High-dose ascorbic acid induces carcinostatic effects through hydrogen peroxide and superoxide anion radical generation-induced cell death and growth arrest in human tongue carcinoma cells

High-dose ascorbic acid (AsA) treatment, known as pharmacological AsA, has been shown to exert carcinostatic effects in many types of cancer cells and in vivo tumour models. Although pharmacological AsA has potential as a complementary and alternative medicine for anticancer treatment, its effects on human tongue carcinoma have not yet been elucidated. In this study, we investigated the effect of AsA treatment on human tongue carcinoma HSC-4 cells compared with non-tumourigenic tongue epithelial dysplastic oral keratinocyte (DOK) cells. Our results show that treatment with 1 and 3?mM of AsA for 60?min preferentially inhibits the growth of human tongue carcinoma HSC-4 over DOK cells. Furthermore, AsA-induced effects were accompanied by increased intracellular oxidative stress and were repressed by treatment with a hydrogen peroxide (H₂O₂) scavenger catalase and a superoxide anion radical (O₂^?) scavenger, tempol. Time-lapse observation and thymidine analog EdU incorporation revealed that AsA treatment induces not only cell death but also suppression of DNA synthesis and cell growth. Moreover, the growth arrest was accompanied by abnormal cellular morphologies whereby cells extended dendrite-like pseudopodia. Taken together, our results demonstrate that AsA treatment can induce carcinostatic effects through induction of cell death, growth arrest, and morphological changes mediated by H₂O₂ and O₂^? generation. These findings suggest that high-dose AsA treatment represents an effective treatment for tongue cancer as well as for other types of cancer cells.     

The role of insulin against hydrogen peroxide-induced oxidative damages in differentiated SH-SY5Y cells

Abstract

Exogenous hydrogen peroxide (H₂O₂) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H₂O₂-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H₂O₂ for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 24?h of 100?µM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca²⁺) in neuronal cells, but insulin can effectively diminish the H₂O₂-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H₂O₂-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H₂O₂-induced oxidative stress related to the Akt/Bcl-2 pathways.     

Hydrogen peroxide enhanced Ca(2+)-activated BK currents and promoted cell injury in human dermal fibroblasts

AimsRecent studies have shown that dermal fibroblasts possess multiple types of voltage-dependent K⁺ channels, and the activation of these channels induces apoptosis. In the present study, we aimed to investigate whether hydrogen peroxide (H₂O₂), an oxidative stress inducer, could modulate these channels or induce human dermal fibroblasts injury.Main methodsThe effects of H₂O₂ on K⁺ currents were studied using a whole-cell recording. Intracellular PKC levels were measured with a direct human PKC enzyme immunoassay kit. Cell viability was assessed using PI staining and apoptotic nuclei were detected with TdT-mediated digoxigenin-dUTP nick-end labelling assay (TUNEL) assay.Key findingsTreatment of cells with 100 μM H₂O₂ resulted in a partially reversible increase in non-inactivating outward K⁺ currents and an alteration in the steady-state activation property of the channels. The H₂O₂-induced increase in K⁺ currents was mimicked by a PKC activator, and was blocked by the PKC inhibitor or the large conductance Ca²⁺-activited K⁺ (BK) channel blockers. The intracellular PKC levels were significantly enhanced by H₂O₂ treatment in a concentration-dependent manner. After exposure to H₂O₂, evaluation of fibroblasts survival rate and damaged cell number with TUNEL-positive nuclei revealed an increased cell injury. Blocking the K⁺ channels with blockers significantly decreased the H₂O₂-induced human dermal fibroblasts injury.SignificanceOur results revealed that H₂O₂ could enhance BK currents by PKC pathway. Increased K⁺ currents might be related to H₂O₂-induced human dermal fibroblasts injury. The results reported here contribute to our understanding of the mechanism underlying H₂O₂-induced human dermal fibroblasts injury.     

Light-induced intracellular hydrogen peroxide generation through genetically encoded photosensitizer KillerRed-SOD1

Abstract

Hydrogen peroxide (H₂O₂) plays an important role in various biological processes in numerous organisms. Depending on the concentration and the distribution within the cell, it can act as stressor or redox signalling molecule. To analyse the effects of H₂O₂ and its diffusion within the cell we developed the new genetically encoded photosensitizer KillerRed-SOD1 which enables a light-induced spatially and temporally controlled generation of H₂O₂ in living cells. The KillerRed-SOD1 is a fusion protein of the photosensitizer KillerRed (KR) and the cytosolic superoxide dismutase isoform 1 (SOD1) connected by a helix-forming peptide linker. Light irradiation at a wavelength of 560?nm induced superoxide radical formation at the KR domain which was transformed to H₂O₂ at the SOD1 domain. H₂O₂ was specifically detected under live cell conditions using the fluorescent sensor protein HyPer. Genetically encoded photosensitizers have the advantage that appropriate tag sequences can determine the localisation of the protein within the cell. Herein, it was exemplarily shown that the peroxisomal targeting sequence 1 directed the photosensitizer KR-SOD1 to the peroxisomes and enabled H₂O₂ formation specifically in these organelles. In summary, with the photosensitizer KR-SOD1 a new valuable tool was established which allows a controlled intracellular H₂O₂ generation for the analysis of H₂O₂ effects on a subcellular level.     

The ginsenoside protopanaxatriol protects endothelial cells from hydrogen peroxide-induced cell injury and cell death by modulating intracellular redox status

Ginsenosides, the active components of the famous Chinese herb ginseng, have been suggested to possess cardiovascular-protective effects. The mechanism of ginsenosides is believed to be associated with their ability to prevent cellular oxidative stress. The purpose of this study was to explore the cytoprotective effects of the ginsenoside protopanaxatriol (PPT) on hydrogen peroxide (H₂O₂)-induced endothelial cell injury and cell death. Pretreatment of human umbilical vein endothelial cells (HUVECs) with PPT for 24 h was able to protect the cells against H₂O₂-induced injury. In addition to cell death, pretreatment with PPT could also reduce H₂O₂-induced DNA damage, overactivation of the DNA repair enzyme PARP-1, and concomitant depletion of the intracellular substrate NAD⁺. Furthermore, PPT could reverse the decrease in ATP/ADP ratio caused by H₂O₂. The metabolism of glutathione was also changed. H₂O₂ could induce a significant decrease in GSH level resulting in a decrease in the GSH/GSSG ratio. This could be prevented by pretreatment with PPT. The action was associated with increasing activities of the GSH-metabolizing enzymes glutathione reductase and glutathione peroxidase. These findings suggest that the ginsenoside PPT could protect HUVECs against H₂O₂-induced cell death via its action against oxidative stress, which may be responsible for the cardiovascular-protective action of ginseng.     

Dosage effects of EGb761 on hydrogen peroxide-induced cell death in SH-SY5Y cells

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, is one of the most popular herbal supplements, taken for its multivalent properties. In this study, dosage effects of EGb761 on hydrogen peroxide (H₂O₂)-induced apoptosis of human neuroblastoma SH-SY5Y cells were investigated. It was found that H₂O₂-induced apoptotic cell death in SH-SY5Y cells, which was revealed in DNA fragmentation, mitochondrial membrane potential depolarization, and activation of Akt, c-Jun N-terminal kinases (JNK) and caspase 3. Low doses of EGb761 (50–100 μg/ml) inhibited H₂O₂-induced cell apoptosis via inactivation of Akt, JNK and caspase 3 while high doses of EGb761 (250–500 μg/ml) enhanced H₂O₂ toxicities via inactivation of Akt and enhancement of activation of JNK and caspase 3. Additional experiments revealed that H₂O₂ decreased intracellular GSH content, which was also inhibited by low concentrations of EGb761 but enhanced after high concentrations of EGb761 treatment. This further suggests to us that dosage effects of EGb761 on apoptotic signaling proteins may be correlated with regulation of cell redox state. Therefore, treatment dosage may be one of the vital factors that determine the specific action of EGb761 on oxidative stress-induced cell apoptosis. To understand the mechanisms of dosage effects of EGb761 may have important clinical implications.     

Hyaluronic acid optimises therapeutic effects of hydrogen peroxide‐induced oxidative stress on breast cancer

Distinguishing the multiple effects of reactive oxygen species (ROS) on cancer cells is important to understand their role in tumour biology. On one side, ROS can be oncogenic by promoting hypoxic conditions, genomic instability and tumorigenesis. Conversely, elevated levels of ROS‐induced oxidative stress can induce cancer cell death. This is evidenced by the conflicting results of research using antioxidant therapy, which in some cases promoted tumour growth and metastasis. However, some antioxidative or ROS‐mediated oxidative therapies have also yielded beneficial effects. To better define the effects of oxidative stress, in vitro experiments were conducted on 4T1 and splenic mononuclear cells (MNCs) under hypoxic and normoxic conditions. Furthermore, hydrogen peroxide (H₂O₂; 10–1,000 μM) was used as an ROS source alone or in combination with hyaluronic acid (HA), which is frequently used as drug delivery vehicle. Our result indicated that the treatment of cancer cells with H₂O₂ + HA was significantly more effective than H₂O₂ alone. In addition, treatment with H₂O₂ + HA led to increased apoptosis, decreased proliferation, and multiphase cell cycle arrest in 4T1 cells in a dose‐dependent manner under normoxic or hypoxic conditions. As a result, migratory tendency and the messenger RNA levels of vascular endothelial growth factor, matrix metalloproteinase‐2 (MMP‐2), and MMP‐9 were significantly decreased in 4T1 cells. Of note, HA treatment combined with 100–1,000 μM H₂O₂ caused more damage to MNCs as compared to treatment with lower concentrations (10–50 μM). Based on these results, we propose to administer high‐dose H₂O₂ + HA (100–1000 μM) for intratumoural injection and low doses for systemic administration. Intratumoural route could have toxic and inhibitory effects not only on the tumour but also on residential myeloid cells defending it, whereas systemic treatment could stimulate peripheral immune responses against the tumour. More in vivo research is required to confirm this hypothesis.     

The actions of leptin on survival and hydrogen peroxide toxicity in primary mixed glial cells of rat

Studies indicate that leptin is involved in not only energy expenditure and food intake, but also in protection against apoptosis, in inflammation and in stimulation of proliferation in many cell types. However, leptin treatment increases the oxidative stress in many cell culture studies. This contradiction evoked a question of whether leptin acts as an oxidant or antioxidant on glial cells. We investigated the effect of leptin on glial cell survival and hydrogen peroxide (H₂O₂)-induced toxicity in vitro. The survival rate of the cells was determined by using 3-(4,5-D-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, thyazolyl blue (MTT) method. The cells obtained from the whole brain of 1–3 day-old rat were treated with 1, 10, 100 and 1000 ng/mL leptin for 24 or 72 h. Either the pretreatment of leptin alone for 5 h or leptin combined simultaneously with H₂O₂ or well known antioxidant glutathione (GSH) were applied to the cells. Malondialdehyde (MDA) levels were measured in cell lysates to which leptin was added for 24 h. The 100 and 1000 ng/mL leptin treatment for 72 h increased the glial viability by 19% and 36%, respectively. The dose of H₂O₂ that killed 75% of the cells was determined as 100 μM. GSH at different doses was applied as a positive control to the cells and the dose of 500 μM completely eliminated toxic effect of 100 μM H₂O₂. Either the pretreatment of leptin alone for 5 h or leptin combined simultaneously with H₂O₂ could not eliminate H₂O₂-caused toxicity. Furthermore, respective leptin doses did not change the glia MDA level. We suggest that leptin can increase glia survival dose dependently, but can not eliminate H₂O₂-induced oxidation in primary mixed glial cell culture.     

Alterations in mRNA expression of ribosomal protein S9 in hydrogen peroxide-treated neurotumor cells and in rat hippocampus after transient ischemia

This study was designed to isolate new genes related to apoptosis in rat pheochromocytoma (PC12) cells treated with hydrogen peroxide (H₂O₂), and to characterize the roles of the genes using both in vitro and in vivo models of oxidative injury. cDNA libraries were prepared from H₂O₂-treated and -untreated PC12 cells, and a ribosomal protein S9 (RPS9) clone was isolated by a differential screening method. Increase of RPS9 expression in both H₂O₂-treated PC12 and neuroblastoma (Neuro-2A) cells was shown by Northern blot analysis. Viability of the antisense-transfected Neuro-2A (RPS9-AS) cells following H₂O₂ treatment was significantly reduced in a dose-dependent manner. In an in vivo model of transient forebrain ischemia, an increase in RPS9 expression was prominent by 1 day postischemia in the granule cell layer neurons of the dentate gyrus. Both activation of caspase-3 and significant recovery of viability following pretreatment with cycloheximide were shown in RPS9-AS cells treated with H₂O₂. These data suggest that RPS9 plays a protective role in oxidative injury of neuronal cells.     

Antisteroidogenic effects of hydrogen peroxide on rat granulosa cells

Abstract

Rat granulosa cells (GCs) were treated with human chorionic gonadotropin (hCG), 8-bromo-adenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), forskolin, phorbol 12-myristate 13-acetate (PMA), A23187 or pregnenolone in the absence or presence of hydrogen peroxide (H₂O₂). Different doses of trilostane were applied to GCs treated with steroidogenic precursors, that is, 25-hydroxy-cholesterol (25-OH-C) in the absence or presence of H₂O₂. Results showed that all of the chemicals stimulated the progesterone (PG) release from rat GCs, but the stimulatory effects were inhibited by H₂O₂ dose-dependently. 25-OH-C stimulated the PG release, which was inhibited by H₂O₂ in the presence of trilostane. H₂O₂ attenuated steroidogenic acute regulatory (StAR) protein expression, but did not alter the expression of cytochrome P450 side chain cleavage (P450scc) in Western blotting. This study indicated that H₂O₂ inhibited PG production by GCs via cAMP pathway, protein kinase C (PKC) and the activities of intracellular calcium, P450scc and StAR protein.     

Long‐term hydrogen peroxide exposure potentiates anoikis resistance and anchorage‐independent growth in lung carcinoma cells

Hydrogen peroxide (H₂O₂) is upregulated in tumour microenvironments and may contribute to effects on metastatic cancer cells. This study demonstrates that treatment of lung carcinoma and melanoma cells with H₂O₂ for 14 days results in an induction of anoikis resistance and growth in an anchorage‐independent condition. H₂O₂ exposure increased the Cav‐1 (caveolin‐1) level through an increase of Cav‐1 mRNA with minimal effect on protein degradation. Upregulation of Cav‐1 induced anoikis resistance and facilitated growth in a detached manner. The findings show a novel role of hydrogen peroxide in the regulation of metastatic potential of cancer cells.     

Interaction of intracellular hydrogen peroxide accumulation with nitric oxide production in abscisic acid signaling in guard cells

ABSTRACT

Reactive oxygen species and nitric oxide (NO?) concomitantly play essential roles in guard cell signaling. Studies using catalase mutants have revealed that the inducible and constitutive elevations of intracellular hydrogen peroxide (H₂O₂) have different roles: only the inducible H₂O₂ production transduces the abscisic acid (ABA) signal leading stomatal closure. However, the involvement of inducible or constitutive NO? productions, if exists, in this process remains unknown. We studied H₂O₂ and NO? mobilization in guard cells of catalase mutants. Constitutive H₂O₂ level was higher in the mutants than that in wild type, but constitutive NO? level was not different among lines. Induced NO? and H₂O₂ levels elicited by ABA showed a high correlation with each other in all lines. Furthermore, NO? levels increased by exogenous H₂O₂ also showed a high correlation with stomatal aperture size. Our results demonstrate that ABA-induced intracellular H₂O₂ accumulation triggers NO? production leading stomatal closure.     

Elevated exhalation of hydrogen peroxide in patients with non-small cell lung cancer is not affected by chemotherapy

Objectives: Reactive oxygen species, which are implicated in the process of carcinogenesis, are also responsible for cell death during chemotherapy (CHT). Therefore, the aim of the study was to evaluate exhaled H₂O₂ levels in non-small cell lung cancer (NSCLC) patients before and after CHT.

Methods: Thirty patients (age 61.3?±?9.3 years) with advanced NSCLC (stage IIIB–IV) and 15 age-matched healthy cigarette smokers were enrolled into the study. Patients received four cycles of cisplatin or carboplatin with vinorelbine every three weeks. Before and after the first, second, and fourth cycle, the concentration of H₂O₂ in exhaled breath condensate was measured with respect to treatment response.

Results: At the baseline, NSCLC patients exhaled 3.8 times more H₂O₂ than the control group (0.49?±?0.14 vs. 0.13?±?0.03?µmol/L, P?2O₂ levels independent of the treatment response (partial remission vs. progressive disease). Pre- and post-CHT cycles of H₂O₂ levels generally correlated positively.

Discussion: The study demonstrated the occurrence of oxidative stress in the airways of advanced NSCLC patients. Exhaled H₂O₂ level was not affected by CHT and independent of treatment results and changes in the number of circulating neutrophils.     

Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Hydrogen peroxide (H₂O₂) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H₂O₂ requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H₂O₂ in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H₂O₂, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H₂O₂. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H₂O₂-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H₂O₂ in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H₂O₂ can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H₂O₂, they cannot be used to monitor transient changes in H₂O₂ concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H₂O₂ and can be targeted to various compartments of the cell, but they are not selective for H₂O₂ because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H₂O₂-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.     

What are the sources of hydrogen peroxide production by heart mitochondria?

Coupled rat heart mitochondria produce externally hydrogen peroxide at the rates which correspond to about 0.8 and 0.3% of the total oxygen consumption at State 4 with succinate and glutamate plus malate as the respiratory substrates, respectively. Stimulation of the respiratory activities by ADP (State 4–State 3 transition) decreases the succinate- and glutamate plus malate-supported H₂O₂ production 8- and 1.3-times, respectively. NH₄⁺ strongly stimulates hydrogen peroxide formation with either substrate without any effect on State 4 and/or State 3 respiration. Rotenone-treated, alamethicin-permeabilized mitochondria catalyze NADH-supported H₂O₂ production at a rate about 10-fold higher than that seen in intact mitochondria under optimal (State 4 succinate-supported respiration in the presence of ammonium chloride) conditions. NADH-supported hydrogen peroxide production by the rotenone-treated mitochondria devoid of a permeability barrier for H₂O₂ diffusion by alamethicin treatment are only partially (～ 50%) sensitive to the Complex I NADH binding site-specific inhibitor, NADH-OH. The residual activity is strongly (～ 6-fold) stimulated by ammonium chloride. NAD⁺ inhibits both Complex I-mediated and ammonium-stimulated H₂O₂ production. In the absence of stimulatory ammonium about half of the total NADH-supported hydrogen peroxide production is catalyzed by Complex I. In the presence of ammonium about 90% of the total hydrogen peroxide production is catalyzed by matrix located, ammonium-dependent enzyme(s).     

New Molecular Mechanisms on the Activation of TRPM2 Channels by Oxidative Stress and ADP-Ribose

The Na⁺ and Ca²⁺-permeable melastatin related transient receptor potential (TRPM2) cation channels can be gated either by ADP-ribose (ADPR) in concert with Ca²⁺ or by hydrogen peroxide (H₂O₂), an experimental model for oxidative stress, and binding to the channel’s enzymatic Nudix domain. Since the mechanisms that lead to TRPM2 inhibiting in response to ADPR and H₂O₂ are not understood, I reviewed the effects of various inhibitors such as flufenamic acid and PARP inhibitors on ADPR, NAD⁺ and H₂O₂-induced TRPM2 currents. In our experimental study, TRPM2 cation channels in chinese hamster ovary transected cells were gated both by ADPR and NAD⁺. In addition, H₂O₂ seems to activate TRPM2 by changing to the hydroxyl radical in the intracellular space after passing the plasma membrane. Experimental studies with respect to patch-clamp and Ca²⁺ imaging, inhibitor roles of antioxidants are also summarized in the review.     

Micromolar concentrations of hydrogen peroxide induce oxidative DNA lesions more efficiently than millimolar concentrations in mammalian cells

Reactive oxygen species produce oxidized bases, deoxyribose lesions and DNA strand breaks in mammalian cells. Previously, we demonstrated that aldehydic DNA lesions (ADLs) were induced in mammalian cells by 10 mM hydrogen peroxide (H₂O₂). Interestingly, a bimodal H₂O₂ dose–response relationship in cell toxicity has been reported for Escherichia coli deficient in DNA repair as well as Chinese hamster ovary (CHO) cells. Furthermore, it has been demonstrated that H₂O₂ causes single-strand breaks in purified DNA in the presence of iron and induces mitochondrial DNA damage in CHO cells with a biphasic dose–response curve. Here we show that H₂O₂ produces ADLs at concentrations as low as 0.06 mM in HeLa cells and that lower concentrations of H₂O₂ were much more efficient at inducing ADLs than higher concentrations. This dose–response curve is strikingly similar to that for cell killing effects in E.coli deficient in DNA repair exposed to H₂O₂. Interestingly, serial treatment of submillimolar levels of H₂O₂ induced a massive accumulation of ADLs. The toxicity arising from H₂O₂ determined by intracellular NAD(P)H in cells correlated well with the formation of ADLs. The addition of dipyridyl, an iron (II)-specific chelator, significantly protected against DNA damage and cell toxicity from submillimolar, but not millimolar, amounts of H₂O₂. These results suggest that ADLs induced by submillimolar levels of H₂O₂ may be due to a Fenton-type reaction between H₂O₂ and intracellular iron ions in mammalian cells.     

Aquaporin as a membrane transporter of hydrogen peroxide in plant response to stresses

Hydrogen peroxide (H₂O₂) is a reactive oxygen species that signals between cells, and H₂O₂ signaling is essential for diverse cellular processes, including stress response, defense against pathogens, and the regulation of programmed cell death in plants. Although plasma membrane intrinsic proteins (PIPs) have been known to transport H₂O₂ across cell membranes, the permeability of each family member of PIPs toward H₂O₂ has not yet been determined in most plant species. In a recent study, we showed that certain isoforms of Arabidopsis thaliana AtPIPs, including AtPIP2;2, AtPIP2;4, AtPIP2;5, and AtPIP2;7, are permeable for H₂O₂ in yeast cells. Since the expression of PIPs is differently modulated in Arabidopsis by abiotic stress or H₂O₂ treatment, it is important to investigate the integrated regulation of aquaporin expression and their physiological significance in H₂O₂ transport and plant response to diverse abiotic stresses.     

Insulin on hydrogen peroxide-induced oxidative stress involves ROS/Ca2+ and Akt/Bcl-2 signaling pathways

Abstract

Oxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H₂O₂) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H₂O₂-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H₂O₂ for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H₂O₂ was selected to establish a model of H₂O₂-induced oxidative stress. Further assays showed that 3 h of 500 μM H₂O₂-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca²⁺) in C6 cells, with insulin able to effectively diminish H₂O₂-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H₂O₂-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway.