Extracellular Hydrogen Peroxide Research Articles

Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players.

Sperm capacitation is a critical process for male fertility. It involves a series of biochemical and physiological changes that occur in the female reproductive tract, rendering the sperm competent for successful fertilization. The precise mechanisms and, specifically, the role of mitochondria, in sperm capacitation remain incompletely understood. Previously, we revealed that in mouse sperm mitochondrial activity (e.g., oxygen consumption, membrane potential, ATP/ADP exchange, and mitochondrial Ca2+ ) increases during capacitation. Herein, we studied mitochondrial function by high-resolution respirometry (HRR) and reactive oxygen species production in capacitated (CAP) and non-capacitated (NC) human spermatozoa. We found that in capacitated sperm from normozoospermic donors, the respiratory control ratio increased by 36%, accompanied by a double oxygen consumption rate (OCR) in the presence of antimycin A. Extracellular hydrogen peroxide (H2 O2 ) detection was three times higher in CAP than in NC sperm cells. To confirm that H2 O2 production depends on mitochondrial superoxide ( ) formation, we evaluated mitochondrial aconitase (ACO2) amount, activity, and role in the metabolic flux from the sperm tricarboxylic acid cycle. We estimated that CAP cells produce, on average by individual, (59 ± 22)% more in the steady-state compared to NC cells. Finally, we analyzed two targets of oxidative stress: lipid peroxidation by western blot against 4-hydroxynonenal and succinate dehydrogenase (SDH) activity by HRR. We did not observe modifications in lipoperoxidation nor the activity of SDH, suggesting that during capacitation, the increase in mitochondrial H2 O2 production does not damage sperm and it is necessary for the normal CAP process.

Increase of the degradation activity of Phanerochaete chrysosporium by hybridization with silica nanoparticles

Silica nanoparticle–hybridized Phanerochaete chrysosporium mycelium pellets were prepared by a simple one-step composite technology–based approach, involving co-culturing of silica nanoparticles with this fungus under mild culture conditions. The hybrid generated displays a significant increase in peroxidase activity, which reaches a maximum on the fifth day of culturing. Hybridization causes a morphology change and significantly increases the rate of crystal violet degradation, the amounts of excreted extracellular manganese peroxidase and hydrogen peroxide, and the rate of glucose consumption. The results show that hybridization with silica nanoparticles significantly increases of the degradation activity of P. chrysosporium cells.

Iron Oxide Nanozymes Enhanced by Ascorbic Acid for Macrophage-Based Cancer Therapy

In recent years, pharmacological ascorbic acid has emerged as a promising therapeutic approach in cancer treatment, owing to its capacity to induce extracellular hydrogen peroxide (H2O2) production in solid tumors....

Sleeve Gastrectomy Provides Cardioprotection from Oxidative Stress In Vitro Due to Reduction of Circulating Myeloperoxidase.

Bariatric surgery, including sleeve gastrectomy (SG), improves systolic and diastolic function, which is independent of weight loss in rodent models. The cause of weight loss-independent improvements in cardiac function are unknown but may originate from the gastrointestinal tract. In this study, we investigated whether a circulating blood factor is a mechanism for acute cardioprotection after SG by testing the utility of rodent SG plasma to reduce metabolic stress in vitro. For the initial experiment, obese male Zucker rats underwent SG, ad lib sham, or pair-fed sham surgeries (n = six SG, n = eight SH, n = eight PF). For all other studies, a second group of Zucker rats underwent SG or ad lib sham surgeries (n = eight SH, n = six SG). Six weeks following surgery, plasma was collected from each group, both in the fasting and post-prandial (pp) state. This plasma was then pooled per surgical group and nutrient state and tested in multiple in vitro cell culture and extra-cellular assays to determine the effect of SG on myotubular metabolic stress compared to the sham surgeries. Post-prandial SG plasma (ppSG), but not fasting SG, pp, or fasting sham plasma, reduced the metabolic stress of the H9c2 cells as measured by lactate dehydrogenase (LDH) release (p < 0.01). Unlike SG, weight reduction through pair-feeding did not prevent H9c2 metabolic stress. The PpSG plasma had the slowest rate of extracellular hydrogen peroxide consumption and peroxidatic activity compared to the pp sham, fasting SG, and fasting sham groups. Redox testing of plasma with aminiobenzoic acid hydrazide and edaravone suggested a pattern supporting myeloperoxidase (MPO), or other peroxidases, as the primary component responsible for reduced metabolic stress with ppSG plasma. The PpSG plasma contained 35% less circulating MPO protein as compared to the pp sham and fasting SG plasma. The plasma from an MPO global knockout rat also prevented metabolic stress of the H9c2 cells, compared to the significant increase in LDH release from the plasma of the WT controls (p < 0.01). The MPO global knockout plasma also had a rate of extracellular hydrogen peroxide consumption and peroxidatic activity comparable to the ppSG plasma. These studies suggest that one of the weight loss-independent mechanisms by which SG improves myocellular function could be a reduced pro-oxidative environment due to lower circulating levels of MPO. It appears that the gastrointestinal tract is of critical importance to these findings, as the MPO levels were only lowered after enteral, nutrient stimulation in the SG rats. If this surgical effect is confirmed in humans, SG may be a unique surgical treatment for multiple diseases with a pathogenesis of inflammation and oxidative damage, including obesity-associated heart failure with preserved ejection fraction.

Pharmacological Ascorbate Elicits Anti-Cancer Activities against Non-Small Cell Lung Cancer through Hydrogen-Peroxide-Induced-DNA-Damage.

Non-small cell lung cancer (NSCLC) poses a significant global health burden with unsatisfactory survival rates, despite advancements in diagnostic and therapeutic modalities. Novel therapeutic approaches are urgently required to improve patient outcomes. Pharmacological ascorbate (P-AscH-; ascorbate at millimolar concentration in plasma) emerged as a potential candidate for cancer therapy for recent decades. In this present study, we explore the anti-cancer effects of P-AscH- on NSCLC and elucidate its underlying mechanisms. P-AscH- treatment induces formation of cellular oxidative distress; disrupts cellular bioenergetics; and leads to induction of apoptotic cell death and ultimately reduction in clonogenic survival. Remarkably, DNA and DNA damage response machineries are identified as vulnerable targets for P-AscH- in NSCLC therapy. Treatments with P-AscH- increase the formation of DNA damage and replication stress markers while inducing mislocalization of DNA repair machineries. The cytotoxic and genotoxic effects of P-AscH- on NSCLC were reversed by co-treatment with catalase, highlighting the roles of extracellular hydrogen peroxide in anti-cancer activities of P-AscH-. The data from this current research advance our understanding of P-AscH- in cancer treatment and support its potential clinical use as a therapeutic option for NSCLC therapy.

Action mechanism of snake venom l-amino acid oxidase and its double-edged sword effect on cancer treatment: Role of pannexin 1-mediated interleukin-6 expression

Snake venom l-amino acid oxidases (svLAAOs) have been recognized as promising candidates for anticancer therapeutics. However, multiple aspects of their catalytic mechanism and the overall responses of cancer cells to these redox enzymes remain ambiguous. Here, we present an analysis of the phylogenetic relationships and active site-related residues among svLAAOs and reveal that the previously proposed critical catalytic residue His 223 is highly conserved in the viperid but not the elapid svLAAO clade. To gain further insight into the action mechanism of the elapid svLAAOs, we purify and characterize the structural, biochemical, and anticancer therapeutic potentials of the Thailand elapid snake Naja kaouthia LAAO (NK-LAAO). We find that NK-LAAO, with Ser 223, exhibits high catalytic activity toward hydrophobic l-amino acid substrates. Moreover, NK-LAAO induces substantial oxidative stress-mediated cytotoxicity with the magnitude relying on both the levels of extracellular hydrogen peroxide (H2O2) and intracellular reactive oxygen species (ROS) generated during the enzymatic redox reactions, but not being influenced by the N-linked glycans on its surface. Unexpectedly, we discover a tolerant mechanism deployed by cancer cells to dampen the anticancer activities of NK-LAAO. NK-LAAO treatment amplifies interleukin (IL)-6 expression via the pannexin 1 (Panx1)-directed intracellular calcium (iCa2+) signaling pathway to confer adaptive and aggressive phenotypes on cancer cells. Accordingly, IL-6 silencing renders cancer cells vulnerable to NK-LAAO-induced oxidative stress together with abrogating NK-LAAO-stimulated metastatic acquisition. Collectively, our study urges caution when using svLAAOs in cancer treatment and identifies the Panx1/iCa2+/IL-6 axis as a therapeutic target for improving the effectiveness of svLAAOs-based anticancer therapies.

Interaction among spring phytoplankton succession, water discharge patterns, and hydrogen peroxide dynamics in the Caloosahatchee River in southwest Florida

Phytoplankton communities are major primary producers in the aquatic realm and are responsible for shaping aquatic ecosystems. The dynamics of algal blooms could be determined by a succession of variable taxonomic groups, which are altered based on complex environmental factors such as nutrient availability and hydraulic factors. In-river structures potentially increase the occurrence of harmful algal blooms (HABs) by increasing water residence time and deteriorating water quality. How flowing water stimulates cell growth and affects the population dynamics of phytoplankton communities is a prioritized question that needs to be addressed for water management tactics. The goal of this study was to determine if an interaction between water flow and water chemistry is present, furthermore, to determine the relationship among phytoplankton community successions in the Caloosahatchee River, a subtropical river strongly influenced by human-controlled water discharge patterns from Lake Okeechobee. Particularly we focused on how phytoplankton community shifts influence the natural abundance of hydrogen peroxide, the most stable reactive oxygen species and a byproduct of oxidative photosynthesis. High-throughput amplicon sequencing using universal primers amplify 23S rRNA gene in cyanobacteria and eukaryotic algal plastids revealed that Synechococcus and Cyanobium were the dominant cyanobacterial genera and their relative abundance ranged between 19.5 and 95.3% of the whole community throughout the monitoring period. Their relative abundance declined when the water discharge increased. On the contrary, the relative abundance of eukaryotic algae sharply increased after water discharge increased. As water temperature increased in May, initially dominant Dolichospermum decreased as Microcystis increased. When Microcystis declined other filamentous cyanobacteria such as Geitlerinema, Pseudanabaena, and Prochlorothreix increased in their relative abundances. Interestingly, a peak of extracellular hydrogen peroxide was observed when Dolichospermum dominance was ended, and M. aeruginosa numbers increased. Overall, phytoplankton communities were strongly impacted by human-induced water discharge patterns.

Expression of the H2O2 Biosensor roGFP-Tpx1.C169S in Fission and Budding Yeasts and Jurkat Cells to Compare Intracellular H2O2 Levels, Transmembrane Gradients, and Response to Metals

Intracellular hydrogen peroxide (H2O2) levels can oscillate from low, physiological concentrations, to intermediate, signaling ones, and can participate in toxic reactions when overcoming certain thresholds. Fluorescent protein-based reporters to measure intracellular H2O2 have been developed in recent decades. In particular, the redox-sensitive green fluorescent protein (roGFP)-based proteins fused to peroxiredoxins are among the most sensitive H2O2 biosensors. Using fission yeast as a model system, we recently demonstrated that the gradient of extracellular-to-intracellular peroxides through the plasma membrane is around 300:1, and that the concentration of physiological H2O2 is in the low nanomolar range. Here, we have expressed the very sensitive probe roGFP2-Tpx1.C169S in two other model systems, budding yeast and human Jurkat cells. As in fission yeast, the biosensor is ~40-50% oxidized in these cell types, suggesting similar peroxide steady-state levels. Furthermore, probe oxidation upon the addition of extracellular peroxides is also quantitatively similar, suggesting comparable plasma membrane H2O2 gradients. Finally, as a proof of concept, we have applied different concentrations of zinc to all three model systems and have detected probe oxidation, demonstrating that an excess of this metal can cause fluctuations of peroxides, which are moderate in yeasts and severe in mammalian cells. We conclude that the principles governing H2O2 fluxes are very similar in different model organisms.

Differential Production of Nitric Oxide and Hydrogen Peroxide among Drosophila melanogaster, Apis mellifera, and Mamestra brassicae Immune-Activated Hemocytes after Exposure to Imidacloprid and Amitraz.

Invertebrates have a diverse immune system that responds differently to stressors such as pesticides and pathogens, which leads to different degrees of susceptibility. Honeybees are facing a phenomenon called colony collapse disorder which is attributed to several factors including pesticides and pathogens. We applied an in vitro approach to assess the response of immune-activated hemocytes from Apis mellifera, Drosophila melanogaster and Mamestra brassicae after exposure to imidacloprid and amitraz. Hemocytes were exposed to the pesticides in single and co-exposures using zymosan A for immune activation. We measured the effect of these exposures on cell viability, nitric oxide (NO) production from 15 to 120 min and on extracellular hydrogen peroxide (H2O2) production after 3 h to assess potential alterations in the oxidative response. Our results indicate that NO and H2O2 production is more altered in honeybee hemocytes compared to D. melanogaster and M. brassicae cell lines. There is also a differential production at different time points after pesticide exposure between these insect species as contrasting effects were evident with the oxidative responses in hemocytes. The results imply that imidacloprid and amitraz act differently on the immune response among insect orders and may render honeybee colonies more susceptible to infection and pests.

Sensitive detection of extracellular hydrogen peroxide using plasmon-enhanced electrochemical activity on Pd-tipped Au nanobipyramids.

The fabrication of electroactive nanostructures with high electron concentration and specific electron transport is crucial for electrochemical sensing. In this study, a plasmon-enhanced electrochemical sensor has been developed for the detection of extracellular hydrogen peroxide (H2O2) from cancer cells, utilizing Pd-tipped Au nanobipyramids (PTA NBPs) as the electrocatalysts. Plasmonic PTA NBPs were synthesized by depositing Pd nanoparticles onto the tips of Au nanobipyramids (Au NBPs). Under excitation of localized surface plasmon resonance (LSPR), the PTA NBPs generate high-energy electron-hole pairs (e-/h+) on their surface. The generated electrons (e-) significantly enhance the electrochemical reduction of H2O2. Based on this, a plasmon-enhanced H2O2 electrochemical sensor is constructed with high sensitivity (986.57 μA mM-1 cm-2), low detection limit (0.02 μM), wide linear range (0.1 μM to 980 μM), and good stability and repeatability. Moreover, this sensor also enables the measurement of extracellular H2O2 derived from cancer cells (MCF-7), highlighting its potential applications in cellular biology and biomedical research.

The Potential Contribution of Hexavalent Chromium to the Carcinogenicity of Chrysotile Asbestos.

Chrysotile asbestos is a carcinogenic mineral that has abundantly been used in industrial and consumer applications. The carcinogenicity of the fibers is partly governed by reactive Fe surface sites that catalyze the generation of highly toxic hydroxyl radicals (HO•) from extracellular hydrogen peroxide (H2O2). Chrysotile also contains Cr, typically in the low mass permille range. In this study, we examined the leaching of Cr from fibers at the physiological lung pH of 7.4 in the presence and absence of H2O2. Furthermore, we investigated the potential of cells from typical asbestos-burdened tissues and cancers to take up Cr leached from chrysotile in PCR expression, immunoblot, and cellular Cr uptake experiments. Finally, the contribution of Cr to fiber-mediated H2O2 decomposition and HO• generation was studied. Chromium readily dissolved from chrysotile fibers in its genotoxic and carcinogenic hexavalent redox state upon oxidation by H2O2. Lung epithelial, mesothelial, lung carcinoma, and mesothelioma cells expressed membrane-bound Cr(VI) transporters and accumulated Cr up to 10-fold relative to the Cr(VI) concentration in the spiked medium. Conversely, anion transporter inhibitors decreased cellular Cr(VI) uptake up to 45-fold. Finally, chromium associated with chrysotile neither decomposed H2O2 nor contributed to fiber-mediated HO• generation. Altogether, our results support the hypothesis that Cr may leach from inhaled chrysotile in its hexavalent state and subsequently accumulate in cells of typically asbestos-burdened tissues, which could contribute to the carcinogenicity of chrysotile fibers. However, unlike Fe, Cr did not significantly contribute to the adverse radical production of chrysotile.

In Situ and Quantitatively Imaging of Heat-Induced Oxidative State and Oxidative Damage of Living Neurons Using Scanning Electrochemical Microscopy.

Central nervous system is sensitive and vulnerable to heat. Oxidative state and oxidative damage of neurons under heat stress are vital for understanding early consequences and mechanisms of heat-related neuronal injury, which remains elusive partly due to the technical challenge of in situ and quantitative monitoring methods. Herein, a temperature-controlled scanning electrochemical microscopy (SECM) platform with programmable pulse potential and depth scan modes is developed for in situ and quantitatively monitoring of oxygen consumption, extracellular hydrogen peroxide level, and cell membrane permeability of neurons under thermal microenvironment of 37-42°C. The SECM results show that neuronal oxygen consumption reaches a maximum at 40°C and then decreases, extracellular H2 O2 level increases from 39°C, and membrane permeability increases from 2.0 ± 0.6 × 10-5 to 7.2 ± 0.8 × 10-5 m s-1 from 39 to 42°C. The therapeutic effect on oxidative damage of neurons under hyperthermia conditions (40-42°C) is further evaluated by SECM and fluorescence methods, which can be partially alleviated by the potent antioxidant edaravone. This work realizes in situ and quantitatively observing the heat-induced oxidative state and oxidative damage of living neurons using SECM for the first time, which results can contribute to a better understanding of the heat-related cellular injury mechanism.

Luteolin and Chrysin Could Prevent E. coli Lipopolysaccharide-Ochratoxin A Combination-Caused Inflammation and Oxidative Stress in In Vitro Porcine Intestinal Model.

Simple SummaryEnvironmental stressors, such as mycotoxins and enteropathogenic bacteria, can exert a detrimental effect on the gastrointestinal tract of the swine on contaminated corn-based feed which leads to the development of retarded growth; weight loss, thus, contributes greatly to lowered meat productivity. Plant-derived compounds such as flavonoid-type chrysin and luteolin were investigated in porcine enterocytes to evaluate their beneficial properties in gut damage. Toxic effects of ochratoxin A mycotoxin and bacterial outer membrane component, lipopolysaccharide could not be alleviated by the applied flavonoids even in combinations. However, luteolin and chrysin could prevent the excessive inflammatory responses by lowering cytokines’ production elicited by mycotoxin and bacterial toxin. Due to their antioxidant properties, these flavonoids were effective in compensating redox homeostasis tipped by combined exposure of ochratoxin A and inflammatory lipopolysaccharide. The porcine intestinal epithelial cell model applied by us was proven to be suitable for studying the harmful effects of the toxins and at the same time to evaluate the protective effects of flavonoids without living animal sacrifices. This study also suggests that mycotoxin-weakened intestinal function might result in the development of enteropathogen-caused secondary infections.Ochratoxin A (OTA) and lipopolysaccharide (LPS) intake can cause gastrointestinal disorders. Polyphenolic chrysin (CHR) and luteolin (LUT) display anti-inflammatory and antioxidant properties. Porcine intestinal epithelial (jejunal) IPEC-J2 cells were treated with OTA (1 µM, 5 µM and 20 µM), E. coli LPS (10 µg/mL), CHR (1 µM) and LUT (8.7 µM) alone and in their combinations. Cell viabilities (MTS assay) and extracellular (EC) hydrogen-peroxide (H2O2) production (Amplex red method) were evaluated. Intracellular (IC) reactive oxygen species (ROS) were assessed using a 2′-7′dichlorodihydrofluorescein diacetate (DCFH-DA) procedure. ELISA assay was used to evaluate IL-6 and IL-8 secretion. OTA decreased cell viabilities (p < 0.001) which could not be alleviated by LUT or CHR (p > 0.05); however, EC H2O2 production was successfully suppressed by LUT in IPEC-J2 cells (p < 0.001). OTA with LPS elevated the IC ROS which was counteracted by CHR and LUT (p < 0.001). IL-6 and IL-8 secretion was elevated by LPS + OTA (p < 0.001) which could be inhibited by LUT (p < 0.01 for IL-6; p < 0.001 for IL-8). Based on our results, CHR and LUT exerted beneficial effects on IC ROS levels and on cytokine secretion (LUT) in vitro; thus, they might be used as dietary and feed supplements to avoid OTA- and LPS-related health risks.

Importance of Locations of Iron Ions to Elicit Cytotoxicity Induced by a Fenton-Type Reaction.

Simple SummaryThe Fenton reaction generates the hydroxyl radical (•OH), which is the most reactive and toxic reactive oxygen species and widely recognized as a key player in oxidative stress. To clarify whether this highly reactive molecule travels to its biological target, the effects of the site of the Fenton reaction on cytotoxicity were investigated. Cytotoxicity induced by the Fenton reaction was affected by the distribution of iron ions surrounding and/or being incorporated into cells. Cytotoxicity was enhanced when the Fenton reaction occurred inside cells. Instead of enhancing cytotoxicity, extracellular iron ions exerted protective effects against the cytotoxicity of extracellular hydrogen peroxide in an ion concentration-dependent manner. Distance had a negative impact on the reactivity of extracellular •OH and biologically effective targets. Furthermore, an assessment of plasmid DNA breakage showed that the Fenton reaction system did not effectively induce DNA breakage.The impact of the site of the Fenton reaction, i.e., hydroxyl radical (•OH) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2), or pre-incubated with FeSO4 and then H2O2 was added after medium was replaced to remove iron ions or after the medium was not replaced. Cell lethality in rat thymocytes was estimated by measuring cell sizes using flow cytometry. High extracellular concentrations of FeSO4 exerted protective effects against H2O2-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO4 enhanced cell lethality induced by H2O2, whereas a pre-incubation with a high concentration of FeSO4 exerted protective effects. FeSO4 distributed extracellularly or on the surface of cells neutralized H2O2 outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of •OH, occurred inside cells. An assessment of plasmid DNA breakage showed that •OH induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated •OH does not appear to be DNA.

The Impact of Quercetin and Its Methylated Derivatives 3-o-Methylquercetin and Rhamnazin in Lipopolysaccharide-Induced Inflammation in Porcine Intestinal Cells.

Oxidative stress in the small intestine can lead to inflammation and barrier malfunction. The present study describes the effect of quercetin (Q), 3-o-methylquercetin (QM), and rhamnazin (R) on cell viability, paracellular permeability, production of intracellular reactive oxygen species (ROS), extracellular hydrogen peroxide (H2O2), and interleukin-6 (IL-6) after challenging jejunal cells (IPEC-J2) with different types (Salmonella enterica ser. Typhimurium, Escherichia coli O111:B4, and E. coli O127:B8) of lipopolysaccharides (LPS) applied in 10 µg/mL concentration. The intracellular ROS level increased after all LPS treatments, which could be decreased by all tested flavonoid compounds in 50 µM concentration. Extracellular H2O2 production significantly increased after Q and R treatment (50 µM). S. Typhimurium LPS could significantly increase IL-6 production of enterocytes, which could be alleviated by Q, QM, and R (50 µM) as well. Using fluorescein isothiocyanate dextran (FD4) tracer dye, we could demonstrate that S. Typhimurium LPS significantly increased the permeability of the cell layer. The simultaneous treatments of S. Typhimurium LPS and the flavonoid compounds showed no alteration in FD4 penetration compared to untreated cells. These results highlight that Q, QM, and R are promising substances that can be used to protect intestinal epithelial cells from the deteriorating effects of oxidative stress.

Redox Potential Correlates with Changes in Metabolite Concentrations Attributable to Pathways Active in Oxidative Stress Response in Swine Traumatic Shock.

Oxidation-reduction (redox) reactions, and the redox potential (RP) that must be maintained for proper cell function, lie at the heart of physiologic processes in critical illness. Imbalance in RP reflects systemic oxidative stress, and whole blood RP measures have been shown to correlate with oxygen debt level over time in swine traumatic shock. We hypothesize that RP measures reflect changing concentrations of metabolites involved in oxidative stress. To test this hypothesis, we compared blood and urine RP with concentrations of multiple metabolites in a swine traumatic shock model to identify meaningful RP-metabolite relationships. Seven swine were subjected to traumatic shock. Mixed venous (MV) RP, urine RP, and concurrent MV and urine metabolite concentrations were assessed at baseline, max O 2 Debt (80 mL/kg), end resuscitation, and 2 h post-resuscitation. RP was measured at collection via open circuit potential using nanoporous gold electrodes with Ag/AgCl reference and a ParstatMC potentiostat. Metabolite concentrations were measured by quantitative 1 H-NMR spectroscopy. MV and urine RP were compared with time-matched metabolites across all swine. LASSO regression with leave-one-out cross validation was used to determine meaningful RP/metabolite relationships. Metabolites had to maintain magnitude and direction of coefficients across 6 or more swine to be considered as having a meaningful relationship. KEGG IDs of these metabolites were uploaded into Metscape for pathway identification and evaluation for physiologic function. Meaningful metabolite relationships (and mean coefficients across cross-validation folds) with MV RP included: choline (-6.27), ATP (-4.39), glycine (5.93), ADP (1.84), glucose (15.96), formate (-13.09), pyruvate (6.18), and taurine (-7.18). Relationships with urine RP were: betaine (4.81), urea (4.14), glycine (-2.97), taurine (10.32), 3-hydroxyisobutyrate (-7.67), N-phenylacetylglycine, PAG (-14.52), hippurate (12.89), and formate (-5.89). These meaningful metabolites were found to scavenge extracellular peroxide (pyruvate), inhibit ROS and activate cellular antioxidant defense (taurine), act as indicators of antioxidant mobilization against oxidative stress (glycine + PAG), and reflect renal hydroxyl radical trapping (hippurate), among other activities. Real-time RP measures demonstrate significant relationships with metabolites attributable to metabolic pathways involved in systemic responses to oxidative stress, as well as those involved in these processes. These data support RP measures as a feasible, biologically relevant marker of oxidative stress. As a direct measure of redox state, RP may be a useful biomarker and clinical tool in guiding diagnosis and therapy in states of increased oxidative stress and may offer value as a marker for organ injury in these states as well.

Assess heavy metals-induced oxidative stress of microalgae by Electro-Raman combined technique

Oxidative stress of aquatic microorganisms under heavy metal stress is closely reflected by metabolite changes in cells but it is very difficult to study due to the fast metabolism process and severe in-situ measurements hurdle. Herein, the oxidative stress of cadmium on Euglena gracilis was systematically studied through multi-combined techniques. In particular, for the first time electrochemical approach was associated with Raman spectroscopy imaging to vividly to investigate temporal-spatially varied oxidative stress and its effects on cells metabolism, in which former real-time measured a volcanic relation of extracellular hydrogen peroxide versus the increase of cadmium stress, while the latter shows the corresponding metabolic changes by Raman imaging of single cells. This work builds a bridge to unravel the mechanism of cellular oxidative stress under harsh conditions in a more systematic and holistic approach, while holding a great promise to construct heavy metal biosensors precisely monitoring high heavy metal tolerance strains for environmental modification.

Live cell imaging of oxidative stress in human airway epithelial cells exposed to isoprene hydroxyhydroperoxide

Exposure to respirable air particulate matter (PM2.5) in ambient air is associated with morbidity and premature deaths. A major source of PM2.5 is the photooxidation of volatile plant-produced organic compounds such as isoprene. Photochemical oxidation of isoprene leads to the formation of hydroperoxides, environmental oxidants that lead to inflammatory (IL-8) and adaptive (HMOX1) gene expression in human airway epithelial cells (HAEC). To examine the mechanism through which these oxidants alter intracellular redox balance, we used live-cell imaging to monitor the effects of isoprene hydroxyhydroperoxides (ISOPOOH) in HAEC expressing roGFP2, a sensor of the glutathione redox potential (EGSH). Non-cytotoxic exposure of HAEC to ISOPOOH resulted in a rapid and robust increase in EGSH that was independent of the generation of intracellular or extracellular hydrogen peroxide. Our results point to oxidation of GSH through the redox relay initiated by glutathione peroxidase 4, directly by ISOPOOH or indirectly by ISOPOOH-generated lipid hydroperoxides. We did not find evidence for involvement of peroxiredoxin 6. Supplementation of HAEC with polyunsaturated fatty acids enhanced ISOPOOH-induced glutathione oxidation, providing additional evidence that ISOPOOH initiates lipid peroxidation of cellular membranes. These findings demonstrate that ISOPOOH is a potent environmental airborne hydroperoxide with the potential to contribute to oxidative burden of human airway posed by inhalation of secondary organic aerosols.

Regulation of the Structural Stability of Erythrocytes by Hydrogen Peroxide: Mathematical Model and Experiment

In this study, the regulatory mechanisms induced by extracellular hydrogen peroxide were analyzed on the basis of a mathematical model that considers the key stages of the formation of methemoglobin and ferrylhemoglobin, as well as their binding to the erythrocyte membrane. Numerical modeling has shown that reversible binding of methemoglobin to the membrane is an adaptive mechanism aimed at stabilizing the lipid bilayer of the membrane. On the other hand, an increase in the concentration of ferrylhemoglobin and its binding to the membrane leads to an increase in pathophysiological processes that reduce the structural stability of cells. The quantity of methemoglobins and ferrylhemoglobins formed depends on the concentration of extracellular hydrogen peroxide and exposition time, the number of cells in the sample, the state of the antioxidant system of erythrocytes, the metabolic activity of cells and external metabolic conditions. Based on numerical modeling, optimal conditions (oxidant concentration and exposition time) have been determined, under which the activation of adaptive processes occurs. Experiments with erythrocyte hemolysis in vitro have shown that hydrogen peroxide at concentrations of 10–200 μM causes an increase in the structural stability of the membrane and a decrease in the proportion of hemolyzed erythrocytes.

Colorimetric Detection of Extracellular Hydrogen Peroxide Using an Integrated Microfluidic Device.

It is well known that hydrogen peroxide (H2O2) is a signaling molecule essential for vital physiological reactions in mammalian cells, such as cell survival, intercellular communication, and cancer metabolism. However, to fully understand the function of H2O2, it is critical to monitor its intracellular and/or extracellular concentrations. Current techniques implemented to address this need require large sample volumes, expensive instrumentation, and long sample preparation and analysis times, inapplicable to inline or online monitoring. In this paper, a new integrated microfluidic device capable of overcoming these limitations is demonstrated for the colorimetric detection of extracellular hydrogen peroxide H2O2. The device contains an optical waveguide to determine absorbance changes and micromixers to enable complete mixing of reagents using a passive approach. This novel H2O2-sensing device has allowed the detection of H2O2 in the range of 0.5-60 μM with a detection limit of 167 ± 5.8 nM and a sensitivity of 13.5 ± 0.1 AU/mM. Proof of concept of the device was demonstrated by quantifying H2O2 release from benign prostatic epithelial (BPH-1) cells upon stimulation with phorbol 12-myristate 13-acetate (PMA). Results show that this integrated device can be potentially utilized to continuously monitor cell-released metabolites autonomously without constant human supervision during the process. Furthermore, this can be achieved without interfering with the cell culture conditions, as only a very small volume of conditioned media (less than 0.4 μL), and not the cells, is required.