tumor cell growth delay, we examined growth inhibition patterns produced by both agents in the NCI-60 tumor panel, and determined expression levels of Nox gene family members across these cell lines. Possible molecular targets were predicted using the COMPARE program. DPI was more potent than DTI (GI50: 10nM versus 10μM); DPI and DTI exposure produced unique patterns of growth inhibition when evaluated against the small molecule anticancer database of the National Cancer Institute. Growth inhibition profiling of DPI revealed a modest positive correlation with Nox1 levels; novel mechanisms of DPI and DTI action, including alterations in Stat, Erk1/2, and Akt pathways, were inferred by correlation with NCI-60 Affymetrix® array data. Exposure of HT-29 colon cancer cells, which express Nox1, to DPI and DTI confirmed their inhibitory effects on steady state ROS levels, and demonstrated decreased Stat, Erk1/2, and Akt signaling mediated by IL-4, IL-6, IL-13, and IL-22, possibly due to a concomitant increase in tumor cell phosphatase activity. These findings suggest that DPI and DTI may act therapeutically by altering ROS-related signal transduction.

Redox-control of matrix metalloproteinase-1: A critical link between free radicals, matrix remodeling and degenerative disease

Many degenerative disease processes associated with aging result from enhanced extracellular matrix (ECM) breakdown. Concomitant with aberrant matrix destruction are alterations in levels of reactive oxygen species (ROS) generating and detoxification systems. ROS function as second messengers due to their ability to react with wide range of biomolecules resulting in modification of an array of signaling networks. ROS can activate upstream kinases (MKK) responsible for MAPK activation and restrict the activity of their inhibitory phosphatases. Here we focus on the redox-sensitive signaling components that control the expression of MMP-1, which is largely responsible for maintaining ECM homeostasis. Numerous disease processes are associated with shifts in steady state ROS levels that influence overall ECM degradation. This review highlights the redox-sensitive regulatory signals that control the expression of the primary initiating protease MMP-1 and provides strong rational for the use of antioxidant based therapies for treatment of degenerative disorders associated with aberrant matrix destruction.

Bcl-2 sustains hormetic response by inducing Nrf-2 nuclear translocation in L929 mouse fibroblasts

Hormesis is the process whereby exposure to a low dose of a chemical agent induces an adaptive effect on the cell or organism. This response evokes the expression of cytoprotective and antioxidant proteins, allowing pro-oxidants to emerge as important hormetic agents. The antiapoptotic protein Bcl-2 is known to protect cells against death induced by oxidants; it has been suggested that Bcl-2 might also modulate steady-state reactive oxygen species levels. The aim of this work was to find out if Bcl-2 might play a role during the hormetic response and in Nrf-2 activation. We have established a model to study the oxidative conditioning hormesis response (OCH) by conditioning the cell line L929 with 50 μM H 2O 2 for 9 h. This condition did not induce oxidative damage nor oxidative imbalance, and OCH cells maintained a 70–80% survival rate after severe H 2O 2 treatment compared to nonconditioned cells. When cells were pretreated with the Bcl-2 inhibitor HA14-1 or were silenced with Bcl-2-siRNA, both the hormetic effect and the Nrf-2 nuclear translocation previously observed were abrogated. Our results suggest a sequence of causal events related to increase in Bcl-2 expression, induction of Nrf-2 activation, and sustained expression of cytoprotective proteins such as GST and γGCS.

Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation

Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose-deprivation-induced cytotoxicity. To determine whether oxidative stress mediated by O(2)(*-) and hydroperoxides contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, the oxidation of DHE (dihydroethidine; for O(2)(*-)) and CDCFH(2) [5- (and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate; for hydroperoxides] was measured in human colon and breast cancer cells (HT29, HCT116, SW480 and MB231) and compared with that in normal human cells [FHC cells, 33Co cells and HMECs (human mammary epithelial cells)]. Cancer cells showed significant increases in DHE (2-20-fold) and CDCFH(2) (1.8-10-fold) oxidation, relative to normal cells, that were more pronounced in the presence of the mitochondrial electron-transport-chain blocker, antimycin A. Furthermore, HCT116 and MB231 cells were more susceptible to glucose-deprivation-induced cytotoxicity and oxidative stress, relative to 33Co cells and HMECs. HT29 cells were also more susceptible to 2DG (2-deoxyglucose)-induced cytotoxicity, relative to FHC cells. Overexpression of manganese SOD (superoxide dismutase) and mitochondrially targeted catalase significantly protected HCT116 and MB231 cells from glucose-deprivation-induced cytotoxicity and oxidative stress and also protected HT29 cells from 2DG-induced cytotoxicity. These results show that cancer cells (relative to normal cells) demonstrate increased steady-state levels of ROS (reactive oxygen species; i.e. O(2)(*-) and H(2)O(2)) that contribute to differential susceptibility to glucose-deprivation-induced cytotoxicity and oxidative stress. These studies support the hypotheses that cancer cells increase glucose metabolism to compensate for excess metabolic production of ROS and that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.

Suppression of Reactive Oxygen Species and Enhanced Stress Tolerance in Rubia cordifolia Cells Expressing the rolC Oncogene

It is known that expression of the Agrobacterium rhizogenes rolC gene in transformed plant cells causes defense-like reactions, such as increased phytoalexin production and expression of pathogenesis-related proteins. In the present study, we examined whether this phenomenon is associated with increased production of reactive oxygen species (ROS). Single-cell assays based on confocal microscopy and fluorogenic dyes (2,7-dichlorofluorescein diacetate and dihydrorhodamine 123) showed reduced steady-state levels of ROS in rolC-expressing Rubia cordifolia cells as compared with normal cells. Paraquat, a ROS inducer, caused significant ROS elevation in normal cells but had little effect on rolC-transformed cells. Likewise, ROS elevation triggered by a light stress was suppressed in transformed cells. Our results indicate that the rolC gene acts as a ROS suppressor in unstressed cells and its expression prevents stress-induced ROS elevations. We detected a two- to threefold increase in tolerance of rolC-transformed cells to salt, heat, and cold treatments. Simultaneously, rolC-transformed cells maintained permanently active defensive status, as found by measuring isochorismate synthase gene expression and anthraquinone production. Thus, the oncogene provoked multiple effects in which ROS production and phytoalexin production were clearly dissociated.

Mitochondrial Dysfunction Increases Oxidative Stress and Decreases Chronological Life Span in Fission Yeast

BackgroundOxidative stress is a probable cause of aging and associated diseases. Reactive oxygen species (ROS) originate mainly from endogenous sources, namely the mitochondria.Methodology/Principal FindingsWe analyzed the effect of aerobic metabolism on oxidative damage in Schizosaccharomyces pombe by global mapping of those genes that are required for growth on both respiratory-proficient media and hydrogen-peroxide-containing fermentable media. Out of a collection of approximately 2700 haploid yeast deletion mutants, 51 were sensitive to both conditions and 19 of these were related to mitochondrial function. Twelve deletion mutants lacked components of the electron transport chain. The growth defects of these mutants can be alleviated by the addition of antioxidants, which points to intrinsic oxidative stress as the origin of the phenotypes observed. These respiration-deficient mutants display elevated steady-state levels of ROS, probably due to enhanced electron leakage from their defective transport chains, which compromises the viability of chronologically-aged cells.Conclusion/SignificanceIndividual mitochondrial dysfunctions have often been described as the cause of diseases or aging, and our global characterization emphasizes the primacy of oxidative stress in the etiology of such processes.

Catalase ameliorates polychlorinated biphenyl-induced cytotoxicity in nonmalignant human breast epithelial cells

Polychlorinated biphenyls (PCBs) are environmental chemical contaminants believed to adversely affect cellular processes. We investigated the hypothesis that PCB-induced changes in the levels of cellular reactive oxygen species (ROS) induce DNA damage resulting in cytotoxicity. Exponentially growing cultures of human nonmalignant breast epithelial cells (MCF10A) were incubated with PCBs for 3 days and assayed for cell number, ROS levels, DNA damage, and cytotoxicity. Exposure to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) or 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of 4-chlorobiphenyl (PCB3), significantly decreased cell number and MTS reduction and increased the percentage of cells with sub-G 1 DNA content. Results from electron paramagnetic resonance (EPR) spectroscopy showed a 4-fold increase in the steady-state levels of ROS, which was suppressed in cells pretreated with catalase. EPR measurements in cells treated with 4-Cl-BQ detected the presence of a semiquinone radical, suggesting that the increased levels of ROS could be due to the redox cycling of 4-Cl-BQ. A dose-dependent increase in micronuclei frequency was observed in PCB-treated cells, consistent with an increase in histone 2AX phosphorylation. Treatment of cells with catalase blunted the PCB-induced increase in micronuclei frequency and H2AX phosphorylation that was consistent with an increase in cell survival. Our results demonstrate a PCB-induced increase in cellular levels of ROS causing DNA damage, resulting in cell killing.