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.     

活性氧不敏感型拟南芥的突变体对H2O2的响应

检测拟南芥ros突变株对H2O2响应的结果表明,此种突变体对H2O2有较强的耐受性,表现为气孔开度对H2O2不敏感和H2O2胁迫时的膜脂过氧化水平较低。采用激光扫描共聚焦显微术(LSCM)并结合H2O2荧光探针H2DCFDA检测外源ABA诱导保卫细胞的结果显示,突变体内荧光强度比野生型拟南芥低,暗示此种突变体消除H2O2的能力可能有提高,从而可增强植株抗氧化胁迫的能力。     

Luminol-Enhanced Chemiluminescent Response of Human Melanocytes and Melanoma Cells to Hydrogen Peroxide Stress

The response of human melanocytes and melanoma cells to hydrogen peroxide stress was measured. Cells were exposed to glucose/glucose oxidase or free H₂O₂ and reactive oxygen species measured by luminol-enhanced chemiluminescence. The response was distinctly different between the two types and the addition of superoxide dismutase to melanoma cells paradoxically enhanced the chemiluminescent signal. These findings coupled with other known differences between the way these two types of cells handle oxidative stress at a molecular level suggests that a therapeutic window may be avail-able for exploitation.     

Action of Cystine in the Cytotoxic Response of Escherichia Coli Cells Exposed to Hydrogen Peroxide

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 μM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems.

In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.

Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.     

Resistance of Serratia marcescens to Hydrogen Peroxide

Irradiation with ultraviolet (u.v.) light (71 J/m²) reduced the viable count of suspenrsions of Serratia marcescens , grown in a glycerol-salts defined medium, to five in 10⁴ cells. Subsequent incubation of irradiated cells in hydrogen peroxide failed to decrease the survivors, but u.v. irradiation in the presence of hydrogen peroxide reduced the viable count to fewer than two in 10⁶ cells. Cells grown in defined medium with added iron had more measurable catalase activity and were more resistant to hydrogen peroxide alone and to simultaneous treatment with u.v. irradiation and hydrogen peroxide. Cells grown in a non-defined medium contained little iron and measurable catalase activity but were more resistant to hydrogen peroxide. Treatment with toluene, heat killing or sonication increased the catalase activity detected in all cell suspensions and showed that resistance to hydrogen peroxide and to u.v. irradiation in hydrogen peroxide was related to the total catalase activity within cells.     

Interpreting Heterogeneity in Response of Cells Expressing a Fluorescent Hydrogen Peroxide Biosensor

Fluorescent, genetically encoded sensors of hydrogen peroxide have enabled visualization of perturbations to the intracellular level of this signaling molecule with subcellular and temporal resolution. Ratiometric sensors hold the additional promise of meaningful quantification of intracellular hydrogen peroxide levels as a function of time, a longstanding goal in the field of redox signaling. To date, studies that have connected the magnitudes of observed ratios with peroxide concentrations have either examined suspensions of cells or small numbers of adherent cells (∼10). In this work, we examined the response of all cells in several microscopic fields of view to an identical perturbation and observed a striking degree of heterogeneity of fluorescence ratios from individual cells. The expression level of the probe and phase within the cell cycle were each examined as potential contributors to the observed heterogeneity. Higher ratiometric responses correlated with greater expression levels of the probe and phase in the cell cycle were also shown to influence the magnitude of response. To aid in the interpretation of experimental observations, we incorporated the reaction of the reduced probe with peroxide and the reactions of the oxidized probe with glutathione and glutaredoxin into a larger kinetic model of peroxide metabolism. The predictions of the kinetic model suggest possible explanations for the experimental observations. This work highlights the importance of a systems-level approach to understanding the output of genetically encoded sensors that function via redox reactions involving thiol and disulfide groups.     

Role of Nitric Oxide and Hydrogen Peroxide During the Salt Resistance Response

Ion homeostasis is essential for plant cell resistance to salt stress. Under salt stress, to avoid cellular damage and nutrient deficiency, plant cells need to maintain adequate K nutrition and a favorable K to Na ratio in the cytosol. Recent observations revealed that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) act as signaling molecules to regulate K to Na ratio in calluses from Populus euphratica under salt stress. Evidence indicated that NO mediating H₂O₂ causes salt resistance via the action of plasma membrane H⁺-ATPase but that activity of plasma membrane NADPH oxidase is dependent on NO. Our study demonstrated the signaling transduction pathway. In this addendum, we proposed a testable hypothesis for NO function in regulation of H₂O₂ mediating salt resistance.Key Words: hydrogen peroxide, nitric oxide, signaling molecule, salt resistanceUnder salinity conditions, tolerant plant cells achieve ion homeostasis by extruding Na to the external medium and/or compartmentalizing into vacuoles, maintaining K uptake and high K and low Na in the cytosol.^1,2 Control of Na movement across the plasma membrane (PM) and tonoplast in order to maintain a low Na concentration in the cytoplasm is a key factor of cellular adaptation to salt stress.^3,4 Na transport across the PM is dependent on the electrochemical gradient created by the PM H⁺-ATPase.^5,6 It has been proven that the activity of the PM H⁺-ATPase is a key index of plant adaptation to salt stress.⁷ Therefore, the regulation of expression of the PM H⁺-ATPase may represent an important cellular mechanism for salt resistance. In contrast to our understanding of the regulation of PM H⁺-ATPase by other factors, the roles of NO and H₂O₂ act as signals under salt stress have been less known.Previous studies have shown that both NO and H₂O₂ function as stress signals in plants, mediating a range of resistance mechanisms in plants under stress conditions.^8–10 We have previously shown that NO serves as a signal in inducing salt resistance by increasing the K to Na ratio, which is dependent on the increased PM H⁺-ATPase activity in calluses from reed.¹¹ Although NO acts as a signal molecule under salt stress and induces salt resistance by increasing PM H⁺-ATPase activity, our research results also indicated NO can not activate purified PM H⁺-ATPase activity, at least in vitro. Subsequently, we set out to find the other signal molecules and factors between NO and PM H⁺-ATPase activity. Since our studies have indicated that NO can not induce salt resistance directly, what roles dose it play in salt resistance in tolerant cells under salt stress? We initially hypothesized ABA or H₂O₂ might be downstream signal molecules to regulate the activity of PM H⁺-ATPase. Further results indicated H₂O₂ content increased greatly under salt stress. Since H₂O₂ might be the candidate downstream signal molecule, we tested PM H⁺-ATPase activity and K to Na ratio in calluses by adding H₂O₂. The results suggested that H₂O₂ inducing an increased PM H⁺-ATPase activity resulted in an increased K to Na ratio. Summing up this new assay that allows us to speculate NO maybe regulate the H₂O₂ generation.Since H₂O₂ is involved in downstream signal molecule of NO, PM NADPH oxidase, the main source of H₂O₂ production, might be the regulated target of NO. We took a pharmacological approach to examine the speculation. The results indicated that PM NADPH oxidase is required for H₂O₂ accumulation and PM NADPH oxidase activity could attribute to NO in calluses under salt stress. These results also raised another question regarding what concentrations of NO can induce such effects. In our experiments, NO content was induced 1.6 times higher than the control values under salt treatment. We speculated there exists an effective balance point in NO signal system similar to previous reports by Delledonne et al.¹² in disease resistance.Further research work is required to decipher the mechanism through which NO and H₂O₂ acts and how K and Na elements uptake might be connected with salt resistance. We would like to propose a simple testable model that accounts for the results reported in this paper (Fig. 1). According to our model, H₂O₂ rather than NO is the major signaling molecular that mediated directly PM H⁺-ATPase under salt stress. Normally, NO generated from nitric oxide synthase (NOS) acts as a signal molecule to regulate other mechanisms. Under salt stress, accumulated NO activates PM NADPH oxidase activity. Then, a number of H₂O₂ is produced from PM NADPH oxidase. The PM H⁺-ATPase is activated greatly by the accumulated H₂O₂. Eventually, the transmembrane electrochemical gradient is created and K to Na ratio increases. The model we have proposed here is testable and should provide further insights into salt resistance mechanism regulated by NO and H₂O₂ signal molecules.Open in a separate windowFigure 1Hypothetical model for the potential function of NO and H₂O₂ as signaling molecules in inducing salt resistance. Salt stress activates a signal transduction cascade that leads to the increased activity of PM H⁺-ATPase, whose expression produces salt resistance. NO is generated by NOS, and H₂O₂ is produced by NADPH oxidase attributed to NO. The activity of PM H⁺-ATPase is regulated by H₂O₂ directly under salt stress. The model is based on the recent results in calluses from P. euphratica¹² and those previously reported on the NO function in reed.¹¹Research on roles of NO and H₂O₂ under stress conditions in plant is advancing rapidly. Further analysis of salt resistance mechanism with novel technology will certainly increase our knowledge in this field.     

Response of Lens Epithelial Cells to Hydrogen Peroxide Stress and the Protective Effect of Caloric Restriction

Hydrogen peroxide (H₂O₂) has been reported to be present at significant levels in the lens and aqueous humor in some cataract patients and suggested as a possible source of chronically inflicted damage to lens epithelial (LE) cells. We measured H₂O₂effects on bovine and mouse LE cells and determined whether LE cells from old calorically restricted mice were more resistant to H₂O₂-induced cellular damage than those of same age ad libitum fed (AL) mice. Bovine lens epithelial cells were exposed to H₂O₂at 40 or 400 μM for 2 h and then allowed to recover from the stress. The cells were assayed for DNA damage, DNA synthesis, cell viability, cell morphology, response to growth stimuli, and proliferation potential. Hydrogen peroxide-treated cells showed an increased DNA unwinding 50% greater than that for untreated controls. These DNA strand breaks appeared to be almost completely rejoined by 30 min following removal of the cells from a 2-h exposure. The 40 μM exposure did not produce a significantly lower DNA synthesis rate than the control, it responded to growth factor stimuli, and it replicated as did the control cells after removal of H₂O₂. The 400 μM H₂O₂severely affected DNA synthesis and replication, as shown by increased cell size and by markedly reduced clonal cell growth. The cells did not respond to growth stimulation by serum or growth factors and lost irreversibly the capacity to proliferate. The responses of LE cells from old adlib diet (AL) and calorically restricted (CR) mice to H₂O₂were significantly different. Exposure of LE cells to 20, 40, or 100 μM H₂O₂for 1 h induces a significant loss of cellular proliferation in cells from old AL mice. LE cells from long-term CR mice of the same strain and age were more resistant to oxidative damage at all three concentrations of H₂O₂than those of both old and young AL mice and showed a significantly higher proliferation potential following treatment. It is concluded that CR results in superior resistance to reactive oxygen radicals in the lens epithelium.     

Resistance of Photosynthesis to Hydrogen Peroxide in Algae

The effects of H₂O₂ on the photosynthetic fixation of CO₂ andon thiol-modulated enzymes involved in the photosynthetic reductionof carbon in algae were studied in a comparison with those inchloroplasts isolated from spinach leaves. In both systems,H₂O₂-scavenging enzymes were inhibited by addition of 0.1 mMNaN₃ 1 h prior to the addition of H₂O₂. A concentration (10^-4M) of H₂O₂ caused strong inhibition of the CO₂ fixation by intactspinach chloroplasts, as observed by Kaiser [(1976) Biochim.Biophys. Acta 440: 476], but not that by Euglena and Chlamydomonascells. The same results were also obtained with cells of thecyanobacteria Synechococcus PCC 7942 and Synechocystis PCC 6803in the presence of 1 mM hydroxylamine. These results indicatethat algal photosynthesis is rather resistant to H₂O₂. The insusceptibilityto H₂O₂ of thiolmodulated enzymes, namely, fructose-1,6-bisphosphatase,NADP-glyceraldehyde-3-phosphate dehydrogenase, and ribulose-5-phosphatekinase, was also observed in the chloroplasts of Euglena andChlamydomonas and in cyanobacterial cells. It seems likely thatthe resistance of photosynthesis to H₂O₂ is due in part to theinsusceptibility of the algal thiol-modulated enzymes to H₂O₂. (Received April 22, 1995; Accepted June 29, 1995)     

Hydrogen Peroxide Metabolism in Yeasts

A catalase-negative mutant of the yeast Hansenula polymorpha consumed methanol in the presence of glucose when the organism was grown in carbon-limited chemostat cultures. The organism was apparently able to decompose the H₂O₂ generated in the oxidation of methanol by alcohol oxidase. Not only H₂O₂ generated intracellularly but also H₂O₂ added extracellularly was effectively destroyed by the catalase-negative mutant. From the rate of H₂O₂ consumption during growth in chemostat cultures on mixtures of glucose and H₂O₂, it appeared that the mutant was capable of decomposing H₂O₂ at a rate as high as 8 mmol · g of cells⁻¹ · h⁻¹. Glutathione peroxidase (EC 1.11.1.9) was absent under all growth conditions. However, cytochrome c peroxidase (CCP; EC 1.11.1.5) increased to very high levels in cells which decomposed H₂O₂. When wild-type H. polymorpha was grown on mixtures of glucose and methanol, the CCP level was independent of the rate of methanol utilization, whereas the level of catalase increased with increasing amounts of methanol in the substrate feed. Also, the wild type decomposed H₂O₂ at a high rate when cells were grown on mixtures of glucose and H₂O₂. In this case, an increase of both CCP and catalase was observed. When Saccharomyces cerevisiae was grown on mixtures of glucose and H₂O₂, the level of catalase remained low, but CCP increased with increasing rates of H₂O₂ utilization. From these observations and an analysis of cell yields under the various conditions, two conclusions can be drawn. (i) CCP is a key enzyme of H₂O₂ detoxification in yeasts. (ii) Catalase can effectively compete with mitochondrial CCP for hydrogen peroxide only if hydrogen peroxide is generated at the site where catalase is located, namely in the peroxisomes.     

Hydrogen Peroxide in Human Blood

Blood and plasma of humans and rats were analyzed for hydrogen peroxide. The samples were analyzed after deproteinization with trichloroacetic acid, immediately after they were withdrawn from human volunteers or rats. A radio-isotopic technique based on peroxide-dependent decarboxylation of 1-¹⁴C-alpha-ketoacids and consequent liberation of ¹⁴CO₂ was used. The results demonstrate the presence ofmicromolar levels of H₂O₂, both, in the plasma as well as in the whole blood. The values in the whole blood were substantially greater than the plasma. This was true for rats as well as humans. The presence of such significant quantities of H₂O₂ in the blood have been demonstrated for the first time. The investigation, therefore, opens a newer avenue of research on diseases purported to be related to the generation of oxygen radicals in vivo.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.     

Hydrogen Peroxide Modification of Human Oxyhemoglobin

The effect of H₂O₂ on the primary structure of OxyHb was studied. Upon treatment of Oxy Hb with H₂O₂ ([Heme]/[H₂O₂] =I), tryptophan and methionine residues of the /-chain were modified. Treatment of ApoHb with H₂O₂ resulted in the modification of histidine and methionine residues in both globin chains. Tryptophan residues were unaffected. Modification of methionine residues in both the β-chain of OxyHb and ApoHb probably results from the direct oxidation of mcthionine by H₂O₂. The modification of histidine residues in ApoHb may be mediated by a metal-catalyzed oxidation system comprised of H₂O₂ and histidine-bound iron. The H₂O₂-mediated modification of tryptophan in the OxyHb β-chain. however, requires the heme moiety.