Epr analysis reveals three tissues responding to endotoxin by increased formation of reactive oxygen and nitrogen species

The excessive formation of reactive oxygen and nitrogen species (RONS) in tissue has been implicated in the development of various diseases. In this study we adopted ex vivo low temperature EPR spectroscopy combined with spin trapping technique to measure local RONS levels in frozen tissue samples. CP-H (1-hydroxy-3-carboxy-pyrrolidine), a new nontoxic spin probe, was used to analyze RONS in vivo. In addition, nitrosyl complexes of hemoglobin were determined to trace nitric oxide released into blood. By this technique we found that RONS formation in tissue of control animals increased in the following order: liver < heart < brain < cerebellum < lung < muscle < blood < ileum < kidney < duodenum < jejunum. We also found that endotoxin challenge, which represents the most common model of septic shock, increased the formation of RONS in rat liver, heart, lung, and blood, but decreased RONS formation in jejunum. We did not find changes in RONS levels in other parts of gut, brain, skeletal muscles, and kidney. Scavenging of RONS by CP-H was accompanied by an increase in blood pressure, indicating that LPS-induced vasodilatation may be due to RONS, but not due to nitric oxide. Experiments with tissue homogenates incubated in vitro with CP-H showed that ONOO⁻ and O₂^•−, as well as other not identified RONS, are detectable by CP-H in tissue. In summary, low-temperature EPR combined with CP-H infusion allowed detection of local RONS formation in tissues. Increased formation of RONS in response to endotoxin challenge is organ specific.     

The reactive oxygen species are involved in resistance responses of wheat to the Russian wheat aphid

The effect of Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), infestation on the hydrogen peroxide (H(2)O(2)) content and NADPH oxidase (EC 1.6.3.1) activity was studied in the resistant (cv. Tugela DN) and near-isogenic susceptible (cv. Tugela) wheat (Triticum aestivum L.). The objective of this study was to investigate the involvement of the reactive oxygen species (ROS) during the resistance responses against the RWA. Infestation significantly induced an early accumulation of the H(2)O(2) and increase of NADPH oxidase activity to higher levels in the resistant than susceptible plants. Results of inhibitory studies using diphenylene iodonium (DPI), a suicide inhibitor of NADPH oxidase, strongly suggested a possible signalling role for H(2)O(2) during RWA resistance response by activation of downstream defence enzymes [intercellular peroxidase (EC 1.11.1.7) and beta-1,3-glucanase (EC 3.2.1.39)].     

Oxygen and nitrogen are pro-carcinogens. Damage to DNA by reactive oxygen, chlorine and nitrogen species: measurement, mechanism and the effects of nutrition.

Humans are exposed to many carcinogens, but the most significant may be the reactive species derived from metabolism of oxygen and nitrogen. Nitric oxide seems unlikely to damage DNA directly, but nitrous acid produces deamination and peroxynitrite leads to both deamination and nitration. Scavenging of reactive nitrogen species generated in the stomach may be an important role of flavonoids, flavonoids and other plant-derived phenolic compounds. Different reactive oxygen species produce different patterns of damage to DNA bases, e.g., such patterns have been used to implicate hydroxyl radical as the ultimate agent in H(2)O(2)-induced DNA damage. Levels of steady-state DNA damage in vivo are consistent with the concept that such damage is a major contributor to the age-related development of cancer and so such damage can be used as a biomarker to study the effects of diet or dietary supplements on risk of cancer development, provided that reliable assays are available. Methodological questions addressed in this article include the validity of measuring 8-hydroxydeoxyguanosine (8OHdG) in cellular DNA or in urine as a biomarker of DNA damage, the extent of artifact formation during analysis of oxidative DNA damage by gas chromatography-mass spectrometry and the levels of oxidative damage in mitochondrial DNA.     

Copper induces apoptosis in BA/F3beta cells: Bax, reactive oxygen species, and NFkappaB are involved

Copper, an essential trace element, can be toxic to some cells when present in excess. But thorough investigations into the cytotoxicity of copper and subsequent molecular mechanisms are rare, although the cytotoxicity of copper has been applied to cancer chemotherapy. The present study demonstrates that Cu(2+) inhibits [(3)H] thymidine incorporation in mouse pro-B cell line BA/F3beta and induces apoptosis. Apoptosis was mainly judged by morphology of cells, quantification of subdiploid DNA contents by flow cytometry, and detection of DNA fragmentation by gel electrophoresis. The apoptotic effect is dose and time dependent. Western blotting shows Bax is upregulated by Cu(2+). Bcl-2 overexpression can partially inhibit this apoptosis. Moreover, Cu(2+) increases the production of reactive oxygen species (ROS) in a dose-dependent manner. The antioxidant N-acetylcysteine (NAC) not only significantly inhibited copper-induced apoptosis but also totally blocked generation of ROS, while Bcl-2 overexpression has no effect on the generation of ROS. Furthermore, our results show that NFkappaB is downregulated by Cu(2+). Bcl-2 overexpression or NAC can sustain the activity of NFkappaB. These data indicate that Cu(2+) might induce apoptosis in BA/F3beta cells via upregulation of Bax and ROS and subsequent inactivation of NFkappaB.     

NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and arabidopsis

Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.     

Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species

Eosinophil recruitment and enhanced production of NO are characteristic features of asthma. However, neither the ability of eosinophils to generate NO-derived oxidants nor their role in nitration of targets during asthma is established. Using gas chromatography-mass spectrometry we demonstrate a 10-fold increase in 3-nitrotyrosine (NO(2)Y) content, a global marker of protein modification by reactive nitrogen species, in proteins recovered from bronchoalveolar lavage of severe asthmatic patients (480 +/- 198 micromol/mol tyrosine; n = 11) compared with nonasthmatic subjects (52.5 +/- 40.7 micromol/mol tyrosine; n = 12). Parallel gas chromatography-mass spectrometry analyses of bronchoalveolar lavage proteins for 3-bromotyrosine (BrY) and 3-chlorotyrosine (ClY), selective markers of eosinophil peroxidase (EPO)- and myeloperoxidase-catalyzed oxidation, respectively, demonstrated a dramatic preferential formation of BrY in asthmatic (1093 +/- 457 micromol BrY/mol tyrosine; 161 +/- 88 micromol ClY/mol tyrosine; n = 11 each) compared with nonasthmatic subjects (13 +/- 14.5 micromol BrY/mol tyrosine; 65 +/- 69 micromol ClY/mol tyrosine; n = 12 each). Bronchial tissue from individuals who died of asthma demonstrated the most intense anti-NO(2)Y immunostaining in epitopes that colocalized with eosinophils. Although eosinophils from normal subjects failed to generate detectable levels of NO, NO(2-), NO(3-), or NO(2)Y, tyrosine nitration was promoted by eosinophils activated either in the presence of physiological levels of NO(2-) or an exogenous NO source. At low, but not high (e.g., >2 microM/min), rates of NO flux, EPO inhibitors and catalase markedly attenuated aromatic nitration. These results identify eosinophils as a major source of oxidants during asthma. They also demonstrate that eosinophils use distinct mechanisms for generating NO-derived oxidants and identify EPO as an enzymatic source of nitrating intermediates in eosinophils.     

Analytical methods for 3-nitrotyrosine as a marker of exposure to reactive nitrogen species: a review.

Nitric oxide (NO*) is a diatomic free radical which has recently been found to have a key role in both normal physiological processes and disease states. The presence of NO in biological systems leads to the formation of reactive nitrogen species (RNS) such as peroxynitrite which reacts avidly with tyrosine residues in proteins to form nitrotyrosine (NTYR). Since peroxynitrite has a very short half-life at neutral pH, the presence of NTYR has been used as a marker of RNS production in various tissues. A number of methods for separation, detection, and quantitation of NTYR in biological samples have been developed. These methods include immunochemical techniques such as immunhistochemistry, ELISA, and Western blotting, high-performance liquid chromatography (HPLC) in combination with various detection systems including UV and electrochemical detection (ECD), gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), and electrospray mass spectrometry. In terms of sensitivity and specificity, it would appear that methods based on combinations of HPLC and various types of ECD are very versatile giving a limit of detection of 20 fmol per injection of protein hydrolysate. They are only limited by the sample quantity and the preparation that is required to achieve acceptable chromatograms. In addition to the detection of NTYR as a marker of RNS, its role in biological systems may be more subtle with nitration of key tyrosine residues likely to profoundly affect cellular function such as signaling cascades. Further advances are likely to be made in the localization of NTYR residues in peptide fragments using mass spectrometry.     

Sex as a response to oxidative stress: a twofold increase in cellular reactive oxygen species activates sex genes

Organisms are constantly subjected to factors that can alter the cellular redox balance and result in the formation of a series of highly reactive molecules known as reactive oxygen species (ROS). As ROS can be damaging to biological structures, cells evolved a series of mechanisms (e.g. cell-cycle arrest, programmed cell death) to respond to high levels of ROS (i.e. oxidative stress). Recently, we presented evidence that in a facultatively sexual lineage--the multicellular green alga Volvox carteri--sex is an additional response to increased levels of stress, and probably ROS and DNA damage. Here we show that, in V. carteri, (i) sex is triggered by an approximately twofold increase in the level of cellular ROS (induced either by the natural sex-inducing stress, namely heat, or by blocking the mitochondrial electron transport chain with antimycin A), and (ii) ROS are responsible for the activation of sex genes. As most types of stress result in the overproduction of ROS, we believe that our findings will prove to extend to other facultatively sexual lineages, which could be indicative of the ancestral role of sex as an adaptive response to stress and ROS-induced DNA damage.     

Sublethal concentrations of salicylic acid decrease the formation of reactive oxygen species but maintain an increased nitric oxide production in the root apex of the ethylene-insensitive Never ripe tomato mutants

The pattern of salicylic acid (SA)-induced production of reactive oxygen species (ROS) and nitric oxide (NO) were different in the apex of adventitious roots in wild-type and in the ethylene-insensitive Never ripe (Nr) mutants of tomato (Solanum lycopersicum L. cv Ailsa Craig). ROS were upregulated, while NO remained at the control level in apical root tissues of wildtype plants exposed to sublethal concentrations of SA. In contrast, Nr plants expressing a defective ethylene receptor displayed a reduced level of ROS and a higher NO content in the apical root cells. In wild-type plants NO production seems to be ROS(H₂O₂)-dependent at cell death-inducing concentrations of SA, indicating that ROS and NO may interact to trigger oxidative cell death. In the absence of significant ROS accumulation, the increased NO production caused moderate reduction in cell viability in root apex of Nr plants exposed to 10⁻³ M SA. This suggests that a functional ethylene signaling pathway is necessary for the control of ROS and NO production induced by SA.Key words: ethylene receptor mutant, never ripe, nitric oxide, reactive oxygen species, root apex, salicylic acid, tomatoSeveral signal molecules, including salicylic acid (SA) have been implicated in the response of plants to biotic^1–3 and abiotic stressors.^4–6 SA was identified as a central regulator of local defense against (hemi)biotophic pathogens inducing a hypersensitive response (HR), which is characterized by the development of lesions that restrict pathogen spread. It has also emerged as a possible signaling component involved in the activation of certain plant defense responses in non-infected part of the plants establishing the systemic acquired resistance (SAR).⁷The SA-induced biotic and abiotic stress adaptation most likely involves reactive oxygen species (ROS) and nitric oxide (NO) in primary signaling events that activate multiple signal transduction pathways. SA-induced ROS is required for the activation of antioxidant defense mechanisms⁴ and if the generation of ROS exceeds the capacity of antioxidant systems, the cells die.⁸ NO is another important player that is required for the induction of defense mechanisms⁹ or for ROS-induced cell death.¹⁰Accumulation of SA, and two other plant hormones, ethylene (ET) and jasmonic acid (JA) are intimately associated with the initiation or spread of cell death. In HR SA and ROS have been proposed to be on a positive feedback loop that amplifies signals and leads to programmed cell death (PCD). Ethylene caused increased spreading of cell death, while lesion containment can be achieved by JA through decreasing the sensitivity of the cells to ethylene and through the suppression of SA biosynthesis and signaling.⁸Ethylene evolution is associated with diverse physiological processes such as leaf and flower senescence, abscission of organs and fruit ripening.¹¹ The biosynthesis of ethylene is stimulated by a variety of abiotic and biotic stress factors. Ethylene overproducing mutants (eto1 and eto3) of Arabidopsis were found to be more sensitive to O₃, an abiotic stressor which induces ROS-dependent cell death.¹² Cadmium-induced cell death was also accompanied by increased production of ethylene and simultaneously by H₂O₂ accumulation in tomato cell suspension, and based on the effect of specific inhibitors of ethylene biosynthesis and action the authors concluded that the cell death process required H₂O₂ production and a functional ethylene signaling pathway.¹³ Ethylene signaling is also required for the susceptible disease response of tomato plants infected with Xanthomonas campestris pv vesicatoria.¹⁴ It was found that the accumulation of SA and increased production of ethylene were important components of the disease symptoms of this pathogen in wild-type plants, while in Never ripe (Nr) mutants, which have a non-functional ethylene receptor, the infected plants failed to accumulate SA, produced less ethylene, and the leaves exhibited reduced necrotic lesions.It has been also shown that SA enhances NO synthesis in a dose-dependent manner.¹⁵ ROS, such as ^·O₂⁻ and H₂O₂ as well as NO can act together in the cell death regulation and propagation.^8,16 The compartment-specific (down)regulation of ROS can be controlled by NO, accordingly, ROS and NO homeostasis may be essential for the induction or for the avoidance of cell death.     

The reactive oxygen species network pathways: an essential prerequisite for perception of pathogen attack and the acquired disease resistance in plants

Availability of complete Arabidopsis(Arabidopsis thaliana) and rice(Oryza sativa) genome sequences, together with molecular recourses of functional genomics and proteomics have revolutionized our understanding of reactive oxygen species (ROS) signalling network mediating disease resistance in plants. So far, ROS have been associated with aging, cellular and molecular alteration in animal and plant cells. Recently, concluding evidences suggest that ROS network is essential to induce disease resistance and even to mediate resistance to multiple stresses in plants. ROS are obligatory by-products emerging as a result of normal metabolic reactions. They have the potential to be both beneficial and harmful to cellular metabolism. Their dual effects on metabolic reactions are dosage specific. In this review we focus our attention on cellular ROS level to trigger beneficial effects on plant cells responding to pathogen attack. By exploring the research related contributions coupled with data of targeted gene disruption, and RNA interference approaches, we show here that ROS are ubiquitous molecules of redox-pathways that play a crucial role in plant defence mechanism. The molecular prerequisites of ROS network to activate plant defence system in response to pathogen infections are here underlined. Bioinformatic tools are now available to scientists for high throughput analysis of cellular metabolisms. These tools are used to illustrate crucial ROS-related genes that are involved in the defence mechanism of plants. The review describes also the emerging findings of ROS network pathways to modulate multiple stress resistance in plants.     

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.     

Similar stress responses are elicited by copper and ultraviolet radiation in the aquatic plant Lemna gibba: implication of reactive oxygen species as common signals

Metals and ultraviolet (UV) radiation are two environmental stressors that can cause damage to plants. These two types of stressors often impact simultaneously on plants and both are known to promote reactive oxygen species (ROS) production. However, little information is available on the potential parallel stress responses elicited by metals and UV radiation. Using the aquatic plant Lemna gibba, we found that copper and simulated solar radiation (SSR, a light source containing photosynthetically active radiation (PAR) and UV radiation) induced similar responses in the plants. Both copper and SSR caused ROS formation. The ROS levels were higher when copper was combined with SSR than when applied with PAR. Higher concentrations of copper plus PAR caused toxicity as monitored by diminished growth and chlorophyll content. This toxicity was more pronounced when copper was combined with SSR. Because the generation of ROS was also higher when copper was combined with SSR, we attributed this enhanced toxicity to elevated levels of ROS. In comparison to PAR-grown plants, SSR treated plants exhibited elevated levels of superoxide dismutase (SOD) and glutathione reductase (GR). These enzyme levels were further elevated under both PAR and SSR when copper was added at concentrations that generated ROS. Interestingly, copper treatment in the absence of SSR (i.e. copper plus PAR) induced synthesis of the same flavonoids as those observed in SSR without copper. Finally, addition of either dimethyl thiourea or GSH (two common ROS scavengers) lowered in vivo ROS production, alleviated toxicity and diminished induction of GR as well as accumulation of UV absorbing compounds. Thus, the potential of ROS being a common signal for acclimation to stress by both copper and UV can be considered.     

The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves

To investigate the role of stress in nitrogen management in plants, the effect of pathogen attack, elicitors, and phytohormone application on the expression of the two senescence-related markers GS1 (cytosolic glutamine synthetase EC 6.3.1.2) and GDH (glutamate dehydrogenase, EC 1.4.1.2) involved in nitrogen mobilization in senescing leaves of tobacco (Nicotiana tabacum L.) plants, was studied. The expression of genes involved in primary nitrogen assimilation such as GS2 (chloroplastic glutamine synthetase) and Nia (nitrate reductase, EC 1.6.1.1) was also analysed. The Glubas gene, coding a beta-1,3-glucanase, was used as a plant-defence gene control. As during natural senescence, the expression of GS2 and Nia was repressed under almost all stress conditions. By contrast, GS1 and GDH mRNA accumulation was increased. However, GS1 and GDH showed differential patterns of expression depending on the stress applied. The expression of GS1 appeared more selective than GDH. Results indicate that the GDH and GS1 genes involved in leaf senescence are also a component of the plant defence response during plant-pathogen interaction. The links between natural plant senescence and stress-induced senescence are discussed, as well as the potential role of GS1 and GDH in a metabolic safeguard process.     

The role of vitamin B6 as an antioxidant in the presence of vitamin B2-photogenerated reactive oxygen species. A kinetic and mechanistic study

We report on the photostability of a mixture of vitamins B6 and B2 (riboflavin, Rf) upon visible light irradiation and on the possible role of the vitamin B6 family (B6D) as deactivators of reactive oxygen species (ROS). The work is a systematic kinetic and mechanistic study under conditions in which only Rf absorbs photoirradiation. Pyridoxine, pyridoxal hydrochloride, pyridoxal phosphate and pyridoxamine dihydrochloride were studied as representative members of the vitamin B6 family. The visible light irradiation of dissolved Rf and B6D in pH 7.4 aqueous medium under aerobic conditions induces photoprocesses that mainly produce B6D degradation. The overall oxidative mechanism involves the participation of ROS. Photogenerated (3)Rf* is quenched either by oxygen, giving rise to O(2)((1)Δ(g)) by electronic energy transfer to dissolved ground state oxygen, or by B6D yielding, through an electron transfer process, the neutral radical RfH˙, and O(2)˙(-) in an subsequent step. B6D act as quenchers of O(2)((1)Δ(g)) and O(2)˙(-), the former in a totally reactive event that also inhibits Rf photoconsumption. The common chromophoric moiety of B6D represented by 3-hydroxypyridine, constitutes an excellent model that mimics the kinetic behavior of the vitamin as an antioxidant towards Rf-generated ROS. The protein lysozyme, taken as an O(2)((1)Δ(g))-mediated oxidizable biological target, is photoprotected by B6D from Rf-sensitized photodegradation through the quenching of electronically excited triplet state of the pigment, in a process that competes with O(2)((1)Δ(g)) generation.