Hydroxyl radicals are not involved in NADPH dependent microsomal lipid peroxidation

Summary NADPH dependent H₂O₂ formation in microsomes in the presence of chelated iron leads to formation of hydroxyl radicals. Enhancement of hydroxyl radical generation (via ferric-EDTA or sodium azide) did not result in a concomitant increase in lipid peroxidation; rather, a decrease was observed. Moreover, the hydroxyl radical scavenger DMSO did not inhibit lipid peroxidation. This comparison of hydroxyl radical formation with lipid peroxidation suggests that hydroxyl radicals do not play a part in NADPH-dependent lipid peroxidation.     

Hydrogen peroxide protects tobacco from oxidative stress by inducing a set of antioxidant enzymes

Tolerance against oxidative stress generated by high light intensities or the catalase inhibitor aminotriazole (AT) was induced in intact tobacco plants by spraying them with hydrogen peroxide (H₂O₂). Stress tolerance was concomitant with an enhanced antioxidant status as reflected by higher activity and/or protein levels of catalase, ascorbate peroxidase, guaiacol peroxidases, and glutathione peroxidase, as well as an increased glutathione pool. The induced stress tolerance was dependent on the dose of H₂O₂ applied. Moderate doses of H₂O₂ enhanced the antioxidant status and induced stress tolerance, while higher concentrations caused oxidative stress and symptoms resembling a hypersensitive response. In stress-tolerant plants, induction of catalase was 1.5-fold, that of ascorbate peroxidase and glutathione peroxidase was 2-fold, and that of guaiacol peroxidases was approximately 3-fold. Stress resistance was monitored by measuring levels of malondialdehyde, an indicator of lipid peroxidation. The levels of malondialdehyde in all H₂O₂-treated plants exposed to subsequent high light or AT stress were similar to those of unstressed plants, whereas lipid peroxidation in H₂O₂-untreated plants stressed with either high light or AT was 1.5- or 2-fold higher, respectively. Although all stress factors caused increases in the levels of reduced glutathione, its levels were much higher in all H₂O₂-pretreated plants. Moreover, significant accumulation of oxidized glutathione was observed only in plants that were not pretreated with H₂O₂. Extending the AT stress period from 1 to 7 days resulted in death of tobacco plants that were not pretreated with H₂O₂, while all H₂O₂-pretreated plants remained little affected by the prolonged treatment. Thus, activation of the plant antioxidant system by H₂O₂ plays an important role in the induced tolerance against oxidative stress. Received 11 December 2001; received after revision 25 January 2002; accepted 4 February 2002     

Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress

We investigated the effects of ischemia duration on the functional response of mitochondria to reperfusion and its relationship with changes in mitochondrial susceptibility to oxidative stress. Mitochondria were isolated from hearts perfused by the Langendorff technique immediately after different periods of global ischemia or reperfusion following such ischemia periods. Rates of O₂ consumption and H₂O₂ release with complex I- and complex II-linked substrates, lipid peroxidation, overall antioxidant capacity, capacity to remove H₂O₂, and susceptibility to oxidative stress were determined. The effects of ischemia on some parameters were time dependent so that the changes were greater after 45 than after 20 min of ischemia, or were significantly different to the nonischemic control only after 45 min of ischemia. Thus, succinate-supported state 3 respiration exhibited a significant decrease after 20 min of ischemia and a greater decrease after 45 min, while pyruvate malate-supported respiration showed a significant decrease only after 45 min of ischemia, indicating an ischemia-induced early inhibition of complex II and a late inhibition of complex I. Furthermore, both succinate and pyruvate malate-supported H₂O₂ release showed significant increases only after 45 min of ischemia. Similarly, whole antioxidant capacity significantly increased and susceptibility to oxidants significantly decreased after 45 min of ischemia. Such changes were likely due to the accumulation of reducing equivalents, which are able to remove peroxides and maintain thiols in a reduced state. This condition, which protects mitochondria against oxidants, increases mitochondrial production of oxyradicals and oxidative damage during reperfusion. This could explain the smaller functional recovery of the tissue and the further decline of the mitochondrial function after reperfusion following the longer period of oxygen deprivation. Received 18 May 2001; received after revision 17 July 2001; accepted 24 July 2001     

The effect of hydrogen peroxide on the cyclin D expression in fibroblasts

Activation of mitogen-activated protein (MAP) kinase is essential for cyclin D1 expression and provides a link between mitogenic signalling and cell cycle progression. Hydrogen peroxide (H₂ O₂ ) activates MAP kinase; however, it is not known whether this leads to cyclin D expression. Sustained expression of cyclin D1 and D2 was observed when Her14 fibroblasts were incu-bated with 3 mM or higher H₂ O₂ concentrations. Similar results were obtained when cells were incubated in the presence of serum (FCS). However, the sustained expres-complex sion of cyclin D1 and D2 upon H₂ O₂ treatment was not due to the MAP kinase pathway, because MAP kinase kinase inhibitors did not inhibit cyclin D expression. Furthermore, cyclin D1 and D2 levels remained constant even after addition of a protein synthesis inhibitor, indicating that the effect of H₂ O₂ was not due to induction of protein synthesis. These results indicate that H₂ O₂ reversibly inhibits the ubiquitin-proteasome dependent degra-dation of cyclin D1 and D2, probably by transiently in-hibiting ubiquitination and/or the proteasome. Received 12 March 2001; received after revision 5 April 2001; accepted 9 April 2001     

Lipid flippases and their biological functions

The typically distinct phospholipid composition of the two leaflets of a membrane bilayer is generated and maintained by bi-directional transport (flip-flop) of lipids between the leaflets. Specific membrane proteins, termed lipid flippases, play an essential role in this transport process. Energy-independent flippases allow common phospholipids to equilibrate rapidly between the two monolayers and also play a role in the biosynthesis of a variety of glycoconjugates such as glycosphingolipids, N-glycoproteins, and glycosylphosphatidylinositol (GPI)-anchored proteins. ATP-dependent flippases, including members of a conserved subfamily of P-type ATPases and ATP-binding cassette transporters, mediate the net transfer of specific phospholipids to one leaflet of a membrane and are involved in the creation and maintenance of transbilayer lipid asymmetry of membranes such as the plasma membrane of eukaryotes. Energy-dependent flippases also play a role in the biosynthesis of glycoconjugates such as bacterial lipopolysaccharide. This review summarizes recent progress on the identification and characterization of the various flippases and the demonstration of their biological functions. Received 12 April 2006; received after revision 22 June 2006; accepted 30 August 2006     

The superoxide-generating NADPH oxidase: structural aspects and activation mechanism

Flavocytochrome b ₅₅₈ is the catalytic core of the respiratory-burst oxidase, an enzyme complex that catalyzes the NADPH-dependent reduction of O₂ into the superoxide anion O₂ ^- in phagocytic cells. Flavocytochrome b ₅₅₈ is anchored in the plasma membrane. It is a heterodimer that consists of a large glycoprotein gp91phox (phox for phagocyte oxidase) (β subunit) and a small protein p22phox (α subunit). The other components of the respiratory-burst oxidase are water-soluble proteins of cytosolic origin, namely p67phox, p47phox, p40phox and Rac. Upon cell stimulation, they assemble with the membrane-bound flavocytochrome b ₅₅₈ which becomes activated and generates O₂ ^-. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections, illustrating the role of O₂ ^- and the derived metabolites H₂O₂ and HOCl in host defense against invading microorganisms. The electron carriers, FAD and hemes b, and the binding site for NADPH are confined to the gp91phox subunit of flavocytochrome b ₅₅₈ . The p22phox subunit serves as a docking site for the cytosolic phox proteins. This review provides an overview of current knowledge on the structural organization of the O₂ ^--generating flavocytochrome b ₅₅₈ , its kinetics, its mechanism of activation and the regulation of its biosynthesis. Homologues of gp91phox, called Nox and Duox, are present in a large variety of non-phagocytic cells. They exhibit modest O₂ ^--generating oxidase activity, and some act as proton channels. Their role in various aspects of signal transduction is currently under investigation and is briefly discussed. Received 28 May 2002; received after revision 20 June 2002; accepted 24 June 2002     

One lipid, multiple functions: how various pools of PI(4,5)P2 are created in the plasma membrane

Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is a minor lipid of the inner leaflet of the plasma membrane that controls the activity of numerous proteins and serves as a source of second messengers. This multifunctionality of PI(4,5)P₂ relies on mechanisms ensuring transient appearance of PI(4,5)P₂ clusters in the plasma membrane. One such mechanism involves phosphorylation of PI(4)P to PI(4,5)P₂ by the type I phosphatidylinositol-4-phosphate 5-kinases (PIP5KI) at discrete membrane locations coupled with PI(4)P delivery/synthesis at the plasma membrane. Simultaneously, both PI(4)P and PI(4,5)P₂ participate in anchoring PIP5KI at the plasma membrane via electrostatic bonds. PIP5KI isoforms are also selectively recruited and activated at the plasma membrane by Rac1, talin, or AP-2 to generate PI(4,5)P₂ in ruffles and lamellipodia, focal contacts, and clathrin-coated pits. In addition, PI(4,5)P₂ can accumulate at sphingolipid/cholesterol-based rafts following activation of distinct membrane receptors or be sequestered in a reversible manner due to electrostatic constrains posed by proteins like MARCKS.     

Oxidative stress triggers neuronal caspase-independent death: Endonuclease G involvement in programmed cell death-type III

To characterize neuronal death, primary cortical neurons (C57/Black 6 J mice) were exposed to hydrogen peroxide (H₂O₂) and staurosporine. Both caused cell shrinkage, nuclear condensation, DNA fragmentation and loss of plasma membrane integrity. Neither treatment induced caspase-7 activity, but caspase-3 was activated by staurosporine but not H₂O₂. Each treatment caused redistribution from mitochondria of both endonuclease G (Endo G) and cytochrome c. Neurons knocked down for Endo G expression using siRNA showed reduction in both nuclear condensation and DNA fragmentation after treatment with H₂O₂, but not staurosporine. Endo G suppression protected cells against H₂O₂-induced cell death, while staurosporine-induced death was merely delayed. We conclude that staurosporine induces apoptosis in these neurons, but severe oxidative stress leads to Endo G-dependent death, in the absence of caspase activation (programmed cell death-type III). Therefore, oxidative stress triggers in neurons a form of necrosis that is a systematic cellular response subject to molecular regulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Über die Aktivitätssteigerung mikrobieller Neuraminidasen bei niedrigen Wasserstoffperoxid-Konzentrationen

Summary The activity of bacterial neuraminidases can be influenced by H₂O₂ secreted by some organisms. Thus, at low H₂O₂ concentrations (0.002–0.01%), an increase of neuraminidase activities was observed, whereas at concentrations 0.1% the enzyme activities were destroyed.     

Wip1 protects hydrogen peroxide-induced colonic epithelial barrier dysfunction

Tight junctions (TJs) create a paracellular permeability barrier. Although reactive oxygen species have been implicated as mediators of inflammation in inflammatory bowel diseases, their influence on the function of colonic epithelial TJs remains unknown. Oxidative stress-mediated colonic epithelial permeability was significantly attenuated by a p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Although the amount of TJ proteins was not altered, hydrogen peroxide (H₂O₂) changed the localization of claudin-4 protein from an NP-40 insoluble fraction to a soluble fraction and from an apical TJ to lateral membrane. The p38 MAP kinase inactivator Wip1 significantly attenuated phosphorylation of p38 MAP kinase, and oxidative stress mediated permeability. H₂O₂-induced changes in claudin-4 localization were abolished by SB203580 pretreatment as well as Wip1-expressing adenovirus infection. This is the first study to demonstrate that exogenous Wip1 functions to protect oxidative stress-mediated colonic mucosal permeability and that H₂O₂-induced claudin-4 dislocalization is abolished by Wip1. Received 14 June 2007; received after revision 8 October 2007; accepted 8 October 2007     

Functional and biochemical characteristics of mitochondrial fractions from rat liver in cold-induced oxidative stress

We determined characteristics of rat liver mitochondrial fractions, resolved at 1000 (M₁), 3000 (M₃), and 10,000 g (M₁₀) after 2 and 10 days cold exposure. In all groups, the M₁ fraction exhibited the highest oxidative capacity, oxidative damage, H₂O₂ production rate, and susceptibility to stress conditions, and the lowest antioxidant levels. Cold exposure increased cytochrome oxidase activity in all fractions and succinate-supported O₂ consumption in the M₁ and M₁₀ fractions during state 3 and state 4 respiration, respectively. With succinate, the H₂O₂ release rate increased in all fractions during state 4 and state 3 respiration, whereas with pyruvate/malate, it increased only during state 4 respiration. Increases in tissue mitochondrial proteins caused a faster H₂O₂ flow from the mitochondrial to cytosolic compartment, which was limited by the reduction in the M₁ fraction. Despite increased liposoluble antioxidant levels, cold also caused enhanced oxidative damage and susceptibility to oxidative challenge and Ca²⁺-induced swelling in all fractions. These changes leading to elimination of H₂O₂-overproducing mitochondria avoided excessive tissue damage. We propose that triiodothyronine, whose levels increase in the cold environment, brings about the biochemical changes producing oxidative damage and those limiting its extent.Received 16 July 2004; received after revision 27 September 2004; accepted 18 October 2004     

Role of phospholipids in the binding activity of vasoactive intestinal peptide receptors

Summary Phospholipase digestion of rat intestinal epithelial cell membranes was performed in order to study the influence of membrane phospholipids on the binding activity of VIP receptors. Phospholipases A₂ and C strougly (ED₅₀4×10^–2 and 4×10^–1 g/ml, respectively) and rapidly reduced¹²⁵I-VIP binding to membranes whereas phospholipase D was ineffective. This suggests an important role of both hydrophobic and hydrophilic groups of phospholipids on VIP receptor binding activity.This work was supported by INSERM (CRL 827017) and the Fondation pour la Recherche Médicale Française.     

Autophagic activity in cortical neurons under acute oxidative stress directly contributes to cell death

Primary neurons undergo insult-dependent programmed cell death. We examined autophagy as a process contributing to cell death in cortical neurons after treatment with either hydrogen peroxide (H₂O₂) or staurosporine. Although caspase-9 activation and cleavage of procaspase-3 were significant following staurosporine treatment, neither was observed following H₂O₂ treatment, indicating a non-apoptotic death. Autophagic activity increased rapidly with H₂O₂, but slowly with staurosporine, as quantified by processing of endogenous LC3. Autophagic induction by both stressors increased the abundance of fluorescent puncta formed by GFP-LC3, which could be blocked by 3-methyladenine. Significantly, such inhibition of autophagy blocked cell death induced by H₂O₂ but not staurosporine. Suppression of Atg7 inhibited cell death by H₂O₂, but not staurosporine, whereas suppression of Beclin 1 prevented cell death by both treatments, suggesting it has a complex role regulating both apoptosis and autophagy. We conclude that autophagic mechanisms are activated in an insult-dependent manner and that H₂O₂ induces autophagic cell death.     

Pressure acclimation of the eel and liver membrane composition

Homeoviscous adaptation of membrane fluidity is known to exist in fish living at great depths. Is this adaptation also present in fish living near the surface but experimentally acclimated to high pressure? The composition of mitochondria-rich fractions extracted from the livers of eels acclimated for 15 days at 101 ATA was determined. The results show that pressure induced a significant increase (+100%) of total phospholipids (PL) and cholesterol without a change in their ratio. The increase of PL content was accompanied by a decrease in phosphatidylcholine in favour of phosphatidyl ethanolamine which, due to its preference for the H₁₁ form, is able to compensate for the loss in fluidity induced by pressure.     

Oxidative stress and hypoxia-like injury cause Alzheimer-type molecular abnormalities in central nervous system neurons

Neuronal loss and neuritic/cytoskeletal lesions (synaptic disconnection and proliferation of dystrophic neurites) represent major dementia-associated abnormalities in Alzheimer’s disease (AD). This study examined the role of oxidative stress as a factor contributing to both the cell death and neuritic degeneration cascades in AD. Primary neuron cultures were treated with H₂O₂ (9–90 μM) or desferrioxamine (2–25 μM) for 24 h and then analyzed for viability, mitochondrial mass, mitochondrial function, and pro-apoptosis and sprouting gene expression. H₂O₂ treatment causes free-radical injury and desferrioxamine causes hypoxia-type injury without free radical generation. The H₂O₂-treated cells exhibited sustained viability but neurite retraction, impaired mitochondrial function, increased levels of the pro-apoptosis gene product CD95/Fas, reduced expression of N2J1-immunoreactive neuronal thread protein and synaptophysin, and reduced distribution of mitochondria in neuritic processes. Desferrioxamine treatment resulted in dose-dependent neuronal loss associated with impaired mitochondrial function, proliferation of neurites, and reduced expression of GAP-43, which has a role in path-finding during neurite outgrowth. The results suggest that oxidative stress can cause neurodegeneration associated with enhanced susceptibility to apoptosis due to activation of pro-apoptosis genes, neurite retraction (synaptic disconnection), and impaired transport of mitochondria to cell processes where they are likely required for synaptic function. In contrast, hypoxia-type injury causes neuronal loss with proliferation of neurites (sprouting), impaired mitochondrial function, and reduced expression of molecules required to form and maintain synaptic connections. Since similar abnormalities occur in AD, both oxidative stress and hypoxic injury can contribute to AD neurodegeneration. Received 24 May 2000; received after revision 7 July 2000; accepted 27 July 2000     

Chronic hydrogen peroxide intake and peroxide metabolizing enzyme activities in some tissues of mice and rats

Summary Chronic daily intake of 0.5% H₂O₂ in drinking water decreased Se-dependent glutathione peroxidase (Se-GSHPx) activity in rat skeletal muscle, kidney and liver. Non-Se GSHPx activity decreased in kidney. Deprivation of drinking water decreased Se-GSHPx activity in kidney and non-Se GSHPx activity in kidney and liver. H₂O₂ intake decreased activity of catalase in rat skeletal muscle. H₂O₂ intake or water deprivation caused no changes in these enzyme activities in mice.     

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease

The amyloid β-peptide (Aβ) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer’s disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Aβ and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Aβ can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Aβ might have a stronger (or weaker) inhibitory effect on the proteasome than does native Aβ. We therefore also investigated the proteasome inhibitory action of Aβ _1–40 (a peptide comprising the first 40 residues of Aβ) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H₂O₂ modification of Aβ _1–40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Aβ _1–40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer’s disease Received 26 September 2000; accepted 26 September 2000     

Histamine binding to H2 receptors stimulates phospholipid methylation in mast cells

Summary Rat peritoneal mast cells incubated in vitro in the presence of L-[methyl-³H] methionine and exposed to histamine undergo a rapid but transient increase in phospholipid methylation. By using specific H₁- and H₂-receptor antagonists, and histamine analogues differing in their H₂-receptor agonist potency, it has been demonstrated that this metabolic event is dependent on histamine binding to H₂-receptors.We are grateful to Smith Kline and French Laboratories for the generous gift of histamine analogues.     

Oxidative stress in the moderately halophilic bacteriumDeleya halophila: Effect of NaCl concentration

The sensitivity ofDeleya halophila to oxidative stress caused by hydrogen peroxide (H₂O₂) was found to vary, depending on the NaCl concentration of the growth medium. Pretreatment of the bacteria at a low concentration of H₂O₂ (50 M) protected the cells against the lethal effects of higher levels (1–2 mM) of H₂O₂. Exposure ofD. halophila cells to 50 M H₂O₂ resulted in the induction of several proteins (hydrogen peroxide-inducible proteins, hips). However, the kinetics of induction, the extent of induction and the number of hips appear to be influenced by the salt concentration of the growth medium. Five of the hips exhibited apparent molecular masses identical to those of five heat shock proteins (hsps).     

Hydrogen peroxide generation inTrypanosoma cruzi

Summary Homogenates from T. cruzi epimastigotes produced 3.4 pmoles H₂O₂/min 10⁶ cells, as detected by the cytochrome c peroxidase assay. Addition of NADH or NADPH increased H₂O₂ production by a factor of 3 and 5, respectively. When supplemented with NADH and NADPH, the mitochondrial, microsomal, and supernatant fractions produced H₂O₂, the soluble fraction and the mitochondrial membranes being apparently the main generators of H₂O₂. The epimastigote homogenates showed cyanide-sensitive superoxide dismutase activity, equivalent to 0.28 g bovine superoxide dismutase per mg homogenate protein.This investigation was supported by grants from Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET) Argentina and the Scientific Office, American States Organization.Career Investigator of CONICET.