Hydrogen Peroxide-mediated Oxidative Stress Research Articles

Exosome-Coated tPA/Catalase Nanoformulation for Thrombolytic Therapy

Current tissue plasminogen-based therapeutic strategies for stroke suffer from systemic side effects and poor efficacy. Hence, novel drug delivery methods are needed to overcome these shortcomings. Exosome-based drug formulations have been shown to have superior therapeutic outcomes compared to conventional systemic drug delivery approaches. In this paper, we report exosome surface-coated tissue plasminogen activator (tPA)/catalase nanoformulations with improved thrombolytic efficacy compared to free tPA, which also reduce side effects. The results showed that the tPA exosome formulations retained tPA activity, improved tPA stability, exhibited significant fibrinolysis, and showed no significant toxicity effects. Further, when combined with antioxidant enzyme catalase, the formulation was able to inhibit hydrogen peroxide-mediated oxidative stress and toxicity. Hence, exosome-based tPA/catalase nanoformulations could have the potential to offer a safer and effective thrombolytic therapy.

Gold Nanoparticles Encapsulated Resveratrol as an Anti-Aging Agent to Delay Cataract Development.

Nanoparticle-based drug delivery systems, which can overcome the challenges associated with poor aqueous solubility and other harmful side effects of drugs, display potent applications in cataract treatment. Herein, we designed a nanosystem of gold nanoparticles containing resveratrol (RGNPs) as an anti-aging agent to delay cataracts. The spherical RGNPs had a superior ability to inhibit hydrogen peroxide-mediated oxidative stress damage, including reactive oxygen species (ROS) production, malondialdehyde (MDA) generation, and glutathione (GSH) consumption in the lens epithelial cells. Additionally, the present data showed that RGNPs could delay cellular senescence induced by oxidative stress by decreasing the protein levels of p16 and p21, reducing the ratio of BAX/BCL-2 and the senescence-associated secretory phenotype (SASP) in vitro. Moreover, the RGNPs could also clearly relieve sodium selenite-induced lens opacity in a rat cataract model. Our data indicated that cell senescence was reduced and cataracts were delayed upon treatment with RGNPs through activating the Sirt1/Nrf2 signaling pathway. Our findings suggested that RGNPs could serve as an anti-aging ingredient, highlighting their potential to delay cataract development.

In vitro anticholinesterase potential of some spices consumed in Cameroon and their protective effects on hydrogen peroxide-mediated oxidative stress damage in SK-N-SH cells.

BackgroundMany neurodegenerative such as Alzheimer's disease (AD) are characterized by cholinergic dysfunction and oxidative stress which is a key event in neuronal death process. Thus, anticholinesterase and anti-oxidation compounds are two promising strategies in the development of AD drugs. Beyond their culinary use, spices are today studies for health purpose. In this study, some spices consumed in Cameroon were evaluated for their anticholinesterase and neuroprotective effects.MethodsColorimetric methods were used to determine total flavonoid and alkaloid content of a combinated extract (hydroethanolic + ethanolic extracts) of different selected spices. Aftermaths, anti-cholinesterase activity of spice extract was carried out using Ellman’s method. Finally, neuroprotective effects performed on human SK-N-SH cells stressed with H2O2 by assessing neuronal survival ( resazurin assay) and neuronal death (LDH assay).ResultsFlavonoid content of spices extract were ranged from 22.94 to 32.01 mg EQ/g DM and alkaloid content were ranged from 320 to 896 mg EQu/g DM. Among the spices studied, Xylopia parviflora presented the greatest acetylcholinesterase inhibition with an IC50 = 14 µg/mL. In Cell culture experiments, pre-incubation of SK-N-SH cell with the selected spices at different concentrations were improved neuronal survival and reduced the percentage of neuronal cells dead.ConclusionThe present results reveal that selected spices consumed in Cameroon have good anticholinesterase activity as well as neuroprotective effect on SK-N-SH which may provide new natural compounds that could help in the management of Alzheimer's disease.

Cytoprotective Effects of Punicalagin on Hydrogen-Peroxide-Mediated Oxidative Stress and Mitochondrial Dysfunction in Retinal Pigment Epithelium Cells.

The retinal pigment epithelium (RPE) is a densely pigmented, monostratified epithelium that provides metabolic and functional support to the outer segments of photoreceptors. Endogenous or exogenous oxidative stimuli determine a switch from physiological to pathological conditions, characterized by an increase of intracellular levels of reactive oxygen species (ROS). Accumulating evidence has elucidated that punicalagin (PUN), the major ellagitannin in pomegranate, is a potent antioxidant in several cell types. The present study aimed to investigate the protective effect of PUN on mitochondrial dysfunction associated with hydrogen peroxide (H2O2)–induced oxidative stress. For this purpose, we used a human RPE cell line (ARPE–19) exposed to H2O2 for 24 h. The effects of PUN pre–treatment (24 h) were examined on cell viability, mitochondrial ROS levels, mitochondrial membrane potential, and respiratory chain complexes, then finally on caspase–3 enzymatic activity. The results showed that supplementation with PUN: (a) significantly increased cell viability; (b) kept the mitochondrial membrane potential (ΔΨm) at healthy levels and limited ROS production; (c) preserved the activity of respiratory complexes; (d) reduced caspase–3 activity. In conclusion, due to its activity in helping mitochondrial functions, reducing oxidative stress, and subsequent induction of cellular apoptosis, PUN might be considered a useful nutraceutical agent in the treatment of oxidation–associated disorders of RPE.

The moonlighting peroxiredoxin-glutaredoxin in Neisseria meningitidis binds plasminogen via a C-terminal lysine residue and contributes to survival in a whole blood model

Neisseria meningitidis is a human-restricted bacterium that can invade the bloodstream and cross the blood-brain barrier resulting in life-threatening sepsis and meningitis. Meningococci express a cytoplasmic peroxiredoxin-glutaredoxin (Prx5-Grx) hybrid protein that has also been identified on the bacterial surface. Here, recombinant Prx5-Grx was confirmed as a plasminogen (Plg)-binding protein, in an interaction which could be inhibited by the lysine analogue ε-aminocapronic acid. rPrx5-Grx derivatives bearing a substituted C-terminal lysine residue (rPrx5-GrxK244A), but not the active site cysteine residue (rPrx5-GrxC185A) or the sub-terminal rPrx5-GrxK230A lysine residue, exhibited significantly reduced Plg-binding. The absence of Prx5-Grx did not significantly reduce the ability of whole meningococcal cells to bind Plg, but under hydrogen peroxide-mediated oxidative stress, the N. meningitidis Δpxn5-grx mutant survived significantly better than the wild-type or complemented strains. Significantly, using human whole blood as a model of meningococcal bacteremia, it was found that the N. meningitidis Δpxn5-grx mutant had a survival defect compared with the parental or complemented strain, confirming an important role for Prx5-Grx in meningococcal pathogenesis.

Integrated Analysis of miRNA-mRNA Interaction Network in Porcine Granulosa Cells Undergoing Oxidative Stress.

Oxidative stress (OS), a common intracellular phenomenon induced by excess reactive oxygen species (ROS) generation, has been shown to be associated with mammalian ovarian follicular development blockage and granulosa cell (GC) impairment. However, the mechanism involved in these effects remains unknown, and the effect of OS on the transcriptome profiles in porcine GCs has not been fully characterized. In this study, we found that hydrogen peroxide-mediated oxidative stress induced porcine GC apoptosis and impaired cell viability. Moreover, RNA-seq analysis showed that oxidative stress induced dramatic changes in gene expression in porcine GCs. A total of 2025 differentially expressed genes (DEGs) were identified, including 1940 DEmRNAs and 55 DEmiRNAs. Functional annotation showed that the DEGs were mainly associated with cell states and function regulation. In addition, multiple hub genes (FOXO1, SOD2, BMP2, DICER1, BCL2L11, FZD4, ssc-miR-424, and ssc-miR-27b) were identified by constructing protein-protein interaction and DEmiRNA-DEmRNA regulatory networks. Furthermore, a gene-pathway-function coregulatory network was established and demonstrated that these hub genes were enriched in FoxO, TGF-β, Wnt, PIK3-Akt, MAPK, and cAMP signaling pathways, which play important roles in regulating cell apoptosis, cell proliferation, stress responses, and hormone secretion. The current research provides a comprehensive perspective of the effects of oxidative stress on porcine GCs and also identifies potential therapeutic targets for oxidative stress-induced female infertility.

Hydrogen peroxide-mediated oxidative stress induces inflammasome activation in term human placental explants.

The placenta is a multifunctional organ that can suffer with imbalances between pro- and antioxidant molecules, contributing for inflammatory imbalance. The inflammation generated by oxidative stress may induce inflammasome activation, an essential complex for pro-inflammatory cytokine production. The aim of this study was to evaluate whether hydrogen peroxide (H2O2) mediated oxidative stress induces inflammasome activation on placental explants. Tissue cultures of placental explants obtained from normotensive pregnant women were performed in different concentrations of H2O2. Gene expressions of NLRP3, caspase-1, IL-1β, TNF-α and IL-10 were evaluated by qPCR. Superoxide dismutase (SOD), catalase, Heat shock protein 70 (Hsp70), Caspase-1, TNF-α, IL-1β, IL-10 and human Chorionic Gonadotropin (hCG) were determined by ELISA. Concentrations of catalase, Hsp70, hCG and SOD were higher in cultures with 100 and 1000 µM H2O2 compared to controls. Gene and protein expressions of TNF-α and IL-1β were elevated in cultures with 1000 μM H2O2 compared to controls. This concentration led to inflammasome activation, by increasing gene expressions of NLRP3, caspase-1 and IL-1β. In contrast, gene and protein expressions of IL-10 were reduced at 100 and 1000 μM H2O2. Protein expression of caspase-1 was higher in cultures of 100 μM H2O2 compared to controls. Treatment with Glybenclamide at 200 μM was used to prevent NLRP3 inflammasome activation. This concentration reduced protein expression of caspase-1 compared to culture with only H2O2 and control cultures. Our results confirm that H2O2 induces oxidative stress on placental explants and demonstrate that cell responses to this stress involve inflammasome activation.

Peroxiredoxin System of Aspergillus nidulans Resists Inactivation by High Concentration of Hydrogen Peroxide-Mediated Oxidative Stress.

Most eukaryotic peroxiredoxins (Prxs) are readily inactivated by a high concentration of hydrogen peroxide (H₂O₂) during catalysis owing to their "GGLG" and "YF" motifs. However, such oxidative stress sensitive motifs were not found in the previously identified filamentous fungal Prxs. Additionally, the information on filamentous fungal Prxs is limited and fragmentary. Herein, we cloned and gained insight into Aspergillus nidulans Prx (An.PrxA) in the aspects of protein properties, catalysis characteristics, and especially H₂O₂ tolerability. Our results indicated that An.PrxA belongs to the newly defined family of typical 2-Cys Prxs with a marked characteristic that the "resolving" cysteine (CR) is invertedly located preceding the "peroxidatic" cysteine (CP) in amino acid sequences. The inverted arrangement of CR and CP can only be found among some yeast, bacterial, and filamentous fungal deduced Prxs. The most surprising characteristic of An.PrxA is its extraordinary ability to resist inactivation by extremely high concentrations of H₂O₂, even that approaching 600 mM. By screening the H₂O₂-inactivation effects on the components of Prx systems, including Trx, Trx reductase (TrxR), and Prx, we ultimately determined that it is the robust filamentous fungal TrxR rather than Trx and Prx that is responsible for the extreme H₂O₂ tolerence of the An.PrxA system. This is the first investigation on the effect of the electron donor partner in the H₂O₂ tolerability of the Prx system.

Neuroprotective Effects of Schisandra chinensis and Ribes fasciculatum Extract on Hydrogen Peroxide-Mediated Oxidative Stress in Neuroblastic SH-SY5Y Cell Line

Neuroprotective Effects of Schisandra chinensis and Ribes fasciculatum Extract on Hydrogen Peroxide-Mediated Oxidative Stress in Neuroblastic SH-SY5Y Cell Line

Crystallographic studies of the extracytoplasmic function σ factor σ(J) from Mycobacterium tuberculosis.

Mycobacterium tuberculosis has multiple σ factors which enable the bacterium to reprogram its transcriptional machinery under diverse environmental conditions. σ(J), an extracytoplasmic function σ factor, is upregulated in late stationary phase cultures and during human macrophage infection. σ(J) governs the cellular response to hydrogen peroxide-mediated oxidative stress. σ(J) differs from other canonical σ factors owing to the presence of a SnoaL_2 domain at the C-terminus. σ(J) crystals belonged to the tetragonal space group I422, with unit-cell parameters a = b = 133.85, c = 75.08 Å. Diffraction data were collected to 2.16 Å resolution on the BM14 beamline at the European Synchrotron Radiation Facility (ESRF).

Transcriptional response of Corynebacterium glutamicum ATCC 13032 to hydrogen peroxide stress and characterization of the OxyR regulon

The aerobic soil bacterium Corynebacterium glutamicum ATCC 13032 has a remarkable natural resistance to hydrogen peroxide. A major player in hydrogen peroxide defense is the LysR type transcriptional regulator OxyR, homologs of which are present in a wide range of bacteria. In this study, the global transcriptional response of C. glutamicum to oxidative stress induced by hydrogen peroxide was examined using whole genome DNA microarrays, demonstrating the dynamic reaction of the regulatory networks. Deletion of oxyR resulted in an increased resistance of the C. glutamicum mutant to hydrogen peroxide. By performing DNA microarray hybridizations and RT-qPCR, differentially expressed genes were detected in the mutant. The direct control by OxyR was verified by electrophoretic mobility shift assays for 12 target regions. The results demonstrated that OxyR in C. glutamicum acts as a transcriptional repressor under non-stress conditions for a total of 23 genes. The regulated genes encode proteins related to oxidative stress response (e.g. katA), iron homeostasis (e.g. dps) and sulfur metabolism (e.g. suf cluster). Besides the regulator of the suf cluster, SufR, OxyR regulated the gene cg1695 encoding a putative transcriptional regulator, indicating the role of OxyR as a master regulator in defense against oxidative stress. Using a modified DNase footprint approach, the OxyR-binding sites in five target promoter regions, katA, cydA, hemH, dps and cg1292, were localized and in each upstream region at least two overlapping binding sites were found. The DNA regions protected by the OxyR protein are about 56bp in length and do not have evident sequence similarities. Still, by giving an insight in the H2O2 stimulon and extending the OxyR regulon this study considerably contributes to the understanding of the response of C. glutamicum to hydrogen peroxide-mediated oxidative stress.

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H2O2-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H2O2. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.

The Yeast Snt2 Protein Coordinates the Transcriptional Response to Hydrogen Peroxide-Mediated Oxidative Stress

Regulation of gene expression is a vital part of the cellular stress response, yet the full set of proteins that orchestrate this regulation remains unknown. Snt2 is a Saccharomyces cerevisiae protein whose function has not been well characterized that was recently shown to associate with Ecm5 and the Rpd3 deacetylase. Here, we confirm that Snt2, Ecm5, and Rpd3 physically associate. We then demonstrate that cells lacking Rpd3 or Snt2 are resistant to hydrogen peroxide (H2O2)-mediated oxidative stress and use chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to show that Snt2 and Ecm5 recruit Rpd3 to a small number of promoters and in response to H2O2, colocalize independently of Rpd3 to the promoters of stress response genes. By integrating ChIP-seq and expression analyses, we identify target genes that require Snt2 for proper expression after H2O2. Finally, we show that cells lacking Snt2 are also resistant to nutrient stress imparted by the TOR (target of rapamycin) pathway inhibitor rapamycin and identify a common set of genes targeted by Snt2 and Ecm5 in response to both H2O2 and rapamycin. Our results establish a function for Snt2 in regulating transcription in response to oxidative stress and suggest Snt2 may also function in multiple stress pathways.

Hydrogen peroxide-mediated oxidative stress and collagen synthesis in cardiac fibroblasts: Blockade by tanshinone IIA

Ethnopharmacological relevanceWe have recently reported that tanshinone IIA attenuated cardiac fibrosis in two-kidney, two-clip renovascular hypertensive rats via inhibiting NAD(P)H oxidase. However, little is known about the cellular and molecular mechanisms of tanshinone IIA mediated anti-fibrotic effects in cardiac fibroblasts after H2O2 stimulation. The present study was performed to investigate whether H2O2 may increase collagen synthesis in cardiac fibroblasts by affecting the expression and activity of NAD(P)H oxidase and whether the effects of H2O2 on cardiac fibroblasts can be blocked by treatment of tanshinone IIA. Materials and methodsCardiac fibroblasts were treated with H2O2 (100μmol/L) in the presence or absence of tanshinone IIA (1μmol/L), NAD(P)H oxidase inhibitors diphenyleneiodonium (10μmol/L), siRNA-p47phox, siRNA-Nox2 and siRNA-Nox4. Collagen synthesis was measured by [3H]proline incorporation, O2− production were determined by flow cytometry and DHE fluorescence microscopy. NAD(P)H oxidase activity was measured by lucigenin-enhanced chemiluminescence. ResultsH2O2 induced the activity of NAD(P)H oxidase, O2− production, collagen synthesis and fibronectin expression in cardiac fibroblasts, and DPI abolished this induction. Exposure of adult rat cardiac fibroblasts to H2O2 had time-dependent increase in the expression of p47phox, Nox2 and Nox4 oxidases. In addition, tanshinone IIA significantly inhibited H2O2-induced collagen synthesis via attenuation of O2− generation and NAD(P)H oxidase activity. Moreover, siRNA-mediated knockdown of p47phox, Nox2 and Nox4 inhibited H2O2-induced NADPH oxidase activity. H2O2-induced collagen synthesis and fibronectin expression were also inhibited by p47phox, Nox2 and Nox4 knock down. ConclusionsOur data show that NAD(P)H oxidase plays a significant role in regulating collagen synthesis in H2O2-stimulated cardiac fibroblasts. Inhibition of NAD(P)H oxidase with tanshinone IIA completely blocked the H2O2-stimulated collagen production, which will raise the experimental basis for using tanshinone IIA to cardiac fibrosis in clinic.

Biological effects of melt spinning fabrics composed of 1% bioceramic material

This study evaluated the usefulness of bioceramic materials (ceramic materials that emit high-performance far-infrared (FIR) rays), processed into fabrics using a traditional manufacturing melt spinning method. Numerous measurements were designed to test the biological functions of 1% bioceramic fabrics. These included physical induction of intracellular nitric oxide (NO) in NIH 3T3 cells (mouse fibroblasts), the effects on cell viability in osteoblastic cells (MC3T3-E1) under hydrogen peroxide-mediated oxidative stress, and the effects on lipopolysaccharide (LPS)-induced cyclo-oxygenase-2 (COX-2) and prostaglandin E2 (PGE2) production in a chondrosarcoma (SW1353) cell line. When compared to the control group, the bioceramic fabrics were capable of inducing further intracellular NO production using NIH 3T3 cells, and maintaining increased viability and against cell intoxication of osteoblastic cells by suppressing cell release of lactate dehydrogenase (LDH) under oxidative stress. In addition, it was found to suppress LPS-induced COX-2 production more significantly in a SW1353 cell line. These processes represent the biomolecular changes occurring during promotion of decline in aging, prevention of osteoporosis, and prevention of inflammatory processes within the human body. Therefore, these bioceramic fabrics are likely to fulfill their claims of having health-promoting benefits.

An in vitro study of the antioxidant activities and effect on human DNA of the Chinese herbal decoction ‘Liu Wei Di Huang’

A Chinese medicinal formulation, ‘Liu Wei Di Huang’, and its individual components have been tested for the genoprotective effect on human DNA by the comet assay. Results showed no DNA protection contributed by this prescription. However, the aqueous extracts of individual herbs, namely Cortex Moutan and Rhizoma Dioscoreae, were able to decrease by 10–15% the DNA stand break from hydrogen peroxide-mediated oxidative stress. This provides insight to further evaluate the interaction among herbs and the search for the active ingredient responsible for the DNA protective effect.

Neuroprotective Effect of Baicalein on Hydrogen Peroxide-Mediated Oxidative Stress and Mitochondrial Dysfunction in PC12 Cells

Oxidative stress plays an important role in many neurodegenerative disorders. In this study, the effect of baicalein, a natural flavonoid isolated from the root of Scutellaria baicalensis G., on hydrogen peroxide (H2O2)-induced cytotoxicity in PC12 cells were investigated. Exposure of PC12 cells to 0.15 mM H2O2 for 20 min induced a significant decrease in cell viability accompanied by increased oxidative stress, mitochondrial dysfunction, downregulation of Bcl-2, upregulation of Bax, and cell apoptosis. Pretreatment of PC12 cells with baicalein inhibited H2O2-induced cell viability loss, intracellular reactive oxygen species generation, and lipid peroxidation in a dose-dependent manner. Meanwhile, baicalein potentially inhibited H2O2-induced cell apoptosis characterized with the DNA fragment. And the mitochondrial pathway involving the mitochondrial dysfunction associated with cell apoptosis including membrane potential loss, the release of cytochrome c, the downregulation of Bcl-2, upregulation of Bax induced by H2O2 were also abrogated in the presence of baicalein. Taken together, these results suggest that baicalein can block H2O2-induced apoptosis by prevention of oxidative stress as well as regulation of Bcl-2 family members and suppression of mitochondria dysfunction, which might be beneficial for the treatment of oxidative stress in aging and age-associated neurodegenerative diseases.

Propofol attenuation of hydrogen peroxide-mediated oxidative stress and apoptosis in cultured cardiomyocytes involves haeme oxygenase-1

Our aim was to investigate the cytoprotective effect of propofol against hydrogen peroxide (H2O2)-mediated injury and the effects on the haeme oxygenase-1 system, which is a possible new cytoprotective pathway of propofol. Primary cultured newborn rat cardiomyocytes were divided into five groups: (1) untreated (Group control); (2) treated with 200 micromol L(-1) H2O2 (Group H) and treated with 200 micromol L(-1) H2O2 in the presence of propofol (25, 50 and 100 micromol L(-1), (3) Group 25P + H, (4) Group 50P + H and (5) Group 100P + H, respectively); added with zinc protoporphyrin IX (ZnPPIX) (10 micromol L(-1)), a potent inhibitor of haeme oxygenase activity, or SC-3060 (0.2 micromol L(-1)), a specific synthetic inhibitor of nuclear factor kappaB. All were incubated for 6 h. The protective effects of propofol were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide cytotoxicity assay, the concentration of malondialdehyde, superoxide dismutase activity and cell apoptosis by enzyme-linked immunosorbent assay (ELISA). Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to detect haeme oxygenase-1 expression. Compared with H2O2, propofol concentrations (ranging from 50 to 100 micromol L(-1)) significantly increased haeme oxygenase-1 expression and decreased cardiomyocytes apoptosis, accompanied with a decrease in malondialdehyde, but with an increase in superoxide dismutase activity and cell activity (P < 0.05 and P < 0.01, respectively). The protective effects of propofol were mitigated by the addition of ZnPPIX. The addition of SC-3060 reversed propofol-induced haeme oxygenase-1 expression. Propofol can protect cardiomyocytes against H2O2-mediated cytotoxicity in a dose-dependent manner and increase haeme oxygenase-1 expression, which may partly mediate the cytoprotective effects of propofol.

Hydrogen peroxide-mediated oxidative stress disrupts calcium binding on calmodulin: More evidence for oxidative stress in vitiligo

Patients with acute vitiligo have low epidermal catalase expression/activities and accumulate 10 −3 M H 2O 2. One consequence of this severe oxidative stress is an altered calcium homeostasis in epidermal keratinocytes and melanocytes. Here, we show decreased epidermal calmodulin expression in acute vitiligo. Since 10 −3 M H 2O 2 oxidises methionine and tryptophan residues in proteins, we examined calcium binding to calmodulin in the presence and absence of H 2O 2 utilising 45calcium. The results showed that all four calcium atoms exchanged per molecule of calmodulin. Since oxidised calmodulin looses its ability to activate calcium ATPase, enzyme activities were followed in full skin biopsies from lesional skin of patients with acute vitiligo ( n = 6) and healthy controls ( n = 6). The results yielded a 4-fold decrease of ATPase activities in the patients. Computer simulation of native and oxidised calmodulin confirmed the loss of all four calcium ions from their specific EF-hand domains. Taken together H 2O 2-mediated oxidation affects calcium binding in calmodulin leading to perturbed calcium homeostasis and perturbed l-phenylalanine-uptake in the epidermis of acute vitiligo.

Influence of lactic acid bacteria and their spent culture supernatant on hydrogen peroxide-induced interleukin-8 synthesis and necrosis of Caco-2 cells

Intestinal epithelial cells are confronted with many noxious stimuli which play an important role in the mucosal immune response. In the present study, Caco-2 cells treated with hydrogen peroxide were used as a model system for studying inflammatory responses and induction of cell death. Live lactobacilli and their concentrated spent culture supernatant (SCS) were applied for studying possible short- and long-term protective effects against hydrogen peroxide-mediated oxidative stress in Caco-2 cells. The secretion of pro-inflammatory cytokine interleukin-8 (IL-8) was investigated in non-filter grown Caco-2 cells, while transepithelial electrical resistance and cell death was studied in filter grown Caco-2 cells. Pre-incubation of Caco-2 cells with Lactobacillus plantarum 2142 did not decrease IL-8 levels induced by 1 mM hydrogen peroxide, nor did lactobacilli suppress IL-8 levels induced by hydrogen peroxide when Caco-2 cells were treated simultaneously with 1 mM hydrogen peroxide and L. plantarum 2142. Thus, lactobacilli did not exert a long-term protective effect against hydrogen peroxide in non-filter grown Caco-2 cells. However, the concentrated SCS of lactobacilli was able to reduce IL-8 levels by more than 6-fold, as determined 24 h after treatment. A short-term effect of lactobacilli was observed in filter grown Caco-2 cells, as they inhibited cell death induced by hydrogen peroxide in the concentration range 10-40 mM. Pre-incubation of epithelial cells with L. plantarum 2142 or simultaneous exposure to hydrogen peroxide and lactobacilli protected Caco-2 cells against cell death. In spite of the presence of lactobacilli, the permeability of membrane increased (transepithelial electrical resistance decreased), and exhibited a similar characteristic pattern as under treatment with hydrogen peroxide alone.