NTBC Treatment of the Pyomelanogenic Pseudomonas aeruginosa Clinical Isolate PA1111 Inhibits Pigment Production and Increases Sensitivity to Oxidative Stress

Pyomelanin is a brown/black extracellular pigment with antioxidant and iron acquisition properties that is produced by a number of different bacteria. Production of pyomelanin in Pseudomonas aeruginosa contributes to increased resistance to oxidative stress and persistence in chronic infections. We demonstrate that pyomelanin production can be inhibited by 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC). This treatment increases sensitivity of pyomelanogenic P. aeruginosa strains to oxidative stress, without altering the growth rate or resistance to aminoglycosides. As such, NTBC has potential to function as an anti-virulence factor in treating pyomelanogenic bacterial infections.     

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H₂O₂ are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.     

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H₂O₂.Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H₂O₂ and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H₂O₂. Analysis of different hallmarks of programmed cell death was also carried out.Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H₂O₂, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H₂O₂ and nitric oxide contents and maintenance of the glutathione redox state were observed.Conclusions A decreased content of endogenous H₂O₂ may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H₂O₂, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.     

Physiological changes induced in four bacterial strains following oxidative stress

In order to study the behavior and resistance of bacteria under extreme conditions, physiological changes associated with oxidative stress were monitored using flow cytometry. The study was conducted to assess the maintenance of membrane integrity and potential as well as the esterase activity, the intracellular pH and the production of superoxide anions in four bacterial strains (Ralstonia metallidurans, Escherichia coli, Shewanella oneidensis and Deinococcus radiodurans). The strains were chosen for their potential use in bioremediation. Suspensions of R. metallidurans, E. coli, S. oneidensis and D. radiodurans were submitted to 1 h of oxidative stress (H₂O₂ at various concentrations from 0 to 880 mM). Cell membrane permeability (propidium iodide) and potential (rhodamine-123,3,3’-dihexyloxacarbocyanine iodide), intracellular esterase activity (fluorescein diacetate), intracellular-reactive oxygen species concentration (hydroethidine) and intracellular pH (carboxy-fluorescein diacetate succinimidyl ester 5-(6)) were monitored to evaluate the physiological state and the overall fitness of individual bacterial cells under oxidative stress. The four bacterial strains exhibited varying sensitivities towards H₂O₂. However, for all the bacterial strains, some physiological damage could already be observed from 13.25 mM H₂O₂ onwards, in particular with regard to their membrane permeability. Depending on the bacterial strains, moderate to high physiological damage could be observed between 13.25 mM and 220 mM H₂O₂. The membrane potential, esterase activity, intracellular pH and production of superoxide anion production were in all four strains considerably modified at high H₂O₂ concentrations. In conclusion, we show that a range of significant physiological alterations occur when bacteria are challenged with H₂O₂ and fluorescent staining methods coupled with flow cytometry are used for monitoring the changes induced not only by oxidative stress, but also by other stresses like temperature, radiation, pressure, pH, etc. The text was submitted by the authors in English.     

Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity

Oxidative stress induced by ciprofloxacin and pyoverdin, a leukotoxic pigment, was studied by comparing their effect in bacteria and leukocytes. Chemiluminescence (CL) assays with lucigenin or luminol were adapted to measure the stimuli of superoxide anion (O₂^?) and other reactive species of oxygen (ROS) in bacteria. Ciprofloxacin principally induced the production of O₂^? in the three species studied: Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Lucigenin CL assay showed high oxidative stress in S. aureus due to its low superoxide dismutase (SOD) activity, whereas E. coli exhibited important SOD activity, responsible for little production of O₂^? in absence or presence of ciprofloxacin. Reduction of nitroblue of tetrazolium (NBT) was applied. This assay indicated that there was higher oxidative stress in S. aureus and E. faecalis than in E. coli. The comparison of oxidative stress generated in bacteria and leukocytes was used to check the selective toxicity of ciprofloxacin in comparison with pyoverdin. Ciprofloxacin did not generate significant stimuli of O₂^? in neutrophils, while pyoverdin duplicated the production of O₂^?. CL and NBT were useful to study the leukotoxicity of ciprofloxacin. Oxidative stress caused by the antibiotic and the leukotoxic pigment was similar in bacteria. Copyright © 2003 John Wiley & Sons, Ltd.     

兼性厌氧细菌硫化氢的产生机理及其生理功能

硫化氢(H_2S)是继一氧化氮(NO)和一氧化碳(CO)后发现的第3种气态信号分子,但其细菌生理学研究才刚刚起步。本文根据作者对奥内达希瓦氏菌的研究,结合新近文献,就细菌的H_2S产生机理及其生理功能作了较为全面的阐述。细菌的H_2S产生途径主要有2条,一是通过降解半胱氨酸产生,二是通过厌氧呼吸产生。产生的H_2S除可为互生性微生物提供能源、供氢体和无机矿质营养外,还具有抑制竞争性微生物的生长,有效占领生态位的作用。H_2S在氧化应答中也起着重要的作用,一方面可抑制过氧化氢酶活性,增加过氧化氢对细菌的杀灭效果;另一方面可作为信号分子激活细菌的氧化应答,诱导拮抗系统的表达,保护细胞免受氧化损伤。这两种看似"矛盾"的作用与H_2S的处理时间有关:短时间处理以抑制为主,而延长处理时间则以保护为主。细菌H_2S产生机理及生理功能的阐明可为硫元素生物地球化学循环规律的揭示和感染性病原细菌的控制提供有益的参考。     

Protective effects of cyclosativene on H2O2-induced injury in cultured rat primary cerebral cortex cells

Sesquiterpenes have attracted much interest with respect to their protective effect against oxidative damage that may be the cause of many diseases including several neurodegenerative disorders and cancer. Our previous unpublished work suggested that cyclosativene (CSV), a tetracyclic sesquiterpene, has antioxidant and anticarcinogenic features. However, little is known about the effects of CSV on oxidative stress induced neurotoxicity. We used hydrogen peroxide (H₂O₂) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of CSV in H₂O₂-induced toxicity in new-born rat cerebral cortex cell cultures for the first time. For this aim, MTT and lactate dehydrogenase release assays were carried out to evaluate cytotoxicity. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to evaluate oxidative changes. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels, the single cell gel electrophoresis (or Comet assay) was also performed for measuring the resistance of neuronal DNA to H₂O₂-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (Comet assay) increased in the H₂O₂ alone treated cultures. But pre-treatment of CSV suppressed the cytotoxicity, genotoxicity and oxidative stress which were increased by H₂O₂. On the basis of these observations, it is suggested that CSV as a natural product with an antioxidant capacity in mitigating oxidative injuries in the field of neurodegenerative disorders.     

Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress

Cytochrome bd is a prokaryotic respiratory quinol:O₂ oxidoreductase, phylogenetically unrelated to the extensively studied heme–copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented in one of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O₂, NO and H₂O₂ is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

A novel 1-Cys thioredoxin peroxidase gene in Apis cerana cerana: characterization of AccTpx4 and its role in oxidative stresses

Thioredoxin peroxidase (Tpx), also named peroxiredoxin (Prx), is an important peroxidase that can protect organisms against stressful environments. AccTpx4, a 1-Cys thioredoxin peroxidase gene from the Chinese honey bee Apis cerana cerana, was cloned and characterized. The AccTpx4 gene encodes a protein that is predicted to contain the conserved PVCTTE motif from 1-Cys peroxiredoxin. Quantitative real-time PCR (Q-PCR) and Western blotting revealed that AccTpx4 was induced by various oxidative stresses, such as cold, heat, insecticides, H₂O₂, and HgCl₂. The in vivo peroxidase activity assay showed that recombinant AccTpx4 protein could efficiently degrade H₂O₂ in the presence of DL-dithiothreitol (DTT). In addition, disc fusion assays revealed that AccTpx4 could function to protect cells against oxidative stresses. These results indicate that AccTpx4 plays an important role in oxidative stress responses and may contribute to the conservation of honeybees.     

Arsenic-induced oxidative stress in the common bean legume,Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide

The adverse effects of arsenic (As) toxicity on seedling growth, root and shoot anatomy, chlorophyll and carotenoid contents, root oxidizability (RO), antioxidant enzyme activities, H₂O₂ content, lipid peroxidation and electrolyte leakage (EL%) in common bean (Phaseolus vulgaris L.) were investigated. The role of exogenous nitric oxide (NO) in amelioration of As-induced inhibitory effect was also evaluated using sodium nitroprusside (100 μM SNP) as NO donor and 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (200 μM PTIO) as NO scavenger in different combinations with 50 μM As. As-induced growth inhibition was associated with marked anomalies in anatomical features, reduction in pigment composition, increased RO and severe perturbations in antioxidant enzyme activities. While activity of superoxide dismutase and catalase increased, levels of ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase decreased significantly and guaiacol peroxidase remained normal. The over-accumulation of H₂O₂ content along with high level of lipid peroxidation and electrolyte leakage indicates As-induced oxidative damage in P. vulgaris seedlings with more pronounced effect on the roots than the shoots. Exogenous addition of NO significantly reversed the As-induced oxidative stress, maintaining H₂O₂ in a certain level through balanced alterations of antioxidant enzyme activities. The role of NO in the process of amelioration has ultimately been manifested by significant reduction of membrane damage and improvement of growth performance in plants grown on As + SNP media. Onset of oxidative stress was more severe after addition of PTIO, which confirms the protective role of NO against As-induced oxidative damage in P. vulgaris seedlings.     

Inhibition of the oxidative stress-induced miR-23a protects the human retinal pigment epithelium (RPE) cells from apoptosis through the upregulation of glutaminase and glutamine uptake

The degeneration of retinal pigment epithelium (RPE) cells in the sub retinal pigment epithelial space and choroid is an initial pathological characteristic for the age-related macular degeneration which is the leading cause of severe vision loss in old people. Moreover, oxidative stress is implicated as a major inducer of RPE cell death. Here, we assessed the correlation between the H₂O₂-induced RPE cell death and glutamine metabolism. We found under low glutamine supply (20 %), the ARPE-19 cells were more susceptive to H₂O₂-induced apoptosis. Moreover, the glutamine uptake and the glutaminase (GLS) were suppressed by H₂O₂ treatments. Moreover, we observed miR-23a was upregulated by H₂O₂ treatments and overexpression of miR-23a significantly sensitized ARPE-19 cells to H₂O₂. Importantly, Western blotting and luciferase assay demonstrated GLS1 is a direct target of miR-23a in RPE cells. Inhibition of the H₂O₂-induced miR-23a by antagomiR protected the RPE cells from the oxidative stress-induced cell death. In addition, recovery of GLS1 expression in miR-23a overexpressed RPE cells rescued the H₂O₂-induced cell death. This study illustrated a mechanism for the protection of the oxidative-induced RPE cell death through the recovery of glutamine metabolism by inhibition of miR-23a, contributing to the discovery of novel targets and the developments of therapeutic strategies for the prevention of RPE cells from oxidative stress.     

A novel method for promoting conidial production by a nematophagous fungus, Pochonia chlamydosporia AS6.8

A novel method was described for promoting conidial production of nematophagous fungus, Pochonia chlamydosporia AS6.8, by hydrogen peroxide (H₂O₂) treatment and two-stage cultivation. The fungi were first grown on potato dextrose agar plates to encourage vegetative growth, after briefly treating with 90 mM concentrations of H₂O₂, and then were transferred to water agar (WA) plates for sporulation. Sporulation of the P. chlamydosporia AS6.8 was significantly enhanced (as much as 15 times) using this method. Enhancement of sporulation by H₂O₂ (oxidative stress) and by transfer to WA (low-nutrient stress) was not synergistic. In order to make sure if the H₂O₂ had enhancing effect on other nematophagous fungi, we also evaluated the effect of H₂O₂ on sporulation of Arthrobotrys oligospora CBS 115.81 and Dactylellina cionopaga CBS 113355 besides P. chlamydosporia. And the result showed that 90 mM concentrations of H₂O₂ had enhancing effect on sporulation of all of the three isolates.     

Different behavior of agmatine in liver mitochondria: Inducer of oxidative stress or scavenger of reactive oxygen species?

Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H₂O₂ generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H₂O₂ and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca²⁺. Instead, 1 mM agmatine generates larger amounts of H₂O₂ than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca²⁺-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed.     

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.     

Assessment of cell death studies by monitoring hydrogen peroxide in cell culture

Hydrogen peroxide (H₂O₂) has been widely used to study the oxidative stress response. However, H₂O₂ is unstable and easily decomposes into H₂O and O₂. Consequently, a wide range of exposure times and treatment concentrations has been described in the literature. In the present study, we established a ferrous oxidation-xylenol orange (FOX) assay, which was originally described for food and body liquids, as a method for the precise quantification of H₂O₂ concentrations in cell culture media. We observed that the presence of FCS and high cell densities significantly accelerate the decomposition of H₂O₂, therefore acting as a protection against cell death by accidental necrosis.     

Regulation of a novel cell differentiation-associated gene,JWA during oxidative damage in K562 and MCF-7 cells

Oxidative stress, or the production of oxygen-centered free radicals, has been hypothesized as the major source of DNA damage that can lead to a variety of diseases including cancer. It is known that 8-hydroxy-deoxyguanosine (8-oxo-dG) is a useful biomarker of oxidative DNA damage. Our recent data showed that JWA, initially being cloned as a novel cell differentiation-associated gene, was also actively responsive to environmental stressors, such as heat-shock, oxidative stress and so on. In the present study, we have applied a modified comet assay and bacterial repair endonucleases system (endonuclease III and formamidopyrimidine glycosylase) to investigate if JWA is involved in hydrogen peroxide (H₂O₂)-induced DNA damage and repair in K562 and MCF-7 cells, and to demonstrate if the damage is associated with 8-oxo-dG. The results from the comet assay have shown that the average tail length and the percentage of the cells with DNA tails are greatly induced by H₂O₂ treatment and further significantly enhanced by the post-treatment of repair endonucleases. The H₂O₂-induced 8-oxo-dG formation in K562 and MCF-7 cells is dose-dependent. In addition, the data have clearly demonstrated that JWA gene expression is actively induced by H₂O₂ treatment in K562 and MCF-7 cells. The results suggest that JWA can be regulated by oxidative stress and is actively involved in the signal pathways of oxidative stress in the cells.     

Quorum sensing enhancement of the stress response promotes resistance to quorum quenching and prevents social cheating

Quorum sensing (QS) coordinates the expression of virulence factors and allows bacteria to counteract the immune response, partly by increasing their tolerance to the oxidative stress generated by immune cells. Despite the recognized role of QS in enhancing the oxidative stress response, the consequences of this relationship for the bacterial ecology remain unexplored. Here we demonstrate that QS increases resistance also to osmotic, thermal and heavy metal stress. Furthermore a QS-deficient lasR rhlR mutant is unable to exert a robust response against H₂O₂ as it has less induction of catalase and NADPH-producing dehydrogenases. Phenotypic microarrays revealed that the mutant is very sensitive to several toxic compounds. As the anti-oxidative enzymes are private goods not shared by the population, only the individuals that produce them benefit from their action. Based on this premise, we show that in mixed populations of wild-type and the mexR mutant (resistant to the QS inhibitor furanone C-30), treatment with C-30 and H₂O₂ increases the proportion of mexR mutants; hence, oxidative stress selects resistance to QS compounds. In addition, oxidative stress alone strongly selects for strains with active QS systems that are able to exert a robust anti oxidative response and thereby decreases the proportion of QS cheaters in cultures that are otherwise prone to invasion by cheats. As in natural environments stress is omnipresent, it is likely that this QS enhancement of stress tolerance allows cells to counteract QS inhibition and invasions by social cheaters, therefore having a broad impact in bacterial ecology.     

Rapid in vivo screening system for anti‐oxidant activity using bacterial redox sensor strains

Aim: To develop a faster and easier in vivo method to screen compounds for anti‐oxidant activity using a microbial system. Methods and Results: Bacterial redox sensor‐based assay systems were applied. The activities of SoxR and OxyR, the bacterial redox sensors, were monitored to probe the intracellular redox status through two reporter strains, Escherichia coli soxS_p‐lacZ and oxyS_p‐lacZ fusions, which specifically respond to paraquat, a superoxide generator, and H₂O₂, respectively, with practically no cross reactivity. For the test screening, 27 natural compounds including phenolics and flavonoids that are putatively considered anti‐oxidant nutritional supplements were collected and assayed for their capability to alleviate oxidative stress in these bacterial systems. Among them, rutin, kaempferol and quercetin had significant anti‐H₂O₂ activity, and betaine, glycyrrhizic acid and baicalin had weak anti‐superoxide activity. While rutin, kaempferol and quercetin significantly reduced the H₂O₂ stress at low concentrations, betaine, glycyrrhizic acid and baicalin required higher concentration for their anti‐superoxide effects. In vitro, only quercetin protected DNA in a metal‐catalysed oxidation system, suggesting that the other compounds might indirectly exert their anti‐oxidant activities through other biological functions. Finally, quercetin, rutin and kaempferol significantly restored the viability of a superoxide dismutase mutant that has limited viability because of defective defence against oxidative stress. Conclusion: These bacterial systems could provide a more efficient method for measuring the activity of compounds affecting cellular oxidative stress and viability. Significance and Impact of the Study: The demand for anti‐oxidant and anti‐ageing activities is increasing in one of the fastest growing segments of the functional food market, but the screening for these activities is currently very laborious, expensive and time consuming. This study suggests a basis for a high throughput screening method for these activities.