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1.
《BBA》2020,1861(12):148290
We hypothesized that NO • is generated in isolated cardiac mitochondria as the source for ONOO − production during oxidative stress. We monitored generation of ONOO − from guinea pig isolated cardiac mitochondria subjected to excess Ca 2+ uptake before adding succinate and determined if ONOO − production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl 2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O 2•−. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO − to form dityrosine (diTyr) in proportion to the ONOO − present. We found that exposing mitochondria to excess CaCl 2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H 2O 2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa 2+ and succinate, increased mitochondrial ONOO − production via NO • and O 2•−. Changes in mitochondrial ONOO − production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO • scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO − production, PTIO decreased production of ONOO −, and TEMPOL decreased ONOO − levels by converting more O 2•− to H 2O 2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO − into the buffer is dependent both on O 2•− released from mitochondria and NO • derived from a mtCa 2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible. 相似文献
2.
Tumor metastasis is the main cause of death in cancer patients. Anoikis resistance is one critical malefactor of metastatic cancer cells to resist current clinical chemotherapeutic treatments. Although endoperoxide-containing compounds have long been suggested as anticancer drugs, few have been clinically employed due to their instability, complex synthesis procedure or low tumor cell selectivity. Herein, we describe a one-pot strategy to synthesize novel amino endoperoxides and their derivatives with good yields and stabilities. In vitro cell-based assays revealed that 4 out of the 14 amino endoperoxides selectively induce metastatic breast carcinoma cells but not normal breast cells to undergo apoptosis, in a dose-dependent manner. Mechanistic studies showed that the most potent amino endoperoxide, 4-Me, is selective for cancer cells expressing a high level of Nox4. The anticancer effects are further shown to be associated with reduced O 2−:H 2O 2 ratio and increased ·OH level in the cancerous cells. Animal study showed that 4-Me impairs orthotopic breast tumor growth as well as tumor cell metastasis to lymph nodes. Altogether, our study suggests that anticancer strategies that focus on redox-based apoptosis induction in tumors are clinically viable. 相似文献
3.
Mitochondrial reactive oxygen species regulate many important biological processes. We studied H 2O 2 formation by nonsynaptic brain mitochondria in response to the addition of low concentrations of glutamate, an excitatory neurotransmitter. We demonstrated that glutamate at concentrations from 10 to 50 μM stimulated the H 2O 2 generation in mitochondria up to 4-fold, in a dose-dependent manner. The effect of glutamate was observed only in the presence of Ca 2+ (20 μM) in the incubation medium, and the rate of calcium uptake by the brain mitochondria was increased by up to 50% by glutamate. Glutamate-dependent effects were sensitive to the NMDA receptor inhibitors MK-801 (10 μM) and D-AP5 (20 μM) and the inhibitory neurotransmitter glycine (5 mM). We have shown that the H 2O 2 formation caused by glutamate is associated with complex II and is dependent on the mitochondrial potential. We have found that nonsynaptic brain mitochondria are a target of direct glutamate signaling, which can specifically activate H 2O 2 formation through mitochondrial respiratory chain complex II. The H 2O 2 formation induced by glutamate can be blocked by glycine, an inhibitory neurotransmitter that prevents the deleterious effects of glutamate in brain mitochondria. 相似文献
5.
Serum albumin (SA) has been shown to act as a heme scavenger in hemolysis and can protect cell against the toxic effect of heme. However, the mechanism of SA in heme detoxification is not well understood. Interestingly, increasing studies indicate that heme/H2O2-dependent reaction is unlikely to be the principal cause of heme toxicity in excessive intravascular hemolysis conditions. Moreover, high levels of NO2? and NO3? were also found in patients with severe hemolytic diseases, which seem to involve in heme toxic effect as well. Therefore, we proposed that studying the protection mechanism of SA against the heme/H2O2/NO2?-induced cytotoxicity may be more consistent with free heme-associated disorder pathologies. In this study, we tested the hypotheses that tyrosine residues of bovine serum albumin (BSA) play a prominent role in detoxifying heme in SH-SY5Y cells. Both BSA and tyrosine modified BSA (BSA-T) were used to explore this protective mechanism. Most of cellular injury (oxidative and nitrative damage) induced by heme/H2O2/NO2? were prevented by pretreatment with an equimolar concentration of BSA or BSA-T, and BSA was found more efficient than BSA-T. Meanwhile, BSA or BSA-T binding to heme is not accompanied by a decrease of heme’s peroxidase activity. Collectively, these data suggest that the protecting effect of BSA against heme-induced damage in the intravascular hemolysis diseases is not accomplished by preventing the primary reactivity of heme with H2O2, but by trapping radical through special residues such as tyrosine to render other important protein less damaged. 相似文献
7.
Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin‐3‐gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H 2O 2 release in cell cultures, and exogenous catalase (CAT) abrogated EGCG‐induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3‐loaded, EGCG‐exposed MMe cells showed significant [Ca 2+] i rise, prevented by CAT, dithiothreitol or the T‐type Ca 2+ channel blockers mibefradil and NiCl 2. Cell loading with dihydrorhodamine 123 revealed EGCG‐induced ROS production, prevented by CAT, mibefradil or the Ca 2+ chelator BAPTA‐AM. Direct exposure of cells to H 2O 2 produced similar effects on Ca 2+ and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Ca v3.2 T‐type Ca 2+ channels in these cells, compared to normal mesothelium. Also, Ca v3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Ca v3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T‐type Ca 2+ channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target. 相似文献
9.
Nitric oxide, a gaseous free radical, is poorly reactive with most biomolecules but highly reactive with other free radicals. Its ability to scavenge peroxyl and other damaging radicals may make it an important antioxidant in vivo, particular in the cardiovascular system, although this ability has been somewhat eclipsed in the literature by a focus on the toxicity of peroxynitrite, generated by reaction of O ·-2 with NO · (or of NO - with O 2). On balance, experimental and theoretical data support the view that ONOO - can lead to hydroxyl radical (OH ·) generation at pH 7.4, but it seems unlikely that OH · contributes much to the cytotoxicity of ONOO -. The cytotoxicity of ONOO - may have been over-emphasized: its formation and rapid reaction with antioxidants may provide a mechanism of using NO · to dispose of excess O ·-2, or even of using O ·-2 to dispose of excess NO ·, in order to maintain the correct balance between these radicals in vivo. Injection or instillation of “bolus” ONOO - into animals has produced tissue injury, however, although more experiments generating ONOO - at steady rates in vivo are required. The presence of 3-nitrotyrosine in tissues is still frequently taken as evidence of ONOO - generation in vivo, but abundant evidence now exists to support the view that it is a biomarker of several “reactive nitrogen species”. Another under-addressed problem is the reliability of assays used to detect and measure 3-nitrotyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artefacts. Exposure of biological material to low pH (e.g. during acidic hydrolysis to liberate nitrotyrosine from proteins) or to H 2O 2 might cause artefactual generation of nitrotyrosine from NO -2 in the samples. This may be the origin of some of the very large values for tissue nitrotyrosine levels quoted in the literature. Nitrous acid causes not only tyrosine nitration but also DNA base deamination at low pH: these events are relevant to the human stomach since saliva and many foods are rich in nitrite. Several plant phenolics inhibit nitration and deamination in vitro, an effect that could conceivably contribute to their protective effects against gastric cancer development. 相似文献
10.
High-dose ascorbic acid (AsA) treatment, known as pharmacological AsA, has been shown to exert carcinostatic effects in many types of cancer cells and in vivo tumour models. Although pharmacological AsA has potential as a complementary and alternative medicine for anticancer treatment, its effects on human tongue carcinoma have not yet been elucidated. In this study, we investigated the effect of AsA treatment on human tongue carcinoma HSC-4 cells compared with non-tumourigenic tongue epithelial dysplastic oral keratinocyte (DOK) cells. Our results show that treatment with 1 and 3?mM of AsA for 60?min preferentially inhibits the growth of human tongue carcinoma HSC-4 over DOK cells. Furthermore, AsA-induced effects were accompanied by increased intracellular oxidative stress and were repressed by treatment with a hydrogen peroxide (H 2O 2) scavenger catalase and a superoxide anion radical (O 2?) scavenger, tempol. Time-lapse observation and thymidine analog EdU incorporation revealed that AsA treatment induces not only cell death but also suppression of DNA synthesis and cell growth. Moreover, the growth arrest was accompanied by abnormal cellular morphologies whereby cells extended dendrite-like pseudopodia. Taken together, our results demonstrate that AsA treatment can induce carcinostatic effects through induction of cell death, growth arrest, and morphological changes mediated by H 2O 2 and O 2? generation. These findings suggest that high-dose AsA treatment represents an effective treatment for tongue cancer as well as for other types of cancer cells. 相似文献
11.
Although reactive oxygen species (ROS) are well-established mediators of oxidative damage and cell demise, the mechanisms by which they trigger specific cell death modalities and the temporal/spatial requirements underlying this phenomenon are largely unknown. Yet, it is well established that most anticancer therapies depend on ROS production for efficient tumor eradication. Using several non-small-cell lung cancer cell lines, we have dissected how the site of ROS production and accumulation in various cell compartments affect cell fate. We demonstrate that high levels of exogenously generated H 2O 2 induce extensive DNA damage, ATP depletion, and severe cytotoxicity. Although these effects were independent of caspase activity, they could—at least in part—be prevented by RIP1 kinase inhibition. In contrast, low levels of exogenously produced H 2O 2 triggered a modest drop in ATP level, delayed toxicity, G2/M arrest, and cell senescence. Mitochondrially produced H 2O 2 induced a reversible ATP drop without affecting cell viability. Instead, the cells accumulated in the G1/S phase of the cell cycle and became senescent. Concomitant inhibition of glycolysis was found to markedly sensitize cells to death in the presence of otherwise nontoxic concentrations of H 2O 2, presumably by the inhibition of ATP-restoring mechanisms. Combined, our data provide evidence that ROS might dictate different cellular consequences depending on their overall concentration at steady-state levels and on their site of generation. 相似文献
12.
Mitochondria are a major source of reactive oxygen species (ROS). Recent studies have estimated that mitochondrial DNA mutations inducing the overproduction of ROS are associated with human cancer. However, a substantial challenge in elucidating their diverse roles in regulating tumorigenesis is the lack of methods for probing ROS in living systems with molecular specificity. In this study, we reported the application of two fluorescent probes, 2‐chloro‐1,3‐dibenzothiazolinecyclohexene and naphthofluorescein disulfonate, which showed high selectivity for superoxide (O 2•−) and hydrogen peroxide (H 2O 2). They were capable of detecting and visualizing O 2•− and H 2O 2 overproduction caused by a mutation in the gene encoding nicotinamide adenine dinucleotide dehydrogenase subunit 6 ( ND6) in HepG2 cells. The levels of O 2•− and H 2O 2 in mitochondria isolated from HepG2 cells were found to be 0·63 ± 0·07 and 1·13 ± 0·05 μM, respectively. Using assays of tumorigenesis in mouse models, we found that treatment of the mice with different ROS scavengers suppressed tumour growth. These findings suggested that ROS generated by ND6 gene mutation do play an important role in regulating tumorigenesis and H 2O 2 may be a key modulator. Copyright © 2011 John Wiley & Sons, Ltd. 相似文献
13.
Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet ( 1Δ g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO ? generated in the diffusion - controlled reaction of cellular NO ? and O. The experiments consist of (i) chemical generation of ONOO ? from NO ? gas and KO 2 powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO ? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of 1O 2 by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of 1O 2 generated in ONOO ?/H + reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H 2O 2/OCl ? reaction that generates 1O 2 with unit efficiency at alkaline pH. 1O 2 was generated with unit efficiency with respect to ONOO ? concentration by the ONOO ?/H + reaction. 相似文献
14.
Menadione-catalyzed H 2O 2 production by viable animal cells was proportional to the viable cell number, and H 2O 2 production decreased with increasing cytotoxic effects after the incubation of cells with cytotoxic compounds. The cytotoxic
effects of food additives, pesticides, antibiotics, heavy metals, phytotoxins, mycotoxins, and marine toxins were estimated
using the above test employingNIH/3T3 and Neuro-2a cells. Synergistic effects of the toxin mixture were observed and acute
cytotoxicity detected 1 h after the incubation of cells with toxins. This menadione-catalyzed H 2O 2production assay is rapid and simple compared to other popular cytotoxicity tests such as the MTT reduction assay and Neutral
red inclusion test, requiring4 h. The menadione-catalyzed H 2O 2 production assay is expected to be a useful food safety test for rapidly detecting toxic compounds having a basic cytotoxic
effect on common animal cells.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
15.
Reactive oxygen species (ROS) can trigger neuronal cell death and has been implicated in a variety of neurodegenerative diseases as well as brain ischemia. Here, we demonstrate that chronic (but not acute) glutamate toxicity in primary cortical neuronal cultures is associated with hydrogen peroxide (H 2O 2) accumulation in the culture medium and that neurotoxicity can be eliminated by external catalase treatment. Neuronal cultures in Ca 2+-free medium or treated with BAPTA showed reduced glutamate-induced H 2O 2 generation, indicating that H 2O 2 generation is Ca 2+-dependent. Pharmacological and genetic approaches revealed that NADPH oxidase plays a role in glutamate-induced H 2O 2 generation and that activation of NMDA and AMPA receptors is involved in this H 2O 2 generation. The Nox4 siRNA reduced NMDA-induced H 2O 2 production by 54% and cytotoxicity in parallel, suggesting that Nox4-containing NADPH oxidase functions NMDA receptor-mediated H 2O 2 production resulting in neurotoxicity. These findings suggest that the modulation of NADPH oxidase can be used as a new therapeutic strategy for glutamate-induced neuronal diseases. 相似文献
16.
Sesquiterpenes have attracted much interest with respect to their protective effect against oxidative damage that may be the cause of many diseases including several neurodegenerative disorders and cancer. Our previous unpublished work suggested that cyclosativene (CSV), a tetracyclic sesquiterpene, has antioxidant and anticarcinogenic features. However, little is known about the effects of CSV on oxidative stress induced neurotoxicity. We used hydrogen peroxide (H 2O 2) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of CSV in H 2O 2-induced toxicity in new-born rat cerebral cortex cell cultures for the first time. For this aim, MTT and lactate dehydrogenase release assays were carried out to evaluate cytotoxicity. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, the single cell gel electrophoresis (or Comet assay) was also performed for measuring the resistance of neuronal DNA to H 2O 2-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (Comet assay) increased in the H 2O 2 alone treated cultures. But pre-treatment of CSV suppressed the cytotoxicity, genotoxicity and oxidative stress which were increased by H 2O 2. On the basis of these observations, it is suggested that CSV as a natural product with an antioxidant capacity in mitigating oxidative injuries in the field of neurodegenerative disorders. 相似文献
17.
Regulation of H 2 utilization, as monitored by the hydrogenase-mediated 3H 2 exchange reaction, was examined among phytoplankton communities in situ and populations in culture. During a 2-year study in the Chowan River, North Carolina, at least 2 major groups of phytoplankton dominated 3H 2 exchange rates. They included N 2 fixing cyanobacteria and NO 3 }- utilizing genera. Utilization of 3H 2 by N 2 fixers was mainly dark-mediated, whereas 3H 2 utilization associated with periods of NO 3 }- abundance revealed an increasing dependence on light. Inhibitors of N 2 fixation (C 2H 2 and NH 4 +) negatively affected 3H 2 utilization, substantiating previous findings that close metabolic coupling of both processes exists among N 2 fixing cyanobacteria. Conversely, NO 3 }- stimulated 3H 2 utilization among N 2 and non-N 2 fixing genera, particularly under illuminated conditions. A variety of environmental factors were shown to control 3H 2 exchange. In addition to the nitrogen sources discussed above, dissolved O 2, photosynthetically available radiation (PAR), temperature, and pH changes altered 3H 2 exchange rates. It is likely that other factors not addressed here could also affect 3H 2 exchange rates. At least 2 ecological benefits from H 2 utilization in natural phytoplankton can be offered. They include the simultaneous generation of adenosine triphosphate (ATP) and consumption of O 2 during the oxidation of H 2 via an oxyhydrogen or “Knallgas” reaction. Both processes could help sustain phytoplankton, and particularly cyanobacterial, bloom intensity under natural conditions when O 2 supersaturation is common in surface waters. H 2 utilization appeared to be a general feature of natural and laboratory phytoplankton populations. The magnitudes of 3H 2 utilization rates were directly related to community biomass. Although it can be shown that utilization rates are controlled by specific environmental factors, the potential relationships between H 2 utilization and phytoplankton primary production remain poorly understood. 相似文献
18.
Catalase is well known as capable of inducing the decomposition of H 2O 2. In this study, a kind of immobilized catalase (entrapped in cross-linked chitosan beads) was dispersed in conventional acetate filter as an antioxidant additive. Quantitative estimation of the free radicals in mainstream cigarette smoke (MCS) was performed to address the effect of this modified filter. It was found that the levels of PBN adduct and NO ?/NO 2? associated with the gas-phase mainstream cigarette smoke (GPCS) were efficiently decreased by ~40% through catalase filtering. Besides, the modified filter was found to lower the MCS-induced adverse biological effects including lipid peroxidation and mutagenicity. This was proved to be substantially attributed to the catalase-dependent breakdown of NO ?, which was stimulated by some of peroxides (most probably being H 2O 2), the dismutation products of tar particulate matters (TPM). These results highlighted a promising approach to reduce the smoking-associated health risks to passive smokers. Moreover, the mechanisms of catalase filtering may be helpful for the development of appropriate immobilized enzyme systems to be applied for reducing health risks associated with gaseous pollutants. 相似文献
19.
Summary. Multidrug resistance (MDR) has been studied extensively because it is one of major problems in cancer chemotherapy. The MDR phenotype is often due to overexpression of P-glycoprotein (P-gp), that acting as an energy-dependent drug efflux pump exports various anticancer drugs out of cells. The major goal of our investigation is to establish whether bovine serum amine oxidase (BSAO), which generates the products H 2O 2 and aldehyde(s), from the polyamine spermine, is able to overcome MDR of human cancer cells. The cytotoxicity of the products was evaluated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. A clonogenic cell survival assay demonstrated that LoVo DX cells were more sensitive than LoVo WT cells. Exogenous catalase protected cells against cytotoxicity mainly due to the formation of H 2O 2. However, spermine-derived aldehyde(s) still induced some cytotoxicity. The cytotoxic effect was totally inhibited in the presence of both enzymes, catalase and NAD-dependent aldehyde dehydrogenase (ALDH). Transmission electron microscopy investigations showed that BSAO and spermine induced evident mitochondria alterations, more pronounced in MDR than in LoVo WT cells. The mitochondrial activity was checked by flow cytometry studies, labelling cells with the probe JC1, that displayed a basal hyperpolarized status of the mitochondria in multidrug-resistant cells. After treatment with amine oxidase in the presence of polyamine-spermine, the cells showed a marked increase in mitochondrial membrane depolarization higher in LoVo DX than in LoVo WT cells. Our findings suggest that toxic oxidation products formed from spermine and BSAO could be a powerful tool in the development of new anticancer treatments, mainly against MDR tumor cells. 相似文献
20.
In this article, a new compound H 2[{Cu(HL)(H 2O)} 2(P 2Mo 5O 23)]·5H 2O ( 1) (HL = 2-acetylpyrazine thiosemicarbazone) has been synthesized and structurally characterized by single-crystal X-ray diffraction of and other detection techniques. Interestingly, the structure of 1 is different from many reported copper-based complexes, in which the [P 2Mo 5O 23] 6−, two Cu 2+ ions and two HL were directly connected by covalent bands. Biological studies demonstrated that 1 indicated moderate antibacterial activity against Escherichia coli ( E. coli) and Staphylococcus aureus ( S. aureus), and a better cytotoxicity against human hepatic cancer line (SMMC-7721) than Mitoxantrone (Mito), the current clinical anticancer drug. Besides, the antibacterial mechanisms of 1 have been studied by the membrane integrity disruption, the destructive reactive oxygen species generation (ROS), the glutathione (GSH) depletion and the depressed enzymatic activity of respiratory chain dehydrogenases (RCD). These results revealed that the combination of HL, Cu 2+, [P 2Mo 5O 23] 6− shows a higher antibacterial and cytotoxic activity. 相似文献
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