Fluorescent Probe Dihydrorhodamine Research Articles

Detection of mammalian 5‐lipoxygenase activity using the fluorescent probe dihydrorhodamine 123

In this article, we describe a new, fast and reliable method for determining 5‐LOX activity using the fluorescent probe dihydrorhodamine 123 (DHR). In the new assay, the products of 5‐LOX, 5S‐hydroperoxy‐6E,8Z,11Z,14Z‐eicosatetraenoic acid, can directly oxidize non‐fluorescent DHR to highly fluorescent rhodamine 123. The fluorescence intensity can be measured using fluorescence spectroscopy at Ex/Em = 500/536 nm. As 5‐LOX requires some cofactors to achieve its optimum activity, the effects of its cofactors on the fluorescence assay are also investigated. Using this new assay, we evaluated the inhibitory activity of two well‐known 5‐LOX inhibitors (NDGA and AA861) in a 384‐well format and the IC50 values of them are consistent with previously references. The fluorescence assay is amenable to both purified 5‐LOX and 5‐LOX in cell lysates and will be widely used for the discovery of novel 5‐LOX inhibitors for clinical application.

Model Combustion-Generated Particulate Matter Containing Persistent Free Radicals Redox Cycle to Produce Reactive Oxygen Species

Particulate matter (PM) is emitted during thermal decomposition of waste. During this process, aromatic compounds chemisorb to the surface of metal-oxide-containing PM, forming a surface-stabilized environmentally persistent free radical (EPFR). We hypothesized that EPFR-containing PM redox cycle to produce ROS and that this redox cycle is maintained in biological environments. To test our hypothesis, we incubated model EPFRs with the fluorescent probe dihydrorhodamine (DHR). Marked increases in DHR fluorescence were observed. Using a more specific assay, hydroxyl radicals ((•)OH) were also detected, and their level was further increased by cotreatment with thiols or ascorbic acid (AA), known components of epithelial lining fluid. Next, we incubated our model EPFR in bronchoalveolar lavage fluid (BALF) or serum. Detection of EPFRs and (•)OH verified that PM generate ROS in biological fluids. Moreover, incubation of pulmonary epithelial cells with EPFR-containing PM increased (•)OH levels compared to those in PM lacking EPFRs. Finally, measurements of oxidant injury in neonatal rats exposed to EPFRs by inhalation suggested that EPFRs induce an oxidant injury within the lung lining fluid and that the lung responds by increasing antioxidant levels. In summary, our EPFR-containing PM redox cycle to produce ROS, and these ROS are maintained in biological fluids and environments. Moreover, these ROS may modulate toxic responses of PM in biological tissues such as the lung.

Heat-induced formation of nitrogen oxides in water

It was found by the fluorimetric method using 2,3-diaminonaphthalene that moderate heating of water (60-80°C, for up to 4h) leads to the fixation of atmospheric nitrogen with the formation of nitrite. The kinetic parameters of this process were determined. The energy of activation of [Formula: see text]formation was estimated to be 139kJ/mol. It was found that the amount of nitrite formed depends on the concentration of dissolved oxygen and nitrogen. It was shown by two independent methods (Griess reagent/VCl3 and 2,3-diaminonaphthalene/nitrate reductase) that heating of water (80°C, 1h) results in the formation of nitrate; with the use of the fluorescent probe dihydrorhodamine 123, the generation of nitrogen dioxide (peroxynitrite) was revealed. Nitrite, nitrate, and nitrogen dioxide are formed in water upon heating in approximately equal amounts. A scheme of reactions proceeding with bidistilled water by the action of heat with the formation of nitrogen oxides is proposed.

Effect of Priming in Subpopulations of Peripheral Neutrophils From Patients with Chronic Periodontitis

Patients with periodontal disease are reported to generate more reactive oxygen species (ROS) than matched controls, suggesting increased inflammatory defense activity. The purpose of this study is to determine whether there are subpopulations of peripheral neutrophils in patients with chronic periodontitis (CP) that generate different levels of intracellular ROS when primed with tumor necrosis factor-α (TNF-α) or the chemokine interleukin-8 (IL-8, CXCL8) compared to controls. Venous blood was collected from 13 patients with CP despite careful maintenance over 2 to 8 years and from 13 healthy age- and sex-matched controls. Neutrophils were separated from whole blood over a Percoll gradient and then activated via the Fcγ receptor with opsonized Staphylococcus aureus after priming with TNF-α or IL-8. The samples were analyzed by flow cytometry using the fluorescent probe dihydrorhodamine 123. Generation of ROS was measured as the intensity of fluorescence (IFL). Two subpopulations were found in both patients and controls: one with low and one with high generation of IFL. The subpopulation with high generation of IFL in patients with CP was more responsive to IL-8 (P <0.05) than the same subpopulation in healthy controls. No other differences in generation of ROS or priming effects were found between patients with CP and controls. Generation of ROS was dependent on nicotinamide adenine dinucleotide phosphate oxidase, and the intracellular ROS was primarily the oxygen anion. Patients with CP had a subpopulation of peripheral neutrophils that were more responsive to IL-8 priming than controls.

Oxidative Metabolism Genes Are Not Responsive to Oxidative Stress in Rodent Beta Cell Lines

Altered expression of oxidative metabolism genes has been described in the skeletal muscle of individuals with type 2 diabetes. Pancreatic beta cells contain low levels of antioxidant enzymes and are particularly susceptible to oxidative stress. In this study, we explored the effect of hyperglycemia-induced oxidative stress on a panel of oxidative metabolism genes in a rodent beta cell line. We exposed INS-1 rodent beta cells to low (5.6 mmol/L), ambient (11 mmol/L), and high (28 mmol/L) glucose conditions for 48 hours. Increases in oxidative stress were measured using the fluorescent probe dihydrorhodamine 123. We then measured the expression levels of a panel of 90 oxidative metabolism genes by real-time PCR. Elevated reactive oxygen species (ROS) production was evident in INS-1 cells after 48 hours (P < 0.05). TLDA analysis revealed a significant (P < 0.05) upregulation of 16 of the 90 genes under hyperglycemic conditions, although these expression differences did not reflect differences in ROS. We conclude that although altered glycemia may influence the expression of some oxidative metabolism genes, this effect is probably not mediated by increased ROS production. The alterations to the expression of oxidative metabolism genes previously observed in human diabetic skeletal muscle do not appear to be mirrored in rodent pancreatic beta cells.

A highly sensitive chemiluminescence assay for superoxide detection and chronic granulomatous disease diagnosis

Reactive oxygen species (ROS) produced by neutrophils are crucial for defense against infectious diseases, and the adequate measurement of ROS levels is an important way to evaluate the possibility of infections. The fluorescent probe dihydrorhodamine 123 has been applied exclusively to the measurement of ROS thus far. We developed a novel method for detecting ROS, which utilizes the chemiluminescent probes Luminol and Diogenes. The new method quantitatively detects ROS produced by as few as 10 to 104 neutrophils. Furthermore, this method can detect ROS levels in one microliter of whole blood or ROS produced by Epstein-Barr immortalized B lymphocytes. This method will be valuable for prompt diagnosis of neonatal chronic granulomatous diseases in which neutrophils aberrantly produce superoxide.

Chloroguaiacols Change Some Antioxidative Parameters and Affect the Activity of Glutathione S-transferase in the Leaves of Reed Canary Grass (Phalaris arudinacea)

In this work, we evaluated the effect of harmful ecotoxins, 4,5,6-trichloroguaicol (4,5,6-TCG) and tetrachloroguaiacol (TeCG), on the oxidation of the fluorescent probe dihydrorhodamine 123, the content of free phenols and the level of the total, oxidized and reduced glutathione in the leaves of reed canary grass (Phalaris arudinacea). Furthermore, the effect on the activity of guaiacol peroxidase and glutathione S-transferase was investigated. Both 4,5,6-TCG and TeCG increased the activity of guaiacol peroxidase and glutathione S-transferase, they also elevated the content of free phenols and the level of the total glutathione. A stronger effect was exerted by tetrachloroguaiacol, which strongly increased the level of the total glutathione and the content of free phenols on the 3rd and 6th day of the experiment. The activity of glutathione S-transferase was more intensively induced by trichloroguaiacol. Both 4,5,6-TCG and TeCG oxidized dihydrorodamine 123 and the effect was stronger in the presence of magnesium ions.

The development and in vitro characterisation of an intracellular nanosensor responsive to reactive oxygen species

Advances in sensor technologies have enhanced our understanding of the roles played by reactive oxygen species (ROS) in a number of physiological and pathological processes. However, high inter-reactivity and short life spans has made real-time monitoring of ROS in cellular systems challenging. Fluorescent dyes capable of intracellular ROS measurements have been reported. However, these dyes are known to be intrinsically cytotoxic and thus can potentially significantly alter cellular metabolism and adversely influence in vitro data. Reported here is the development and in vitro application of a novel ROS responsive nanosensor, based on PEBBLE (Probes Encapsulated By Biologically Localised Embedding) technology. The ROS sensitive fluorescent probe dihydrorhodamine 123 (DHR 123) was employed as the sensing element of the PEBBLE through entrapment within a porous, bio-inert polyacrylamide nanostructure enabling passive monitoring of free radical flux within the intracellular environment. Successful delivery of the nanosensors into NR8383 rat alveolar macrophage cells via phagocytosis was achieved. Stimulation of PEBBLE loaded NR8383 cells with phorbol-12-myristate-13-acetate (PMA) enabled real time monitoring of ROS generation within the cell without affecting cellular viability. These data suggest that PEBBLE nanosensors could offer significant advantages over existing technologies used in monitoring the intracellular environment.

Analysis of 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced Proteome Changes in 5L Rat Hepatoma Cells Reveals Novel Targets of Dioxin Action Including the Mitochondrial Apoptosis Regulator VDAC2

As part of a comprehensive survey of the impact of the environmental pollutant and hepatocarcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the proteome of hepatic cells, we have performed a high resolution two-dimensional gel electrophoresis study on the rat hepatoma cell line 5L. 78 protein species corresponding to 73 different proteins were identified as up- or down-regulated following exposure of the cells to 1 nm TCDD for 8 h. There was an overlap of only nine proteins with those detected as altered by TCDD in our recent study using the non-gel-based isotope-coded protein label method (Sarioglu, H., Brandner, S., Jacobsen, C., Meindl, T., Schmidt, A., Kellermann, J., Lottspeich, F., and Andrae, U. (2006) Quantitative analysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced proteome alterations in 5L rat hepatoma cells using isotope-coded protein labels. Proteomics 6, 2407-2421) indicating a strong complementarity of the two approaches. For the majority of the altered proteins, an effect of TCDD on their abundance or posttranslational modifications had not been known before. Several observations suggest that a sizable fraction of the proteins with altered abundance was induced as an adaptive response to TCDD-induced oxidative stress that was demonstrated using the fluorescent probe dihydrorhodamine 123. A prominent group of these proteins comprised various enzymes for which there is evidence that their expression is regulated via the Keap1/Nrf2/antioxidant response element pathway. Other proteins included several involved in the maintenance of mitochondrial energy production and the regulation of the mitochondrial apoptotic pathway. A particularly intriguing finding was the up-regulation of the mitochondrial outer membrane pore protein, voltage-dependent anion channel-selective protein 2 (VDAC2), which was dependent on the presence of a functional aryl hydrocarbon receptor. The regulatability of VDAC2 protein abundance has not been described previously. In view of the recently discovered central role of VDAC2 as an inhibitor of the activation of the proapoptotic protein BAK and the mitochondrial apoptotic pathway, the present data point to a hitherto unrecognized mechanism by which TCDD may affect cellular homeostasis and survival.

Fluorescent approach to quantitation of reactive oxygen species in mainstream cigarette smoke.

A novel approach to monitoring of mainstream smoke reactive oxygen species (ROS) has been developed and applied to the quantitation of smoke oxidants. Redox-active fluorescent probe dihydrorhodamine 6G (DHR-6G) was selected as the molecular probe because it is sensitive to typical smoke ROS. The experimental system couples an automatic cigarette smoke machine fiber-optic fluorometer for real-time monitoring of the reaction progress between cigarette smoke and DHR-6G. Quantitation was achieved based on the amount of rhodamine 6G, which is the sole product from DHR-6G oxidation. With the optimization of the trapping efficiency, we detected 391 nmol of ROS/cigarette in the mainstream CS for a standard cigarette 2R4F under standard Federal Trade Commission smoking protocol. Applying this method, we quantified the ROS of selected cigarettes and found that the cigarettes made of burley tobacco have much ( approximately 10 times) higher ROS content in the smoke than that in the tobacco made of bright tobacco. The smokeless cigarette, Eclipse, has comparable ROS with cigarettes made of bright tobacco.

Production of reactive oxygen species and 8-hydroxy-2 ′deoxyguanosine in KB cells co-exposed to benzo[ a]pyrene and UV-A radiation

Previous studies have shown that ultraviolet (UV) A light and the polycyclic aromatic hydrocarbon benzo[ a]pyrene (BaP) can synergistically enhance the formation of 8-hydroxy-2 ′deoxyguanosine (8-OHdG) in living cells. It has been postulated that the underlying mechanism is production of reactive oxygen species (ROS) via photosensitization, but direct evidence supporting this hypothesis has been lacking. This study examined intracellular ROS production in living cells co-exposed to UV-A and BaP as well as the relationship between intracellular production of ROS and formation of 8-OHdG. KB cells were exposed to BaP for 24 h, followed by exposure to UV-A (365 nm) or UV-B (312 nm). The levels of intracellular ROS were directly measured by use of the fluorescent probe dihydrorhodamine 123 (DHR-123) in flow cytometry. Levels of 8-OHdG were measured by high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD). The results demonstrated that UV-B itself induced a much greater level of intracellular ROS than did UV-A alone under the same dose of energy (0.10 mW/cm 2, 20 min). The presence of BaP (13.3 μM) substantially increased ROS production in UV-A-treated cells (2.9-fold), but only slightly enhanced ROS production in UV-B-treated cells (1.3-fold). These results show that BaP acts mainly as a photosensitizer of UV-A, but not UV-B. Furthermore, greater intracellular ROS production was proportional to both BaP concentration and UV-A dosage. There was a linear relationship between ROS production and 8-OHdG formation in cells co-exposed to BaP and UV-A. Results of this study suggest that UV-A and BaP act synergistically to enhance ROS production and formation of 8-OHdG, resulting in increased DNA damage.

Enhanced Host Defense After Gene Transfer in the Murine p47phox-Deficient Model of Chronic Granulomatous Disease

AbstractThe p47phox−/− mouse exhibits a phenotype similar to that of human chronic granulomatous disease (CGD) and, thus, is an excellent model for the study of gene transfer technology. Using the Moloney murine leukemia virus–based retroviral vector MFG-S encoding the human form of p47phox, we performed ex vivo gene transfer into Sca-1+ p47phox−/− marrow progenitor cells without conditioning of donors with 5-fluorouracil. Transduced progenitors were transplanted into moderately irradiated (500 cGy), G-CSF preconditioned sibling p47phox−/− mice. Using the fluorescent probe dihydrorhodamine 123 (DHR), in vivo biochemical correction of the superoxide-generating NADPH oxidase system was detected by flow cytometry in 12.3% ± 0.9% of phorbol myristate acetate–stimulated peripheral blood neutrophils at 4 weeks and 2.6% ± 1.0% at 14 weeks after transplantation. Following gene therapy, mice were challenged with the CGD pathogen Burkholderia (formerly Pseudomonas) cepacia and bacteremia levels were assessed at 24 hours and 7 days after inoculation. At both time points, bacteremia levels in gene corrected p47phox−/− mice were significantly lower than untreated p47phox−/− mice (0.89 ± 0.30 colonies v 237.7 ± 83.6 colonies at 24 hours, P < .02; 4.0 ± 2.0 colonies v 110.2 ± 26.5 colonies at 7 days, P < .0014). More importantly, Kaplan-Meier survival analysis showed a significant survival advantage of gene corrected versus untreated p47phox−/− mice (P < .001). Thus, stem-cell–directed ex vivo gene therapy is capable of restoring phagocyte oxidant-dependent host-defense function in this mouse model of a human immune-system disorder.

Effects of an intensive 12-wk training program by elite swimmers on neutrophil oxidative activity

The effects of an intensive 12-wk training program by 12 national-level swimmers on neutrophil oxidative activity were studied. Eleven sedentary (untrained) subjects (6 males and 5 females) served as environmental controls. Blood samples (10 ml) were taken at rest from an antecubital vein and neutrophils isolated by standard separation techniques. The oxidative burst activity of isolated neutrophils was assessed with an in vitro flow cytometric assay that used the fluorescent probe dihydrorhodamine 123. Two-way ANOVA (repeated measures) showed that oxidative activity was lower (P < 0.05) in the elite swimmers compared with the sedentary control group across the 12-wk period. Analysis of cells from swimmers in training was made: repeated measures ANOVA provided evidence of a significant decline (P < 0.05) in the number of cells responding positively ito in vitro challenge. Despite this decline, there was no significant difference in self-reported upper respiratory tract infection rate between the swimmers and sedentary individuals. These data show that: (i) elite swimmers undertaking intensive training have a significantly lower neutrophil oxidative activity at rest than do age- and sex-matched sedentary individuals; (ii) aspects of oxidative activity in swimmers are further suppressed during periods of strenuous training, and (iii) the extent of the suppression does not appear to be of clinical significance.