Isotope-reinforced polyunsaturated fatty acids protect mitochondria from oxidative stress

Polyunsaturated fatty acid (PUFA) peroxidation is initiated by hydrogen atom abstraction at bis-allylic sites and sets in motion a chain reaction that generates multiple toxic products associated with numerous disorders. Replacement of bis-allylic hydrogens of PUFAs with deuterium atoms (D-PUFAs), termed site-specific isotope reinforcement, inhibits PUFA peroxidation and confers cell protection against oxidative stress. We demonstrate that structurally diverse deuterated PUFAs similarly protect against oxidative stress-induced injury in both yeast and mammalian (myoblast H9C2) cells. Cell protection occurs specifically at the lipid peroxidation step, as the formation of isoprostanes, immediate products of lipid peroxidation, is drastically suppressed by D-PUFAs. Mitochondrial bioenergetics function is a likely downstream target of oxidative stress and a subject of protection by D-PUFAs. Pretreatment of cells with D-PUFAs is shown to prevent inhibition of maximal uncoupler-stimulated respiration as well as increased mitochondrial uncoupling, in response to oxidative stress induced by agents with diverse mechanisms of action, including t-butylhydroperoxide, ethacrynic acid, or ferrous iron. Analysis of structure–activity relationships of PUFAs harboring deuterium at distinct sites suggests that there may be a mechanism supplementary to the kinetic isotope effect of deuterium abstraction off the bis-allylic sites that accounts for the protection rendered by deuteration of PUFAs. Paradoxically, PUFAs with partially deuterated bis-allylic positions that retain vulnerable hydrogen atoms (e.g., monodeuterated 11-D₁-Lin) protect in a manner similar to that of PUFAs with completely deuterated bis-allylic positions (e.g., 11,11-D₂-Lin). Moreover, inclusion of just a fraction of deuterated PUFAs (20–50%) in the total pool of PUFAs preserves mitochondrial respiratory function and confers cell protection. The results indicate that the therapeutic potential of D-PUFAs may derive from the preservation of mitochondrial function.     

Adaptation to oxidative stress induced by polyunsaturated fatty acids in yeast

To create a conditional system for molecular analysis of effects of polyunsaturated fatty acids (PUFA) on cellular physiology, we have constructed a strain of yeast (Saccharomyces cerevisiae) that functionally expresses, under defined conditions, the Delta12 desaturase gene from the tropical rubber tree, Hevea brasiliensis. This strain produces up to 15% PUFA, exclusively under inducing conditions resulting in production of 4-hydroxy-2-nonenal, one of the major end products of n-6 polyunsaturated fatty acid peroxidation. The PUFA-producing yeast was initially more sensitive to oxidative stress than the wild-type strain. However, over extended time of cultivation it became more resistant to hydrogen peroxide indicating adaptation to endogenous oxidative stress caused by the presence of PUFA. Indeed, PUFA-producing strain showed an increased concentration of endogenous ROS, while initially increased hydrogen peroxide sensitivity was followed by an increase in catalase activity and adaptation to oxidative stress. The deletion mutants constructed to be defective in the catalase activity lost the ability to adapt to oxidative stress. These data demonstrate that the cellular synthesis of PUFA induces endogenous oxidative stress which is overcome by cellular adaptation based on the catalase activity.     

Oxygenation of polyunsaturated fatty acids and oxidative stress within blood platelets

The oxygenation metabolism of arachidonic acid (ArA) has been early described in blood platelets, in particular with its conversion into the potent labile thromboxane A₂ that induces platelet aggregation and vascular smooth muscle cells contraction. In addition, the primary prostaglandins D₂ and E₂ have been mainly reported as inhibitors of platelet function. The platelet 12-lipoxygenase (12-LOX) product, i.e. the hydroperoxide 12-HpETE, appears to stimulate platelet ArA metabolism at the level of its release from membrane phospholipids through phospholipase A₂ (cPLA₂) and cyclooxygenase (COX-1) activities, the first enzymes in prostanoid production cascade. Also, 12-HpETE may regulate the oxygenation of other polyunsaturated fatty acids (PUFA) by platelets, especially that of eicosapentaenoic acid (EPA). On the other hand, the reduced product of 12-HpETE, 12-HETE, is able to antagonize TxA₂ action. This is even more obvious for the 12-LOX end-products from docosahexaenoic acid (DHA), 11- and 14-HDoHE. In addition, 12-HpETE plays a key role in platelet oxidative stress as observed in pathophysiological conditions, but may be regulated by DHA with a bimodal way according to its concentration. Other oxygenated products of PUFA, especially omega-3 PUFA, produced outside platelets may affect platelet functions as well.     

Tyrphostins protect neuronal cells from oxidative stress

Tyrphostins are a family of tyrosine kinase inhibitors originally synthesized as potential anticarcinogenic compounds. Because tyrphostins have chemical structures similar to those of the phenolic antioxidants, we decided to test the protective efficacy of tyrphostins against oxidative stress-induced nerve cell death (oxytosis). Many commercially available tyrphostins, at concentrations ranging from 0.5 to 200 microm, protect both HT-22 hippocampal cells and rat primary neurons from oxytosis brought about by treatment with glutamate, as well as by treatment with homocysteic acid and buthionine sulfoximine. The tyrphostins protect nerve cells by three distinct mechanisms. Some tyrphostins, such as A25, act as antioxidants and eliminate the reactive oxygen species that accumulate as a result of glutamate treatment. These tyrphostins also protect cells from hydrogen peroxide and act as antioxidants in an in vitro assay. In contrast, tyrphostins A9 and AG126 act as mitochondrial uncouplers, collapsing the mitochondrial membrane potential and thereby reducing the generation of reactive oxygen species from mitochondria during glutamate toxicity. Finally, the third group of tyrphostins does not appear to be effective as antioxidants but rather protects cells by increasing the basal level of cellular glutathione. Therefore, the effects of tyrphostins on cells are not limited to their ability to inhibit tyrosine kinases.     

Metabolic pathway that produces essential fatty acids from polymethylene-interrupted polyunsaturated fatty acids in animal cells

Sciadonic acid (20:3 Delta-5,11,14) and juniperonic acid (20:4 Delta-5,11,14,17) are polyunsaturated fatty acids (PUFAs) that lack the Delta-8 double bond of arachidonic acid (20:4 Delta-5,8,11,14) and eicosapentaenoic acid (20:5 Delta-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-derived PUFAs are metabolized to essential fatty acids in animal cells. When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid (18:2 Delta-9,12) accumulated in the cells to an extent dependent on the concentration of sciadonic acid. At the same time, a small amount of 16:2 Delta-7,10 appeared in the cellular lipids. Both 16:2 Delta-7,10 and linoleic acid accumulated in sciadonic acid-supplemented CHO cells, but not in peroxisome-deficient CHO cells. We confirmed that 16:2 Delta-7,10 was effectively elongated to linoleic acid in rat liver microsomes. These results indicate that sciadonic acid was partially degraded to 16:2 Delta-7,10 by two cycles of beta-oxidation in peroxisomes, then elongated to linoleic acid in microsomes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n-3 analogue of sciadonic acid, induced accumulation of alpha-linolenic acid (18:3 Delta-9,12,15) in cellular lipids, suggesting that juniperonic acid was metabolized in a similar manner to sciadonic acid. This PUFA remodeling is thought to be a process that converts unsuitable fatty acids into essential fatty acids required by animals.     

Treatment with n-3 polyunsaturated fatty acids reverses endothelial dysfunction and oxidative stress in experimental menopause

Menopause is associated with endothelial dysfunction and oxidative stress. In this condition, reduced n-3 polyunsaturated fatty acids (n-3 PUFAs) contribute to cardiovascular disease. We investigated whether treatment with n-3 PUFA reverses endothelial dysfunction and oxidative stress in experimental menopause. Thirty female rats underwent either sham-surgery or bilateral ovariectomy or bilateral ovariectomy+oral n-3 PUFA (0.8 g kg^-1 day^-1 for 2 months).Ovariectomy caused endothelial dysfunction to acetylcholine, which was reversed by superoxide scavenger Tiron. Erythrocyte membrane lipid composition was characterized by reduced n-3 PUFA total content and omega-3 index, and by concomitant increase in n-6:n-3 PUFA ratio. Ovariectomy-related oxidative stress, demonstrated by both enhanced superoxide production and 3-nitrotyrosine expression in aorta, was associated with increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX-4 protein expression. Endothelial nitric oxide synthase (eNOS) functional inhibition by l-NG-nitroarginine methyl ester, protein expression and activity did not change.In ovariectomized rats, treatment with n-3 PUFA increased n-3 PUFA total content and omega-3 index and decreased n-6:n-3 PUFA ratio in erythrocyte membrane, reversed vascular oxidative stress, endothelial dysfunction, aortic 3-nitrotyrosine and markedly lowered NOX-4 protein expression; eNOS protein expression also increased, paralleled by reversal of inhibitory binding to Caveolin-1, while ex-vivo functional inhibition and NOS synthesis were unchanged.These findings demonstrate in vivo a therapeutic benefit of n-3 PUFA on menopause-associated endothelial dysfunction by reversal of alterations in membrane lipid composition induced by ovariectomy and by reduction of vascular oxidative stress. In this setting they also identify NOX-4 as a potential target to reduce oxidative stress-mediated vascular complications.     

Haemoglobin-induced oxidative stress is associated with both endogenous peroxidase activity and H2O2 generation from polyunsaturated fatty acids

Patients with increased haemolytic haemoglobin (Hb) have 10-20-times greater incidence of cardiovascular mortality. The objective of this study was to evaluate the role of Hb peroxidase activity in LDL oxidation. The role of Hb in lipid peroxidation, H(2)O(2) generation and intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed using NaN(3), a peroxidase inhibitor, catalase, a H(2)O(2) decomposing enzyme and human umbilical vein endothelial cells (HUVECs), respectively. Hb induced H(2)O(2) production by reacting with LDL, linoleate and cell membrane lipid extracts. Hb-induced LDL oxidation was inhibited by NaN(3) and catalase. Furthermore, Hb stimulated ICAM-1 and VCAM-1 expression, which was inhibited by the antioxidant, probucol. Thus, the present study suggests that the peroxidase activity of Hb produces atherogenic, oxidized LDL and oxidized polyunsaturated fatty acids (PUFAs) in the cell membrane and reactive oxygen species (ROS) formation mediated Hb-induced ICAM-1 and VCAM-1 expression.     

Flavonoids protect neuronal cells from oxidative stress by three distinct mechanisms

Flavonoids are a family of antioxidants found in fruits and vegetables as well as in popular beverages such as red wine and tea. Although the physiological benefits of flavonoids have been largely attributed to their antioxidant properties in plasma, flavonoids may also protect cells from various insults. Nerve cell death from oxidative stress has been implicated in a variety of pathologies, including stroke, trauma, and diseases such as Alzheimer's and Parkinson's. To determine the potential protective mechanisms of flavonoids in cell death, the mouse hippocampal cell line HT-22, a model system for oxidative stress, was used. In this system, exogenous glutamate inhibits cystine uptake and depletes intracellular glutathione (GSH), leading to the accumulation of reactive oxygen species (ROS) and an increase in Ca(2+) influx, which ultimately causes neuronal death. Many, but not all, flavonoids protect HT-22 cells and rat primary neurons from glutamate toxicity as well as from five other oxidative injuries. Three structural requirements of flavonoids for protection from glutamate are the hydroxylated C3, an unsaturated C ring, and hydrophobicity. We also found three distinct mechanisms of protection. These include increasing intracellular GSH, directly lowering levels of ROS, and preventing the influx of Ca(2+) despite high levels of ROS. These data show that the mechanism of protection from oxidative insults by flavonoids is highly specific for each compound.     

The Rho1 GTPase acts together with a vacuolar glutathione S-conjugate transporter to protect yeast cells from oxidative stress

Maintenance of redox homeostasis is critical for the survival of all aerobic organisms. In the budding yeast Saccharomyces cerevisiae, as in other eukaryotes, reactive oxygen species (ROS) are generated during metabolism and upon exposure to environmental stresses. The abnormal production of ROS triggers defense mechanisms to avoid the deleterious consequence of ROS accumulation. Here, we show that the Rho1 GTPase is necessary to confer resistance to oxidants in budding yeast. Temperature-sensitive rho1 mutants (rho1(ts)) are hypersensitive to oxidants and exhibit high accumulation of ROS even at a semipermissive temperature. Rho1 associates with Ycf1, a vacuolar glutathione S-conjugate transporter, which is important for heavy metal detoxification in yeast. Rho1 and Ycf1 exhibit a two-hybrid interaction with each other and form a bimolecular fluorescent complex on the vacuolar membrane. A fluorescent-based complementation assay suggests that the GTP-bound Rho1 associates with Ycf1 and that their interaction is enhanced upon exposure to hydrogen peroxide. The rho1(ts) mutants also exhibit hypersensitivity to cadmium, while cells carrying a deletion of YCF1 or mutations in a component of the Pkc1-MAP kinase pathway exhibit little or minor sensitivity to oxidants. We thus propose that Rho1 protects yeast cells from oxidative stress by regulating multiple downstream targets including Ycf1. Since both Rho1 and Ycf1 belong to highly conserved families of proteins, similar mechanisms may exist in other eukaryotes.     

Why most insects have very low proportions of C20 polyunsaturated fatty acids: The oxidative stress hypothesis

Eicosanoids, a group of C20 oxygenated polyunsaturated fatty acids (PUFAs), mediate various physiological processes, such as immunity, reproduction, excretion, and metabolism in insects. Arachidonic acid (AA) is used for the main precursor for the production of various eicosanoids. However, most terrestrial insects possess relatively low AA levels. Insects are presumed to be evolved since the Paleozoic era, at which oxygen levels might be much higher than current conditions. Compared with other animals, they exhibit relatively high metabolic rates with the well-developed tracheal system, which directly supply enough oxygen to active tissues like flight muscles. This might allow insects to be susceptible to reactive oxygen species (ROS) generated from high oxidative catabolism. Long-chain PUFAs including AA is usually reacted with ROS and become peroxidized. Peroxidized PUFAs cause various cellular damage. Thus, we propose a hypothesis that terrestrial insects minimize AA levels to minimize oxidative stress.     

Inhibitors of catalase-amyloid interactions protect cells from beta-amyloid-induced oxidative stress and toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.     

Heterologous ferredoxin reductase and flavodoxin protect Cos-7 cells from oxidative stress

Background

Ferredoxin-NADP(H) reductase (FNR) from Pisum sativum and Flavodoxin (Fld) from Anabaena PCC 7119 have been reported to protect a variety of cells and organisms from oxidative insults. In this work, these two proteins were expressed in mitochondria of Cos-7 cells and tested for their efficacy to protect these cells from oxidative stress in vitro.

Principal Findings

Cos-7/pFNR and Cos-7/pFld cell lines expressing FNR and Fld, respectively, showed a significantly higher resistance to 24 h exposure to 300–600 µM hydrogen peroxide measured by LDH retention, MTT reduction, malondialdehyde (MDA) levels and lipid peroxide (LPO; FOX assay) levels. However, FNR and Fld did not exhibit any protection at shorter incubation times (2 h and 4 h) to 4 mM hydrogen peroxide or to a 48 h exposure to 300 µM methyl viologen. We found enhanced methyl viologen damage exerted by FNR that may be due to depletion of NADPH pools through NADPH-MV diaphorase activity as previously observed for other overexpressed enzymes.

Significance

The results presented are a first report of antioxidant function of these heterologous enzymes of vegetal and cyanobacterial origin in mammalian cells.     

Correlation between medium C-chain fatty acids and polyunsaturated fatty acids

The medium C-chain fatty acids increased in the muscle, lungs, pancreas and adipose tissue (and not in the liver) of the rats injected with CCl4 or nourished with "balanced" diet for the lipids. When CCl4 and balanced diet are furnished together, these acids decrease strongly: the discussion of the results is difficult.     

Procyanidins protect Fao cells against hydrogen peroxide-induced oxidative stress

In this paper, we evaluate the extent to which flavonoids in red wine (catechin, epicatechin, quercetin and procyanidins) protect against hydrogen peroxide-induced oxidative stress in Fao cells. When cells were exposed to H(2)O(2), malondialdehyde (MDA) levels, oxidized glutathione (GSSG) levels and lactate dehydrogenase (LDH) release increased, indicating membrane damage and oxidative stress. All the flavonoids studied, and in particular epicatechin and quercetin, protected the plasma membrane. Only procyanidins lowered MDA levels and LDH leakage, maintained a higher reduced/oxidized glutathione ratio, and increased catalase/superoxide dismutase and glutathione peroxidase/superoxide dismutase ratios, and glutathione reductase and glutathione transferase activities. These results show that the procyanidin mixture has a greater antioxidant effect than the individual flavonoids studied, probably due to its oligomer content and/or the additive/synergistic effect of its compounds. This suggests that the mixture of flavonoids found in wine has a greater effect than individual phenols, which may explain many of the healthy effects attributed to wine.     

Decrease in adhesion of cells cultured in polyunsaturated fatty acids

The addition of long chain unsaturated fatty acids (linoleic, linolenic and arachidonic acids) to BHK cells reduces the cell to substrate adhesion, causes morphological changes and alters the cellular growth properties. The new characteristics are similar to those of transformed cells. The data indicate that the effects are probably due to actual changes in the surface membrane lipids and not due to prostaglandin synthesis.     

Vimentin intermediate filaments protect mitochondria from oxidative stress

The role of intermediate filaments (IFs) in eukaryotic cells is still unclear. The disturbance of mitochondria distribution and function, in particular the enhanced production of reactive oxygen species (ROS) and decreased membrane potential, is observed in cells devoid of IFs. The aim of this work was to study the dependence of mitochondria sensitivity to oxidative stress on the presence of vimentin IFs. It was found that mitochondria are less sensitive to ROS in cells containing vimentin than in cells devoid of vimentin. Besides, mitochondrial membrane potential was demonstrated to increase upon regeneration of vimentin IFs in the cells. Substitution of Pro-57 by Arg in N-terminal part of the vimentin molecule abandoned its protective ability and the effect on mitochondrial membrane potential.     

Mitochondrial and cytosolic expression of human peroxiredoxin 5 in Saccharomyces cerevisiae protect yeast cells from oxidative stress induced by paraquat

Human peroxiredoxin 5 is a recently discovered mitochondrial, peroxisomal and cytosolic thioredoxin peroxidase able to reduce hydrogen peroxide and alkyl hydroperoxides. To gain insight into peroxiredoxin 5 antioxidant role in cell protection, we investigated the resistance of yeast cells expressing human peroxiredoxin 5 in mitochondria or in the cytosol against oxidative stress induced by paraquat. The herbicide paraquat is a redox active drug known to generate superoxide anions in mitochondria and the cytosol of yeast and mammalian cells leading to the formation of several reactive oxygen species. Here, we report that mitochondrial and cytosolic human peroxiredoxin 5 protect yeast cells from cytotoxicity and lipid peroxidation induced by paraquat.