Suppression of Fas ligand expression on endothelial cells by arsenite through reactive oxygen species

Exposure of rats to high doses of quartz and other insoluble isometric particles can produce lung tumors. In contrast, after exposure of such particles in hamsters no tumor outcome has been observed. Recent studies have demonstrated that the tumorigenic effect of particles is closely linked to the induction of inflammatory processes and the subsequent formation and persistence of mutagenic oxidative DNA-modifications. Species-specific differences in sensitivity to particles should therefore be reflected in the molecular reaction of the lung cells. We exposed rats and hamsters to two different doses of quartz (0.3 mg, 1.2 mg/100 g body weight) by intratracheal instillation and characterized the dose-related pattern of pulmonary inflammation (neutrophil recruitment, TNF), toxicity (protein content, surfactant phospholipids), antioxidant defence (glutathione content), mutagenicity (8-oxoguanine, p53) and proliferation. Our results clearly demonstrate a significantly higher response of the rat to quartz exposure for all determined molecular and cellular parameters. Therefore the examination of these parameters in humans would contribute to the evaluation of the relevance of rats or hamsters as models to predict particle-induced human lung cancer risk.     

Enhancement of nuclear factor-kappaB activation and protein tyrosine phosphorylation by a tyrosine phosphatase inhibitor, pervanadate, involves reactive oxygen species in silica-stimulated macrophages

Reactive oxygen species (ROS) and phosphorylation events mediated by tyrosine kinase are involved in silica-induced nuclear factor-kappa B (NF-kappaB) activation. Protein tyrosine phosphatase (PTPase) acts to limit protein tyrosine phosphorylation. In the present study, we investigated the role of PTPase in NF-kappaB activation and tyrosine phosphorylation in silica-stimulated macrophages, and the involvement of ROS in these responses. Treatment of mouse peritoneal macrophages (RAW264.7 cells) with a PTPase inhibitor, pervanadate, markedly enhanced the DNA-binding activity of NF-kappaB in the presence or absence of silica. The stimulatory effect of pervanadate on NF-kappaB activation was also demonstrated in LPS-stimulated macrophages. A specific inhibitor of protein tyrosine kinase (PTK), genistein, prevented the NF-kappaB activation induced by pervanadate in the presence of silica while inhibitors of protein kinase A or C, such as staurosporine or H7, had no inhibitory effect on NF-kappaB activation. A variety of antioxidants, such as catalase, superoxide dismutase, N-acetyl cysteine (NAC), and pyrrolidine dithiocarbamate, inhibited NF-kappaB activation induced by pervanadate in the presence of silica. Furthermore, pervanadate markedly enhanced silica- or LPS-induced protein tyrosine phosphorylation in cells. Treatment of macrophages with NAC abolished the increase in tyrosine phosphorylation in cells stimulated with the combination of pervanadate and either silica or LPS or with silica alone. The results suggest that PTPase may play a crucial role in the negative regulation of silica-signaling pathways leading to NF-kappaB activation in macrophages. Furthermore, ROS appear to be involved in downstream signaling between PTPase inhibition and NF-kappaB activation.     

Suppression of rat neutrophil reactive oxygen species production and adhesion by the diterpenoid lactone andrographolide

The present study was to examine whether andrographolide, a diterpenoid lactone isolated from the anti-inflammatory herbal medicine Andrographis paniculata (Burm. f.) Nees. (Acanthaceae), has the ability to prevent phorbol-12-myristate-13-acetate (PMA)-induced reactive oxygen species (ROS) production, as well as N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced adhesion by rat neutrophils. Results demonstrated that PMA (100 ng/ml) induced rapid accumulation of H2O2 and O2. in neutrophils within 30 minutes. Andrographolide (0.1 to 10 microM) pretreatment (10 min, 37 degrees C) significantly attenuated the accumulation of these two oxygen radical metabolites. Administration of andrographolide also significantly prevented fMLP-induced neutrophil adhesion. These data suggest that preventing ROS production and neutrophils adhesion may confer andrographolide the ability to be an anti-inflammatory drug.     

Aminoferrocene-based prodrugs activated by reactive oxygen species

Cancer cells generally generate higher amounts of reactive oxygen species than normal cells. On the basis of this difference, prodrugs have been developed (e.g., hydroxyferrocifen), which remain inactive in normal cells, but become activated in cancer cells. In this work we describe novel aminoferrocene-based prodrugs, which, in contrast to hydroxyferrocifen, after activation form not only quinone methides (QMs), but also catalysts (iron or ferrocenium ions). The released products act in a concerted fashion. In particular, QMs alkylate glutathione, thereby inhibiting the antioxidative system of the cell, whereas the iron species induce catalytic generation of hydroxyl radicals. Since the catalysts are formed as products of the activation reaction, it proceeds autocatalytically. The most potent prodrug described here is toxic toward cancer cells (human promyelocytic leukemia (HL-60), IC(50) = 9 μM, and human glioblastoma-astrocytoma (U373), IC(50) = 25 μM), but not toxic (up to 100 μM) toward representative nonmalignant cells (fibroblasts).     

Biphasic regulation of angiogenesis by reactive oxygen species

Reactive oxygen species (ROS) are believed to be important molecules in the regulation of angiogenesis. However, direct evidence is obtained from hydrogen peroxide only. The comparison of superoxide anion (O2-), hydrogen peroxide (H202) and hydroxyl radical (HO*) effects on angiogenesis in one angiogenic model were studied. Tube formation, migration and adhesion of endothelial cells were enhanced with a low concentration of O2 generated by 500 [microM xanthine (X) and 1 mU/ml xanthine oxidase (XO), but significantly inhibited as the XO increased to 10 mU/ml or more. Low concentrations of H2O2 (0.01-1 microM) induced tube formation and the maximal tube formation was achieved at 0.1 microM which also induced cell migration and adhesion, while high concentrations of H2O2 (100 microM) inhibited tube formation and cell migration. Both H2O2 and O2 inhibited cell proliferation at high concentration only. HO* at low concentration neither inhibited nor stimulated the tube formation, cell proliferation and migration but inhibited at high concentration. The effects of O2 were significantly abolished by catalase (CAT) alone or in combination with superoxide dismutase (SOD), but not by inactive CAT or SOD alone. Active CAT, but not inactive CAT, also reversed the effects of H2O2. Pretreatment with GSH effectively reversed the inhibitory effects of HO*. Therefore, our results suggest that ROS have biphasic effects on angiogenesis, which indicated that pharmacologically regulating cellular ROS levels might serve as an anti-angiogenic or angiogenic principles. They also provide a theoretical basis for the development and rational use of novel angiogenic and anti-angiogenic drugs.     

Intracellular generation of reactive oxygen species by mitochondria

Mitochondria have bioenergetic properties that strongly suggest their involvement in the cellular formation of reactive oxygen species (ROS). Apparent confirmation of this process has come from work with isolated mitochondria, which have been shown to produce H(2)O(2) from dismutating superoxide radicals. Two different sites were reported to shuttle single electrons to oxygen out of the normal respiratory sequence. However, the mechanisms for ROS formation at these two sites are controversial. Arguments against mitochondrial ROS formation in the living cell are based on the fact that bioenergetic alterations may result from the mechanical removal of mitochondria from their natural environment. Furthermore, the invasive detection methods that are generally used may be inappropriate because of the possible interaction of the detection system with mitochondrial constituents. The use of non-invasive detection methods has proved that ROS formation does not occur unless changes in the physical state of the membrane are established. The aim of this commentary is to discuss critically the arguments in favor of mitochondria as the main intracellular source of ROS. The pros and cons of working with isolated mitochondria, as well as the detection methodology are carefully analyzed to judge whether or not the above assumption is correct. The conclusion that mitochondria are the main ROS generators in the cell contradicts the fact that ROS release was not observed. However, if electron flow from ubiquinol to the bc(1) complex is hindered due to changes in lipid fluidity, single electrons may transfer to dioxygen and produce H(2)O(2) via superoxide radicals.     

活性氧对P-糖蛋白调节作用的研究进展

P-糖蛋白是由多药耐药基因(MDR1)编码的跨膜糖蛋白,介导ATP依赖的多药耐药,能把多种亲脂性外源物质排出胞外。P-gp过表达介导的多药耐药是目前多药耐药发生的主要机制。活性氧(reactive oxygen species,ROS)是细胞内的信号分子,能被多种物质调节升高。近有研究显示多种ROS诱导剂可以下调P-gp的表达,推测某些ROS诱导剂有望成为P-gp抑制剂而成为肿瘤多药耐药逆转剂研究的新方向。     

Ezetimibe and reactive oxygen species

Ezetimibe is a potent inhibitor of cholesterol absorption that has been approved for the treatment of hypercholesterolemia. Statin, 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, is an inhibitor of cholesterol synthesis. Statin is the first-choice drug to reduce low-density lipoprotein (LDL)-cholesterol for patients with hypercholesterolemia, due to its strong effect to lower the circulating LDL-cholesterol levels. Because a high dose of statins causes concern about rhabdomyolysis, it is sometimes difficult to achieve the guideline-recommended levels of LDL-cholesterol in patients with high LDL-cholesterol treated with statin monotherapy. Ezetimibe has been reported to reduce LDL-cholesterol safely with both monotherapy and combination therapy with statins. Ezetimibe is especially expected to be the best pharmacological option for the treatment of patients unable to achieve LDL-cholesterol goals with statins. Reactive oxygen species (ROS) are produced at low levels to maintain physiological redox balance. Oxidative stress results when ROS production exceeds the ability of cells to detoxify ROS. Overproduction of ROS damages cellular components, including lipids, leading to decline in physiological function and cell death. Oxidative stress exacerbates atherosclerosis, the major risk factor for coronary artery disease and ischemic stroke, at every step involves the accumulation of oxidized LDL in the arteries, leading to foam cell formation, plaque development, and plaque rupture. This review focuses on the recent findings of ezetimibe-related atheroprotective effects in vasculature. Moreover, known and proposed mechanisms of how ezetimibe could improve ROS-induced pro-atherosclerotic conditions in vasculature are discussed; these effects may help to explain the mechanisms by which ezetimibe may protect vascular from atherosclerosis.     

Regulation of NMDA receptor function by Fyn-mediated tyrosine phosphorylation]

The ionnotropic glutamate receptor, N-methyl-D-aspartate (NMDA) receptor, is a prominent ligand-gated and voltage-gated ion channel in excitatory synaptic transmission in the mammalian central nervous system. The NMDA channel is also regulated by its phosphorylation. We have shown that an Src family kinase Fyn phosphorylates NR2A and NR2B subunits of the NMDA receptor. The phosphorylation events are facilitated by the presence of PSD-95, which is quite likely due to the complex formation of Fyn, PSD-95, and the NMDA receptor: Fyn interacts with PSD-95 and PSD-95 interacts with the NMDA receptor. We have identified tyrosine phosphorylation sites on NR2A and NR2B. A phosphorylation of one of the sites on NR2B (Tyr1472) is largely dependent on Fyn and is elevated upon the LTP induction of hippocampal CA1 neurons. The data suggest that Tyr-1472 phosphorylation of NR2B is important for synaptic plasticity. A phosphorylation of the other tyrosine residues of NR2A and NR2B would also be involved in brain development and function.     

活性氧类物质在化学物诱导的细胞凋亡中的调节作用(英文)

细胞凋亡是在生理或病理条件下 ,为维持内环境的稳定的一种程序性细胞死亡。诱导细胞凋亡的因素可分为物理性、化学性和生物性因素。很多实验数据表明氧化应激在细胞凋亡的发生过程中具有重要作用。各种活性氧类物质如超氧阴离子、过氧化氢、羟自由基和一氧化氮均与细胞凋亡的发生有关。但是活性氧类物质诱导凋亡发生的机理尚未阐明。本综述在对细胞凋亡和氧化应激描述的基础上着重讨论活性氧类物质诱导细胞凋亡发生的可能机理。在以后的研究中 ,将着重于探讨化学物质通过产生活性氧类物质而影响细胞凋亡的机理及建立相关的生物标志物。     

Silica-induced nuclear factor-kappaB activation: involvement of reactive oxygen species and protein tyrosine kinase activation

Nuclear factor-kappaB (NF-kappaB) is a multiprotein complex that may regulate a variety of inflammatory cytokines involved in the initiation and progression of silicosis. The present study documents the ability of in vitro silica exposure to induce DNA-binding activity of NF-kappaB in a mouse peritoneal macrophage cell line (RAW264.7 cells) and investigates the role of reactive oxygen species (ROS) and/or protein tyrosine kinase in this activation. In vitro exposure of mouse macrophages to silica (100 microg/ml) resulted in a twofold increase in ROS production, measured as the generation of chemiluminescence (CL), and caused activation of NF-kappaB. Silica-induced CL was inhibited 100% by superoxide dismutase (SOD) and 75% by catalase, while NF-kappaB activation was inhibited by a variety of antioxidants (catalase, superoxide dismutase, alpha-tocopherol, pyrrolidine dithiocarbamate, or N-acetylcysteine). Further evidence for the involvement of ROS in NF-kappaB activation is that 1 mM H2O2 enhanced NF-kappaB/DNA binding and that this activation was inhibited by catalase. Specific inhibitors of protein tyrosine kinase, such as herbimycin A, genistein, and AG-494, prevented NF-kappaB activation in silica-treated cells. Genistein and AG-494 also reduced NF-kappaB activation in H2O2-treated cells. Results confirm that tyrosine phosphorylation of several cellular proteins (approximate molecular mass of 39, 58-70, and 103 kD) was increased in silica-exposed macrophages and that genistein inhibited this silica-induced phosphorylation. In contrast, inhibitors of protein kinase A or C, such as H89, staurosporin, calphostin C, and H7, had no marked inhibitory effect on silica-induced NF-kappaB activation. The results suggest that ROS may play a role in silica-induced NF-kappaB activation in macrophages and that phosphorylation events mediated by tyrosine kinase may be involved in this activation.     

Physico-chemical aspects of reactive oxygen species

Reactive Oxygen Species (ROS), namely hydroxyl (*OH) and superoxide (O2*-) free radicals and hydrogen peroxide (H2O2), are involved in all oxidative stress phenomena. These latter occur in numerous pathological disorders such as, for example, cardiovascular diseases, diabetes or neurodegenerative diseases. Knowledge of thermodynamic (reduction potentials) and kinetic (rate constants) properties of ROS allows to draw up a rigorous overview of the chemical reactivity of these species. Hydroxyl free radicals (*OH) are powerful oxidants (very toxic species) which attack all the biomolecular targets (DNA, proteins, lipids...), giving other free radicals localized on the targets. Superoxide free radicals (O2*-) have a more graduated reactivity, since they don't directly react with biological molecules. However, some toxicity would be attributed to them via secondary radicular reactions. Other free radicals (of peroxyl RO2* and alkoxyl RO* types), belonging also to the ROS family, contribute by enhancement to oxidative stress.     

Agonist-stimulated reactive oxygen species formation regulates beta2-adrenergic receptor signal transduction

Generation of reactive oxygen species (ROS) can occur upon agonist stimulation of surface receptors to modulate downstream signaling processes. Here, we show that activation of the beta2 adrenergic receptor (beta2AR) by stimulation with the agonist isoproterenol leads to generation of ROS that is required for beta2AR signal transduction. Specifically, we show that inhibition of NADPH oxidase with diphenyliodonium chloride, inhibition of the small GTPase Rac1 with NSC23766, and inhibition of formed ROS with the antioxidant N-acetyl-L-cysteine decreases beta2AR-mediated cAMP formation, protein kinase A activation, and receptor phosphorylation and internalization, but does not impact ligand binding. The results also show that inhibition of ROS attenuates active beta2AR-mediated binding of GTP to alpha subunits of heterotrimeric G proteins. Based on these results, we propose that agonist-dependent ROS formation is needed for beta2AR signal transduction, perhaps through stabilization of active receptor conformers by redox-mediated modification of receptor and/or Galpha proteins cysteine residues.     

Cytotoxic effects of B lymphocytes mediated by reactive oxygen species

Reactive oxygen species (ROS) are produced in all mammalian cells as a result of norman cellular metabolism and due to the activation of oxidant-producing enzymes in response to exogenous stimuli. The balance between ROS production and antioxidant defenses determines the degree of oxidative stress. Generation of ROS has been associated with cell signaling, stress responses, cell proliferation, aging and cancer development. The ability of ROS to induce cellular damage and to cause cell death opens the possibility to exploit this property in the treatment of cancer through a fee radical-mediated mechanism.     

Scavenging of reactive oxygen species by Eriobotrya japonica seed extract

We have clarified that Eriobotrya japonica seed extract has strong antioxidative activity, and is effective for the prevention and treatment of various diseases, such as hepatopathy and nephropathy. In this study, to investigate the influences of components of Eriobotrya japonica seed extract on its antioxidative activity, extracts were prepared using various solvents (n-hexane (Hex), ethyl acetate (EtOAc), n-butanol (n-BuOH), methanol (MeOH) and H2O) and the antioxidative activity of the solvent fractions and components was evaluated based on the scavenging of various radicals (DPPH and O2(-)) measured by the ESR method and the inhibition of Fe3+-ADP induced NADPH dependent lipid peroxidation in rat liver microsomes. The radical scavenging activities and inhibitory activities on lipid peroxidation differed among the solvent fractions and components. In the n-BuOH, MeOH and H2O fractions, radical scavenging activity and inhibitory activity on lipid peroxidation were high. In addition, these fractions contained abundant polyphenols, and the radical scavenging activity increased with the polyphenol content. In the low-polar Hex and EtOAc fractions, the radical scavenging activity was low, but the lipid peroxidation inhibition activity was high. These fractions contained beta-sitosterol, and the inhibitory activity on lipid peroxidation was high. Based on these findings, the antioxidative activity of Eriobotrya japonica seed extract may be derived from many components involved in a complex mechanism, resulting in high activity.     

Regulation of the expression of soluble guanylyl cyclase by reactive oxygen species

BACKGROUND AND PURPOSE: Superoxide anions produced during vascular disease scavenge nitric oxide (NO), thereby reducing its biological activity. The aim of the present study was to investigate whether reactive oxygen species (ROS) have a direct effect on soluble guanylyl cyclase (sGC) subunit levels and function and to ascertain the mechanism(s) involved. EXPERIMENTAL APPROACH: Rat aortic smooth muscle cells (RASM) or freshly isolated vessels were exposed to reactive oxygen species (ROS)-generating agents and sGC subunit expression was determined at the mRNA and/or protein level. cGMP accumulation was also determined in RASM exposed to ROS. KEY RESULTS: Incubation of smooth muscle cells with H(2)O(2), xanthine/xanthine oxidase (X/XO) or menadione sodium bisulphite (MSB) significantly decreased protein levels of alpha1 and beta1 subunits of sGC and reduced SNP-induced cGMP formation. Similarly, sGC expression was reduced in freshly isolated vessels exposed to ROS-generating agents. The ROS-triggered inhibition of alpha1 and beta1 levels was not blocked by proteasome inhibitors, suggesting that decreased sGC protein was not due to protein degradation through this pathway. Real time RT-PCR analysis demonstrated a 68% reduction in steady state mRNA levels for the alpha1 subunit following exposure to H(2)O(2). In addition, alpha1 promoter-driven luciferase activity in RASM decreased by 60% after H(2)O(2) treatment. CONCLUSION AND IMPLICATIONS: We conclude that oxidative stress triggers a decrease in sGC expression and activity that results from reduced sGC steady state mRNA levels. Altered sGC expression is expected to contribute to the changes in vascular tone and remodeling observed in diseases associated with ROS overproduction.     

多硫代二酮哌嗪类化合物C87体外对肿瘤细胞生长的抑制作用及对活性氧产生的影响

目的研究多硫代二酮哌嗪类化合物C87对肿瘤细胞生长的影响及其可能机制。方法用磺酰罗丹明B法观察C87 0.05~1μmol·L-1与A549,HCT116,He La和SMMC7721作用24,48和72 h对肿瘤细胞存活率的影响,并计算50%生长抑制浓度(GI50);用流式细胞术检测C87 0.1~2.5μmol·L-1作用HCT116和He La细胞6 h,C87 2.5μmol·L-1作用0~6 h活性氧(ROS)的生成量;用流式细胞术检测C872.5μmol·L-1伴随给予抗氧化剂N-乙酰半胱氨酸(NAC)作用HCT116和He La细胞6 h ROS的生成量;用SRB法观察C87 0.05~1μmol·L-1伴随给予NAC作用24和48 h对HCT116细胞存活率的影响。结果C87 0.05~1μmol·L-1作用24,48和72 h,可明显抑制A549,HCT116,He La和SMMC7721细胞存活(P<0.05),且具有浓度依赖性(72 h,r2=0.946,0.989,0.973和0.984,P<0.05);C87 1μmol·L-1与上述4种肿瘤细胞作用24,48和72 h能时间依赖性地抑制细胞存活(r2=0.983,0.956,0.951和0.873,P<0.05)。C87 0.25~2.5μmol·L-1与He La细胞和HCT116细胞作用6 h,可诱发细胞内ROS产生,且呈浓度依赖性(r2=0.760,P=0.045;r2=0.987,P=0.001);C87 2.5μmol·L-1作用0.5~6 h,诱导ROS的产生呈时间依赖性(r2=0.886,P=0.017;r2=0.994,P=0.000)。给予抗氧化剂NAC后能明显抑制C87引起的He La细胞和HCT116细胞ROS生成(P<0.05),NAC 5和10 mmol·L-1可明显逆转C87引起的HCT116细胞死亡,GI50值明显增大,作用24 h时GI50为1.446和1.134μmol·L-1(C87处理组为0.513μmol·L-1);处理48 h时GI50为0.882和1.166μmol·L-1(C87处理组为0.333μmol·L-1)。结论化合物C87对A549,HCT116,He La和SMMC7721肿瘤细胞的生长具有抑制作用,其机制可能与其诱导细胞内氧化应激有关。     

Models of reactive oxygen species in cancer

Increased generation of reactive oxygen species (ROS) has been observed in cancer, degenerative diseases, and other pathological conditions. ROS can stimulate cell proliferation, promote genetic instability, and induce adaptive responses that enable cancer cells to maintain their malignant phenotypes. However, when cellular redox balance is severely disturbed, high levels of ROS may cause various damages leading to cell death. The studies of ROS effects on biological systems, their underlying mechanisms and therapeutic implications largely depend on proper experimental models. Here we review several in vitro and in vivo models for ROS research.