H2O2 Release Research Articles

Exposure of Iron Nanoparticles to Arabidopsis thaliana Enhances Root Elongation by Triggering Cell Wall Loosening

In this study, we investigated the effect of nZVI on plant root elongation in Arabidopsis thaliana and showed, for the first time, that nZVI enhanced root elongation by inducing OH radical-induced cell wall loosening. Exposure of plants to 0.5 g/L nZVI enhanced root elongation by 150-200% over that in the control, and further mechanistic studies showed that this occurred via nZVI-mediated OH radical-induced cell wall loosening. The oxidation capacity of nZVI, leading to release of H2O2, allowed it to cause OH radical-induced cell wall loosening in roots. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometers (MALDI-TOFMS)-based analysis clearly revealed that pectin-polysaccharides in roots were degraded; they are one of the main matrix-polysaccharide-connecting and load-bearing polymers in cell walls. Rapid root elongation led to structural changes in root cell walls: reduction of cell wall thickness and a bias on the orientation of cellulose microfibrils. Additionally, the asymmetrical distribution of tensional strength resulted from the OH radical-induced cell wall loosening enhanced endocytosis. These findings emphasize that OH radical-induced cell wall loosening is important for mechanical regulation of the cell wall and provide new insights into the cellular responses of plants exposed to reactive metal nanoparticles.

Advances around technologies investigating mitochondrial function and insights gained by their applications.

Diabetes mellitus is defined as a state of persistently high levels of blood glucose and insulin deficiency, and resistance to it is considered as the cause(s). However, the metabolic abnormalities of diabetes are not limited to glucose, but encompass the entire metabolism. Insulin controls not only glucose, but fat and protein metabolism as well. Furthermore, body structures of patients with this disease become abnormal, termed complications, which eventually lead to the death of patients.

Mammalian flavin-containing monooxygenase (FMO) as a source of hydrogen peroxide

Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30–50% of O2 consumed as H2O2 upon addition of NADPH. Addition of substrate had little effect on H2O2 production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H2O2. FMO2 S195L exhibited higher “leakage”, producing much greater amounts of H2O2, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H2O2 generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H2O2 release. Catalase did not reduce levels of H2O2 with either FMO2.1 or FMO3 but inhibited H2O2 generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP+ binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H2O2.

The cardioprotectant 3′,4′-dihydroxyflavonol inhibits opening of the mitochondrial permeability transition pore after myocardial ischemia and reperfusion in rats

The study aimed to determine the effect of 3′,4′-dihydroxyflavonol (DiOHF) on mitochondrial function, in particular opening of the mitochondrial permeability transition pore (mPTP), respiratory function and reactive oxygen species (ROS) production, in isolated cardiac mitochondria after coronary artery occlusion and reperfusion in vivo. Opening of the mPTP, oxygen consumption and ROS production (assessed by measurement of H2O2) was determined in mitochondria isolated from normal hearts or from the ischemic zone of rat hearts subjected to 30min coronary artery occlusion and 15min reperfusion. Treatment of sham rats with DiOHF (10mgkg−1 iv) significantly increased the concentration of Ca2+ required to stimulate mPTP opening. This was accompanied by increased state 3 oxygen consumption and decreased H2O2 release. Ischemia and reperfusion (IR) significantly decreased the concentration of Ca2+ required to stimulate mPTP opening, decreased state 3 oxygen consumption and increased H2O2 release, when pyruvate plus malate was provided as a substrate. Treatment with DiOHF prevented IR-induced changes in mPTP opening, state 3 oxygen consumption and H2O2 release so that there was no difference compared to sham. In isolated cardiac mitochondria from normal rats DiOHF had no effect on mPTP opening or on state 3 respiration but caused a small increase in state 4 respiration and decreased the respiratory control ratio.DiOHF, administered during ischemia just before reperfusion, inhibits mPTP opening and preserves mitochondrial function through a mechanism likely to be independent of its antioxidant activity or any direct effect on the mPTP.

Inhibition of Delta-6 Desaturase Reverses Cardiolipin Remodeling and Prevents Contractile Dysfunction in the Aged Mouse Heart Without Altering Mitochondrial Respiratory Function

Aging results in a redistribution of polyunsaturated fatty acids (PUFAs) in myocardial phospholipids. In particular, a selective loss of linoleic acid (18:2n6) with reciprocal increases of long-chain PUFAs (eg, arachidonic and docosahexaenoic acids) in the mitochondrial phospholipid cardiolipin correlates with cardiac mitochondrial dysfunction and contractile impairment in aging and related pathologies. In this study, we demonstrate a reversal of this aged-related PUFA redistribution pattern in cardiac mitochondria from aged (25 months) C57Bl/6 mice by inhibition of delta-6 desaturase, the rate limiting enzyme in long-chain PUFA biosynthesis. Interestingly, delta-6 desaturase inhibition had no effect on age-related mitochondrial respiratory dysfunction, H2O2 release, or lipid peroxidation but markedly attenuated cardiac dilatation, hypertrophy, and contractile dysfunction in aged mice. Taken together, our studies indicate that PUFA metabolism strongly influences phospholipid remodeling and cardiac function but dissociates these processes from mitochondrial respiratory dysfunction and oxidant production in the aged mouse heart.

Evidence for necrosis, but not apoptosis, in human hepatoma cells with knockdown of mitochondrial aquaporin-8

We previously found that mitochondrial aquaporin-8 (mtAQP8) channels facilitate mitochondrial H2O2 release in human hepatoma HepG2 cells and that their knockdown causes oxidant-induced mitochondrial dysfunction and loss of viability. Here, we studied whether apoptosis or necrosis is involved as the mode of cell death. We confirmed that siRNA-induced mtAQP8 knockdown significantly decreased HepG2 viability by MTT assay, LDH leakage, and trypan blue exclusion test. Analysis of mitochondrial proapoptotic Bax-to-antiapoptotic BclXL ratio, mitochondrial cytochrome c release and caspase-3 activation showed no alterations in mtAQP8-knockdown cells. This indicates a primary mechanism of cell death other than the intrinsic mitochondrial apoptotic pathway. Thus, nuclear staining with DAPI did not reveal any increase of apoptotic features, i.e. chromatin condensation or nuclear fragmentation. Flow cytometry studies after double cell staining with annexin V and propidium iodide confirmed lack of apoptosis and suggested necrosis as the primary mechanism of death in mtAQP8-knockdown HepG2 cells. Necrosis was further supported by the increased nuclear delocalization and extracellular release of the High Mobility Group Box 1 protein. The knockdown of mtAQP8 in another human hepatoma-derived cell line, i.e. HuH-7 cells, also induced necrotic but not apoptotic death. Our data suggest that mtAQP8 knockdown induces necrotic cell death in human neoplastic hepatic cells, a finding that might be relevant to therapeutic strategies against hepatoma cells.

Glucagon-like peptide-1 receptor agonist activation ameliorates venous thrombosis-induced arteriovenous fistula failure in chronic kidney disease.

High shear stress that develops in the arteriovenous fistula of chronic kidney diseases (CKD) may increase H2O2 and thromboxane A2 (TXA2) release, thereby exacerbating endothelial dysfunction, thrombosis, and neointimal hyperplasia. We investigated whether glucagon-like peptide-1 receptor agonist/exendin-4, a potentially cardiovascular protective agent, could improve TXA2-induced arteriovenous fistula injury in CKD. TXA2 administration to H2O2-exposed human umbilical vein endothelial cells increased apoptosis, senescence, and detachment; these phenotypes were associated with the downregulation of phosphorylated endothelial nitric oxide synthase/heme oxygenase-1 (eNOS/HO-1) signalling. Exendin-4 reduced H2O2/TXA2-induced endothelial injury via inhibition of apoptosis-related mechanisms and restoration of phosphorylated eNOS/HO-1 signalling. Male Wistar rats subjected to right common carotid artery-external jugular vein anastomosis were treated with exendin-4 via cervical implant osmotic pumps for 16-42 days. High shear stress induced by the arteriovenous fistula significantly increased venous haemodynamics, blood and tissue H2O2 and TXB2 levels, macrophage/monocyte infiltration, fibrosis, proliferation, and adhesion molecule-1 expression. Apoptosis was also increased due to NADPH oxidase gp91 activation and mitochondrial Bax translocation in the proximal end of the jugular vein of CKD rats. Exendin-4-treatment of rats with CKD led to the restoration of normal endothelial morphology and correction of arteriovenous fistula function. Exendin-4 treatment or thromboxane synthase gene deletion in CKD mice markedly reduced ADP-stimulated platelet adhesion to venous endothelium, and prevented venous occlusion in FeCl3-injured vessels by upregulation of HO-1. Together, these data reveal that the use of glucagon-like peptide-1 receptor agonists is an effective strategy for treatment of CKD-induced arteriovenous fistula failure.

Honey in dermatology and skin care: a review

Honey is a bee-derived, supersaturated solution composed mainly of fructose and glucose, and containing proteins and amino acids, vitamins, enzymes, minerals, and other minor components. Historical records of honey skin uses date back to the earliest civilizations, showing that honey has been frequently used as a binder or vehicle, but also for its therapeutic virtues. Antimicrobial properties are pivotal in dermatological applications, owing to enzymatic H2 O2 release or the presence of active components, like methylglyoxal in manuka, while medical-grade honey is also available. Honey is particularly suitable as a dressing for wounds and burns and has also been included in treatments against pityriasis, tinea, seborrhea, dandruff, diaper dermatitis, psoriasis, hemorrhoids, and anal fissure. In cosmetic formulations, it exerts emollient, humectant, soothing, and hair conditioning effects, keeps the skin juvenile and retards wrinkle formation, regulates pH and prevents pathogen infections. Honey-based cosmetic products include lip ointments, cleansing milks, hydrating creams, after sun, tonic lotions, shampoos, and conditioners. The used amounts range between 1 and 10%, but concentrations up to 70% can be reached by mixing with oils, gel, and emulsifiers, or polymer entrapment. Intermediate-moisture, dried, and chemically modified honeys are also used. Mechanisms of action on skin cells are deeply conditioned by the botanical sources and include antioxidant activity, the induction of cytokines and matrix metalloproteinase expression, as well as epithelial-mesenchymal transition in wounded epidermis. Future achievements, throwing light on honey chemistry and pharmacological traits, will open the way to new therapeutic approaches and add considerable market value to the product.

Role of enzymatic and non-enzymatic processes in H2O2 removal by rat liver and heart mitochondria

We compared the capacity of rat liver and heart mitochondria to remove exogenously produced H2O2, determining their ability to decrease fluorescence generated by H2O2 detector system. In the absence of substrates, liver and heart mitochondria removed H2O2 at similar rates. Respiratory substrate addition increased removal rates, indicating a respiration-dependent process. Moreover, the rates were higher with pyruvate/malate than with succinate and in heart than in liver mitochondria. Generally, the changes in H2O2 removal rates mirrored those of H2O2 release rates excluding the possibility that endogenous and exogenous H2O2 competed for the removing system. This idea was supported by the observation that the heaviest of three liver mitochondrial fractions exhibited the highest rates of both H2O2 release and removal. Pharmacological inhibition showed tissue-linked differences in antioxidant enzyme contribution to H2O2 removal which were consistent with the differences in antioxidant system activities. The enzymatic processes accounted only in part for net H2O2 removal and the non-enzymatic ones participated to H2O2 scavenging to a degree that was higher for heart than for liver mitochondria. The idea that non-enzymatic scavenging was due in great part to hemoproteins action was consistent with observation that the concentration of cytochromes, in particular cytochrome c, was higher in heart mitochondria. Indirect support was also obtained by a technique of enhanced luminescence, utilizing the capacity of cytochrome c/H2O2 to catalyze the luminol oxidation, which showed that luminescence response to an oxidative challenge was higher in heart mitochondria.

Pro-oxidative effects of aggregated transthyretin in human Schwannoma cells

Neurotoxicity mechanisms of amyloidogenic polypeptides such as transthyretin (TTR) are not well understood. Misfolded and aggregated TTRs (agTTR) lead to age-related diseases such as senile systemic amyloidosis and familial amyloid polyneuropathy (FAP). Among other clinical manifestations in TTR amyloidic disease, peripheral nerve tissue, including Schwann cell, degeneration has been observed. In this study, we examined potential toxic effects of agTTR in human Schwannoma cells (sNF94.3 peripheral nerve sheath line). Cells were treated with agTTR (2.4μM pre-aggregation concentration) or, as controls, normal, soluble TTR (2.4μM) or no-TTR treatment, and then analyzed for different pro-oxidant and anti-oxidant markers: hydrogen peroxide (H2O2), catalase (CAT), glutathione (GSH), and more generalized cellular antioxidant capacity. In the latter case, cytosolic fractions were prepared after agTTR (or control) treatments and analyzed in oxidation assays. Relative to treatment with normal soluble TTR, cells treated with agTTR increase their release of H2O2. Residual CAT activity is decreased after agTTR treatment. The Schwannoma cells also exhibit significantly lower levels of GSH after agTTR treatment (p<0.05, relative to controls). More generally, cytosols from agTTR-treated cells exhibited a lower capacity to prevent oxidation relative to those from control cells (TTR-treated, or non-TTR-treated). These results suggest that agTTR (a) stimulates production of reactive oxygen species, (b) leads to lower levels of endogenous antioxidants, and (c) decreases overall cellular antioxidant capacity, in Schwannoma cells.

Response to Marinelli and Marchissio

We are glad that our colleagues Marinelli and Marchissio found our recent article ‘‘Tyrosine kinase signal modulation: a matter of H2O2 membrane permeability?’’ (1) of interest. Owing to the strict space limitations of News & Views articles, we could not present the RT-PCR data showing the presence of aquaporin-8 (AQP8) in our HeLa cells and its strong reduction upon silencing. In our model, the entry of H2O2 into mitochondria was efficient also in cells in which AQP8 was knocked down and—as a consequence—transport across the plasma membrane was severely impaired (1). Thus, the conclusion that AQP8 is dispensable for mitochondrial H2O2 import remains valid, even though this aquaporin may facilitate the release of H2O2 from these organelles, when present (3). Clearly, more has to be learned on the role of intracellular aquaporins in transporting substances other than water. In our opinion, a matter of particular interest is how AQP8 can be targeted to the exocytic pathway and/or to mitochondria in cells, in which a mitochondrial function has been described. As an example of alternative intracellular routing, the cotranslational translocation of PrP and other proteins normally destined to the secretory pathway is inhibited in cells undergoing ER stress (2,4). Finally, we entirely agree that the mechanisms controlling H2O2 transport across membranes deserve further studies, in view of their paramount pathophysiological relevance.

Abstract 418: Angiotensin II Evokes Acute ATP and H 2 O 2 Release in the Kidney

The study of the functional processes in the whole kidney presents a tremendous experimental challenge due to the structural and functional heterogeneity of the kidney tissues and rapid transient changes in the concentrations of the signaling molecules. The purines, like adenosine-5’-triphosphate (ATP) as well as reactive oxygen species including H 2 O 2 are among the most important paracrine signaling agents. Angiotensin II (AngII) directly regulates many processes in the body and is strongly linked to the hypertension. We recently developed a novel approach based on the use of enzymatic biosensors suitable for real-time measurements of endogenous substances release in freshly isolated rat kidney. AngII-induced changes of interstitial concentration of ATP and H 2 O 2 were determined in Sprague Dawley (SD) and Dahl salt sensitive (SS) rats. Freshly isolated kidneys of the SD or SS rats were infused with AngII (1 μM) under constant laminar flow. The maximum amplitude of the AngII induced H 2 O 2 responses averaged 70 ± 15, 189 ± 58 and 301 ± 91 nM for SD and SS rats fed a low (0.4%) and high salt (4%; 3 weeks) diets, respectively. The H 2 O 2 response exhibited a slow increase to a stable maximal level that lasted during the rest of the perfusion time. The average production rate was 67 ± 17, 187 ± 55 and 294 ± 81 nM/s for SD, and SS rats fed low and high salt diets. The kinetics of ATP release had different characteristics compared to H 2 O 2 release with rapid increase in concentration triggered by AngII and followed by a slow decay of the signal due to ATP conversion to adenosine. The maximum amplitudes averaged 449 ± 145, 630 ± 49 and 1087 ± 96 nM for SD rats and SS rats fed low and high salts. The ATP production rates were 257 ± 100, 496 ± 202 and 829 ± 319 nM/s for the corresponding groups. To test AT receptor subtype mediated the acute release of ATP and H 2 O 2 in response to AngII, we used an AT 1 receptor antagonist losartan. Losartan (10 μM) inhibited the release of both ATP and H 2 O 2 in response to AngII application. Thus, these experiments reveal that established approach can be successfully used to analyze acute changes in endogenous substances in response to drug perfusion. Importantly, these data demonstrate that perfusion of the kidney with AngII rapidly induced release of both ATP and H 2 O 2 .

Histamine Stimulates Hydrogen Peroxide Production by Bronchial Epithelial Cells via Histamine H1 Receptor and Dual Oxidase

Oxidative stress has been implicated in the pathogenesis of bronchial asthma. Besides granulocytes, the airway epithelium can produce large amounts of reactive oxygen species and can contribute to asthma-related oxidative stress. Histamine is a major inflammatory mediator present in large quantities in asthmatic airways. Whether histamine triggers epithelium-derived oxidative stress is unknown. We therefore aimed at characterizing human airway epithelial H2O2 production stimulated by histamine. We found that air-liquid interface cultures of primary human bronchial epithelial cells (BECs) and an immortalized BEC model (Cdk4/hTERT HBEC) produce H2O2 in response to histamine. The main source of airway epithelial H2O2 is an NADPH dual oxidase, Duox1. Out of the four histamine receptors (H1R-H4R), H1R has the highest expression in BECs and mediates the H2O2-producing effects of histamine. IL-4 induces Duox1 gene and protein expression levels and enhances histamine-induced H2O2 production by epithelial cells. Using HEK-293 cells expressing Duox1 or Duox2 and endogenous H1R, histamine triggers an immediate intracellular calcium signal and H2O2 release. Overexpression of H1R further increases the oxidative output of Duox-expressing HEK-293 cells. Our observations show that BECs respond to histamine with Duox-mediated H2O2 production. These findings reveal a mechanism that could be an important contributor to oxidative stress characteristic of asthmatic airways, suggesting novel therapeutic targets for treating asthmatic airway disease.

Dectin-1, a C-type lectin receptor, participates in the killing of Candida albicans by human neutrophils: an experimental study

Objective To investigate whether human neutrophils kill Candida albicans through recognition of insoluble β-glucan in cell walls of C.albicans (CalG) by dectin-1,a C-type lectin receptor.Methods Neutrophils were obtained from peripheral blood of healthy human subjects and cultured in vitro.Real-time PCR was carried out to quantify the mRNA expressions of dectin-1 and Toll-like receptor 2 (TLR2) in neutrophils challenged with CaIG of 100 mg/L for 1,6,and 24 hours.A Fluoro hydrogen peroxide (H2O2) detection kit was used to determine H2O2 levels in some neutrophils exposed to CaIG (100 mg/L) for 15 minutes,2 hours,6 hours,as well as in some neutrophils preincubated with laminarin (a dectin-1 inhibitor) of 100 mg/L and 50 mg/L for 30 minutes followed by challenge with CaIG of 100 mg/L for 2 hours.Colony forming units (CFUs) were counted after the incubation of C.albicans with neutrophils pretreated with laminarin of 100 mg/L and 50 mg/L for 30 minutes.Results The relative mRNA expression level of dectin-1 was 2.8195 + 0.1669,5.4859 + 0.7244 and 3.6041 + 0.5372 in neutrophils challenged with CaIG for 1,6 and 24 hours,respectively,significantly higher than that in unchallenged neutrophils at these corresponding time points (all P ＜ 0.01).The level of H2O2 was (64.55 + 15.67),(290.34 + 30.56),and (208.54 ± 26.88) μ mol/L respectively in neutrophils treated with CaIG for 15 minutes,2 hours,and 6 hours respectively,compared to (22.05 ± 3.99) μmol/L in untreated neutrophils (all P ＜ 0.01).The pretreatment with laminarin of 100 and 50 mg/L attenuated the release of H2O2 in CaIG-treated neutrophils by 73％ ((80.45 + 22.41) μ mol/L,P＜ 0.01) and 45％ ((130.42 + 44.55) μmol/L,P＜ 0.01),respectively,compared with neutrophils treated with CaIG only.The fungicidal activity of neutrophils against C.albicans was also significantly inhibited by pretreatment with laminarin of 50 and 100 mg/L (both P＜ 0.01).Conclusions Dectin-1 may be involved in the secretion of H2O2 as well as killing of C.albicans by human neutrophils,which may provide a preliminary evidence for adoptive transfer of neutrophils as an approach to the treatment of systemic C.albicans infection. Key words: Candida albicans; Neutrophils; beta-Glucans; Lectins, c-type; Hydrogen peroxide

Evidence for inflammation-mediated memory dysfunction in gastropods: putative PLA2and COX inhibitors abolish long-term memory failure induced by systemic immune challenges

BackgroundPrevious studies associate lipid peroxidation with long-term memory (LTM) failure in a gastropod model (Lymnaea stagnalis) of associative learning and memory. This process involves activation of Phospholipase A2 (PLA2), an enzyme mediating the release of fatty acids such as arachidonic acid that form the precursor for a variety of pro-inflammatory lipid metabolites. This study investigated the effect of biologically realistic challenges of L. stagnalis host defense response system on LTM function and potential involvement of PLA2, COX and LOX therein.ResultsSystemic immune challenges by means of β-glucan laminarin injections induced elevated H2O2 release from L. stagnalis circulatory immune cells within 3 hrs of treatment. This effect dissipated within 24 hrs after treatment. Laminarin exposure has no direct effect on neuronal activity. Laminarin injections disrupted LTM formation if training followed within 1 hr after injection but had no behavioural impact if training started 24 hrs after treatment. Intermediate term memory was not affected by laminarin injection. Chemosensory and motor functions underpinning the feeding response involved in this learning model were not affected by laminarin injection. Laminarin’s suppression of LTM induction was reversed by treatment with aristolochic acid, a PLA2 inhibitor, or indomethacin, a putative COX inhibitor, but not by treatment with nordihydro-guaiaretic acid, a putative LOX inhibitor.ConclusionsA systemic immune challenge administered shortly before behavioural training impairs associative LTM function in our model that can be countered with putative inhibitors of PLA2 and COX, but not LOX. As such, this study establishes a mechanistic link between the state of activity of this gastropod’s innate immune system and higher order nervous system function. Our findings underwrite the rapidly expanding view of neuroinflammatory processes as a fundamental, evolutionary conserved cause of cognitive and other nervous system disorders.

The cardioprotectant 3',4'-dihydroxyflavonol inhibits the mitochondrial permeability transition pore

Purpose: 3',4'-Dihydroxyflavonol (DiOHF) reduces injury caused by myocardial ischaemia and reperfusion (IR) in association with a reduction in oxidative stress. Oxidative stress contributes to the opening of the mitochondrial permeability transition pore (mPTP), a key event in myocardial IR injury. The aim of this study was to determine the effect of DiOHF on mPTP, as well as mitochondrial respiration and the generation of reactive oxygen species (ROS) by cardiac mitochondria after IR in anaesthetised rats. Methods: Male Wistar rats were anaesthetised with pentobarbitone (70 mg/kg iv), intubated and ventilated. Through a left thoracotomy the left main coronary artery was occluded for 30 min and then reperfused for 15 minutes. The heart was then rapidly removed, cooled and the area at risk of ischaemia homogenised and centrifuged for the isolation of mitochondria. In sham experiments the rats were anaesthetised but not subject to ischaemia. DiOHF (10 mg/kg iv) or vehicle (50% DMSO) was injected intravenously 5 minutes before reperfusion. mPTP opening was measured by monitoring mitochondrial Ca2+ retention capacity. Oxygen consumption of isolated mitochondria was measured in the presence of pyruvate (5 mM) and malate (5 mM) with a Clark-type electrode and ROS generation was assessed by measuring the rate of H2O2 production detected by amplex red. Results: Treatment of sham rats with DiOHF significantly increased the concentration of Ca2+ required to stimulate mPTP opening (sham 87±6; sham+DiOHF 120±9 μM). This was accompanied by an increase in state 3 oxygen consumption (sham 352±55; sham+DiOHF 572±21 nmol O2/min/mg protein) and a decrease in H2O2 release (sham 0.028±0.002; sham+DiOHF 0.019±0.002 nmol/min/mg protein). IR significantly decreased the concentration of Ca2+ required to stimulate mPTP opening (IR 44±5 μM), decreased state 3 oxygen consumption (IR 232±15 nmol O2/min/mg protein) and increased H2O2 release (IR 0.034±0.001 nmol/min/mg protein) compared to sham. Treatment with DiOHF prevented IR-induced changes in mPTP opening (IR+DiOHF 78±7 μM), state 3 oxygen consumption (IR+DiOHF 375±30 nmol O2/min/mg protein) and H2O2 release (IR 0.028±0.002 nmol/min/mg protein) so that there was no difference compared to sham. Conclusions: In normal rats DiOHF inhibits mPTP opening and decreases mitochondrial ROS production. Importantly, DiOHF administration before reperfusion prevents IR-induced mPTP opening, impairment of state 3 respiration and increases in ROS production. The beneficial actions of DiOHF on mitochondria are likely to make a major contribution to its cardioprotective actions.

Insulin-Dependent H2O2 Production Is Higher in Muscle Fibers of Mice Fed with a High-Fat Diet

Insulin resistance is defined as a reduced ability of insulin to stimulate glucose utilization. C57BL/6 mice fed with a high-fat diet (HFD) are a model of insulin resistance. In skeletal muscle, hydrogen peroxide (H2O2) produced by NADPH oxidase 2 (NOX2) is involved in signaling pathways triggered by insulin. We evaluated oxidative status in skeletal muscle fibers from insulin-resistant and control mice by determining H2O2 generation (HyPer probe), reduced-to-oxidized glutathione ratio and NOX2 expression. After eight weeks of HFD, insulin-dependent glucose uptake was impaired in skeletal muscle fibers when compared with control muscle fibers. Insulin-resistant mice showed increased insulin-stimulated H2O2 release and decreased reduced-to-oxidized glutathione ratio (GSH/GSSG). In addition, p47phox and gp91phox (NOX2 subunits) mRNA levels were also high (~3-fold in HFD mice compared to controls), while protein levels were 6.8- and 1.6-fold higher, respectively. Using apocynin (NOX2 inhibitor) during the HFD feeding period, the oxidative intracellular environment was diminished and skeletal muscle insulin-dependent glucose uptake restored. Our results indicate that insulin-resistant mice have increased H2O2 release upon insulin stimulation when compared with control animals, which appears to be mediated by an increase in NOX2 expression.

Zinc Dioxide Nanoparticulates: A Hydrogen Peroxide Source at Moderate pH

Solid peroxides are a convenient source of hydrogen peroxide, which once released can be readily converted to active oxygen species or to dissolved dioxygen. A zinc peroxide nanodispersion was synthesized and characterized, and its solubility was determined as a function of pH and temperature. We show that zinc peroxide is much more stable in aqueous solutions compared to calcium and magnesium peroxides and that it retains its peroxide content down to pH 6. At low pH conditions H2O2 release is thermodynamically controlled and its dissolution product, Zn(2+), is highly soluble, and thus, hydrogen peroxide release can be highly predictable. The Gibbs free energy of formation of zinc peroxide was found to be -242.0 ± 0.4 kJ/mol and the enthalpy of formation was -292.1 ± 0.7 kJ/mol, substantially higher than theoretically predicted before. The biocidal activity of zinc peroxide was determined by inactivation studies with Escherichia coli cultures, and the activity trend agrees well with the thermodynamic predictions.

Macrophage activity and histopathology of the lymphohematopoietic organs in male Wistar rats orally exposed to single or mixed pesticides

The noxious effects of low or effective dose exposure to single or mixed pesticides on macrophage activity and the lymphohematopoietic organs were investigated. Male Wistar rats were orally exposed to dichlorvos, dicofol, endosulfan, dieldrin and permethrin, either as single or combined mixtures during a 28-day study containing eight groups: one group received a semipurified diet (non-treated); two groups received a semipurified diet containing low dose mixture (dieldrin 0.025 mg/kg, endosulfan, 0.6 mg/kg, dicofol 0.22 mg/kg, dichlorvos 0.23 mg/kg, permethrin 5 mg/kg) or an effective dose mixture (dichlorvos 2.3 mg/kg, dicofol 2.5 mg/kg, endosulfan 2.9 mg/kg, dieldrin 0.05 mg/kg and permethrin 25.0 mg/kg), respectively; the other five groups received a semipurified diet containing each single pesticide in effective doses. At sacrifice, the thymus, spleen, mesenteric lymph nodes, Payer's patches and bone marrow were removed for histological analysis. Peritoneal macrophages were obtained to determine the phagocytosis and spreading indexes and tumoral necrosis factor alpha (TNF-α), nitric oxide (NO) and H2O2 production. Exposure to pesticide mixtures did not alter the percentage of macrophage phagocytosis and spreading, TNF-α production or the NO and H2O2 release when compared to the non-treated group. Neither was there any apparent evidence that a pesticide mixture at low or effective doses altered the histological structure of the lymphohematopoietic organs. The findings indicate that short-term treatment with pesticide mixtures did not induce an apparent immunotoxic effect in male Wistar rats.

A spontaneous mutation in the nicotinamide nucleotide transhydrogenase gene of C57BL/6J mice results in mitochondrial redox abnormalities

NADPH is the reducing agent for mitochondrial H2O2 detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP+ ratio. This enzyme catalyzes the reduction of NADP+ at the expense of NADH oxidation and H+ reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-NntMUT) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-NntMUT mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-NntW) and from B6J-NntMUT mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-NntMUT mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H2O2 release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca2+-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-NntMUT mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-NntW mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-NntMUT mice in biomedical research should not be overlooked.