Dependent Reactive Oxygen Species Production Research Articles

Control of vascular smooth muscle function by Src-family kinases and reactive oxygen species in health and disease.

Reactive oxygen species (ROS) are now recognised as second messenger molecules that regulate cellular function by reversibly oxidising specific amino acid residues of key target proteins. Amongst these are the Src-family kinases (SrcFKs), a multi-functional group of non-receptor tyrosine kinases highly expressed in vascular smooth muscle (VSM). In this review we examine the evidence supporting a role for ROS-induced SrcFK activity in normal VSM contractile function and in vascular remodelling in cardiovascular disease. VSM contractile responses to G-protein-coupled receptor stimulation, as well as hypoxia in pulmonary artery, are shown to be dependent on both ROS and SrcFK activity. Specific phosphorylation targets are identified amongst those that alter intracellular Ca(2+) concentration, including transient receptor potential channels, voltage-gated Ca(2+) channels and various types of K(+) channels, as well as amongst those that regulate actin cytoskeleton dynamics and myosin phosphatase activity, including focal adhesion kinase, protein tyrosine kinase-2, Janus kinase, other focal adhesion-associated proteins, and Rho guanine nucleotide exchange factors. We also examine a growing weight of evidence in favour of a key role for SrcFKs in multiple pro-proliferative and anti-apoptotic signalling pathways relating to oxidative stress and vascular remodelling, with a particular focus on pulmonary hypertension, including growth-factor receptor transactivation and downstream signalling, hypoxia-inducible factors, positive feedback between SrcFK and STAT3 signalling and positive feedback between SrcFK and NADPH oxidase dependent ROS production. We also discuss evidence for and against the potential therapeutic targeting of SrcFKs in the treatment of pulmonary hypertension.

Mcl-1 promotes lung cancer cell migration by directly interacting with VDAC to increase mitochondrial Ca2+ uptake and reactive oxygen species generation.

Mcl-1 is an antiapoptotic member of the Bcl-2 family frequently upregulated in non-small cell lung carcinoma (NSCLC). We now report the physiological significance of an interaction between Mcl-1 and the mitochondrial outer membrane-localized voltage-dependent anion channel (VDAC) in NSCLC cell lines. Mcl-1 bound with high affinity to VDAC1 and 3 isoforms but only very weakly to VDAC2 and binding was disrupted by peptides based on the VDAC1 sequence. In A549 cells, reducing Mcl-1 expression levels or application of VDAC-based peptides limited Ca2+ uptake into the mitochondrial matrix, the consequence of which was to inhibit reactive oxygen species (ROS) generation. In A549, H1299 and H460 cells, both Mcl-1 knockdown and VDAC-based peptides attenuated cell migration without affecting cell proliferation. Migration was rescued in Mcl-1 knockdown cells by experimentally restoring ROS levels, consistent with a model in which ROS production drives increased migration. These data suggest that an interaction between Mcl-1 and VDAC promotes lung cancer cell migration by a mechanism that involves Ca2+-dependent ROS production.

Oxidative Stress and Atrial Fibrillation

Atrial fibrillation (AF), one of the most prevalent cardiac arrhythmias, afflicts >3 million people in the United States.1 Because the risk of developing AF increases with age, the prevalence of AF is projected to be 7.6 million in 2050,1 when 1 in 5 Americans will be over the age of 65.2 AF not only causes debilitating symptoms and functional impairments owing to worsened hemodynamics and embolic stroke, it also increases the risk of mortality up to 1.5- to 1.9-fold.3 Despite the significant morbidity and mortality burden incurred with AF, there are limited therapeutic options that may improve the outcomes of AF patients, and these options are associated with significant AF recurrence rates.4 Article see p 1748 One possible explanation for the limitations of current therapies is that they do not address effectively or completely the underlying causes of AF. Evidence has been mounting that AF is associated with systemic and cardiac oxidation.4,5 Risk factors for AF are similar to those of atherosclerosis, a disease known to be perpetuated by oxidative stress. These risk factors, such as hypertension, aging, diabetes mellitus, and coronary artery bypass surgery, have each been associated with increases in the systemic markers of oxidation.4 In addition, there is also evidence of increased cardiac oxidation of myofibrillar protein5 and membrane lipids6 with AF or with risk factors linked to AF. Although it is not clear whether cardiac oxidation leads to systemic markers of oxidation or whether systemic oxidation leads to cardiac oxidation, the association of oxidative stress and AF is robust and suggests that AF is possibly a manifestation of a systemic disease. Despite the link between oxidative stress and AF, systemic antioxidant therapy for arrhythmias has not met with much success in clinical trials.7, …

188: Dengue infection generates an early NADPH-oxidase dependent ROS production required for antiviral and inflammatory responses

The rapid host response to virus infection is crucial for establishment of the antiviral state and for the release of pro-inflammatory mediators that coordinate the adaptive immune response. Induction of antiviral and inflammatory genes is achieved largely through recognition of viral nucleic acid motifs by pattern recognition receptors. Dengue virus (DENV) is a significant human pathogen that causes a wide spectrum of clinical symptoms, ranging from mild dengue fever to severe hemorrhagic fever and shock syndrome. The cytosolic receptors RIG-I/MDA5 as well as the membrane-bound TLR3/TLR7 localized in the endosomes are important sensors of DENV infection. The role of reactive oxygen species (ROS) in the activation of biological processes such as autophagy, necrosis, and inflammasome activation adds another layer of complexity to the regulation of immune sensing. Transcriptome analysis of DENV-infected human monocyte-derived dendritic cells (Mo-DC) revealed the activation of distinct patterns of gene transcription, with involvement of pathways including the antiviral response, the TNF superfamily and late activation of death receptor signaling. Interestingly, a dominant early gene network associated with the Nrf2-dependent antioxidant response was also identified; DENV infection induced an early production of ROS that was dependent on NADPH-oxidase, and was essential for the activation of inflammatory and antiviral programs, through NF-κB and IRF-3 transcription factors, respectively. Using a high throughput qPCR assay, we demonstrated that interfering with NADPH-oxidase inhibited DENV-induced antiviral and inflammatory responses. We also demonstrated that the antioxidant pathway controlled by the transcription factor Nrf2 is a pivotal upstream link to the regulation of DENV-induced antiviral and inflammatory responses under oxidative conditions. In conclusion, these findings demonstrate that ROS production is crucial for effective activation of pathways leading to innate immune responses against DENV infection in human Mo-DC.

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro-inflammatory cytokines that up-regulate iNOS contribute to pre-eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre-eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham-operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N-[3-(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8-isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ∼30 mmHg in RUPP rats, and 1400 W attenuated this increase by ∼50% (P < 0.05). While RUPP increased plasma 8-isoprostane levels, aortic ROS levels, and NADPH-dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre-eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.

A low temperature-inducible protein AtSRC2 enhances the ROS-producing activity of NADPH oxidase AtRbohF

Reactive oxygen species (ROS) produced by NADPH oxidases play critical roles in plant environmental responses. Arabidopsis thaliana NADPH oxidase AtRbohF-mediated ROS-production is involved in abiotic stress responses. Because overproduction of ROS is highly toxic to cells, the activity of AtRbohF needs to be tightly regulated in response to diverse stimuli. The ROS-producing activity of AtRbohF is activated by Ca2+ and protein phosphorylation, but other regulatory factors for AtRbohF are mostly unknown. In this study, we screened for proteins that interact with the N-terminal cytosolic region of AtRbohF by a yeast two-hybrid screen, and isolated AtSRC2, an A. thaliana homolog of SRC2 (soybean gene regulated by cold-2). A co-immunoprecipitation assay revealed that AtSRC2 interacts with the N-terminal region of AtRbohF in plant cells. Intracellular localization of GFP-tagged AtSRC2 was partially overlapped with that of GFP-tagged AtRbohF at the cell periphery. Co-expression of AtSRC2 enhanced the Ca2+-dependent ROS-producing activity of AtRbohF in HEK293T cells, but did not affect its phosphorylation-dependent activation. Low-temperature treatment induced expression of the AtSRC2 gene in Arabidopsis roots in proportion to levels of ROS production that was partially dependent on AtRbohF. Our findings suggest that AtSRC2 is a novel activator of Ca2+-dependent AtRbohF-mediated ROS production and may play a role in cold responses.

Melamine activates NFκB/COX-2/PGE2 pathway and increases NADPH oxidase–dependent ROS production in macrophages and human embryonic kidney cells

Melamine is a wildly used compound in manufactures of plastics and resins. A variety of toxic effects from melamine, including nephrolithiasis, chronic kidney inflammation, and bladder carcinoma, have been mentioned. Oxidative stress is considered to be an important pathogenic mechanism of kidney disease which may develop from an increasing free radical production through inflammation. The aim of this study is to investigate melamine-induced oxidative stress and inflammation in macrophage-like cell line RAW 264.7 and human embryonic kidney cell line HEK293. Results indicated melamine activated nuclear factor (NF)-κB through increasing IκB-α degradation and NF-κB p65/p50 DNA-binding activity. In addition, melamine significantly increased COX-2 expression and prostaglandin E2 (PGE2) production. Moreover, melamine activated NADPH oxidase (NOX), including NOX1, NOX2 and NOX4, accompanied with an increase in reactive oxygen species (ROS) production. Furthermore, melamine-induced ROS production could be attenuated by apocynin, a NOX inhibitor. In conclusion, our findings suggest melamine increased inflammation and oxidative stress via activation of NF-κB/COX-2 and NOX/ROS pathway, and first revealed the critical role of NOX in melamine-induced ROS production, suggesting the potential of NOX inhibitor against melamine toxicity.

Rho GTPases and Nox dependent ROS production in skin. Is there a connection?

Rho GTPases are a family of small GTP binding proteins most commonly known for the regulation of many cellular processes, including actin cytoskeleton re-organisation, cell proliferation, signal transduction and regulation of apoptosis. Additionally, a link between Rho GTPases and reactive oxygen species (ROS) has been shown. In line with the growing interest in the role of ROS in cell biology, the relevance of this connection is becoming increasingly clearer. ROS production is classically associated with oxidative metabolic pathways (e.g. respiratory chain, arachidonic acid). During these metabolic pathways, ROS are produced as by-products and these can be potentially toxic. However, numerous cell types contain dedicated enzymatic complexes, i.e., NADPH oxidase (Nox) complexes, for regulated production of ROS. This regulated production of ROS seems to be important for a number of fundamental cell biological processes, including cell growth, differentiation, migration, angiogenesis, aimed at maintaining tissue homeostasis. Data suggests that skin cells are capable of a regulated ROS production via Nox complexes. Members of the Rho GTPase family have been found to play a central regulatory role in Nox activity. In the present review we will focus on the involvement of Rho GTPases in regulated production of ROS with special emphasis on the skin. We will also discuss the possibility that some in vivo effects of the deletion of members of the Rho GTPase family in skin cells could potentially be linked to a reduced ability of regulated ROS production.

Uncoupling protein 1 dependent reactive oxygen species production by thymus mitochondria

We have previously shown that uncoupling protein 1 is present in thymus and has a role in T-cell development. As reactive oxygen species have been implicated in T-cell development, we set out to determine whether uncoupling protein 1 had the potential to regulate reactive oxygen species production in mitochondria isolated from thymus. This was achieved by inhibiting proton leak through uncoupling protein 1 using the purine nucleotide GDP and through ablation of uncoupling protein 1, measuring the amplex red sensitive reactive oxygen species production by mitochondria. In this work we demonstrate, for the first time, that uncoupling protein 1 has the potential to regulate reactive oxygen species production in thymus mitochondria. We also show that reverse electron transport is possible in thymus mitochondria respiring on succinate and glycerol-3-phosphate. The implications of this regulatory role for uncoupling protein 1 are discussed in the context of thymus function.This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

An injury-response mechanism conserved across kingdoms determines entry of the fungus Trichoderma atroviride into development

A conserved injury-defense mechanism is present in plants and animals, in which the production of reactive oxygen species (ROS) and lipid metabolism are essential to the response. Here, we describe that in the filamentous fungus Trichoderma atroviride, injury results in the formation of asexual reproduction structures restricted to regenerating cells. High-throughput RNA-seq analyses of the response to injury in T. atroviride suggested an oxidative response and activation of calcium-signaling pathways, as well as the participation of lipid metabolism, in this phenomenon. Gene-replacement experiments demonstrated that injury triggers NADPH oxidase (Nox)-dependent ROS production and that Nox1 and NoxR are essential for asexual development in response to damage. We further provide evidence of H(2)O(2) and oxylipin production that, as in plants and animals, may act as signal molecules in response to injury in fungi, suggesting that the three kingdoms share a conserved defense-response mechanism.

HSP90 inhibition by 17-DMAG attenuates oxidative stress in experimental atherosclerosis

Reactive oxygen species (ROS) participate in atherogenesis through different mechanisms including oxidative stress and inflammation. Proteins implicated in both processes, such as mitogen-activated protein kinase kinase (MEK) and some NADPH oxidase (NOX) subunits, are heat shock protein-90 (HSP90) client proteins. In this work, we investigated the antioxidant properties of the HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) in experimental atherosclerosis. Treatment of ApoE(-/-) mice with 17-DMAG (2 mg/kg every 2 days for 10 weeks) decreased ROS levels and extracellular signal-regulated kinase (ERK) activation in aortic plaques compared with control animals. Accordingly, treatment of rat vascular smooth muscle cells (VSMCs) with 17-DMAG increased HSP27 and HSP70 and inhibited ERK activation. Interestingly, 17-DMAG diminished NADPH oxidase dependent ROS production in VSMCs and monocytes. In addition, a marked reduction in NADPH oxidase dependent ROS production was observed with HSP90siRNA and the opposite pattern with HSP70siRNA. 17-DMAG also diminished the expression of Nox1 and Nox organizer-1 (Noxo1) in VSMCs and monocytes. Interestingly, 17-DMAG was able to modulate ROS-induced monocyte to macrophage differentiation. Finally, higher expression of Nox1 and Noxo1 was found in the inflammatory region of human atherosclerotic plaques, colocalizing with VSMCs, macrophages, and ROS-producing cells. Our results suggest that HSP90 inhibitors interfere with oxidative stress and modulate experimental atherosclerosis development through reduction in pro-oxidative factors.

Nox2 dependent modulation of skeletal muscle Excitation Contraction Coupling

Generation of reactive oxygen species (ROS) under physiological conditions is required for normal force production in skeletal muscle. However, high levels of ROS promote contractile dysfunction, resulting in muscle weakness and fatigue. Our recent studies suggest that sub-cellular site-specific ROS production governs the beneficial vs. damaging effects of ROS. In this study we investigated the role of the enzyme complex NADPH oxidase (Nox2) in skeletal muscle EC coupling. Using sub-cellular site-specific redox probes we show that cytosolic glutathione redox potential becomes more oxidized during contractile activity while mitochondrial redox potential does not change. Increased contractile activity promotes Nox2 dependent ROS production and activates the non-tyrosine kinase Src. These data suggest that Src acts as a redox switch to activate Nox2. Nox2 dependent ROS production could decrease sarcolemmal Ca2+ influx and decrease sarcoplasmic reticulum refilling, contributing to the development of fatigue. As trials of general antioxidant therapy to combat increased ROS production have not been convincingly beneficial, understanding the sub-cellular signaling pathways by which oxidants influence muscle function will allow for the development of targeted therapeutic interventions to combat the deleterious effects of sustained contractile activity as well as skeletal muscle diseases. Supported by NIH/NIAMS K01 AR-051519.

Nox2 Dependent Modulation of Skeletal Muscle EC Coupling

Generation of reactive oxygen species (ROS) under physiological conditions is required for normal force production in skeletal muscle. However, high levels of ROS promote contractile dysfunction, resulting in muscle weakness and fatigue. Our recent studies suggest that sub-cellular site-specific ROS production governs the beneficial vs. damaging effects of ROS. NADPH oxidase (Nox2) is an enzyme complex that generates ROS. In this study we investigated the role of Nox2 in skeletal muscle EC coupling. Using sub-cellular site-specific redox probes we show that cytosolic glutathione redox potential becomes more oxidized during contractile activity while mitochondrial redox potential does not change. We show that increased contractile activity promotes Nox2 dependent ROS production. Interestingly, we have found that the non-tyrosine kinase Src is activated in response to increased contractile activity. These data support a hypothesis in which Src acts as a redox switch to activate Nox2. Increased ROS production during strenuous exercise would decrease sarcolemmal Ca2+ influx and decrease sarcoplasmic reticulum refilling, which could contribute to the development of fatigue. As trials of general antioxidant therapy to combat increased ROS production have not been convincingly beneficial, understanding the sub-cellular signaling pathways by which oxidants influence muscle function will allow for the development of targeted therapeutic interventions to combat the deleterious effects of sustained contractile activity as well as skeletal muscle diseases.

Effect of diallyl disulfide on Ca2+ movement and viability in PC3 human prostate cancer cells

The effect of diallyl disulfide (DADS) on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells is unclear. This study explored whether DADS changed [Ca2+]i in PC3 cells by using fura-2. DADS at 50–1000μM increased [Ca2+]i in a concentration-dependent manner. The signal was reduced by removing Ca2+. DADS-induced Ca2+ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators; but was inhibited by aristolochic acid. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished DADS-induced [Ca2+]i rise. Incubation with DADS inhibited thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter DADS-induced [Ca2+]i rise. At 500–1000μM, DADS killed cells in a concentration-dependent manner. The cytotoxic effect of DADS was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Propidium iodide staining suggests that DADS (500μM) induced apoptosis in a Ca2+-independent manner. Annexin V/PI staining further shows that 10μM and 500μM DADS both evoked apoptosis. DADS also increased reactive oxygen species (ROS) production. Collectively, in PC3 cells, DADS induced [Ca2+]i rise probably by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive channels. DADS induced Ca2+-dependent cell death, ROS production, and Ca2+-independent apoptosis.

Effect of ferric ions on reactive oxygen species formation, cervical cancer cell lines growth and E6/E7 oncogene expression

As iron ions may participate in the pathogenesis of cancer and viral infections, the aim of this study was to monitor their influence on proliferation, E6 and E7 oncogene expression and reactive oxygen species (ROS) production in two human papilloma virus (HPV) positive cervical carcinoma cell lines (HeLa and SiHa) and one HPV negative vulvar cell line (A431). The anti-anaemic drug, ferric–sorbitol–citric acid complex (FSC) as a source of Fe III ions was used. Cells were treated with FSC at the concentrations between 0.001 and 1 mM Fe III for different time periods. Fe III ions inhibited the viability of HeLa and A431 cells while it had no influence on SiHa cells. Furthermore, Fe III treatment showed a time-dependent and a higher stimulatory effect on E6/E7 expression in SiHa cells than in HeLa cells. Fe III ion treatment with concentrations lower than 0.1 mM showed a time and a concentration dependent intracellular ROS production in all tested cell lines, while the treatment with 1 mM concentration decreased ROS production in all tested cell lines. In conclusion, Fe III ion treatment apart from having an anti-tumour effect, as we previously described, enhances survival of HPV 16-positive cells and might be associated with HPV oncogenesis.

Redox Remodeling Allows and Controls B-Cell Activation and Differentiation

During their differentiation to antibody-secreting plasma cells, B lymphocytes undergo dramatic changes in metabolism, structure, and function. Here we show that this transition entails extensive intra- and extracellular redox changes. Lipopolysaccharide (LPS)-driven activation and differentiation of naïve murine B splenocytes is paralleled by increased production of reactive oxygen species (ROS) from different sources, followed by a strong antioxidant response. This response includes upregulation of thioredoxin and of the cystine transporter xCT, and increased production and extracellular release of nonprotein thiols, mainly glutathione (GSH) and cysteine. Although ROS levels are higher in late-differentiating B cells, an early oxidative step is likely required to start the differentiation program, because inhibition of NADPH oxidase-dependent early ROS production impairs B-cell activation and differentiation. Addition of reducing agents such as 2-ME results in increased IgM secretion per cell, suggesting that the antioxidant response not only is aimed at restoring the redox homeostasis but also plays a functional role. A highly reduced environment coincident with the presence of large ROS-producing cells is observed in histologic sections of spleens from immunized mice, indicating that the redox modifications observed in LPS-induced B-cell differentiation in vitro occur also in vivo during physiologic immune responses.

β-Aminobutyric Acid Primes an NADPH Oxidase–Dependent Reactive Oxygen Species Production During Grapevine-Triggered Immunity

The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by beta-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase-dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem.

Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells

The immunotoxicity of three generations of polyamidoamine (PAMAM) dendrimers (G-4, G-5 and G-6) was evaluated in mouse macrophage cells in vitro. Using the Alamar blue and MTT assays, a generation dependent cytotoxicity of the PAMAM dendrimers was found whereby G-6 > G-5 > G-4. The toxic response of the PAMAM dendrimers correlated well with the number of surface primary amino groups, with increasing number resulting in an increase in toxic response. An assessment of intracellular ROS generation by the PAMAM dendrimers was performed by measuring the increased fluorescence as a result of intracellular oxidation of Carboxy H 2DCFDA to DCF both quantitatively using plate reader and qualitatively by confocal laser scanning microscopy. The inflammatory mediators macrophage inflammatory protein-2 (MIP-2), tumour necrosis factor-α (TNF-α) and interleukin-6, (IL-6) were measured by the enzyme linked immunosorbant assay (ELISA) following exposure of mouse macrophage cells to PAMAM dendrimers. A generation dependent ROS and cytokine production was found, which correlated well with the cytotoxicological response and therefore number of surface amino groups. A clear time sequence of increased ROS generation (maximum at ∼ 4 h), TNF-α and IL-6 secretion (maximum at ∼ 24 h), MIP-2 levels and cell death (∼ 72 h) was observed. The intracellular ROS generation and cytokine production induced cytotoxicity point towards the mechanistic pathway of cell death upon exposure to PAMAM dendrimers.

Role of the cytoskeleton in Cd2+-induced death of mouse mesangial cellsThis article is one of a selection of papers published in a Special Issue on Oxidative Stress in Health and Disease.

Cadmium induces apoptotic cell death in mouse mesangial cells that is in part dependent on reactive oxygen species (ROS). Cadmium also activates multiple kinases in these cells, including the Ca2+/calmodulin-dependent protein kinase II (CaMK-II) and p38 kinase, and also leads to disruption of the filamentous actin cytoskeleton. We investigated the role of the cytoskeleton in Cd2+-induced cell death. Cell viability was decreased by Cd2+ and two types of apoptotic death, defined by flow cytometry, were increased. Disruption of actin filaments with cytochalasin D was partially protective, whereas stabilization of the cytoskeleton with jasplakinolide was without effect, indicating that cytoskeletal disruption contributes to, but is not necessary for, induction of apoptosis. Inhibition of CaMK-II and p38 kinase, mitochondrial stabilization with cyclosporine A, and the antioxidant N-acetyl cysteine all protected against apoptosis and prevented disruption of the cytoskeleton. Cytochalasin D decreased Cd2+-dependent ROS production, reduced the decline in mitochondrial membrane potential, and decreased phosphorylation of p38 kinase. We conclude that Cd2+-dependent actin disruption is a downstream event facilitating apoptotic death. Cadmium-dependent cell death involves actin-dependent mitochondrial changes, ROS production, and p38 activation.

Reactive Oxygen Species Mediate Liver Injury Through Parenchymal Nuclear Factor-κB Inactivation in Prolonged Ischemia/Reperfusion

Nuclear factor (NF)-kappaB participates in ischemia/reperfusion (I/R) hepatic signaling, stimulating both protective mechanisms and the generation of inflammatory cytokines. After analyzing NF-kappaB activation during increasing times of ischemia in murine I/R, we observed that the nuclear translocation of p65 paralleled Src and IkappaB tyrosine phosphorylation, which peaked after 60 minutes of ischemia. After extended ischemic periods (90 to 120 minutes) however, nuclear p65 levels were inversely correlated with the progressive induction of oxidative stress. Despite this profile of NF-kappaB activation, inflammatory genes, such as tumor necrosis factor (TNF) and interleukin (IL)-1beta, predominantly induced by Kupffer cells, increased throughout time during ischemia (30 to 120 minutes), whereas protective NF-kappaB-dependent genes, such as manganese superoxide dismutase (Mn-SOD), expressed in parenchymal cells, decreased. Consistent with this behavior, gadolinium chloride pretreatment abolished TNF/IL-1beta up-regulation during ischemia without affecting Mn-SOD levels. Interestingly, specific glutathione (GSH) up-regulation in hepatocytes by S-adenosylmethionine increased Mn-SOD expression and protected against I/R-mediated liver injury despite TNF/IL-1beta induction. Similar protection was achieved by administration of the SOD mimetic MnTBAP. In contrast, indiscriminate hepatic GSH depletion by buthionine-sulfoximine before I/R potentiated oxidative stress and decreased both nuclear p65 and Mn-SOD expression levels, increasing TNF/IL-1beta up-regulation and I/R-induced liver damage. Thus, the divergent role of NF-kappaB activation in selective liver cell populations underlies the dichotomy of NF-kappaB in hepatic I/R injury, illustrating the relevance of specifically maintaining NF-kappaB activation in parenchymal cells.