Aerenchyma formation and associated oxygen movement in seminal and nodal roots of wheat

Abstract The present paper describes the effects of growth of roots of wheat (Triticum aestivum cv. Gamenya) in hypoxic nutrient solutions on acrenchyma formation and O₂ movement from shoots to roots. Two types of roots were investigated: (1) seminal roots of 4–7-d-old seedlings, and (2) seminal and nodal roots of 10–28-d-old plants. Gas-filled porosity of seminal and nodal roots increased from 3 to 12% and from 5–7 to 11–15%, respectively, when the roots emerged in stagnant or N₂-flushed solutions (0.003 mol m ^?3 O₂) compared with growth in continuously acrated solutions (0.26 mol m ^?3 O₂). However, neither root type increased in porosity when they were longer than 100–200 mm at the start of the exposure to these stagnant or N₂-flushed treatments. A vernier microscope and cylindrical platinum-electrode were used to examine the relationship between root extension and transport of O₂ from shoots to roots via the gas spaces. Measurements were made when the roots were in an anoxic medium and were dependent solely on O₂ supplied from the shoots. For seminal roots of 5–7-d-old seedlings raised in stagnant solutions (90–100 mm), internal O₂ transport was sufficient to support a rate of root elongation in the O₂-free medium of between 0.03 and 0.17 mm h^?1. When the O₂ pressure around the shoots was increased from 20 to 100 kPa O₂, the O₂ concentrations at the walls of the expanding zone (2–7 mm from the tip) of these roots increased from 0.006 mol m^?3 to between 0.04 and 0.26 mol m^?3, and the rate of root extension increased five-fold. Oxygen transport to roots grown continuously in acrated solutions was considerably less than for roots raised in stagnant solutions; this difference was greater for seminal than for nodal roots. When the acrated seminal roots were longer than 100 mm and transferred to an O₂-free root medium, O₂ concentration became zero at the root tip causing elongation to cease. After 24 h of anoxia, none of these roots were able to resume elongation following a return to acrated solutions.     

Accumulation of reactive oxygen species in arbuscular mycorrhizal roots

We investigated the accumulation of reactive oxygen species (ROS) in arbuscular mycorrhizal (AM) roots from Medicago truncatula, Zea mays and Nicotiana tabacum using three independent staining techniques. Colonized root cortical cells and the symbiotic fungal partner were observed to be involved in the production of ROS. Extraradical hyphae and spores from Glomus intraradices accumulated small levels of ROS within their cell wall and produced ROS within the cytoplasm in response to stress. Within AM roots, we observed a certain correlation of arbuscular senescence and H₂O₂ accumulation after staining by diaminobenzidine (DAB) and a more general accumulation of ROS close to fungal structures when using dihydrorhodamine 123 (DHR 123) for staining. According to electron microscopical analysis of AM roots from Z. mays after staining by CeCl₃, intracellular accumulation of H₂O₂ was observed in the plant cytoplasm close to intact and collapsing fungal structures, whereas intercellular H₂O₂ was located on the surface of fungal hyphae. These characteristics of ROS accumulation in AM roots suggest similarities to ROS accumulation during the senescence of legume root nodules.     

Visualization of reactive oxygen species formation by phagocytizing macrophages

Activated peritoneal macrophages exhibiting phagocytosing capacity produced an electron-dense precipitate of formazan in contact sites of macrophage plasmalemma and phagocytosed yeast cells. No production of formazan occurred, when non-opsonized latex particles were ingested by macrophages. Formazan precipitation could be prevented by anaerobiosis but not by addition of cyanide.     

The coleoptile sheath of wheat impedes oxygen transport to seminal roots

Abstract Wheat, Triticum aestivum cv. Gamenya, seedlings were grown for 4d in acrated 0.5 mol m^?3 CaSO₄ solution. A cylindrical platinum-electrode and vernier microscope were used to examine the effects of the colcoptile sheath on O₂ transport from shoots to seminal roots via the internal gas-space system, and on the clongation of the root tip. By removing the colcoptile sheath, O₂ concentrations at the root tip in an O₂-free medium at 5°C increased from 0.017 to 0.17 mol m^?3 O₂ when 100 kPa O₂ was around the shoots. When the shoots were in air, the separation of the colcoptile sheath from the primary leaf caused the rate of root clongation in the anoxic medium to increase by two fold.     

Lysigenous aerenchyma formation in maize root is confined to cortical cells by regulation of genes related to generation and scavenging of reactive oxygen species

To adapt to waterlogging, maize (Zea mays) forms lysigenous aerenchyma in root cortex as a result of ethylene-promoted programmed cell death (PCD). Respiratory burst oxidase homolog (RBOH) gene encodes a homolog of gp91^phox in NADPH oxidase, and has a role in the generation of reactive oxygen species (ROS). Recently, we found that during aerenchyma formation, RBOH was upregulated in all maize root tissues examined, whereas an ROS scavengingrelated metallothionein (MT) gene was downregulated specifically in cortical cells. Together these changes should lead to high accumulations of ROS in root cortex, thereby inducing PCD for aerenchyma formation. As further evidence of the involvement of ROS in root aerenchyma formation, the PCD was inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor. Based on these results, we propose a model of cortical cell-specific PCD for root aerenchyma formation.Key words: aerenchyma, ethylene, laser microdissection, maize (Zea mays), metallothionein, programmed cell death, reactive oxygen species, respiratory burst oxidase homologIn both wetland and non-wetland plants, lysigenous aerenchyma is formed in roots by creating gas spaces as a result of death and subsequent lysis of some cortical cells, and allows internal transport of oxygen from shoots to roots under waterlogged soil conditions.^1–3 In rice (Oryza sativa) and some other wetland plant species, lysigenous aerenchyma is constitutively formed under aerobic conditions, and is further enhanced under waterlogged conditions.⁴ On the other hand, in non-wetland plants, including maize (Zea mays), lysigenous aerenchyma does not normally form under well-drained soil conditions, but is induced by waterlogging.⁵ Ethylene is involved in lysigenous aerenchyma formation,^1–3,6,7 but the molecular mechanisms are unclear.We recently identified two reactive oxygen species (ROS)-related genes that were specifically regulated in maize root cortex by waterlogged conditions, but not in the presence of an ethylene perception inhibitor 1-methylcyclopropene (1-MCP).⁵ One was respiratory burst oxidase homolog (RBOH), which has a role in ROS generation and the other was metallothionein (MT), which has a role in ROS scavenging. These results suggest that ROS has a role in ethylene signaling in the PCD that occurs during lysigenous aerenchyma formation.     

Histone acetylation and reactive oxygen species are involved in the preprophase arrest induced by sodium butyrate in maize roots

Histone acetylation plays a critical role in controlling chromatin structure, and reactive oxygen species (ROS) are involved in cell cycle progression. To study the relationship between histone acetylation and cell cycle progression in plants, sodium butyrate (NaB), a histone deacetylase (HDAC) inhibitor that can cause a significant increase in histone acetylation in both mammal and plant genomes, was applied to treat maize seedlings. The results showed that NaB had significant inhibition effects on different root zones at the tissue level and caused cell cycle arrest at preprophase in the root meristem zones. This effect was accompanied by a dramatic increase in the total level of acetylated lysine 9 on histone H3 (H3K9ac) and acetylated lysine 5 on histone H4 (H4K5ac). The exposure of maize roots in NaB led to a continuous rise of intracellular ROS concentration, accompanied by a higher electrolyte leakage ratio and malondialdehyde (MDA) relative value. The NaB-treated group displayed negative results in both TdT-mediated dUTP nick end labelling (TUNEL) and γ-H2AX immunostaining assays. The expression of topoisomerase genes was reduced after treatment with NaB. These results suggested that NaB increased the levels of H3K9ac and H4K5ac and could cause preprophase arrest accompanied with ROS formation leading to the inhibition of DNA topoisomerase.     

Replicative senescence induced by Romo1-derived reactive oxygen species

Persistent accumulation of DNA damage induced by reactive oxygen species (ROS) is proposed to be a major contributor toward the aging process. Furthermore, an increase in age-associated ROS is strongly correlated with aging in various species, including humans. Here we showed that the enforced expression of the ROS modulator 1 (Romo1) triggered premature senescence by ROS production, and this also contributed toward induction of DNA damage. Romo1-derived ROS was found to originate in the mitochondrial electron transport chain. Romo1 expression gradually increased in proportion to population doublings of IMR-90 human fibroblasts. An increase in ROS production in these cells with high population doubling was blocked by the Romo1 knockdown using Romo1 small interfering RNA. Romo1 knockdown also inhibited the progression of replicative senescence. Based on these results, we suggest that age-related ROS levels increase, and this contributes to replicative senescence, which is directly associated with Romo1 expression.     

Hypoxia-induced reactive oxygen species formation in skeletal muscle.

The existence of hypoxia-induced reactive oxygen species (ROS) production remains controversial. However, numerous observations with a variety of methods and in many cells and tissue types are supportive of this idea. Skeletal muscle appears to behave much like heart in that in the early stages of hypoxia there is a transient elevation in ROS, whereas in chronic exposure to very severe hypoxia there is evidence of ongoing oxidative stress. Important remaining questions that are addressed in this review include the following. Are there levels of PO2 in skeletal muscle, typical of physiological or mildly pathophysiological conditions, that are low enough to induce significant ROS production? Does the ROS associated with muscle contractile activity reflect imbalances in oxygen uptake and demand that drive the cell to a more reduced state? What are the possible molecular mechanisms by which ROS may be elevated in hypoxic skeletal muscle? Is the production of ROS in hypoxia of physiological significance, both with respect to cell signaling pathways promoting cell function and with respect to damaging effects of long-term exposure? Discussion of these and other topics leads to general conclusions that hypoxia-induced ROS may be a normal physiological response to imbalance in oxygen supply and demand or environmental stress and may play a yet undefined role in normal response mechanisms to these stimuli. However, in chronic and extreme hypoxic exposure, muscles may fail to maintain a normal redox homeostasis, resulting in cell injury or dysfunction.     

Hypoxia induced non-apoptotic cellular changes during aerenchyma formation in rice (Oryza sativa L.) roots

The stress of low oxygen concentrations in a waterlogged environment is minimized in some plants that produce aerenchyma, a tissue characterized by prominent intercellular spaces. It is produced by the predictable collapse of root cortex cells, indicating a programmed cell death (PCD) and facilitates gas diffusion between root and the aerial environment. The objective of this study was to characterize the cellular changes take place during aerenchyma formation in root of rice that accompany PCD. Scanning electron microscopy and transmission electron microscopy were used for cellular analysis of roots. Aerenchyma development was observed in both aerobic and flooded conditions. Structural changes in membranes and organelles were examined during development of root cortex cells to compare with previous examples of PCD. There was an initial collapse which started at a specific position in the mid cortex, indicating loss of turgor, and the cytoplasm became more electron dense. These cells were distinct in shape from those located towards the periphery. Mitochondria and endoplasmic reticulum appeared normal at this early stage though the tonoplast lost its integrity. Subsequently it underwent further degeneration while the plasmalemma retracted from the cell wall followed by death of neighboring cells followed a radial path. However, pycnosis of the nucleus, blebbing of plasma membrane and production of apoptotic bodies were not found which in turn indicated nonapoptotic PCD during aerenchyma formation in rice.     

Epidermal growth factor rescues trophoblast apoptosis induced by reactive oxygen species

Pre-eclampsia and intrauterine growth restriction are associated with increased apoptosis of placental villous trophoblast. This may result from placental hypoperfusion, leading to the generation of reactive oxygen species (ROS). Apoptosis can be induced in villous trophoblast following exposure to oxidative stress. Epidermal growth factor (EGF) reduces trophoblast apoptosis resulting from exposure to hypoxia. We hypothesised that exposure to hydrogen peroxide, a potent generator of ROS, would induce apoptosis in term placental villous explants and that this could be reduced by treatment with EGF. Placental explants were taken from normal term pregnancies and exposed to increasing doses of hydrogen peroxide (0–1,000 μM) or to a combination of increasing doses of hydrogen peroxide and EGF (0–100 ng/ml) for either 6 or 48 h. Apoptosis was assessed by TUNEL, proliferation by Ki-67 immunostaining, necrosis by lactate dehydrogenase activity and trophoblast differentiation by human chorionic gonadotrophin (hCG) secretion in conditioned culture media. Immunoperoxidase staining was performed to identify phosphorylated-AKT (p-AKT) and phosphorylated-PI3 kinase (p-PI3k). Exposure to 1,000 μM hydrogen peroxide for 48 h induced apoptosis in placental explants. The increase in TUNEL positive nuclei predominantly localised to syncytiotrophoblast. The amount of apoptosis was reduced to control levels by treatment with 10 and 100 ng/ml EGF. Proliferation of cytotrophoblasts within villous explants was significantly reduced following exposure to 1,000 μM hydrogen peroxide, this was restored to control levels by simultaneous treatment with 10 or 100 ng/ml EGF. Neither exposure to hydrogen peroxide or EGF altered the amount of necrosis. There was increased immunostaining for pPI3K following treatment with EGF. This study shows that apoptosis may be induced in villous trophoblast following exposure to ROS, and demonstrates the anti-apoptotic effect of EGF in trophoblast, the maintenance of which is essential for normal pregnancy.     

Zinc induces mitogen-activated protein kinase activation mediated by reactive oxygen species in rice roots.

It is well known that zinc (Zn) is one of the micronutrients essential for normal growth and development of plants. However, the molecular mechanisms responsible for the regulation of plant growth by Zn are still not completely understood. The aim of this study was to investigate the signalling transduction pathways activated by Zn. We show that Zn elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot analysis, we suggest that Zn-activated 40- and 42-kDa MBP kinases are mitogen-activated protein kinases (MAPK). Pre-treatment of rice roots with reactive oxygen species (ROS) scavenger, sodium benzoate, was able to effectively prevent Zn-induced MAPK activation. However, phosphoinositide 3-kinase (PI-3K) inhibitor, LY294002, was unable to inhibit Zn-induced MAPK activation. These results suggest that the ROS may function in the Zn-triggered MAPK signalling pathway in rice roots.     

Signal transduction by reactive oxygen species

Although historically viewed as purely harmful, recent evidence suggests that reactive oxygen species (ROS) function as important physiological regulators of intracellular signaling pathways. The specific effects of ROS are modulated in large part through the covalent modification of specific cysteine residues found within redox-sensitive target proteins. Oxidation of these specific and reactive cysteine residues in turn can lead to the reversible modification of enzymatic activity. Emerging evidence suggests that ROS regulate diverse physiological parameters ranging from the response to growth factor stimulation to the generation of the inflammatory response, and that dysregulated ROS signaling may contribute to a host of human diseases.     

Temperature effects on the reactive oxygen species formation and antioxidant defence in roots of two cucurbit species with contrasting root zone temperature optima

Suboptimal root zone temperature (14°C) was imposed on chilling-sensitive cucumber (Cucumis sativus L.) and chilling-tolerant figleaf gourd (Cucurbita ficifolia Bouché) plants. Exposure of roots to low temperature for up to 10 days caused a strong growth inhibition in cucumber compared with figleaf gourd. Physiological analysis showed that generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion was significantly induced in cucumber plants as fast as 1 day after low root zone temperature treatment. In addition to the significant induction of antioxidant superoxide dismutase activity, low root zone temperature also increased the mitochondrial electron transport allocated to alternative pathway while decreased cytochrome pathway salicylhydroxamic acid-resistant respiration. However, these defense responses could not compensate for the ROS production, resulting in membrane lipid peroxidation and loss of root cell viability in the low root zone temperature treated cucumber roots. In contrast, 14°C root zone temperature had no significant effects on figleaf gourd plant growth, antioxidant enzymes, ROS levels and alternative respiratory pathway. Hence, difference in ROS metabolism would be associated with the remarkable difference in adaptability of cucumber and figleaf gourd plants in response to suboptimal root zone temperature condition.     

Apoptosis development pathways in human lymphocytes induced by UV light and reactive oxygen species

We have studied alterations in the structural state of DNA, the level of membrane Fas-receptor expression, functional activity of caspase-3, the concentration of Ca²⁺, p53 and cytochrome c proteins in human lymphocyte cells in the dynamics of apoptosis, induced by UV light (240–390 nm) at doses of 151, 1510, and 3020 J/m² and reactive oxygen species (ROS): superoxide anion radical, hydroxyl radical, hydrogen peroxide, and singlet oxygen. It was established that UV light and ROS induce lymphocyte DNA fragmentation after the incubation of a modified cell for 20 h. It was shown that in 1–5 h after UV light and ROS exposure on lymphocytes, an increase is observed in the level of membrane death Fas-receptors as compared to intact cells. Enhancement was revealed in the functional activity of lymphocyte caspase-3 4 h after the generation of singlet oxygen, hydroxyl radical, and the addition of hydrogen peroxide, as well as 8 and 24 h and 6 and 8 h of UV irradiation of cells at doses of 151 and 1510 J/m², respectively. Using the DNA comet approach, it was revealed that DNA damage (single-stranded breaks) appears approximately 15–20 min after UV irradiation of lymphocytes at doses of 1510 and 3020 J/m² and the addition of hydrogen peroxide at a concentration of 10⁻⁶ mol/L (comets of the C1 type) and reaches its maximum 6 h after cell modification (comets of the C2 and C3 types). Six hours after exposure of lymphocytes to hydrogen peroxide and UV light at doses of 1510 and 3020 J/m², it was established that the p53 level increased in the investigated cells. It was established that under UV light exposure and exogenous generation of reactive oxygen species, the increase in the calcium level in lymphocyte cytoplasm is determined by Ca²⁺ efflux from the intracellular depots as a result of activation of the components of the phosphoinositide information transmission mechanism to a cell. A hypothesis was proposed on the correlation between changes in the calcium level and initiation of programmed cell death in human lymphocytes after UV light and ROS exposure. It was concluded that the lead role is played by receptor-mediated (Fas-dependent) caspase and p53-dependent pathways in the development of lymphocyte apoptosis induced by exposure to UV light at doses of 151 and 1510 J/m² and reactive oxygen metabolites. A scheme is presented which considers possible intracellular events leading to apoptotic death of lymphocytes after UV irradiation.     

Mutagenicity induced by UVC in Escherichia coli cells: reactive oxygen species involvement

We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen ((1)O(2)) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an (1)O(2) chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that (1)O(2) plays a predominant role in UVC-induced mutagenesis. (1)O(2) generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC-TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC-TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC-TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.     

Ways of realizing apoptosis of human lymphocytes induced by UV-light and reactive oxygen species

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.     

Stimulation of ethylene production and gas-space (aerenchyma) formation in adventitious roots of Zea mays L. by small partial pressures of oxygen

Adventitious roots of two to four-weekold intact plants of Zea mays L. (cv. LG11) were shorter but less dense after extending into stagnant, non-aerated nutrient solution than into solution continuously aerated with air. Dissolved oxygen in the non-aerated solutions decreased from 21 kPa to 3–9 kPa within 24 h. When oxygen partial pressures similar to those found in non-aerated solutions (3, 5 and 12 kPa) were applied for 7 d to root systems growing in vigorously bubbled solutions, the volume of gas-space in the cortex (aerenchyma) was increased several fold. This stimulation of aerenchyma was associated with faster ethylene production by 45-mm-long apical root segments. When ethylene production by roots exposed to 5 kPa oxygen was inhibited by aminoethoxyvinylglycine (AVG) dissolved in the nutrient solution, aerenchyma formation was also retarded. The effect of AVG was reversible by concomitant applications of 1-aminocyclopropane-1-carboxylic acid, an immediate precursor of ethylene. Addition of silver nitrate, an inhibitor of ethylene action, to the nutrient solution also prevented the development of aerenchyma in roots given 5 kPa oxygen. Treating roots with only 1 kPa oxygen stimulated ethylene production but failed to promote gas-space formation. These severely oxygen-deficient roots seemed insensitive to the ethylene produced since a supplement of exogeneous ethylene that promoted aerenchyma development in nutrient solution aerated with air (21 kPa oxygen) failed to do so in nutrient solution supplied with 1 kPa oxygen. Both ethylene production and aerenchyma formation were almost completely halted when roots were exposed to nutrient solutions devoid of oxygen. Thus both processes require oxygen and are stimulated by oxygen-deficient surroundings in the 3-to 12-kPa range of oxygen partial pressures when compared with rates observed in air (21 kPa oxygen).Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine     

Detection of reactive oxygen species induced by radiation in cells using the dichlorofluorescein assay

The goal of this study was to determine the amount of reactive oxygen species (ROS) that arises inside cells irradiated in medium containing blood serum using the 2'7'-dichlorofluorescein (DCF) assay. DCF fluorescence in cells and medium was recorded on an MF44 Perkin Elmer fluorimeter, and fluorescence in cells only was recorded on a Partec flow-through cytometer. Human larynx tumor HEp-2 cells and lympholeukosis P388 cells were irradiated with X rays at a dose rate of 1.12 Gy/min. The factors (temperature, pH, serum concentration) affecting the oxidation of 2'7'-dichlorofluorescin (DCFH) to DCF were studied, and errors in the dichlorofluorescein assay of ROS were minimized. The amount of ROS registered by the DCF assay in cells was found to depend on the concentration of serum in the medium during irradiation. In the presence of 10% serum, radiation had no effect on the amount of detectable ROS. The effect of radiation on the formation of intracellular ROS was almost completely abolished if the irradiated medium was removed immediately after radiation exposure. The increase in the formation of ROS in cells irradiated in medium with a low serum content is due mainly to the radiolytic products of water that arise in medium and oxidize DCFH located in cells.     

Ras regulation by reactive oxygen and nitrogen species

Ras GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. We have previously shown that both NO/O(2) (via nitrogen dioxide, (*)NO(2)) and superoxide radical anion (O(2)(*)(-)) promote Ras guanine nucleotide dissociation. We now show that hydrogen peroxide in the presence of transition metals (i.e., H(2)O(2)/transition metals) and peroxynitrite also trigger radical-based Ras guanine nucleotide dissociation. The primary redox-active reaction species derived from H(2)O(2)/transition metals and peroxynitrite is O(2)(*)(-) and (*)NO(2), respectively. A small fraction of hydroxyl radical (OH(*)) is also present in both. We also show that both carbonate radical (CO(3)(*)(-)) and (*)NO(2), derived from the mixture of peroxynitrite and bicarbonate, facilitate Ras guanine nucleotide dissociation. We further demonstrate that NO/O(2) and O(2)(*)(-) promote Ras GDP exchange with GTP in the presence of a radical-quenching agent, ascorbate, or NO, and generation of Ras-GTP promotes high-affinity binding of the Ras-binding domain of Raf-1, a downstream effector of Ras. S-Nitrosylated Ras (Ras-SNO) can be formed when NO serves as a radical-quenching agent, and hydroxyl radical but not (*)NO(2) or O(2)(*)(-) can further react with Ras-SNO to modulate Ras activity in vitro. However, given the lack of redox specificity associated with the high redox potential of OH(*), it is unclear whether this reaction occurs under physiological conditions.