Hydroethidine Research Articles

An improved method for the detection of myeloperoxidase chlorinating activity in biological systems using the redox probe hydroethidine

Conversion of the redox probe hydroethidine (HE) to 2-chloroethidium (2-Cl-E+) by myeloperoxidase (MPO)-derived hypochlorous acid (HOCl) provides comparable specificity and superior sensitivity to measurement of 3-chlorotyrosine (3-Cl-Tyr), the gold standard biomarker for MPO chlorinating activity in biological systems. However, a limitation of the former method is the complex mixture of products formed by the reaction of HE with reagent HOCl, coupled with the difficult purification of 2-Cl-E+ from this mixture for analytical purposes. This limitation prompted us to test whether 2-Cl-E+ could be formed by reaction of HE with the strong and widely used chlorinating agent, N-chlorosuccinimide (NCS). Unexpectedly, such reaction yielded 2-chlorohydroethidine (2-Cl-HE) as the major product in addition to 2-Cl-E+, as assessed by high performance liquid chromatography (HPLC), mass spectrometry (MS), and nuclear magnetic resonance (NMR). 2-Cl-HE was also observed to be the major chlorination product formed from HE with both reagent and enzymatically generated HOCl, just as it was formed ex vivo in different healthy and diseased mouse and human tissues upon incubation with glucose/glucose oxidase to generate a flux of hydrogen peroxide (H2O2). Quantification of 2-Cl-HE plus 2-Cl-E+ improved the sensitivity of the HE-based method compared with measurement of only 2-Cl-E+. Moreover, 2-chlorodimidium (2-Cl-D+) was developed as a practical internal standard instead of the previously used internal standard, deuterated 2-Cl-E+ (d5-2-Cl-E+). Overall, the present study describes an improved method for the detection of MPO/chlorinating activity in biological systems of health and disease.

Titanium dioxide nanoparticle-based hydroxyl and superoxide radical production for oxidative stress biological simulations

The present study introduces a TiO2 nanoparticle-based (TiO2-NP) system for the generation of •OΗ and O2•− upon visible photo-excitation, in order to be used for high oxidative stress biological simulations in vitro. The main novelties of TiO2-NP system are: It is set to produce •OΗ and O2•− alone or both (in contrast to •OΗ-based common use of TiO2), as these options cover all possible generation means of these radicals in biological systems in vivo. Moreover, the known non-specific electrostatic interactions of TiO2-NP with H2O and various biological systems (e.g., cells, membrane proteins, even drugs) simulate direct/distant interactions of any in vivo•OΗ, O2•− source with extra/intracellular biological targets taking place in a densely packed biomolecular environment. The TiO2-NP system can use any commercially available TiO2-NP source (dispersion, nanopowder, crystal type), as long as TiO2-NPs’ concentrations in use meet the critical criterion to produce •OΗ levels linearly proportional to irradiation time, set for a given simulation study. The TiO2-NP system is calibrated by a standardized protocol developed to be applicable to most biological systems, offering the option of TiO2-NP removal via coagulation when needed. The production rates of •OΗ and O2•− by the TiO2-NP system are specifically calibrated with the respective specific probes terephthalic acid (TPA) and hydroethidine (HE), and tested in comparison to the •OΗ-producing Fenton system. The reaction kinetics of •OΗ and O2•− with TPA and HE is found to be in competition with their generating source, the TiO2-NP system. Similar ROS source competition phenomena with biological targets (simulated by TPA/HE) are very common in biological systems. In contrast, the Fenton system is shown not to exhibit such competition kinetics. The TiO2-NP system can be used to study •OΗ/O2•− dose–response-depended oxidative modifications in biological simulations. This stems from the fact that •OΗ and O2•− linear production rates (60 min and up to 8 min, respectively) can be controlled by varying (i) TiO2 concentration, (ii) light-source photon emission energy (decreasing from 370 to 410 nm), and (iii) light intensity (as a function of the inverse of squared distance from the irradiated sample). In contrast, •OΗ production by the Fenton system reaches steady state in ∼5 s regardless of varying Fe-II concentration, rendering it inappropriate for •OΗ simulation studies on biological systems. The biological simulating potential of the TiO2-NP system, as producer of both •OΗ and O2•−, is also experimentally verified on indicative biological examples selected to structurally represent most biological systems: BSA, a model hydrophilic protein; LDL, structurally resembling most of the biological systems (cells, membranes, organelles, lipoproteins, proteins, lipids). The TiO2-NP system causes a linear increase of all the tested oxidative modifications on both BSA and LDL for irradiation exposure 20 to 40 min, which strongly suggests that they are mainly •OΗ dose-proportional. In contrast, the Fenton system does not display •OΗ dose-associated oxidative modifications on BSA.

Flow Cytometric Analysis of Oxidative Stress in Escherichia coli B Strains Deficient in Genes of the Antioxidant Defence.

The detection of reactive oxygen species (ROS) and the analysis of oxidative stress are frequent applications of functional flow cytometry. Identifying and quantifying the ROS species generated during oxidative stress are crucial steps for the investigation of molecular mechanisms underlying stress responses. Currently, there is a wide availability of fluorogenic substrates for such purposes, but limitations in their specificity and sensitivity may affect the accuracy of the analysis. The aim of our work was to validate a new experimental model based in different strains of Escherichia coli B deficient in key genes for antioxidant defense, namely oxyR, sodA and sodB. We applied this model to systematically assess issues of specificity in fluorescent probes and the involvement of different ROS in a bacterial model of oxidative stress, as the probes can react with a variety of oxidants and free radical species. Our results confirm the higher sensitivity and specificity of the fluorescent probe mitochondrial peroxy yellow 1 (MitoPY1) for the detection of H2O2, and its very low capacity for organic hydroperoxides, thus extending MitoPY1′s specificity for H2O2 in mammalian cells to a bacterial model. On the contrary, the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA) is more sensitive to organic peroxides than to H2O2, confirming the lack of selectivity of H2DCF-DA to H2O2. Treatment with organic peroxides and H2O2 suggests a superoxide-independent oxidation of the fluorescent probe Hydroethidine (HE). We found a positive correlation between the lipophilicity of the peroxides and their toxicity to E. coli, suggesting greater quantitative importance of the peroxidative effects on the bacterial membrane and/or greater efficiency of the protection systems against the intracellular effects of H2O2 than against the membrane oxidative stress induced by organic peroxides. Altogether, our results may aid in preventing or minimizing experimental errors and providing recommendations for the proper design of cytometric studies of oxidative stress, in accordance with current recommendations and guidelines.

Inhibition of peroxidases and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in intact barley root tips.

NBT and HE may be efficiently used for the detection of superoxide, while DCDHF-DA and DHR123 for the detection of peroxynitrite in intact barley root tips, only ifPRXs and oxidoreductases are inhibited to avoid false-positive reactions. Strong peroxidase (PRX) and oxidoreductase activities were observed in the barley root tips that were markedly inhibited by NaN3. Rapid and strong nitro-blue tetrazolium chloride (NBT) reduction is associated mainly with the vital functions of root cells but not with superoxide formation. In turn, the inhibition of root surface redox activity by NaN3 strongly reduced the formation of formazan, but its slight accumulation, observed in the root elongation zone, was a result of NADPH oxidase-mediated apoplastic superoxide formation. A longer staining time period with NBT was required for the detection of antimycin A-mediated superoxide formation inside the cells. This antimycin A-induced superoxide was clearly detectable by hydroethidine (HE) after the inhibition of PRXs by NaN3, and it was restricted into the root transition zone. TEMPOL, a superoxide scavenger, strongly inhibited both NBT reduction and HE oxidation in the presence of NaN3. Similarly, the DCDHF-DA and DHR123 oxidation was markedly reduced after the inhibition of apoplastic PRXs by NaN3 and was detectable mainly in the root transition zone. This fluorescence signal was not influenced by the application of pyruvate but was strongly reduced by urea, a peroxynitrite scavenger. The presented results suggest that if the root PRXs and oxidoreductases are inhibited, both NBT and HE detect mainly superoxide, whereas both DCDHF-DA and DHR123 may be efficiently used for the detection of peroxynitrite in intact barley root tips. The inhibition of PRXs and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in the intact barley root tip.

Impaired Formation of Neutrophil Extracellular Traps (NETs) in Patients with Myelodysplastic Syndrome (MDS)

Introduction Increased susceptibility to infections in MDS is due to neutropenia as well as functional impairment of neutrophils. We investigated whether neutrophil dysfunction includes diminished formation of neutrophil extracellular traps (NETs). The latter are networks of extracellular fibers, primarily composed of DNA, which bind pathogens. This component of the immune system's first line of defense was first described in 2004 by Zychlinsky and coworkers. Formation of NETS is triggered by reactive oxygen species (ROS), which in neutrophils are mainly produced by NADPH-oxidase and myeloperoxidase (MPO). Methods Fluorescence microscopy was used to show NET formation in patients with MDS (n=12) (1 MDS del(5q), 1 MDS-RS-SLD, 1 MDS-RS-MLD, 5 MDS-MLD, 2 MDS-EB-1, 2 MDS-EB-2) in comparison to an age-adjusted normal control group (n=15). Neutrophils were isolated from peripheral blood by density gradient centifugation, sedimented on cover slips in 24-well plates, and incubated with phorbol-12-myristate-13-acetate (PMA) for 30 and 180 minutes to stimulate NET formation. Neutrophil elastase was detected by primary anti-NE (Abcam) plus secondary goat anti-rabbit Alexa 488 (FITC), histones by primary anti-histone (Millipore) plus secondary goat anti-mouse Alexa 568 (PE), and cell nuclei by DNA staining with DAPI. Fluorescent images were loaded into Image J/FIJI Software (https://imagej.nih.gov/ij/) for automatic cell detection and assessment of the proportion of cells undergoing NET formation, detectable by positive histone staining. Flow cytometry was used to further characterize NET formation and demonstrate the production of ROS by NADPH-oxidase and MPO. Intracellular MPO staining was positive for 96.7% and 99.1% of neutrophils in controls and MDS patients, respectively. Reactive oxygen species (ROS) were detected using dihydro-rhodamin 123 (DHR) for hydrogen peroxide, hydroethidine (HE) for superoxide anion, and 3`-(p-aminophenyl) fluorescein (APF) and 3`-(hydroxyphenyl) fluorescein (HPF) for hypochloride (HOCl). In order to block certain components of cellular ROS production, the following inhibitors were used: 4-aminobenzoic hydrazide (ABAH) to block MPO; 4-dimethylamino-antipyrine (AP) to block MPO and quench ROS; and diphenylen-iodonium chloride (DPI) to inhibit NADPH oxidase and mitochondrial respiratory complex I. Flow cytometry data were analysed using CellQuest Software (Becton Dickinson) and FCS Express Reader (De Novo Software). Results By fluorescence microscopy, marked morphological abnormalities of NET formation were observed. PMA-stimulated neutrophils of patients with MDS generated significantly fewer NETs than neutrophils from controls (increase from 17% to 67% vs. increase from 17% to 85% of cells, respectively) (p=0.02), see Fig. 1. MDS neutrophils showed significantly less cellular swelling after stimulation with PMA (p=0.04). Histone staining showed a more condensed chromatin in MDS neutrophils prior to stimulation (smaller mean fluorescence intensity (MFI) for PE; p=0.05). Upon stimulation with PMA, the decrease in MFI of DAPI, indicating chromatin decondensation, was significantly less in MDS neutrophils than controls (delta MFI 3467 vs. 4687, p=0.03). In addition, assessment of neutrophil elastase showed that its release from cytoplasmic granules (decreasing MFI for FITC) was diminished in patients with MDS (p=0.00002). On flow cytometry, forward scatter confirmed a significant difference in cell swelling after PMA stimulation (p=0.02). Sideward scatter showed a smaller decrease in granularity after stimulation with H2O2 (p=0.002). The morphological abnormalities did not seem to be attributable to diminished ROS production since measurement of PMA-stimulated ROS production using HE, and assessing oxidative burst activity using DHR did not reveal significant differences. However, H202-stimulated production of HOCl by MPO was lower in MDS patients, albeit only when MPO activity was simultaneously impeded by inhibitors (ABAH and AP). Inhibition of MPO activity was more easily achieved in patients with MDS (p=0.01), corroborating the notion of a partial MPO defect in MDS. Conclusions Our results indicate that NET formation is significantly impaired in MDS neutrophils. Although we found abnormalities of MPO-dependent generation of HOCl, impaired ROS production may not be the only cause of deficient NETosis in MDS. Figure Disclosures Gattermann: Novartis: Honoraria, Research Funding.

Oxidation of ethidium-based probes by biological radicals: mechanism, kinetics and implications for the detection of superoxide

Hydroethidine (HE) and hydropropidine (hbox {HPr}^{+}) are fluorogenic probes used for the detection of the intra- and extracellular superoxide radical anion (hbox {O}_{ {2}}^{bullet -}). In this study, we provide evidence that HE and hbox {HPr}^{+} react rapidly with the biologically relevant radicals, including the hydroxyl radical, peroxyl radicals, the trioxidocarbonate radical anion, nitrogen dioxide, and the glutathionyl radical, via one-electron oxidation, forming the corresponding radical cations. At physiological pH, the radical cations of the probes react rapidly with hbox {O}_{ {2}}^{bullet -}, leading to the specific 2-hydroxylated cationic products. We determined the rate constants of the reaction between hbox {O}_{ {2}}^{bullet -} and the radical cations of the probes. We also synthesized N-methylated analogs of hbox {HPr}^{+} and HE which were used in mechanistic studies. Methylation of the amine groups was not found to prevent the reaction between the radical cation of the probe and the superoxide, but it significantly increased the lifetime of the radical cation and had a substantial effect on the profiles of the oxidation products by inhibiting the formation of dimeric products. We conclude that the N-methylated analogs of HE and hbox {HPr}^{+} may be used as a scaffold for the design of a new generation of probes for intra- and extracellular superoxide.

Are Hydroethidine-Based Probes Reliable for Reactive Oxygen Species Detection?

Detection and quantification of the highly reactive and short-lived superoxide (O•2−) can be challenging. Here, we present a new mass spectrometry (MS)-based method to detect and quantify O•2− using three fluorogenic hydroethidine probes: hydroethidine (HE), mito-hydroethidine (mito-HE), and hydropropidine (HPr+), which measure cytosolic, mitochondrial, and extracellular O•2−, respectively. The probes and their oxidation products were simultaneously quantified by applying multiple reaction monitoring (MRM) with MS that allowed the specific measurement of reactive oxygen species (ROS) distribution within the cell. The advantage of this liquid chromatography–tandem mass spectrometry (LC-MS/MS) method is that coeluting compounds can be precisely distinguished using specific precursor and fragment masses. This method overcomes limitations from spectral overlap of O•2−-specific and nonspecific products in fluorescence spectra or the low specificity associated with chromatography-based approaches. However, our experiments showed that these HE probes can be prone to autoxidation during incubation at 37°C in Hank's solution. Cell treatments with strong oxidants did not significantly increase levels of the O•2− radical. Thus, subtle changes in ROS levels in cell culture experiments might not be quantifiable. Our findings raise the question of whether HE-based probes can be used for the reliable detection of O•2− radicals in cell culture.

P-370 - The pulse radiolytic study of one-electron oxidation of hydropropidine and hydroethidine – implications for the detection of superoxide radical anion in biological systems

Hydroethidine (HE) is a fluorogenic probe widely used for the detection of superoxide radical anion in cellular studies. Recently, we have synthesized cell-impermeant analogue of HE called hydropropidine (HPr +). HPr+ is a water-soluble molecule, possessing highly localized positive charge on the nitrogen atom of the alkyl group, that prevents its cellular uptake. Analogously to its precursor, HPr+ reacts with superoxide forming a specific fluorescent product 2-hydroxypropidine (2-OH-Pr++). According to the proposed mechanism of superoxide-mediated oxidation of HE and HPr+, the probe undergoes one-electron oxidation to the radical cation, and next, in the presence of superoxide, is converted to the appropriate hydroxylation product. Here, we show the spectroscopic characetrization of radical cation of HPr+ and HE by pulse radiolysis technique. The reactivity of both probes toward biologically relevant radicals, e.g. nitrogen dioxide (•NO2) and carbonate (CO3•−) radicals (products of peroxynitrite decomposition), as well as glutathionyl radicals (GS•), will be presented. These results will be discussed in relation to the mechanism of oxidation of hydroethidine based probes in biological milieu.

Neuroprotective effects of curcumin on spinal cord injury mice by nuclear factor E2-related factor 2 pathway

Objective To investigate the neuroprotective effect of curcumin (CCM) on spinal cord injury (SCI) mice by nuclear factor related factor 2 (Nrf2)/ARE pathway. Methods The SCI mouse model was prepared by modified Allen’s hitting method. At 3 days after SCI, the number of necrotic cells near the injured area was detected by propidium iodide (PI) staining. Hydroethidine (HEt) staining was used to detect reactive oxygen species (ROS) production. enzyme linked immunosorbent assay (ELISA) kit was used to detect the expression of tumor necrosis factor-α (TNF-α) and interleukin- 10 (IL- 10). At 28 days after SCI, Basso Mouse Scale (BMS) score was used to detect the hindlimb motor function. Sucorse preference test was used to evaluate the depressive status of mice. The expression of Nrf2, SOD1 and quinone oxidoreductase gene (NQO1) were detected by Western blotting. Results (1) The spinal cord water content (75.540±1.564) in the CCM group was significantly lower than that in the Veh group (85.500±1.297), the difference was statistically significant (t=4.903, P=0.001). (2) BMS scores showed that CCM treatment group 7, 14, 21 and 28 d scores of hindlimb locomotor function [(2.400±0.894), (3.400±0.894), (4.200±0.837) and (4.800±0.837) scores] compared with the Veh Group [(1.000±0.707), (1.800±0.447), (2.200±0.837), (3.000±0.707) scores, P= 0.004]. (3) Sucorse preference test showed that the difference between the CMM treatment group and the Veh group was statistically significant [(55.330±1.124)% and (45.740±1.965)%, t=4.314, P=0.002]. (4) Compared with Veh group, CCM group of anti- inflammatory cytokines IL- 10 expression (674.20±6.94) was significantly higher than that of group Veh (525.00±6.43), and proinflammatory factor TNF- α (763.20±16.24) was significantly lower than that of Veh group (1 091.00±29.02), P=0.000, 0.000. (5) The fluorescence intensity of ROS in CCM group (1 377.00±27.06) was significantly lower than Veh group (1 750.00±42.45), the difference was statistically significant (t=7.410, P=0.000). (6) Compared with Veh group (72.00±3.81), the number of necrotic cells in CCM group was significantly decreased (53.60±3.20), and the difference was statistically significant (t=3.690, P=0.006). (7) Compared with Veh group, the expression of SOD1、Nrf2 and NQO1 in CCM group were up- regulated (0.763±0.039, 0.442±0.026, 0.636±0.023 and 0.937±0.030, 0.692±0.034, 0.898±0.025, P=0.025, 0.004, 0.002). Conclusion Curcumin protects neurons in spinal cord injury by Nrf2 pathway. Key words: Curcumin; Spinal cord injury; Nuclear factor related factor 2 pathway; Neuroprotection

P 061 - N,N.N′,N′-tetramethylhydroethidine (TMHE) - in search for better probes for the detection of superoxide radical anion

The superoxide radical anion is an important reactive species produced by one-electron reduction of molecular oxygen. The main sources of superoxide radical anion in vivo are the enzymes NADPH oxidases (NOX) and mitochondria (mitochondrial electron transport chain). Superoxide radical anion reacts rapidly with nitric oxide with the formation of peroxynitrite (ONOO−). It can also undergo spontaneous or SOD-catalyzed dismutation with the formation of hydrogen peroxide (H2O2) and thus it is a precursor of other biologically relevant oxidants playing an important role in various pathologies. Due to its reactivity and short lifetime in vivo, its detection and quantitation is difficult and demands special and sensitive techniques. One of the approaches is the use of fluorogenic probes, the compounds which themselves are not fluorescent but in the reaction with superoxide are oxidized to the fluorescent products that can be directly detected. Among various profluorescent probes available hydroethidine (HE) seems to be a gold standard for detection of superoxide in biological systems. In the presence of superoxide radical anion, HE undergoes oxidative transformation into 2-hydroxyethidium (2-OH-E+), a specific marker of superoxide radical anion production. In the reaction with other radical, one-electron oxidants, HE is oxidatively transformed into ethidine and dimeric products. Here we present a spectroscopic and chemical characterization of new analogue of hydroethidine - N,N,N,N-tetramethylhydroethidine (TMHE).

Recent developments in detection of superoxide radical anion and hydrogen peroxide: Opportunities, challenges, and implications in redox signaling

In this review, some of the recent developments in probes and assay techniques specific for superoxide (O2–) and hydrogen peroxide (H2O2) are discussed. Over the last decade, significant progress has been made in O2– and H2O2 detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O2–. In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O2– and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H2O2, forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O2– and H2O2 in different cellular compartments will improve our understanding of their role in redox signaling.

FITC Doped Rattle-Type Silica Colloidal Particle-Based Ratiometric Fluorescent Sensor for Biosensing and Imaging of Superoxide Anion.

Fluorescent nanosensors have been widely applied in recognition and imaging of bioactive small molecules; however, the complicated surface modification process and background interference limit their applications in practical biological samples. Here, a simple, universal method was developed for ratiometric fluorescent determination of general small molecules. Taking superoxide anion (O2(•-)) as an example, the designed sensor was composed of three main moieties: probe carrier, rattle-type silica colloidal particles (mSiO2@hmSiO2 NPs); reference fluorophore doped into the core of NPs, fluorescein isothiocyanate (FITC); fluorescent probe for superoxide anion, hydroethidine (HE). In the absence of O2(•-), the sensor just emitted green fluorescence of FITC at 518 nm. When released HE was oxidized by O2(•-), the oxidation product exhibited red fluorescence at 570 nm and the intensity was linearly associated with the concentration of O2(•-), while that of reference element remained constant. Accordingly, ratiometric determination of O2(•-) was sensitively and selectively achieved with a linear range of 0.2-20 μM, and the detection limit was calculated as low as 80 nM. Besides, the technique was also successfully applied for dual-emission imaging of O2(•-) in live cells and realized visual recognition with obvious fluorescence color change in normal conditions or under oxidative stress. As long as appropriate reference dyes and sensing probes are selected, ratiometric biosensing and imaging of bioactive small molecules would be achieved. Therefore, the design could provide a simple, accurate, universal platform for biological applications.

Complex I and complex III inhibition specifically increase cytosolic hydrogen peroxide levels without inducing oxidative stress in HEK293 cells

Inhibitor studies with isolated mitochondria demonstrated that complex I (CI) and III (CIII) of the electron transport chain (ETC) can act as relevant sources of mitochondrial reactive oxygen species (ROS). Here we studied ROS generation and oxidative stress induction during chronic (24h) inhibition of CI and CIII using rotenone (ROT) and antimycin A (AA), respectively, in intact HEK293 cells. Both inhibitors stimulated oxidation of the ROS sensor hydroethidine (HEt) and increased mitochondrial NAD(P)H levels without major effects on cell viability. Integrated analysis of cells stably expressing cytosolic- or mitochondria-targeted variants of the reporter molecules HyPer (H2O2-sensitive and pH-sensitive) and SypHer (H2O2-insensitive and pH-sensitive), revealed that CI- and CIII inhibition increased cytosolic but not mitochondrial H2O2 levels. Total and mitochondria-specific lipid peroxidation was not increased in the inhibited cells as reported by the C11-BODIPY581/591 and MitoPerOx biosensors. Also expression of the superoxide-detoxifying enzymes CuZnSOD (cytosolic) and MnSOD (mitochondrial) was not affected. Oxyblot analysis revealed that protein carbonylation was not stimulated by CI and CIII inhibition. Our findings suggest that chronic inhibition of CI and CIII: (i) increases the levels of HEt-oxidizing ROS and (ii) specifically elevates cytosolic but not mitochondrial H2O2 levels, (iii) does not induce oxidative stress or substantial cell death. We conclude that the increased ROS levels are below the stress-inducing level and might play a role in redox signaling.

Pharmacological antagonism of interleukin-8 receptor CXCR2 inhibits inflammatory reactivity and is neuroprotective in an animal model of Alzheimer's disease.

BackgroundThe chemokine interleukin-8 (IL-8) and its receptor CXCR2 contribute to chemotactic responses in Alzheimer’s disease (AD); however, properties of the ligand and receptor have not been characterized in animal models of disease. The primary aim of our study was to examine effects of pharmacological antagonism of CXCR2 as a strategy to inhibit receptor-mediated inflammatory reactivity and enhance neuronal viability in animals receiving intrahippocampal injection of amyloid-beta (Aβ1–42).MethodsIn vivo studies used an animal model of Alzheimer’s disease incorporating injection of full-length Aβ1–42 into rat hippocampus. Immunohistochemical staining of rat brain was used to measure microgliosis, astrogliosis, neuronal viability, and oxidative stress. Western blot and Reverse Transcription PCR (RT-PCR) were used to determine levels of CXCR2 in animal tissue with the latter also used to determine expression of pro-inflammatory mediators. Immunostaining of human AD and non-demented (ND) tissue was also undertaken.ResultsWe initially determined that in the human brain, AD relative to ND tissue exhibited marked increases in expression of CXCR2 with cell-specific receptor expression prominent in microglia. In Aβ1–42-injected rat brain, CXCR2 and IL-8 showed time-dependent increases in expression, concomitant with enhanced gliosis, relative to controls phosphate-buffered saline (PBS) or reverse peptide Aβ42–1 injection. Administration of the competitive CXCR2 antagonist SB332235 to peptide-injected rats significantly reduced expression of CXCR2 and microgliosis, with astrogliosis unchanged. Double staining studies demonstrated localization of CXCR2 and microglial immunoreactivity nearby deposits of Aβ1–42 with SB332235 effective in inhibiting receptor expression and microgliosis. The numbers of neurons in granule cell layer (GCL) were reduced in rats receiving Aβ1–42, compared with PBS, with administration of SB332235 to peptide-injected animals conferring neuroprotection. Oxidative stress was indicated in the animal model since both 4-hydroxynonenal (4-HNE) and hydroethidine (HEt) were markedly elevated in Aβ1–42 vs PBS-injected rat brain and diminished with SB332235 treatment.ConclusionOverall, the findings suggest critical roles for CXCR2-dependent inflammatory responses in an AD animal model with pharmacological modulation of the receptor effective in inhibiting inflammatory reactivity and conferring neuroprotection against oxidative damage.

Seasonal variation in sperm quality parameters in Swedish red dairy bulls used for artificial insemination

The aim of the present study was to investigate the extent to which seasonal factors such as temperature, atmospheric pressure or visible light length affect dairy bull sperm quality. Straws from 10 dairy bulls, from semen collections in three different seasons, were available for this study. The following quality parameters were assessed: motility, measured by computer assisted sperm motility analysis (SpermVision motility analyzer), membrane integrity (staining with SYBR14/PI (Propidium Iodide)with flow cytometric measurement of fluorescence), content of reactive oxygen species (ROS) measured using HE(Hydroethidine) and DCFDA (2′, 7′-dichlorodihydrofluorescein diacetate), mitochondrial membrane potential (using JC-1 fluorescence stain), acrosome reaction (measured with fluorescein isothiocyanate-PNA (FITC-PNA) labeling combined with calcium ionophore A23187 and PI), DNA fragmentation index (%DFI, Sperm Chromatin Structure Assay) as well as morphology, using the William׳s staining protocol. Data was analyzed by analysis of variance (PROC MIXED), using SAS software. The proportion (%) (mean±SD) of living, dying and dead sperm cells varied between seasons, with the proportion of living spermatozoa being lowest in summer, although a significant difference (P<0.05) was only observed between spring and summer. The %DFI assessed by SCSA was lowest in spring and differed significantly (p <0.04) from summer. A trend towards significance was observed between spring and summer for the motility parameters linearity (LIN) (P=0.068) and straightness (STR) (P=0.062).

Intracellular oxidation of hydroethidine: Compartmentalization and cytotoxicity of oxidation products

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.

Carbon-dot-based ratiometric fluorescent probe for imaging and biosensing of superoxide anion in live cells.

In this article, a ratiometric fluorescent biosensor for O2(•-) was developed, by employing carbon dots (C-Dots) as the reference fluorophore and hydroethidine (HE), a specific organic molecule toward O2(•-), playing the role as both specific recognition element and response signal. The hybrid fluorescent probe CD-HE only emitted at 525 nm is ascribed to C-Dots, while HE was almost nonfluorescent, upon excitation at 488 nm. However, after reaction with O2(•-), a new emission peak ascribed to the reaction products of HE and O2(•-) was clearly observed at 610 nm. Meanwhile, this peak gradually increased with the increasing concentration of O2(•-) but the emission peak at 525 nm stayed constant, leading to a ratiometric detection of O2(•-). The inorganic-organic fluorescent sensor exhibited high sensitivity, a broad dynamic linear range of ~5 × 10(-7)-1.4 × 10(-4) M, and low detection limit down to 100 nM. The present probe also showed high accuracy and excellent selectivity for O2(•-) over other reactive oxygen species (ROS), metal ions, and so on. Moreover, the C-Dot-based inorganic-organic probe demonstrated long-term stability against pH changes and continuous light illumination, good cell-permeability, and low cytotoxicity. Accordingly, the developed fluorescent biosensor was eventually applied for intracellular bioimaging and biosensing of O2(•-) changes upon oxidative stress.

Nitric oxide production by polymorphonuclear leucocytes in infected cystic fibrosis sputum consumes oxygen

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by persisting mucoid biofilms in hypoxic endobronchial mucus. These biofilms are surrounded by numerous polymorphonuclear leucocytes (PMNs), which consume a major part of present molecular oxygen (O(2)) due to production of superoxide (O(2)(-)). In this study, we show that the PMNs also consume O(2) for production of nitric oxide (NO) by the nitric oxide synthases (NOS) in the infected endobronchial mucus. Fresh expectorated sputum samples (n = 28) from chronically infected CF patients (n = 22) were analysed by quantifying and visualizing the NO production. NO production was detected by optode measurements combined with fluorescence microscopy, flow cytometry and spectrophotometry. Inhibition of nitric oxide synthases (NOS) with N(G) -monomethyl-L-arginine (L-NMMA) resulted in reduced O(2) consumption (P < 0·0008, n = 8) and a lower fraction of cells with fluorescence from the NO-indicator 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) (P < 0·002, n = 8). PMNs stained with DAF-FM and the superoxide indicator hydroethidine (HE) and host cells with inducible NOS (iNOS) were identified in the sputum. In addition, the production of the stable end-products of NO in CF sputum was correlated with the concentration of PMNs; NO(3)(-) (P < 0·04, r = 0·66, n = 10) and NO(2)(-) (P< 0·006, r = 0·78, n = 11). The present study suggests that besides consumption of O(2) for production of reactive oxygen species, the PMNs in CF sputum also consume O(2) for production of NO.

Cytometric measurement of in vitro inhibition of Plasmodium falciparum field isolates by drugs: a new approach for re-invasion inhibition study

BackgroundA flow cytometric method is proposed to study in vitro drug sensitivity of Plasmodium falciparum. Standard [3H]-hypoxanthine incorporation assay gives only information on inhibition of maturation by drugs. This method is usable on field isolates and provides data on both inhibition of maturation and re-invasion.MethodsThe method is based on the staining of parasites with hydroethidine (HE) and thiazole orange (TO) which allow differential identification of early, trophozoite and late stage of the parasite by flow cytometry. Late stages of the parasites are obtained by incubation in culture for 24 hours. Reinvasion is followed by culturing parasitized red blood cells for 24 h more.ResultsCompared to the standard [3H]-hypoxanthine incorporation assay, it gave similar results as expressed by 50% inhibitory concentrations for chloroquine of laboratory strains and “field” isolates. The effect of quinine on the schizont-ring transition was also explored using this method. First data on the inhibition of re-invasion induced by quinine are presented for both P. falciparum-cultured strains and field isolates.DiscussionThis method is simple to use event for field isolate study. It is suitable to analyse effect of drugs on steps of the parasite life cycle different for the maturation one. Using this method quinine was found to have a inhibitory effect on re-invasion of red cells by Plasmodium.

Assessment of Myeloperoxidase Activity by the Conversion of Hydroethidine to 2-Chloroethidium

Oxidants derived from myeloperoxidase (MPO) contribute to inflammatory diseases. In vivo MPO activity is commonly assessed by the accumulation of 3-chlorotyrosine (3-Cl-Tyr), although 3-Cl-Tyr is formed at low yield and is subject to metabolism. Here we show that MPO activity can be assessed using hydroethidine (HE), a probe commonly employed for the detection of superoxide. Using LC/MS/MS, (1)H NMR, and two-dimensional NOESY, we identified 2-chloroethidium (2-Cl-E(+)) as a specific product when HE was exposed to hypochlorous acid (HOCl), chloramines, MPO/H2O2/chloride, and activated human neutrophils. The rate constant for HOCl-mediated conversion of HE to 2-Cl-E(+) was estimated to be 1.5 × 10(5) M(-1)s(-1). To investigate the utility of 2-Cl-E(+) to assess MPO activity in vivo, HE was injected into wild-type and MPO-deficient (Mpo(-/-)) mice with established peritonitis or localized arterial inflammation, and tissue levels of 2-Cl-E(+) and 3-Cl-Tyr were then determined by LC/MS/MS. In wild-type mice, 2-Cl-E(+) and 3-Cl-Tyr were detected readily in the peritonitis model, whereas in the arterial inflammation model 2-Cl-E(+) was present at comparatively lower concentrations (17 versus 0.3 pmol/mg of protein), and 3-Cl-Tyr could not be detected. Similar to the situation with 3-Cl-Tyr, tissue levels of 2-Cl-E(+) were decreased substantially in Mpo(-/-) mice, indicative of the specificity of the assay. In the arterial inflammation model, 2-Cl-E(+) was absent from non-inflamed arteries and blood, suggesting that HE oxidation occurred locally in the inflamed artery. Our data suggest that the conversion of exogenous HE to 2-Cl-E(+) may be a useful selective and sensitive marker for MPO activity in addition to 3-Cl-Tyr.