Doxorubicin-efflux pump in Haloferax voleanii is energized by ATP

Abstract Treatment of Candida albicans with low concentrations of either hydrogen peroxide or menadione (a Superoxide generating agent) induces an adaptive response which protects cells from the lethal effects of a subsequent challenge with higher concentrations of these oxidants. Pre-treatment with either menadione or hydrogen peroxide is protective against cell killing by either oxidant. This suggests that the pathogenic yeast C. albicans (unlike the budding yeast Saccharomyces cerevisiae which has separate responses) possesses an adaptive response that responds to both these oxidants. In addition, we found that C. albicans showed a greater level of resistance to oxidants, both H₂O₂ and redox-cycling agents, compared to that observed with S. cerevisiae . In an attempt to characterise the oxidative stress response in more detail we have analysed the effect of oxidants on the activities of a number of enzymes with known antioxidant activity.     

Atypical adaptive and cross-protective responses against peroxide killing in a bacterial plant pathogen, Agrobacterium tumefaciens

Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens. Exposure of A. tumefaciens to sublethal concentrations of H2O2 induced adaptive protection to lethal concentrations of H2O2. Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants. Pretreatment of A. tumefaciens with an inducing concentration of menadione conferred cross-protection against H2O2, but not to tert-butyl hydroperoxide (tBOOH), killing. The menadione induced cross-protection to H2O2 was due to the compound's ability to highly induce the peroxide scavenging enzyme, catalase. The levels of catalase directly correlated with the bacterium's ability to survive H2O2 treatment. Some aspects of the oxidative stress response of A. tumefaciens differ from other bacteria, and these differences may be important in plant/microbe interactions.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H2O2, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pre-treatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H2O2, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Tolerance of the yeast Yarrowia lipolytica to oxidative stress

The adaptive response of the yeast Yarrowia lipolytica to the oxidative stress induced by the oxidants hydrogen peroxide, menadione, and juglone has been studied. H₂O₂, menadione, and juglone completely inhibited yeast growth at concentrations higher than 120, 0.5, and 0.03 mM, respectively. The stationary-phase yeast cells were found to be more resistant to the oxidants than the exponential-phase cells. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.3 mM), menadione (0.05 mM), and juglone (0.005 mM) made the cells more resistant to high concentrations of these oxidants. The adaptation of yeast cells to H₂O₂, menadione, and juglone was associated with an increase in the activity of cellular catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase, the main enzymes involved in cell defense against oxidative stress.     

Interaction of menadione (2-methyl-1,4-naphthoquinone) with glutathione

The interaction of menadione with reduced glutathione (GSH) led to a removal of menadione and formation of menadione-GSH conjugate and glutathione disulfide (GSSG). The changes in thiol level were essentially biphasic with an initial rapid decrease in GSH and appearance of GSSG (less than 1 min) followed by secondary less pronounced changes. The interaction of menadione and GSH caused an oxygen uptake and both superoxide anion radical and hydrogen peroxide were produced during the reaction, the amount dependent on the GSH/menadione ratio. Catalase did not protect against the initial decrease in GSH level but markedly inhibited the secondary changes while superoxide dismutase had little effect. These results suggest that the initial changes in thiol level are the result in part of a redox reaction between menadione and GSH as well as conjugate formation, whilst the secondary changes reflect conjugate formation and the activity of other oxidants such as hydrogen peroxide. The potential biological significance of this reaction was investigated using hepatocytes depleted of reduced pyridine nucleotides and thus not able to perform enzyme-catalyzed reduction of menadione. In these cells menadione induced GSSG formation at a rate similar to that observed in control cells. This suggests that quinone-induced oxidative challenge caused by the chemical interactions of a quinone and glutathione may have biological relevance.     

Oxidative stress response in eukaryotes: effect of glutathione, superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H2O2 did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H2O2 adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H2O2 when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H2O2 achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H2O2 was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H2O2 promoted an increase in catalase activity.     

Atypical Adaptive and Cross-Protective Responses Against Peroxide Killing in a Bacterial Plant Pathogen, <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Physiological adaptive and cross-protection responses to oxidants were investigated in Agrobacterium tumefaciens. Exposure of A. tumefaciens to sublethal concentrations of H₂O₂ induced adaptive protection to lethal concentrations of H₂O₂. Similar treatments with organic peroxide and menadione did not produce adaptive protection to subsequent exposure to lethal concentrations of these oxidants. Pretreatment of A. tumefaciens with an inducing concentration of menadione conferred cross-protection against H₂O₂, but not to tert-butyl hydroperoxide (tBOOH), killing. The menadione induced cross-protection to H₂O₂ was due to the compounds ability to highly induce the peroxide scavenging enzyme, catalase. The levels of catalase directly correlated with the bacteriums ability to survive H₂O₂ treatment. Some aspects of the oxidative stress response of A. tumefaciens differ from other bacteria, and these differences may be important in plant/microbe interactions. Received: 12 November 2002 / Accepted: 13 December 2002     

Exposure of phytopathogenic Xanthomonas spp. to lethal concentrations of multiple oxidants affects bacterial survival in a complex manner

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1, 000-fold; conversely, treatment of cells with menadione plus H(2)O(2) resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H(2)O(2) and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H(2)O(2) and tert-butyl hydroperoxide plus H(2)O(2). These data suggest that H(2)O(2) is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H(2)O(2)-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.     

Low concentration of oxidant and nitric oxide donors stimulate proliferation of human endothelial cells in vitro

Proliferation of human umbilical vein endothelial cells in vitro was inhibited by high concentrations of oxidants and nitric oxide donors but stimulated by low (micromolar or submicromolar) concentrations of hydrogen peroxide, menadione, tert-butyl hydroperoxide, AAPH, nitroglycerin, SIN-1 and sodium nitroprusside. The stimulation seems to be dependent upon generation of secondary reactive oxygen species as inferred from attenuation of cell proliferation by superoxide dismutase and catalase. These results point to another type of possible artefact of cell culture, viz. stimulation of cell proliferation by low concentrations of oxidants.     

Oxidative stress response in eukaryotes: effect of glutathione,superoxide dismutase and catalase on adaptation to peroxide and menadione stresses in Saccharomyces cerevisiae

Abstract

Aiming to clarify the mechanisms by which eukaryotes acquire tolerance to oxidative stress, adaptive and cross-protection responses to oxidants were investigated in Saccharomyces cerevisiae. Cells treated with sub-lethal concentrations of menadione (a source of superoxide anions) exhibited cross-protection against lethal doses of peroxide; however, cells treated with H₂O₂ did not acquire tolerance to a menadione stress, indicating that menadione response encompasses H₂O₂ adaptation. Although, deficiency in cytoplasmic superoxide dismutase (Sod1) had not interfered with response to superoxide, cells deficient in glutathione (GSH) synthesis were not able to acquire tolerance to H₂O₂ when pretreated with menadione. These results suggest that GSH is an inducible part of the superoxide adaptive stress response, which correlates with a decrease in the levels of intracellular oxidation. On the other hand, neither the deficiency of Sod1 nor in GSH impaired the process of acquisition of tolerance to H₂O₂ achieved by a mild pretreatment with peroxide. Using a strain deficient in the cytosolic catalase, we were able to conclude that the reduction in lipid peroxidation levels produced by the adaptive treatment with H₂O₂ was dependent on this enzyme. Corroborating these results, the pretreatment with low concentrations of H₂O₂ promoted an increase in catalase activity.     

Characterization of an inducible oxidative stress response in Vitreoscilla C1

Vitreoscilla becomes resistant to killing by hydrogen peroxide and heat shock when pretreated with nonlethal levels of hydrogen peroxide. The pretreated Vitreoscilla cells (60 microM hydrogen peroxide for 120 min) significantly increased survival of the lethal dose of 20 mM hydrogen peroxide or heat shock (22 degrees C --> 37 degrees C). This indicates the existence of an adaptive response to oxidative stress. However, cells pretreated with 60 microM hydrogen peroxide became nonresistant to a lethal dose of a menadione. This result shows that hydrogen peroxide does not induce cross-resistance to menadione in Vitreoscilla. Furthermore, Vitreoscilla treated with hydrogen peroxide, heat shock, and menadione showed a change in the protein composition, as monitored by a two-dimensional gel analysis. During adaptation to hydrogen peroxide, 12 proteins were induced. Also, 18 new proteins synthesized in response to heat shock were detected by a 2-D gel analysis. The redox-cycling agents also elicited the synthesis of 6 other proteins that were unseen with hydrogen peroxide.     

Adaptive Response of Bacillus sp. F26 to Hydrogen Peroxide and Menadione

The adaptive and cross-protection responses to oxidants were investigated in Bacillus sp. F26. The cells were treated with sublethal concentrations of either H₂O₂ or menadione (a superoxide-generating agent) to induce an adaptive response. The results showed that the cells treated with menadione exhibited cross-protection against, but in another case, those cells treated with H₂O₂ did not show significant resistance to menadione. It suggests that Bacillus sp. F26 possesses two separate adaptive responses that respond to the two different kinds of oxidants. The adaptability is regarded as that which is accompanied by the inductions of some antioxidant enzymes. It was found that catalase (CAT) production was increased about 1.6-fold after treatment with 600 μM H₂O₂, whereas the presence of 50 μM menadione induced CAT, superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PD), and glutathione reductase (GR) by 2-, 2-, 2-, and 1.6-fold, respectively. The results can be used to explain why menadione-treated cells have higher adaptability to lethal concentrations of oxidants than that of those H₂O₂-treated. In addition, it was found that growing Bacillus sp. F26 in high-salinity media causes it to become more resistant to H₂O₂ and menadione stress, which may be partially due to the induction of CAT and SOD production under high NaCl concentration.     

Bacterial defenses against oxidative stress

Bacteria treated with low doses of oxidants such as hydrogen peroxide adapt to subsequent high doses of these oxidants by inducing the expression of numerous genes. The study of these genes and the roles they play in defending bacteria against oxidative damage has given general insights into what oxidants are hazardous to cells, what cell constituents are damaged by oxidants, and how cells sense and respond to oxidative stress.     

Adaptation of the phytopathogenic fungus Fusarium decemcellulare to oxidative stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H2O2 and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H2O2 (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H2O2 and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main oxidative stress defense of enzymes.     

Exposure of Phytopathogenic Xanthomonas spp. to Lethal Concentrations of Multiple Oxidants Affects Bacterial Survival in a Complex Manner

During plant-microbe interactions and in the environment, Xanthomonas campestris pv. phaseoli is likely to be exposed to high concentrations of multiple oxidants. Here, we show that simultaneous exposures of the bacteria to multiple oxidants affects cell survival in a complex manner. A superoxide generator (menadione) enhanced the lethal effect of an organic peroxide (tert-butyl hydroperoxide) by 1,000-fold; conversely, treatment of cells with menadione plus H₂O₂ resulted in 100-fold protection compared to that for cells treated with the individual oxidants. Treatment of X. campestris with a combination of H₂O₂ and tert-butyl hydroperoxide elicited no additive or protective effect. High levels of catalase alone are sufficient to protect cells against the lethal effect of menadione plus H₂O₂ and tert-butyl hydroperoxide plus H₂O₂. These data suggest that H₂O₂ is the lethal agent responsible for killing the bacteria as a result of these treatments. However, increased expression of individual genes for peroxide (alkyl hydroperoxide reductase, catalase)- and superoxide (superoxide dismutase)-scavenging enzymes or concerted induction of oxidative stress-protective genes by menadione gave no protection against killing by a combination of menadione plus tert-butyl hydroperoxide. However, X. campestris cells in the stationary phase and a spontaneous H₂O₂-resistant mutant (X. campestris pv. phaseoli HR) were more resistant to killing by menadione plus tert-butyl hydroperoxide. These findings give new insight into oxidant killing of Xanthomonas spp. that could be generally applied to other bacteria.     

Group IV cytosolic phospholipase A2 mediates arachidonic acid release in H9c2 rat cardiomyocyte cells in response to hydrogen peroxide

Damaging reactive oxygen species are released during episodes of ischemia and reperfusion. Some cellular adaptive responses are triggered to protect the injured organ, while other cascades are triggered which potentiate the damage. In these studies, we demonstrate that rat cardiomyocte H9c2 cells release arachidonic acid in response to hydrogen peroxide. In H9c2 cells, arachidonic acid release is attenuated by methyl arachidonyl fluorophosphonate (MAFP) and pyrrophenone, indicating that a phospholipase A2 Group IV enzyme mediates arachidonic acid mobilization. Moreover, hydrogen peroxide alters the cellular morphology of the H9c2 cells, causing drastic cell shrinkage. Because MAFP and pyrrophenone prevent the morphological alterations caused by hydrogen peroxide, these studies show that phospholipase A2 Group IV activity is likely integrally involved in the damage initiated by hydrogen peroxide.     

Application of a YHB1-GFP reporter to detect nitrosative stress in yeast

Yeast flavohemoglobin (Yhb1p) plays a pivotal role in NO(*) detoxification and has also been implicated in oxidative/reductive stress responses. In this study, we have used a YHB1-GFP reporter expressing a full-length chromosome-tagged Yhb1-GFP fusion protein to monitor changes in flavohemoglobin levels after cell treatment with oxidants, antioxidants and nitric oxide donors. Only nitric oxide donors were found to induce a dose-dependent increase in Yhb1-GFP expression while hydrogen peroxide, menadione and diamide caused a moderate diminution of YHB1-GFP fluorescence. Additionally, the levels of endogenous and hydroperoxide-induced ROS production in the Deltayhb1 mutant were comparable to those in the isogenic wild-type strain. Although peroxides increased NO(*) generation while nitrite and nitric oxide donors augmented ROS levels in yeast cells, their effects were generally not more pronounced in Deltayhb1 than in wild-type cells. Taken together, these data suggest that yeast flavohemoglobin does not contribute to cross-protection against oxidative and nitrosative stress.     

An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.     

Induction of peroxide and superoxide protective enzymes and physiological cross-protection against peroxide killing by a superoxide generator in Vibrio harveyi

Vibrio harveyi is a causative agent of destructive luminous vibriosis in farmed black tiger prawn (Penaeus monodon). V. harveyi peroxide and superoxide stress responses toward elevated levels of a superoxide generated by menadione were investigated. Exposure of V. harveyi to sub-lethal concentrations of menadione induced high expression of genes in both the OxyR regulon (e.g., a monofunctional catalase or KatA and an alkyl hydroperoxide reductase subunit C or AhpC), and the SoxRS regulon (e.g., a superoxide dismutase (SOD) and a glucose-6-phosphate dehydrogenase). V. harveyi expressed two detectable, differentially regulated SOD isozymes, [Mn]-SOD and [Fe]-SOD. [Fe]-SOD was expressed constitutively throughout the growth phase while [Mn]-SOD was expressed at the stationary phase and could be induced by a superoxide generator. Physiologically, pre-treatment of V. harveyi with menadione induced cross-protection against subsequent exposure to killing concentrations of H(2)O(2). This induced cross-protection required newly synthesized proteins. However, the treatment did not induce significant protection against exposures to killing concentrations of menadione itself or cross-protect against an organic hydroperoxide (tert-butyl hydroperoxide). Unexpectedly, growing V. harveyi in high-salinity media induced protection against menadione killing. This protection was independent of SOD induction. Stationary-phase cells were more resistant to menadione killing than exponential-phase cells. The induction of oxidative stress protective enzymes and stress-altered physiological responses could play a role in the survival of this bacterium in the host marine crustaceans.