Chemistry, physiology and pathology of free radicals

The superoxide anion radical and other reactive oxygen species (ROS) are formed in all aerobic organisms by enzymatic and nonenzymatic reactions. ROS arise in both physiological and pathological processes, but efficient mechanisms have evolved for their detoxification. Similarly, reactive nitrogen intermediates (RNI) have physiological activity, but can also react with different types of molecules, including superoxide, to form toxic products. ROS and RNI participate in the destruction of microorganisms by phagocytes, as in the formation of a myeloperoxidase-hydrogen peroxide-chloride/iodide complex which can destroy many cells, including bacteria. It is known that the cellular production of ROS and RNI is controlled by different mechanisms. These free radicals can react with key cellular structures and molecules, thus altering their biological function. An imbalance between the systems producing and removing ROS and RNI may result in pathological consequences.     

Histochemical Detection of Quinone Reductase Activity In Situ Using LY 83583 Reduction and Oxidation

Abstract: The application of enzymatic staining techniques, using tetrazolium dyes, to aldehyde-treated brain sections has revealed the presence of NADPH-diaphorase activity attributed to nitric oxide synthase. When evaluating the specificity of the putative guanylyl cyclase inhibitor LY 83583, a robust and novel staining pattern was noted in epithelial, endothelial, and astrocytic cells when LY 83583 was included in the NADPH-diaphorase histochemical reaction. This LY 83583-dependent staining could be blocked by the NAD(P)H:quinone oxidoreductase inhibitor dicumarol. Based on its quinone structure, we hypothesized that LY 83583 was a substrate for the enzyme NAD(P)H:quinone oxidoreductase. Transfection of human embryonic kidney 293 cells with the rat liver isoform of NAD(P)H:quinone oxidoreductase resulted in robust NADPH- and LY 83583-dependent staining that was completely blocked by dicumarol and was not observed in untransfected cells. Analysis of transfected cell extracts and brain homogenates indicated that LY 83583 was a substrate for NAD(P)H:quinone oxidoreductase, with a K _m similar to the well-characterized substrate menadione. Sensitivity of the nitroblue tetrazolium reduction to superoxide dismutase indicated that the reduction of LY 83583 by NAD(P)H:quinone oxidoreductase leads to superoxide generation. The localization of NAD(P)H:quinone oxidoreductase activity to astrocytic cells suggests a role for glia in combating oxidative insults to brain and in activating quinone-like drugs such as LY 83583.     

Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ sensitization

Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca²⁺ ([Ca²⁺]_i), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC₂₀) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF_2α-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF₂α-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca²⁺]_i, but caused a Y27632-sensitive constriction in α-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC₂₀ at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca²⁺ sensitization and vasoconstriction independent of hydrogen peroxide.     

Superoxide formation as a result of interaction of L-lysine with dicarbonyl compounds and its possible mechanism

The EPR signal recorded in reaction medium containing L-lysine and methylglyoxal is supposed to come from the anion radical (semidione) of methylglyoxal and cation radical of methylglyoxal dialkylimine. These free radical inter-mediates might be formed as a result of electron transfer from dialkylimine to methylglyoxal. The EPR signal was observed in a nitrogen atmosphere, whereas only trace amounts of free radicals were registered under aerobic conditions. It has been established that the decay of methylglyoxal anion radical on aeration of the medium is inhibited by superoxide dismutase. Using the methods of EPR spectroscopy and lucigenin-dependent chemiluminescence, it has been shown that nonenzymatic generation of free radicals including superoxide anion radical takes place during the interaction of L-lysine with methylglyoxal — an intermediate of carbonyl stress — at different (including physiological) pH values. In the course of analogous reaction of L-lysine with malondialdehyde (the secondary product of the free radical derived oxidation of lipids), the formation of organic free radicals or superoxide radical was not observed.     

The one-electron reduction of uroporphyrin I by rat hepatic microsomes

Uroporphyrin I, which accumulates in body tissues of congenital erythropoietic porphyria patients, can undergo an enzymatic one-electron reduction to the porphyrin anion radical when a suitable reducing cofactor is present. We have demonstrated, in the absence of light, that anaerobic microsomal incubations containing NADPH and uroporphyrin I give an electron spin resonance spectrum consistent with the enzymatic formation of a porphyrin anion free radical. This radical undergoes a second-order decay (k2 approximately 10(5) M-1 s-1) due to nonenzymatic disproportionation of the radical. Aerobic microsomal incubations were also investigated for the reduction of oxygen to superoxide by monitoring oxygen consumption and the spin-trapping of superoxide. These experiments demonstrate that electron transfer from the porphyrin radical to molecular oxygen does occur, but due to the slow formation of the radical anion, no oxygen consumption above the basal level could be detected in the microsomal incubations. The photoreduction of uroporphyrin I in aerobic and anaerobic incubations was also investigated.     

The TrkAIII Oncoprotein Inhibits Mitochondrial Free Radical ROS-Induced Death of SH-SY5Y Neuroblastoma Cells by Augmenting SOD2 Expression and Activity at the Mitochondria,within the Context of a Tumour Stem Cell-like Phenotype

The developmental and stress-regulated alternative TrkAIII splice variant of the NGF receptor TrkA is expressed by advanced stage human neuroblastomas (NBs), correlates with worse outcome in high TrkA expressing unfavourable tumours and exhibits oncogenic activity in NB models. In the present study, we report that constitutive TrkAIII expression in human SH-SY5Y NB cells inhibits Rotenone, Paraquat and LY83583-induced mitochondrial free radical reactive oxygen species (ROS)-mediated death by stimulating SOD2 expression, increasing mitochondrial SOD2 activity and attenuating mitochondrial free radical ROS production, in association with increased mitochondrial capacity to produce H₂O₂, within the context of a more tumour stem cell-like phenotype. This effect can be reversed by the specific TrkA tyrosine kinase inhibitor {"type":"entrez-nucleotide","attrs":{"text":"GW441756","term_id":"315858226","term_text":"GW441756"}}GW441756, by the multi-kinase TrkA inhibitors K252a, CEP-701 and Gö6976, which inhibit SOD2 expression, and by siRNA knockdown of SOD2 expression, which restores the sensitivity of TrkAIII expressing SH-SY5Y cells to Rotenone, Paraquat and LY83583-induced mitochondrial free radical ROS production and ROS-mediated death. The data implicate the novel TrkAIII/SOD2 axis in promoting NB resistance to mitochondrial free radical-mediated death and staminality, and suggest that the combined use of TrkAIII and/or SOD2 inhibitors together with agents that induce mitochondrial free radical ROS-mediated death could provide a therapeutic advantage that may also target the stem cell niche in high TrkA expressing unfavourable NB.     

Neuroprotective Effect of Fucoidan on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis in PC12 Cells Via Activation of PI3K/Akt Pathway

One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the neuroprotective effect of fucoidan on H(2)O(2)-induced apoptosis in PC12 cells and the possible signaling pathways involved. The results showed that fucoidan inhibited the decrease of cell viability, scavenged ROS formation and reduced lactate dehydrogenase release in H(2)O(2)-induced PC12 cells. These changes were associated with an increase in superoxide dismutase and glutathione peroxidase activity, and reduction in malondialdehyde. In addition, fucoidan treatment inhibited apoptosis in H(2)O(2)-induced PC12 cells by increasing the Bcl-2/Bax ratio and decreasing active caspase-3 expression, as well as enhancing Akt phosphorylation (p-Akt). However, the protection of fucoidan on cell survival, p-Akt, the Bcl-2/Bax ratio and caspase-3 activity were abolished by pretreating with phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In consequence, fucoidan might protect the neurocytes against H(2)O(2)-induced apoptosis via reducing ROS levels and activating PI3K/Akt signaling pathway.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Photoinduced production of reactive oxygen species by retinal derivatives and conjugates

Formation of reactive oxygen species (ROS) during all-trans-retinal (ATR) illumination was studied by EPR. The quantity of ROS was estimated from the EPR signal of a spin adduct of DEPMPO (5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide). The EPR signal was found to be a superposition of the adducts: 80% superoxide anion radical, 15% hydroxyl radical, and 5% unknown radical. Albumin at an equimolar concentration decreased the relative quantum yield of ROS five times, and a 4-fold albumin excess decreased ROS production 30 times. It is supposed that ATR and A2E in the photoreceptor membrane are the sources of photodamage induced by ROS. Since ATR in the cell is known to be transported by interphotoreceptor retinal-binding proteins of the albumin family, its binding to protein is supposed to play an important protective role, preventing ROS production in the photoreceptor cell.     

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H₂DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.     

Production of reactive oxygen species by photosystem II

Photosysthetic cleavage of water molecules to molecular oxygen is a crucial process for all aerobic life on the Earth. Light-driven oxidation of water occurs in photosystem II (PSII) — a pigment-protein complex embedded in the thylakoid membrane of plants, algae and cyanobacteria. Electron transport across the thylakoid membrane terminated by NADPH and ATP formation is inadvertently coupled with the formation of reactive oxygen species (ROS). Reactive oxygen species are mainly produced by photosystem I; however, under certain circumstances, PSII contributes to the overall formation of ROS in the thylakoid membrane. Under limitation of electron transport reaction between both photosystems, photoreduction of molecular oxygen by the reducing side of PSII generates a superoxide anion radical, its dismutation to hydrogen peroxide and the subsequent formation of a hydroxyl radical terminates the overall process of ROS formation on the PSII electron acceptor side. On the PSII electron donor side, partial or complete inhibition of enzymatic activity of the water-splitting manganese complex is coupled with incomplete oxidation of water to hydrogen peroxide. The review points out the mechanistic aspects in the production of ROS on both the electron acceptor and electron donor side of PSII.     

Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells

Silibinin mostly has been used as hepatoprotectants, but it has other interesting activities, e.g. anti-cancer, cardial protective and brain-protective activities. A previous study demonstrated that silibinin protected amyloid β (Aβ)-induced mouse cognitive disorder by behavioural pharmacological observation. This study assessed the effect of silibinin on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. Subsequent morphologic observation, flow cytometric analysis and Western blot analysis indicated that treatment with SNP significantly induced apoptosis in PC12 cells. However, silibinin eliminated the apoptotic effect by reactive oxygen species (ROS) generation, especially hydroxyl free radical. Silibinin-induced autophagy through ROS generation when exerting a protective effect and silibinin-induced autophagy also enhanced the ROS generation since 3-methyladenine (3-MA), a specific autophagy inhibitor, decreased the ROS generation and rapamycin, an autophagy inducer, enhanced the ROS generation. Therefore, there exists a positive feedback loop between autophagy and ROS generation. Autophagy prevented SNP-induced apoptosis, since the addition of 3-MA significantly eliminated the protective effect of silibinin. This protective effect was attributed to the generation of ROS and its two downstream Ras/PI3K/NF-κB and Ras/Raf/MEK/ERK pathways. Both prevented PC12 cells from apoptosis. The PI3K/NF-κB pathway induced autophagy to protect PC12 cells, but the Raf/MEK/ERK pathway directly protected PC12 cells bypassing the autophagic effect.     

Protective effect of a synthetic anti-oxidant on neuronal cell apoptosis resulting from experimental hypoxia re-oxygenation injury

Oxidative stress is associated with the pathology of acute and chronic neurodegenerative disease. Cultured neuronal cells exposed to hypoxia-reoxygenation (H/R) injury, as a model for stroke, yield a burst of reactive oxygen species (ROS) as measured with electron paramagnetic resonance (EPR) spectroscopy in combination with spin trapping. Added superoxide dismutase inhibited spin-adduct formation verifying that superoxide radical anion was formed in neuronal cells following H/R injury. The intracellular ADP/ATP ratio increased rapidly over the first 5 h following injury and this was due primarily to the decreased cellular pools of ATP, consistent with the notion that H/R promotes mitochondrial dysfunction leading to decreased ATP reserve and increased ROS formation. As an early response to the enhanced oxidative stress, genes encoding for hypoxia-inducible factor 1-alpha (HIF1-alpha), inducible haemoxygenase-1 (HO-1), and the oxygen-sensor neuroglobin increased significantly. Up-regulation of the HO-1 gene was paralleled by increased HO protein expression and activity. Despite this cellular response, apoptosis increased significantly following H/R injury indicating that the endogenous anti-oxidant defenses were unable to protect the cells. In contrast, addition of a phenolic anti-oxidant, bisphenol (BP), prior to H/R injury, inhibited ROS production and gene regulation and significantly decreased neuronal cell apoptosis. Added BP was converted stoichiometrically to the corresponding diphenoquinone indicating the synthetic anti-oxidant effectively decreased oxidative stress through a radical scavenging mechanism. Together, these data indicate that BP has the potential to act as a neuro-protective drug.     

Reactive oxygen species, antioxidants and signaling in plants

Several reactive oxygen species (ROS) are continuously produced in plants as byproducts of many metabolic reactions, such as photosynthesis, photo respiration and respiration, Depending on the nature of the ROS species, some are highly toxic and rapidly detoxified by various cellular enzymatic and nonenzymatic mechanisms. Oxidative stress occurs when there is a serious imbalance between the production of ROS and antioxidative defence. ROS participate in signal transduction, but also modify cellular components and cause damage. ROS is highly reactive molecules and can oxidize all types of cellular components. Various enzymes involved in ROS-scavenging have been manipulated and over expressed or down regulated. An overview of the literature is presented in terms of primary antioxidant free radical scavenging and redox signaling in plant cells. Special attention is given to ROS and ROS-anioxidant interaction as a metabolic interface for different types of signals derived from metabolisms and from the changing environment.     

Cyclic adenosine 5′‐diphosphoribose (cADPR) cyclic guanosine 3′,5′‐monophosphate positively function in Ca2+ elevation in methyl jasmonate‐induced stomatal closure,cADPR is required for methyl jasmonate‐induced ROS accumulation NO production in guard cells

Methyl jasmonate (MeJA) signalling shares several signal components with abscisic acid (ABA) signalling in guard cells. Cyclic adenosine 5′‐diphosphoribose (cADPR) and cyclic guanosine 3′,5′‐monophosphate (cGMP) are second messengers in ABA‐induced stomatal closure. In order to clarify involvement of cADPR and cGMP in MeJA‐induced stomatal closure in Arabidopsis thaliana (Col‐0), we investigated effects of an inhibitor of cADPR synthesis, nicotinamide (NA), and an inhibitor of cGMP synthesis, LY83583 (LY, 6‐anilino‐5,8‐quinolinedione), on MeJA‐induced stomatal closure. Treatment with NA and LY inhibited MeJA‐induced stomatal closure. NA inhibited MeJA‐induced reactive oxygen species (ROS) accumulation and nitric oxide (NO) production in guard cells. NA and LY suppressed transient elevations elicited by MeJA in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in guard cells. These results suggest that cADPR and cGMP positively function in [Ca²⁺]_cyt elevation in MeJA‐induced stomatal closure, are signalling components shared with ABA‐induced stomatal closure in Arabidopsis, and that cADPR is required for MeJA‐induced ROS accumulation and NO production in Arabidopsis guard cells.     

Role of ROS in the protective effect of silibinin on sodium nitroprusside-induced apoptosis in rat pheochromocytoma PC12 cells

Abstract

Silibinin mostly has been used as hepatoprotectants, but it has other interesting activities, e.g. anti-cancer, cardial protective and brain-protective activities. A previous study demonstrated that silibinin protected amyloid β (Aβ)-induced mouse cognitive disorder by behavioural pharmacological observation. This study assessed the effect of silibinin on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. Subsequent morphologic observation, flow cytometric analysis and Western blot analysis indicated that treatment with SNP significantly induced apoptosis in PC12 cells. However, silibinin eliminated the apoptotic effect by reactive oxygen species (ROS) generation, especially hydroxyl free radical. Silibinin-induced autophagy through ROS generation when exerting a protective effect and silibinin-induced autophagy also enhanced the ROS generation since 3-methyladenine (3-MA), a specific autophagy inhibitor, decreased the ROS generation and rapamycin, an autophagy inducer, enhanced the ROS generation. Therefore, there exists a positive feedback loop between autophagy and ROS generation. Autophagy prevented SNP-induced apoptosis, since the addition of 3-MA significantly eliminated the protective effect of silibinin. This protective effect was attributed to the generation of ROS and its two downstream Ras/PI3K/NF-κB and Ras/Raf/MEK/ERK pathways. Both prevented PC12 cells from apoptosis. The PI3K/NF-κB pathway induced autophagy to protect PC12 cells, but the Raf/MEK/ERK pathway directly protected PC12 cells bypassing the autophagic effect.     

Claulansine F suppresses apoptosis induced by sodium nitroprusside in PC12 cells

Abstract

Reactive oxygen species (ROS) are known to be involved in many neurodegenerative diseases. This study assessed the effect of Claulansine F, a new carbazole isolated from Clausena lansium, on sodium nitroprusside (SNP)-treated rat pheochromocytoma PC12 cells. First, it was found that Claulansine F showed more potential on inhibiting the programmed death of PC12 cells than edaravone by cell viability, morphologic observation, and flow cytometric analysis. Further results also showed that Claulansine F attenuated the production of total intracellular ROS formation and lipid peroxidation in PC12 cells, inhibited the mitochondrial membrane potential (MMP) loss, and prevented the programmed cell death event via the P53/Bcl-2 family pathway. Its protective effect was likely medicated by the hydroxyl radical (·OH) scavenging ability, as it appeared to be not involved in the natural antioxidant system. These results suggested a promising potential for Claulansine F as a ROS scavenger in pathologies, where an oxidative stress is involved.     

Neuregulin rescues PC12-ErbB4 cells from cell death induced by H(2)O(2). Regulation of reactive oxygen species levels by phosphatidylinositol 3-kinase

Neuregulins (NRGs), a large family of transmembrane polypeptide growth factors, mediate various cellular responses depending on the cell type and receptor expression. We previously showed that NRG mediates survival of PC12-ErbB4 cells from apoptosis induced by serum deprivation or tumor necrosis factor-alpha treatment. In the present study we show that NRG induces a significant protective effect from H(2)O(2)-induced death. This effect of NRG is mediated by the phosphatidylinositol 3-kinase (PI3K)-signaling pathway since NRG failed to rescue cells from H(2)O(2) insult in the presence of the PI3K inhibitor, LY294002. Furthermore, the downstream effector of PI3K, protein kinase B/AKT, is activated by NRG in the presence of H(2)O(2), and protein kinase B/AKT activation is inhibited by LY294002. In addition, our results demonstrate that reactive oxygen species (ROS) elevation induced by H(2)O(2) is inhibited by NRG. LY294002, which blocks NRG-mediated rescue, increases ROS levels. Moreover, both H(2)O(2)-induced ROS elevation and cell death are reduced by expression of activated PI3K. These results suggest that PI3K-dependent pathways may regulate toxic levels of ROS generated by oxidative stress.     

Oxygen-sensitive and -insensitive nitroreduction by Escherichia coli and rat hepatic microsomes.

Nitrofurazone is shown to undergo an initial 1-electron (oxygen-sensitive) or 2- or more electron (oxygen-insensitive) reduction by partially purified nitroreductases from Escherichia coli. Nitrofurazone (50 micronM) is reduced by the oxygen-sensitive reductase to a nitro anion free radical as indicated by ESR and visible spectroscopy. The visible spectrum of the nitro anion free radical is characterized by an increase in absorption at 406 nm. In the presence of the oxygen-sensitive reductase, nitrofurazone stimulates superoxide formation and oxygen consumption. This enzyme gives a steady state radical concentration which is proportional to the square root of the enzyme concentration, suggesting that the nitrofurazone anion radical is an obligate intermediate in the reduction and that the radical decays by a nonenzymatic second order process. The oxygen-insensitive reductase does not form the nitro anion free radical nor in the presence of nitrofurazone does it stimulate oxygen consumption. Visible spectroscopy shows that nitrofurazone is reduced by the oxygen-sensitive reductase to a species with an absorption maximum at 335 nm, which has been previously identified as the amine. The oxygen-insensitive reductase reduces nitrofurazone to a previously identified cyano derivative with an absorption maximum at 280 nm. Rat hepatic microsomes appear to metabolize nitrofurazone in a manner similar to the oxygen-sensitive E. coli reductase.