Diverse subcellular locations of cryptogein-induced reactive oxygen species production in tobacco Bright Yellow-2 cells

Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report real-time H2O2 accumulation in cell populations, we show that the internal signal for H2O2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H2O2 was also followed in individual cells using the 2',7'-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H2O2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.     

2,7‐DICHLOROFLUORESCIN OXIDATION AND REACTIVE OXYGEN SPECIES: WHAT DOES IT MEASURE?

Intracellular 2,7-dichlorofluorescin (H(2)DCF) oxidation is often used to measure the production of reactive oxygen species (ROS) within cells. The rate of H(2)DCF oxidation depends on the concentration of glutathione, which is an alternative target for ROS. Our results suggest that increased rate of H(2)DCF oxidation be interpreted as an indication of general oxidative stress due to a variety of reasons, including depletion of antioxidants, rather than as a specific proof of augmented ROS formation.     

Effect of extracellular Mg(2+) on ROS and Ca(2+) accumulation during reoxygenation of rat cardiomyocytes

The effects of Mg(2+) on reactive oxygen species (ROS) and cell Ca(2+) during reoxygenation of hypoxic rat cardiomyocytes were studied. Oxidation of 2',7'-dichlorodihydrofluorescein (DCDHF) to dichlorofluorescein (DCF) and of dihydroethidium (DHE) to ethidium (ETH) within cells were used as markers for intracellular ROS levels and were determined by flow cytometry. DCDHF/DCF is sensitive to H(2)O(2) and nitric oxide (NO), and DHE/ETH is sensitive to the superoxide anion (O(2)(-).), respectively. Rapidly exchangeable cell Ca(2+) was determined by (45)Ca(2+) uptake. Cells were exposed to hypoxia for 1 h and reoxygenation for 2 h. ROS levels, determined as DCF fluorescence, were increased 100-130% during reoxygenation alone and further increased 60% by increasing extracellular Mg(2+) concentration to 5 mM at reoxygenation. ROS levels, measured as ETH fluorescence, were increased 16-24% during reoxygenation but were not affected by Mg(2+). Cell Ca(2+) increased three- to fourfold during reoxygenation. This increase was reduced 40% by 5 mM Mg(2+), 57% by 10 microM 3,4-dichlorobenzamil (DCB) (inhibitor of Na(+)/Ca(2+) exchange), and 75% by combining Mg(2+) and DCB. H(2)O(2) (25 and 500 microM) reduced Ca(2+) accumulation by 38 and 43%, respectively, whereas the NO donor S-nitroso-N-acetyl-penicillamine (1 mM) had no effect. Mg(2+) reduced hypoxia/reoxygenation-induced lactate dehydrogenase (LDH) release by 90%. In conclusion, elevation of extracellular Mg(2+) to 5 mM increased the fluorescence of the H(2)O(2)/NO-sensitive probe DCF without increasing that of the O(2)(-).-sensitive probe ETH, reduced Ca(2+) accumulation, and decreased LDH release during reoxygenation of hypoxic cardiomyocytes. The reduction in LDH release, reflecting the protective effect of Mg(2+), may be linked to the effect of Mg(2+) on Ca(2+) accumulation and/or ROS levels.     

Refinement of the dichlorofluorescein assay for flow cytometric measurement of reactive oxygen species in irradiated and bystander cell populations

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.     

Detection of reactive oxygen species induced by radiation in cells using the dichlorofluorescein assay

The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2'7'-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2'7'-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.     

Biochemical identification of a hydroperoxide derivative of the free 8-oxo-7,8-dihydroguanine base

8-Oxo-7,8-dihydroguanine is one the most abundant base lesions in pro- and eukaryotic DNA. In mammalian cells, it is excised by the 8-oxoguanine DNA glycosylase (OGG1) during DNA base-excision repair, and the generated free 8-oxoG base is one of the DNA-derived biomarkers of oxidative stress in biological samples. The modification of 8-oxoG in the context of nucleoside and DNA has been the subject of many studies; however, the oxidative transformation of the free 8-oxoG base has not been described. By using biochemical and cell biological assays, we show that in the presence of molecular oxygen, the free 8-oxoG base transforms to a highly reactive hydroperoxide (8-oxoG*). Specifically, 8-oxoG* oxidizes Amplex red to resorufin, H(2)DCF to DCF, Fe(2+) to Fe(3+), and GSH to GSSG. This property of 8-oxoG* was diminished by treatment with catalase and glutathione peroxidase, but not superoxide dismutase. 8-OxoG* formation was prevented by reducing agents or nitrogen atmosphere. Its addition to CM-H(2)DCF-DA-loaded cells rapidly increased intracellular DCF fluorescence. There were no such properties observed for 8-oxodeoxyguanosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2'-deoxyguanosine, guanine, adenine, guanosine, and 8-hydroxyadenine. These data imply that a free 8-oxoG base is more susceptible to oxidation than is its nucleoside form and, consequently, it stands as unique among intact and oxidatively modified purines.     

Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes

It is currently believed that oxidative stress and inflammation play a significant role in atherogenesis. Artichoke extract exhibits hypolipemic properties and contains numerous active substances with antioxidant properties in vitro. We have studied the influence of aqueous and ethanolic extracts from artichoke on intracellular oxidative stress stimulated by inflammatory mediators (TNFalpha and LPS) and ox-LDL in endothelial cells and monocytes. Oxidative stress which reflects the intracellular production of reactive oxygen species (ROS) was followed by measuring the oxidation of 2', 7'-dichlorofluorescin (DCFH) to 2', 7'-dichlorofluorescein (DCF). Agueous and ethanolic extracts from artichoke were found to inhibit basal and stimulated ROS production in endothelial cells and monocytes in dose dependent manner. In endothelial cells, the ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production by 60% (p<0,001) while aqueous extract (50 microg/ml) by 43% (p<0,01). The ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production in monocytes by 76% (p<0,01). Effective concentrations (25-100 microg/ml) were well below the cytotoxic levels of the extracts which started at 1 mg/ml as assessed by LDH leakage and trypan blue exclusion. Penetration of some active substances into the cells was necessary for inhibition to take place as juged from the effect of preincubation time. These results demonstrate that artichoke extracts have marked protective properties against oxidative stress induced by inflammatory mediators and ox-LDL in cultured endothelial cells and monocytes.     

Adaptation of the dichlorofluorescein assay for detection of radiation-induced oxidative stress in cultured cells

The oxidation of 2'7'-dichlorofluorescin (DCFH) to 2'7'-dichlorofluorescein (DCF), a fluorescent DCFH oxidation product, is a highly sensitive indicator that is used to measure oxidative stress in cells. In the present study, a DCF assay has been adapted to quantify oxidative stress in human breast epithelial cell cultures after exposure to gamma rays. The results demonstrate that the sensitivity and specificity of the DCF assay is strongly influenced by the timing of DCFH diacetate (DCFH-DA) substrate loading in relation to radiation exposure and by the matrix in which the cells were loaded with DCFH-DA substrate. Under the conditions optimized in this study, the DCF assay is capable of detecting increased DCFH oxidation in cell cultures irradiated with gamma rays at a dose as low as 1.5 cGy. The increase in fluorescence was directly proportional to the radiation dose, which ranged from 0 to 2 Gy, and a minimal level of fluorescence was observed in sham-irradiated cells. These results indicate that the DCF assay optimized in this study is highly sensitive, linear and specific for measuring oxidative stress in irradiated cells.     

Active oxygen chemistry within the liposomal bilayer. Part IV: Locating 2',7'-dichlorofluorescein (DCF), 2',7'-dichlorodihydrofluorescein (DCFH) and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer

2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) is commonly used to detect the generation of reactive oxygen intermediates and for assessing the overall oxidative stress in toxicological phenomenon. It has been suggested that DCFH-DA crosses the cell membrane, subsequently undergoing deacetylation by intracellular esterases. The resulting 2',7'-dichlorodihydrofluorescein (DCFH) is proposed to react with intracellular hydrogen peroxide or other oxidizing ROS to give the fluorescent 2',7'-dichlorofluorescein (DCF). Using an NMR chemical shift-polarity correlation, we have determined that DCFH-DA and DCFH are located well within the lipid bilayer and certainly not at the interface. These results, therefore, put into serious question the proposed ability of DCFH to come in contact with the aqueous phase and thereby interact with aqueous intracellular ROS and components. However, H2O2 and superoxide can cross or at least penetrate the lipid bilayer and react with certain lipophilic substrates. This may well describe the mode of reaction of these and other ROS with DCFH.     

Direct oxidation of 2',7'-dichlorodihydrofluorescein by pyocyanin and other redox-active compounds independent of reactive oxygen species production

Formation of dichlorofluorescein (DCF), the fluorescent oxidation product of 2',7'-dichlorodihydrofluorescein (DCFH2), in cells loaded with the latter compound is often used to detect ROS formation. We previously found that exposure of DCFH2-loaded A549 cells to the Pseudomonas aeruginosa secretory product pyocyanin results in DCF formation, consistent with ROS production. However, since pyocyanin directly accepts electrons from NAD(P)H, we hypothesized that pyocyanin might directly oxidize DCFH2 to DCF without an ROS intermediate. Incubation of DCFH2 with pyocyanin rapidly resulted in DCF formation, the rate of which was proportional to the [pyocyanin] and was not inhibited by SOD or catalase. Phenazine methosulfate, a pyocyanin analog, was more effective than pyocyanin in generating DCF. Mitoxantrone and ametantrone also produced DCF. However, menadione, paraquat, plumbagin, streptonigrin, doxorubicin, daunorubicin, and 5-iminodaunorubicin did not. Pyocyanin, phenazine methosulfate, mitoxantrone, and ametantrone also oxidized dihydrofluorescein and 5- (and 6-) -carboxy-2',7'-dichlorodihydrofluorescein, whereas dihydrorhodamine was oxidized only by pyocyanin or phenazine methosulfate. Under aerobic conditions, the interaction of DCFH2 with pyocyanin or phenazine methosulfate (but not mitoxantrone or ametantrone) produced superoxide, as detected by spin trapping. Direct oxidation of the fluorescent probes needs to be controlled for when employing these compounds to assess ROS formation by biological systems exposed to redox active compounds.     

Protein Phosphatases but not Reactive Oxygen Species Play Functional Role in Acute Amphetamine-Mediated Dopamine Release

Drug abuse-induced neurodegeneration can be triggered by elevated production of reactive oxygen species (ROS). Involvement of oxidative stress in acute amphetamine (AMPH)-mediated dopamine (DA) release, however, has not been completely understood yet. In order to elucidate the dopaminergic response of PC12 cells to a single dose of 10 μM AMPH, ROS production was measured as related to the extracellular DA level. Due to the spontaneous oxidation of peroxide-sensitive fluorophore 2′,7′-dichlorofluorescin diacetate (DCFH-DA) to 2′,7′-dichlorofluorescein (DCF), the increase in fluorescence could not be unambiguously attributed to AMPH-triggered ROS production. Based on Amplex Red fluorescence, no ROS production was detected after acute AMPH application. Our data strongly suggest that ROS development was not the main triggering factor for immediate DA release after acute AMPH treatment. On the other hand, AMPH-induced elevation of DA levels in rat brain striatal slices was quenched by the water soluble antioxidant, N-acetylcysteine (NAC) at 10 mM. In this study, we also investigated the contribution of protein phosphatases to the AMPH-induced rat brain striatal dopaminergic response. The experimental protocol, double AMPH challenge was applied for screening the effect of NAC and cantharidin on AMPH-mediated DA release. Here we show that AMPH-mediated DA release increased nearly twofold in striatal rat brain slices pretreated for 30 min with 1000 μM cantharidin, a selective PP1 and PP2A inhibitor. These findings prove the lack of ROS inhibitory action on protein phosphatase activity in acute AMPH-mediated DA efflux.     

Oxidation pathways underlying the pro-oxidant effects of apigenin

Apigenin, a natural flavone, is emerging as a promising compound for the treatment of several diseases. One of the hallmarks of apigenin is the generation of intracellular reactive oxygen species (ROS), as judged by the oxidation of reduced dichlorofluorescein derivatives seen in many cell types. This study aimed to reveal some mechanisms by which apigenin can be oxidized and how apigenin-derived radicals affect the oxidation of 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein (H₂DCF), a probe usually employed to detect intracellular ROS. Apigenin induced a rapid oxidation of H₂DCF in two different immortalized cell lines derived from rat and human hepatic stellate cells. However, apigenin did not generate ROS in these cells, as judged by dihydroethidium oxidation and extracellular hydrogen peroxide production. In cell-free experiments we found that oxidation of apigenin leads to the generation of a phenoxyl radical, which directly oxidizes H₂DCF with catalytic amounts of hydrogen peroxide. The net balance of the reaction was the oxidation of the probe by molecular oxygen due to redox cycling of apigenin. This flavonoid was also able to deplete NADH and glutathione by a similar mechanism. Interestingly, H₂DCF oxidation was significantly accelerated by apigenin in the presence of horseradish peroxidase and xanthine oxidase, but not with other enzymes showing peroxidase-like activity, such as cytochrome c or catalase. We conclude that in cells treated with apigenin oxidation of reduced dichlorofluorescein derivatives does not measure intracellular ROS and that pro- and antioxidant effects of flavonoids deduced from these experiments are inconclusive and must be confirmed by other techniques.     

Photooxidation of Amplex red to resorufin: Implications of exposing the Amplex red assay to light

The Amplex Red assay, a fluorescent assay for the detection of H(2)O(2), relies on the reaction of H(2)O(2) and colorless, nonfluorescent Amplex Red with a 1:1 stoichiometry to form colored, fluorescent resorufin, catalyzed by horseradish peroxidase (HRP). We have found that resorufin is artifactually formed when Amplex Red is exposed to light. In the absence of H(2)O(2) and HRP, the absorption and fluorescence spectra of Amplex Red changed during exposure to ambient room light or instrumental excitation light, clearly indicating that the fluorescent product resorufin had formed. This photochemistry was initiated by trace amounts of resorufin that are present in Amplex Red stock solutions. ESR spin-trapping studies demonstrated that superoxide radical was an intermediate in this process. Oxygen consumption measurements further confirmed that superoxide and H(2)O(2) were artifactually produced by the photooxidation of Amplex Red. The artifactual formation of resorufin was also significantly increased by the presence of superoxide dismutase or HRP. This photooxidation process will result in a less sensitive assay for H(2)O(2) under ambient light exposure and potentially invalid measurements under high energy exposure such as UVA irradiation. In general, precautions should be taken to minimize exposure to light during measurement of oxidative stress with Amplex Red.     

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.     

Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings

Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure, expanding from a few minutes to 24 h, were studied. Intracellular oxidative stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H(2)O(2) formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings.     

Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation

We report here that reduced pyridine nucleotides and reduced glutathione result in an oxidation of Amplex Red by dioxygen that is dependent on the presence of horseradish peroxidase (HRP). Concentrations of NADH and glutathione typically found in biological systems result in the oxidation of Amplex Red at a rate comparable to that produced, for example, by respiring mitochondria. The effects of NADH and glutathione in this assay system are likely to be the result of H(2)O(2) generation via a superoxide intermediate because both catalase and superoxide dismutase prevent the oxidation of Amplex Red. These results suggest caution in the assay of H(2)O(2) production in biological systems using the Amplex Red/HRP because the assay will also report the mobilization of NADH or glutathione. However, the interruption of this process by the addition of superoxide dismutase offers a simple and reliable method for establishing the source of the oxidant signal.     

HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H(2)O(2), the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H(2)O(2) to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O(2)(-)) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H(2)O(2) directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.     

Detecting,Visualizing and Quantitating the Generation of Reactive Oxygen Species in an Amoeba Model System

Reactive oxygen species (ROS) comprise a range of reactive and short-lived, oxygen-containing molecules, which are dynamically interconverted or eliminated either catalytically or spontaneously. Due to the short life spans of most ROS and the diversity of their sources and subcellular localizations, a complete picture can be obtained only by careful measurements using a combination of protocols. Here, we present a set of three different protocols using OxyBurst Green (OBG)-coated beads, or dihydroethidium (DHE) and Amplex UltraRed (AUR), to monitor qualitatively and quantitatively various ROS in professional phagocytes such as Dictyostelium. We optimised the beads coating procedures and used OBG-coated beads and live microscopy to dynamically visualize intraphagosomal ROS generation at the single cell level. We identified lipopolysaccharide (LPS) from E. coli as a potent stimulator for ROS generation in Dictyostelium. In addition, we developed real time, medium-throughput assays using DHE and AUR to quantitatively measure intracellular superoxide and extracellular H₂O₂ production, respectively.     

Effect of diazoxide on flavoprotein oxidation and reactive oxygen species generation during ischemia-reperfusion: a study on Langendorff-perfused rat hearts using optic fibers

This study analyzed the oxidant generation during ischemia-reperfusion protocols of Langendorff-perfused rat hearts, preconditioned with a mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) opener (i.e., diazoxide). The autofluorescence of mitochondrial flavoproteins, and that of the total NAD(P)H pool on the one hand and the fluorescence of dyes sensitive to H(2)O(2) or O(2)(*-) [i.e., the dihydrodichlorofluoroscein (H(2)DCF) and dihydroethidine (DHE), respectively] on the other, were noninvasively measured at the surface of the left ventricular wall by means of optic fibers. Isolated perfused rat hearts were subjected to an ischemia-reperfusion protocol. Opening mitoK(ATP) with diazoxide (100 microM) 1) improved the recovery of the rate-pressure product after reperfusion (72 +/- 2 vs. 16.8 +/- 2.5% of baseline value in control group, P < 0.01), and 2) attenuated the oxidant generation during both ischemic (-46 +/- 5% H(2)DCF oxidation and -40 +/- 3% DHE oxidation vs. control group, P < 0.01) and reperfusion (-26 +/- 2% H(2)DCF oxidation and -23 +/- 2% DHE oxidation vs. control group, P < 0.01) periods. All of these effects were abolished by coperfusion of 5-hydroxydecanoic acid (500 microM), a mitoK(ATP) blocker. During the preconditioning phase, diazoxide induced a transient, reversible, and 5-hydroxydecanoic acid-sensitive flavoprotein and H(2)DCF (but not DHE) oxidation. In conclusion, the diazoxide-mediated cardioprotection is supported by a moderate H(2)O(2) production during the preconditioning phase and a strong decrease in oxidant generation during the subsequent ischemic and reperfusion phases.     

Advanced glycation end-products disrupt human endothelial cells redox homeostasis: new insights into reactive oxygen species production

Advanced glycation end-products (AGEs) trigger multiple metabolic disorders in the vessel wall that may in turn lead to endothelial dysfunction. The molecular mechanisms by which AGEs generate these effects are not completely understood. Oxidative stress plays a key role in the development of deleterious effects that occur in endothelium during diabetes. Our main objectives were to further understand how AGEs contribute to reactive oxygen species (ROS) overproduction in endothelial cells and to evaluate the protective effect of an antioxidant plant extract. The human endothelial cell line EA.hy926 was treated with native or modified bovine serum albumin (respectively BSA and BSA-AGEs). To monitor free radicals formation, we used H₂DCF-DA, dihydroethidium (DHE), DAF-FM-DA and MitoSOX Red dyes. To investigate potential sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial inhibitors were used. The regulation of different types of ROS by the polyphenol-rich extract from the medicinal plant Doratoxylon apetalum was also studied for a therapeutic perspective. BSA-AGEs exhibited not only less antioxidant properties than BSA, but also pro-oxidant effects. The degree of albumin glycoxidation directly influenced oxidative stress through a possible communication between NADPH oxidase and mitochondria. D. apetalum significantly decreased intracellular hydrogen peroxide and superoxide anions mainly detected by H₂DCF-DA and DHE respectively. Our results suggest that BSA-AGEs promote a marked oxidative stress mediated at least by NADPH oxidase and mitochondria. D. apetalum plant extract appeared to be an effective antioxidant compound to protect endothelial cells.