Endogenous H2O2 Production Research Articles

Abstract 3270: SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme cells

Abstract Glioblastoma (GBM), a malignant brain tumor, has poor survival outcomes due to recurrence or drug resistance. In our previous studies, it was found that superoxide dismutase 2 (SOD2) was associated with temozolomide (TMZ) resistance. Moreover, SOD2 can convert O2 •− to H2O2, and H2O2 redox signaling is vital for physiological reactions and promotes tumor progression. We used cell lines, patient primary cells and pharmacological inhibitors/activators to confirm the significance of H2O2 signaling. We used the cultured cells to verify roles of H2O2 signaling in the plasticity of cell fate and did animal experiments to identify the optimal treatment strategy. In the present study, SOD2 was found to be the upstream mediator, and combining SOD inhibitor and TMZ, the cells showed reduced SH3GLB1 and autophagy levels. We then found that SH3GLB1 was regulated by H2O2 through AKT signaling using experiments of regulating redox homeostasis. The change of intracellular H2O2 levels in mitochondria following the treatments is similar to that in cytosol, however, the cell fate of parental or resistant cells was quite diverse. Notably, CCCP, a mitochondrial membrane potential disrupter, increased endogenous H2O2 production, and TMZ-increased SH3GLB1 levels can be reversed by HgCl2, reflecting the aquaporin function. In animal models, a combination of TMZ and 4-hydroxynonenal reduced growth of the SH3GLB1-knockdown resistant cells, along with suppressed autophagy levels. Our results characterizing the mechanism of H2O2-SH3GLB1 axis signaling in acquired TMZ resistance of GBM cells will provide new therapeutic strategies against the disease in the future. Citation Format: Chia-Hung Chien, Kwang-Yu Chang. SH3GLB1 is involved with hydrogen peroxide/akt signaling in glioblastoma multiforme cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3270.

H2O2 promotes trimming-induced tillering by regulating energy supply and redox status in bermudagrass.

Tillering/branching pattern plays a significant role in determining the structure and diversity of grass, and trimming has been found to induce tillering in turfgrass. Recently, it has been reported that hydrogen peroxide (H2O2) regulates axillary bud development. However, the role of H2O2 in trimming-induced tillering in bermudagrass, a kind of turfgrass, remains unclear. Our study unveils the significant impact of trimming on promoting the sprouting and growth of tiller buds in stolon nodes, along with an increase in the number of tillers in the main stem. This effect is accompanied by spatial-temporal changes in cytokinin and sucrose content, as well as relevant gene expression in axillary buds. In addition, the partial trimming of new-born tillers results in an increase in sucrose and starch reserves in their leaves, which can be attributed to the enhanced photosynthesis capacity. Importantly, trimming promotes a rapid H2O2 burst in the leaves of new-born tillers and axillary stolon buds. Furthermore, exogenous application of H2O2 significantly increases the number of tillers after trimming by affecting the expression of cytokinin-related genes, bolstering photosynthesis potential, energy reserves and antioxidant enzyme activity. Taken together, these results indicate that both endogenous production and exogenous addition of H2O2 enhance the inductive effects of trimming on the tillering process in bermudagrass, thus helping boost energy supply and maintain the redox state in newly formed tillers.

A computational model of endogenous hydrogen peroxide metabolism in hepatocytes, featuring a critical role for GSH

This paper presents an ordinary differential equation (ODE) model of endogenous H2O2metabolism in hepatocytes that is unique, at the time of writing, in its ability to accurately compute intracellular H2O2concentration during incidents of oxidative stress and in its usefulness for constructing PBPK/PD models for ROS-generating xenobiotics. Versions of the model are presented for rat hepatocytes in vitro and mouse liver in vivo. A generic method is given for using the model to create PBPK/PD models which predict intracellular H2O2concentration and oxidative-stress-induced hepatocyte death; these are identifiable from in vitro data sets reporting cell mortality following xenobiotic exposure at various levels. The procedure is demonstrated for the trivalent arsenical dimethylarsinous acid (DMAIII), which is produced in liver as part of the arsenic elimination pathway. This is the first model of H2O2metabolism in hepatocytes to feature values for the endogenous rates of H2O2production by mitochondria and other organelles which are inferred from the physiology literature, and to feature a detailed, realistic treatment of GSH metabolism; the latter is achieved by incorporating a minimal version of Reed and coworkers’ pioneering model of GSH metabolism in liver. Model simulations indicate that critical GSH depletion is the immediate trigger for intracellular H2O2rising to concentrations associated with apoptosis (>1μM), that this may only occur hours after the xenobiotic concentration peaks (“delay effect”), that when critical GSH depletion does occur, H2O2concentration rises rapidly in a sequence of two boundary layers, characterized by the kinetics of glutathione peroxidase (first boundary layer) and catalase (second boundary layer), and that intracellular H2O2concentration >1μM implies critical GSH depletion. There has been speculation that ROS levels in the range associated with apoptosis simply indicate, rather than cause, an apoptotic milieu. Model simulations are consistent with this view. In a result of interest to the wider physiology community, the delay effect is shown to provide a GSH-based mechanism by which cells can distinguish transient elevations in H2O2concentration, of use in intracellular signaling, from persistent ones indicative of either pathology or the presence of toxins, the second state of affairs eventually triggering apoptosis.

Mild-temperature responsive nanocatalyst for controlled drug release and enhanced catalytic therapy

Owing to the advantages of the in situ production of toxic agents through catalytic reactions, nanocatalytic therapy has arisen as a highly potential strategy for cancer therapeutics in recent years. However, the insufficient amount of endogenous hydrogen peroxide (H2O2) in the tumor microenvironment commonly limits their catalytic efficacy. Here, we employed carbon vesicle nanoparticles (CV NPs) with high near-infrared (NIR, 808 nm) photothermal conversion efficiency as carriers. Ultrafine platinum iron alloy nanoparticles (PtFe NPs) were grown in situ on the CV NPs, where the highly porous nature of the resultant CV@PtFe NPs was employed to encapsulate a drug, β-lapachone (La), and phase-change material (PCM). As a multifunctional nanocatalyst CV@PtFe/(La-PCM) NPs can exhibit a NIR-triggered photothermal effect and activate cellular heat shock response, which upregulates the downstream NQO1 via HSP70/NQO1 axis to facilitate bio-reduction of the concurrently melted and released La. Moreover, sufficient oxygen (O2) is supplied by CV@PtFe/(La-PCM) NPs catalyzed at the tumor site to reinforce the La cyclic reaction with abundant H2O2 generation. This promotes the bimetallic PtFe-based nanocatalysis, which breaks H2O2 down into highly toxic hydroxyl radicals (•OH) for catalytic therapy. Our results show that this multifunctional nanocatalyst can be used as a versatile synergistic therapeutic agent with NIR-enhanced nanocatalytic tumor therapy by tumor-specific H2O2 amplification and mild-temperature photothermal therapy, which holds promising potential for targeted cancer treatment. Statement of significanceWe present a multifunctional nanoplatform with mild-temperature responsive nanocatalyst for controlled drug release and enhanced catalytic therapy. This work aimed at not only reduce the damage to normal tissues caused by photothermal therapy, but also improves the efficiency of nanocatalytic therapy by stimulating endogenous H2O2 production through photothermal heat. In vitro and in vivo confirmed that CV@PtFe/(La-PCM) NPs exhibited powerful and overall antitumor effects. This formulation may provide an alternative strategy for the development of the mild- photothermal enhanced nanocatalytic therapy effect in solid tumor.

IDH3γ functions as a redox switch regulating mitochondrial energy metabolism and contractility in the heart

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H2O2) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H2O2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H2O2-dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.

Abscisic Acid Mediates Salicylic Acid Induced Chilling Tolerance of Grafted Cucumber by Activating H2O2 Biosynthesis and Accumulation.

Grafting is widely applied to enhance the tolerance of some vegetables to biotic and abiotic stress. Salicylic acid (SA) is known to be involved in grafting-induced chilling tolerance in cucumber. Here, we revealed that grafting with pumpkin (Cucurbita moschata, Cm) as a rootstock improved chilling tolerance and increased the accumulation of SA, abscisic acid (ABA) and hydrogen peroxide (H2O2) in grafted cucumber (Cucumis sativus/Cucurbita moschata, Cs/Cm) leaves. Exogenous SA improved the chilling tolerance and increased the accumulation of ABA and H2O2 and the mRNA abundances of CBF1, COR47, NCED, and RBOH1. However, 2-aminoindan-2-phosphonic acid (AIP) and L-a-aminooxy-b-phenylpropionic acid (AOPP) (biosynthesis inhibitors of SA) reduced grafting-induced chilling tolerance, as well as the synthesis of ABA and H2O2, in cucumber leaves. ABA significantly increased endogenous H2O2 production and the resistance to chilling stress, as proven by the lower electrolyte leakage (EL) and chilling injury index (CI). However, application of the ABA biosynthesis inhibitors sodium tungstate (Na2WO4) and fluridone (Flu) abolished grafting or SA-induced H2O2 accumulation and chilling tolerance. SA-induced plant response to chilling stress was also eliminated by N,N'-dimethylthiourea (DMTU, an H2O2 scavenger). In addition, ABA-induced chilling tolerance was attenuated by DMTU and diphenyleneiodonium (DPI, an H2O2 inhibitor) chloride, but AIP and AOPP had little effect on the ABA-induced mitigation of chilling stress. Na2WO4 and Flu diminished grafting- or SA-induced H2O2 biosynthesis, but DMTU and DPI did not affect ABA production induced by SA under chilling stress. These results suggest that SA participated in grafting-induced chilling tolerance by stimulating the biosynthesis of ABA and H2O2. H2O2, as a downstream signaler of ABA, mediates SA-induced chilling tolerance in grafted cucumber plants.

Self-propelled nanojets an interfacial Schottky junctions modulated oxygen vacancies enriched for enhanced photo-Fenton degradation of organic contaminant: Improving H2O2 generation, Fe3+/Fe2+ cycle and enhancing plant metabolism

The study reports an innovative approach on sunlit driven heterostructure photocatalytic generation of H2O2 and removal of cefixime. In the present work, we have fabricated Mn/Mg doped CoFe2O4 modified CaCr2O4 decorated by Ag3PO4 quantum dots (Ag3PO4 QDs), a p-n-p nano heterojunction. The study promotes the photocatalytic production of H2O2 and self-Fenton photocatalytic degradation of cefixime. Egg white-assisted synthesis of Mn-doped CoFe2O4 causes the lattice oxygen defect, which enhances the photocatalytic activity. Lattice oxygen defect enable the adsorption of O2, which enable the conversion of •O2 in the valence band of CoFe2O4 for the endogenous production of H2O2. The higher in the surface area enhance the photocatalytic activity under visible light irradiation. Mn–CoFe2O4–CaCr2O4–Ag3PO4 QDs enables the complete photocatalytic degradation of cefixime (99.9%) and the complete removal was determined by total organic carbon (TOC) removal and it was around 99.4%. Meanwhile the photocatalytic degradation pathway of cefixime was determined by LC-MS/MS. Reusability of the nano heterojunction was determined by six cycle test, and the reusability of the nano heterojunction was 99.8%. Further, the toxicity of the nanomaterial was studied in maize plant and the results shows that the nanoheterojunction enhances the maize growth. The study systematically reveals the robust activity of nano heterojunction for sustainable water treatment.

Insights into H2O2-induced signaling in Jurkat cells from analysis of gene expression

Hydrogen peroxide (H2O2) is a ubiquitous oxidant produced in a regulated manner by various enzymes in mammalian cells. H2O2 reversibly oxidizes thiol groups of cysteine residues to mediate intracellular signaling. While examples of H2O2-dependent signaling have been reported, the exact molecular mechanism(s) of signaling and the pathways affected are not well understood. Here, the transcriptomic response of Jurkat T cells to H2O2 was investigated to determine global effects on gene expression. With a low H2O2 concentration (10 µM) that did not induce an oxidative stress response or cell death, extensive changes in gene expression occurred after 4 h (6803 differentially expressed genes). Of the genes with a greater then 2-fold change in expression, 85% were upregulated suggesting that in a physiological setting H2O2 predominantly activates gene expression. Pathway analysis identified gene expression signatures associated with FOXO and NTRK signaling. These signatures were associated with an overlapping set of transcriptional regulators. Overall, our results provide a snapshot of gene expression changes in response to H2O2, which, along with further studies, will lead to new insights into the specific pathways that are activated in response to endogenous production of H2O2, and the molecular mechanisms of H2O2 signaling.

Plasma membrane anchored nanosensor for quantifying endogenous production of H2O2 in living cells

Hydrogen peroxide (H2O2) is one of the main second messengers involved in signaling pathways controlling cell metabolism. During tumorigenesis H2O2 is generated on the extracellular space by membrane-associated NADPH oxidases and superoxide dismutase to stimulate cell proliferation and preservation of the transformed state. Accordingly, a characteristic feature of malignant cells is overproduction of H2O2 in the extracellular milieu and the subsequent absorption in the cytosol. Since the most significant gradients of endogenous extracellular H2O2 can be observed only in a very shallow region of the fluid in contact with the plasma membrane, we show here the use of a newly designed nanosensor anchored to the outer cell surface and capable of quantifying H2O2 at nanometer distance from the membrane proteins responsible for its production. This biosensor is built upon gold nanoparticles functionalized with a H2O2-sensitive boronate compound that is probed using surface enhanced Raman spectroscopy (SERS). The highly localized information obtained on the cell surface by SERS analysis is combined with analytical methods of redox biology to estimate the associated levels of intracellular H2O2 responsible for cell signaling. The results obtained from A549 lung cancer cell line show localized spots on the cell surface at concentration up to 12 μM, associated to intracellular concentration up to 5.1 nM.

Designing an ESIPT-based fluorescent probe for imaging of hydrogen peroxide during the ferroptosis process

Hydrogen peroxide (H2O2), depending on its levels, plays a crucial role in either modulating various biological processes as a signal molecule, or mediating oxidative damage as a toxin. Therefore, monitoring intracellular H2O2 levels is pivotal for exploring its physiological and pathological roles. Using a modified 2-(2′-hydroxyphenyl) benzothiazole (HBT) as the fluorophore, and a pinacol phenylborate ester as the responsive group, herein we developed an excited-state intramolecular proton transfer (ESIPT)-based probe BTFMB. The probe exhibited turn-on fluorescence response, large Stokes shift (162 nm) and low detection limit (109 nM) toward H2O2, and was successfully applied for monitoring exogenous and endogenous production of H2O2, and identifying accumulation of H2O2 during the ferroptosis process.

Gold nanoparticles doped metal-organic frameworks as near-infrared light-enhanced cascade nanozyme against hypoxic tumors

We report gold nanoparticles (AuNPs) doped iron-based metal-organic frameworks (GIM) which displays near-infrared light (NIR)-enhanced cascade nanozyme against hypoxic tumors. Due to the strong protein adsorption-induced surface passivation, AuNPs suffer from the loss of glucose oxidase (GOx) activity. However, GIM could protect the GOx-like activity of AuNPs with the satisfactory shield capability. In addition, GIM exhibited excellent photothermal conversion ability and unique NIR light-enhanced GOx-like activity, which could efficiently increase the endogenous H2O2 production. Meanwhile, as the produced H2O2 is converted by GIM into O2 and highly toxic OH. Thus, GIM-catalyzed cascade reactions with NIR light irradiation not only offer the O2 but also promote the reactive oxygen species (ROS) generation at tumor sites. The produced O2 could be further applied to AuNPs catalytic oxidation of glucose and relieve hypoxic condition of tumor microenvironment (TME). As a proof-of-concept study, GIM demonstrates the admirable tumor ablation under NIR irradiation in vivo.

Surplus spermine does not protect salmon cells against lipopolysaccharide (LPS)‐induced stress, but excess spermine is eliminated through spermidine/spermine N1‐acetyltransferase (SSAT)

The present trials tested the efficiency of surplus spermine to reduce inflammation and oxidative stress following LPS‐induced stress using an in vitro model of head kidney and liver cells isolated from Atlantic salmon. Spermine did not protect cells from LPS‐induced inflammatory response at either 0.3, 0.6 or 0.9 mM. However, as the gene expression of spermidine/spermine N1‐acetyltransferase (SSAT) increased with increasing spermine concentration, we addressed possible oxidative effects of the increased SSAT using its activator DENSPM or inhibitor of polyamine oxidation of the acetylated polyamines using MDL72527 at a spermine concentration of 0.6 mM. There was no significant effect of DENSPM, but MDL72527 decreased gene expression of GPX‐3 (p = .04), while gene expression of catalase and MnSOD was unaffected by treatment (p = .30 and p = .48, respectively). In conclusion, spermine did not protect cells from LPS‐provoked inflammation. The higher the spermine concentration, the more SSAT producing acetylated spermine occurred. Inhibiting the acetylated polyamine oxidases by MDL72527 improved oxidation status as expected due to a lower endogenous production of H₂O₂ by polyamine and acetylated polyamine oxidases. Probably care should be taken using polyamines or arginine as functional ingredients to avoid any increased oxidation within cells.

A Simple and Novel Colorimetric Optimized Assay for Determination of Hydrogen Peroxide Concentration Under Oxidative Stress Conditions

Background: Reactive oxygen species are formed through the electron transfer reactions in the mitochondria and chloroplasts and rapidly converted to H2O2.Therefore, H2O2 as a more stable ROS can be considered as an indicator of cellular stress and it can be used in a steady state to monitor intracellular stress level. In this regard, the increasing use of various nanoparticles, most of which are associated with biological systems, are essential to be studied for their possible adverse effects. We measured the concentration of hydrogen peroxide in samples collected before and after the treatment with silver nanoparticles by a novel method and optimized this method for the living tissue. Methods: In this study, we evaluated the endogenous H2O2 production from Pyricularia oryzae tissue under normal and stress conditions (such as after treatment with nanoparticles) by spectrophotometric assay. The method used is based on instant reaction of hydrogen peroxide with vanadium pentoxide in sulfuric acid solution, forming a peroxovanadate complex that has a maximum absorption at 454 nm. This method was also compared with other methods. Results: The results of this method compared with other methods in the same tissue showed that the method is simple, inexpensive and more efficient, and the complex is stable for several hours and can be used for a variety of H2O2 concentrations. Also, the detection range of the mentioned method equals with high-sensitivity methods such as available commercial kits. Furthermore, this method can also measure higher values of H2O2. Conclusion: The optimized methods for measuring the H2O2 concentration with vanadium pentoxide in sulfuric acid solution by the colorimetric method are simple, efficient, rapid, accurate, cost-effective and do not have problems of other methods. The measurements using this method in Pyricularia oryzae under oxidative stress showed that the created oxidative stress caused by the use of silver nanoparticles increased H2O2 in fungal tissue. H2O2 is the SOD reaction product that is later decomposed by CAT. This method is able to measure H2O2 in different ranges and under normal and stress conditions which are indicative of antioxidant defense. Therefore, we recommend it to the researchers in similar conditions.

Synthesis and application of a ratiometric probe for hydrogen peroxide.

Molecular imaging of biological analytes provides detailed insights into signaling processes. Ratiometric probes are particularly attractive due to the ability to quantify analyte production. However, design strategies for ratiometric probes can be hindered by spectral overlap of the product and reactant species. In this chapter, we provide protocols for the synthesis and application of RF620, a ratiometric probe for H2O2 displaying dramatic changes in both excitation and emission wavelengths, designed using an approach we term chemoselective alteration of fluorophore scaffolds. The probe contains a chemoselective functional group within a fluorescent xanthene scaffold, resulting in the in situ synthesis of a new fluorophore upon reaction with H2O2. Under physiological conditions, RF620 exhibits far-red to near-infrared excitation and emission, and upon reaction with H2O2, RF620 is chemically converted into tetramethylrhodamine, producing a significant (~66nm) blue-shift in excitation and emission. RF620 can be used for ratiometric, molecular imaging of endogenous H2O2 production in living cells.

A novel fluorescent glycopolymer for endogenous hydrogen peroxide imaging in living cells in a fully aqueous environment

The development of H2O2-responsive fluorescent glycopolymers with high specificity, sensitivity, and water solubility is a promising strategy for developing probes to detect the production and delivery of endogenous H2O2. Herein, two series of fluorescent glycopolymers (boronic acid derivatives, PG-PFE, and borate derivatives, PG-PFW) were synthesized through esterification and the Williamson reaction, respectively. To increase the sensitivity of the fluorescent glycopolymers, the effect of the reaction conditions on the grafting ratio was investigated in detail. Compared with the PG-PFE samples, the PG-PFW samples had a higher grafting ratio due to the highly efficient Williamson reaction. The structure and properties of the fluorescent glycopolymers were characterized by using nuclear magnetic resonance spectroscopy, gel permeation chromatography, ultraviolet spectroscopy, and photoluminescent spectroscopy. The obtained results showed that all of these fluorescent glycopolymers had good water solubility, low cytotoxicity, and selective responsiveness toward H2O2. HPG-PFW-5, with the highest grafting ratio, displayed a higher sensitivity toward H2O2 and higher stability during hydrolysis than the other materials. In confocal laser scanning microscope images, all of the fluorescent glycopolymers were observed only in cellular mitochondria, and HPG-PFW-5 exhibited a high degree of overlap with Mitotracker Red because of its high sensitivity. Two series of fluorescent glycopolymers (boronic acid derivatives, PG-PFE, and borate derivatives, PG-PFW) were synthesized through different methods. Compared with the PG-PFE samples, the PG-PFW samples had a higher grafting ratio due to the highly efficient Williamson reaction. The fluorescent glycopolymers possessed good water solubility, low cytotoxicity and selective responsiveness toward H2O2. Meantime, the fluorescent glycopolymers were imaged only in cellular mitochondria based on the fact that endogenous H2O2 is mainly distributed in cellular mitochondria.

Oligomers of carrageenan regulate functional activities and artemisinin production in Artemisia annua L. exposed to arsenic stress.

Recently, a promising technique has come forward in field of radiation-agriculture in which the natural polysaccharides are modified into useful oligomers after depolymerization. Ionizing radiation technology is a simple, pioneering, eco-friendly, and single step degradation process which is used in exploiting the efficiency of the natural polysaccharides as plant growth promoters. Arsenic (As) is a noxious and toxic to growth and development of medicinal plants. Artemisinin is obtained from the leaves of Artemisia annua L., which is effective in the treatment of malaria. The present study was undertaken to find out possible role of oligomers of irradiated carrageenan (IC) on two varieties viz. 'CIM-Arogya' (As-tolerant) and 'Jeevan Raksha' (As-sensitive) of A. annua exposed to As. The treatments applied were 0 (control), 40 IC (40mgL-1 IC), 80 IC (80mgL-1 IC), 45 As (45mgkg-1 soil As), 40 IC + 45 As (40mgL-1 IC + 45mgkg-1 soil As), and 80 IC + 45 As (80mgL-1 IC + 45mgkg-1 soil As). The present study was based on various parameters namely plant fresh weight (FW), dry weight (DW), leaf area index (LAI), leaf yield (LY), chlorophyll and carotenoid content, net photosynthetic rate (PN), stomatal conductance (Gs), carbonic anhydrase activity (CA), proline content (PRO), lipid peroxidation (TBARS), endogenous ROS production (H2O2 content), catalase activity (CAT), peroxidase activity (POX), superoxide dismutase activity (SOD), ascorbate peroxidase activity (APX), As content, and artemisinin content in leaves. Plant growth and other physiological and biochemical parameters including enzymatic activities, photosynthetic activity, and its related pigments were negatively affected under As stress. Leaf-applied IC overcame oxidative stress generated due to As in plants by activating antioxidant machinery. Interestingly, leaf-applied IC enhanced the production (content and yield) of artemisinin under high As stress regardless of varieties. The oligomers of IC and As were found to be responsible for the production of endogenous H2O2 which has a pivotal role in the biosynthesis of artemisinin in A. annua.

Caloric restriction induces H2O2 formation as a trigger of AMPK-eNOS-NO pathway in obese rats: Role for CAMKII

Caloric restriction (CR) improves endothelial function through the upregulation of adenosine monophosphate-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Moreover, hydrogen peroxide (H2O2) is upregulated in yeast subjected to CR. Our aim was to assess if mild short-term CR increases vascular H2O2 formation as a link with AMPK and eNOS activation.Twelve-week old Zucker obese (fa/fa) and control Zucker lean male rats were fed a standard chow either ad libitum (AL, n=10) or with a 20% CR (CR, n=10) for two weeks. CR significantly improved relaxation to ACh in fa/fa rats because of an enhanced endogenous production of H2O2 in aortic rings (H2O2 levels fa/faAL=0.5 ± 0.05 nmol/mg vs. H2O2 levels fa/faCR=0.76 ± 0.07 nmol/mg protein; p<0.05). Expression of mitochondrial superoxide dismutase (Mn-SOD) and total SOD activity were increased in aorta from fa/fa animals after CR. In cultured aortic endothelial cells, serum deprivation or 2-deoxy-d-glucose induced a significant increase in: i) superoxide anion and H2O2 levels, ii) p-AMPK/AMPK and p-eNOS/eNOS expression and iii) nitric oxide levels. This effect was reduced by catalase and strongly inhibited by Ca2+/calmodulin-dependent kinase II (CamkII) silencing.In conclusion, we propose that mild short-term CR might be a trigger of mechanisms aimed at protecting the vascular wall by the increase of H2O2, which then activates AMPK and nitric oxide release, thus improving endothelium-dependent relaxation. In addition, we demonstrate that CAMKII plays a key role in mediating CR-induced AMPK activation through H2O2 increase.

Real-time quantification of hydrogen peroxide production in living cells using NiCo2S4@CoS2 heterostructure

The real-time monitoring of hydrogen peroxide (H2O2) production in living cells is of paramount importance; however, the high complexity of biological fluids and low concentrations of in-vivo H2O2 are limiting the sensor’s sensitivity and selectivity. Here, we described a carbon cloth (CC) modified nickel cobalt sulfide/cobalt sulfide nanostructured arrays (NiCo2S4@CoS2 NAs) as a highly sensitive and selective electrochemical transducer for tracking endogenous H2O2 production in real-time. A hydrothermal synthetical procedure was adopted to deposit the nanostructured arrays. NiCo2S4, an important class of binary transition metal dichalcogenide holds outstanding electrocatalytic activity thanks to its redox properties, enlarged electrocatalytic sites due to mixed valence states, and high conductivity. The growth of NiCo2S4 in association with cobalt disulfide CoS2 on a 3D conducting backbone leads to the nanostructured arrays. The structure, size of the particles, crystallinity, and composition of the arrays were analyzed by the state-of-the-art high-vacuum surface techniques. The NiCo2S4@CoS2 NAs@CC was found to have excellent electrocatalytic performance to reduce H2O2. The electrode displayed a rapid, sensitive, and durable amperometric sensing characteristics, over a linear range of 12.64 nM–2104 μM. The detection limit was 2 nM (S/N = 3). The sensor was able to quantify the amount of endogenously produced H2O2 released from Raw 264.7 cells. Thus, the binary TMDs based nanostructures have promising potential in live cell biosensing applications.

Exogenous GABA enhances muskmelon tolerance to salinity-alkalinity stress by regulating redox balance and chlorophyll biosynthesis

BackgroundSalinity-alkalinity stress is one of the major abiotic stresses affecting plant growth and development. γ-Aminobutyrate (GABA) is a non-protein amino acid that functions in stress tolerance. However, the interactions between cellular redox signaling and chlorophyll (Chl) metabolism involved in GABA-induced salinity-alkalinity stress tolerance in plants remains largely unknown. Here, we investigated the role of GABA in perceiving and regulating chlorophyll biosynthesis and oxidative stress induced by salinity-alkalinity stress in muskmelon leaves. We also evaluated the effects of hydrogen peroxide (H2O2), glutathione (GSH), and ascorbate (AsA) on GABA-induced salinity-alkalinity stress tolerance.ResultsSalinity-alkalinity stress increased malondialdehyde (MDA) content, relative electrical conductivity (REC), and the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR). Salinity-alkalinity stress decreased shoot dry and fresh weight and leaf area, reduced glutathione and ascorbate (GSH and AsA) contents, activities of glutathione reductase (GR) and monodehydroascorbate reductase (MDAR). By contrast, pretreatment with GABA, H2O2, GSH, or AsA significantly inhibited these salinity-alkalinity stress-induced effects. The ability of GABA to relieve salinity-alkalinity stress was significantly reduced when the production of endogenous H2O2 was inhibited, but was not affected by inhibiting endogenous AsA and GSH production. Exogenous GABA induced respiratory burst oxidase homologue D (RBOHD) genes expression and H2O2 accumulation under normal conditions but reduced the H2O2 content under salinity-alkalinity stress. Salinity-alkalinity stress increased the accumulation of the chlorophyll synthesis precursors glutamate (Glu), δ-aminolevulinic acid (ALA), porphobilinogen (PBG), uroporphyrinogen III (URO III), Mg-protoporphyrin IX (Mg-proto IX), protoporphyrin IX (Proto IX), protochlorophyll (Pchl), thereby increasing the Chl content. Under salinity-alkalinity stress, exogenous GABA increased ALA content, but reduced the contents of Glu, PBG, URO III, Mg-proto IX, Proto IX, Pchl, and Chl. However, salinity-alkalinity stress or GABA treated plant genes expression involved in Chl synthesis had no consistent trends with Chl precursor contents.ConclusionsExogenous GABA elevated H2O2 may act as a signal molecule, while AsA and GSH function as antioxidants, in GABA-induced salinity-alkalinity tolerance. These factors maintain membrane integrity which was essential for the ordered chlorophyll biosynthesis. Pretreatment with exogenous GABA mitigated salinity-alkalinity stress caused excessive accumulation of Chl and its precursors, to avoid photooxidation injury.

Hydrogen peroxide facilitates Arabidopsis seedling establishment by interacting with light signalling pathway in the dark.

Light is essential for the plant establishment. Arabidopsis seedlings germinated in the dark cannot grow leaf and only have closed cotyledons. However, exogenous application of H2 O2 can induce leaves (establishment) in the dark. Comparative transcriptomic analysis revealed that light-responsive genes were activated by H2 O2 treatment. These genes are functionally correlated with photosynthesis, photorespiration, and components of photosystem, such as antenna proteins and light-harvesting chlorophyll proteins. We further found that application of H2 O2 facilitates cell cycle by accelerating G2 -M checkpoint transition in shoot apical meristem. Phytochrome-mediated light signalling pathway was also involved in the H2 O2 -facilitated establishment process. The constitutive photomorphogenesis 1 and phytochrome interacting factor 3 proteins were shown to be down-regulated by H2 O2 treatment and accordingly removed their inhibitory effects on photomorphogenesis in the dark. The crosstalk between oxidation and light signal pathways explains the mechanism that H2 O2 regulates plant dark establishment. The endogenous photorespiratory H2 O2 production was mimicked by overexpression of glycolate oxidase genes and supplement of substrate glycolate. As expected, seedling establishment was also induced by the endogenously produced H2 O2 under dark condition. These findings also suggest that photorespiratory H2 O2 production is at least partially involved in postgermination establishment.