Phosphatidylserine Peroxidation Research Articles

Correction of the Mitochondrial NADH Oxidase Activity, Peroxidation and Phospholipid Metabolism by Haplogenin-7-Glucoside in Hypoxia and Ischemia

In this study, it was used haplogenin-7-glucoside flavonol (C16H12O8) isolated from the plant Haplophyllum perforatum. It was studied the effect of haplogenin-7-glycoside on NADH oxidase activity, peroxidation, and phospholipid metabolism in rat liver mitochondria under in vitro conditions. Incubation of mitochondria at 37 ℃ leads to disruption of the integrity of mitochondrial membranes, as a result, in cytochrome с desorption from the inner membrane into the intermembrane space, which leads to a decrease in the activity of rotenone-sensitive NADH oxidase and an increase in the activity of rotenone-insensitive NADH oxidase. Haplogenin-7-glucoside exhibits membrane stabilization by reducing these alterations. Under ischemia conditions, the effect of haplogenin-7-glucoside on the LPO process in mitochondria decreases LPO in mitochondria. Quantitative changes in phosphatidylserine in mitochondrial membranes under LPO conditions during incubation of mitochondria at 37 ℃ were observed to accelerate controlled (auto-oxidation) LPO over time (0 - 90 min). Peroxidation of phosphatidylserine in mitochondria when the addition of haplogenin-7-glucoside to the incubation medium at 30, 60 and 90 min, LPO was found to be reduced. The increasing amount of phosphatidylinositol has not happened depend on time. However, a slight increase was found under the influence of haplogenin-7-glucoside.
 HIGHLIGHTS
 
 Cytochrome c desorption from the inner membrane into the interstitial space leads to a decrease in the activity of rotenone-sensitive NADH-oxidase and an increase in the activity of non-rotenone-sensitive NADH-oxidase
 Haplogenin-7-glycoside showed membrane-stabilizing properties by significantly reducing these changes; the addition of haplogenin-7-glucoside to mitochondria leads to a decrease for MDA, a decrease in the LPO process
 At incubation of mitochondria at 37℃, the amount of phosphatidic acid and lysophosphatidic acid in mitochondria under LPO conditions was also found to be less than control under the influence of haplogenin-7-glucoside. Thus, haplogenin-7-glucoside reduces the rate of peroxidation of phospholipids in liver mitochondria and has a corrective effect
 
 GRAPHICAL ABSTRACT

Global Phospholipidomics Analysis Reveals Selective Pulmonary Peroxidation Profiles upon Inhalation of Single-Walled Carbon Nanotubes

It is commonly believed that nanomaterials cause nonspecific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in the two most abundant phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This nonrandom peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H(2)O(2)/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria-targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung.

Oxidative Lipidomics of γ-Radiation-Induced Lung Injury: Mass Spectrometric Characterization of Cardiolipin and Phosphatidylserine Peroxidation

Oxidative damage plays a significant role in the pathogenesis of γ-radiation-induced lung injury. Endothelium is a preferred target for early radiation-induced damage and apoptosis. Given the newly discovered role of oxidized phospholipids in apoptotic signaling, we performed oxidative lipidomics analysis of phospholipids in irradiated mouse lungs and cultured mouse lung endothelial cells. C57BL/6NHsd female mice were subjected to total-body irradiation (10 Gy, 15 Gy) and euthanized 24 h thereafter. Mouse lung endothelial cells were analyzed 48 h after γ irradiation (15 Gy). We found that radiation-induced apoptosis in vivo and in vitro was accompanied by non-random oxidation of phospholipids. Cardiolipin and phosphatidylserine were the major oxidized phospholipids, while more abundant phospholipids (phosphatidylcholine, phosphatidylethanolamine) remained non-oxidized. Electrospray ionization mass spectrometry analysis revealed the formation of cardiolipin and phosphatidylserine oxygenated molecular species in the irradiated lung and cells. Analysis of fatty acids after hydrolysis of cardiolipin and phosphatidylserine by phospholipase A(2) revealed the presence of mono-hydroperoxy and/or mono-hydroxy/mono-epoxy, mono-hydroperoxy/mono-oxo molecular species of linoleic acid. We speculate that cyt c-driven oxidations of cardiolipin and phosphatidylserine associated with the execution of apoptosis in pulmonary endothelial cells are important contributors to endothelium dysfunction in γ-radiation-induced lung injury.

Oxidative lipidomics of hyperoxic acute lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation

Reactive oxygen species have been shown to play a significant role in hyperoxia-induced acute lung injury, in part, by inducing apoptosis of pulmonary endothelium. However, the signaling roles of phospholipid oxidation products in pulmonary endothelial apoptosis have not been studied. Using an oxidative lipidomics approach, we identified individual molecular species of phospholipids involved in the apoptosis-associated peroxidation process in a hyperoxic lung. C57BL/6 mice were killed 72 h after exposure to hyperoxia (100% oxygen). We found that hyperoxia-induced apoptosis (documented by activation of caspase-3 and -7 and histochemical terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of pulmonary endothelium) was accompanied by nonrandom oxidation of pulmonary lipids. Two anionic phospholipids, mitochondria-specific cardiolipin (CL) and extramitochondrial phosphatidylserine (PS), were the two major oxidized phospholipids in hyperoxic lung. Using electrospray ionization mass spectrometry, we identified several oxygenation products in CL and PS. Quantitative assessments revealed a significant decrease of CL and PS molecular species containing C(18:2), C(20:4), C(22:5), and C(22:6) fatty acids. Similarly, exposure of mouse pulmonary endothelial cells (MLEC) to hyperoxia (95% oxygen; 72 h) resulted in activation of caspase-3 and -7 and significantly decreased the content of CL molecular species containing C(18:2) and C(20:4) as well as PS molecular species containing C(22:5) and C(22:6). Oxygenated molecular species were found in the same two anionic phospholipids, CL and PS, in MLEC exposed to hyperoxia. Treatment of MLEC with a mitochondria-targeted radical scavenger, a conjugate of hemi-gramicidin S with nitroxide, XJB-5-131, resulted in significantly lower oxidation of both CL and PS and a decrease in hyperoxia-induced changes in caspase-3 and -7 activation. We speculate that cytochrome c driven oxidation of CL and PS is associated with the signaling role of these oxygenated species participating in the execution of apoptosis and clearance of pulmonary endothelial cells, thus contributing to hyperoxic lung injury.

Oxidative lipidomics of γ-irradiation-induced intestinal injury

Although γ-irradiation-induced tissue injury has been associated with lipid peroxidation, the individual phospholipid molecular targets have not been identified. We employed oxidative lipidomics to qualitatively and quantitatively characterize phospholipid peroxidation in a radiosensitive tissue, the small intestine, of mice exposed to total body irradiation (TBI) (10 and 15 Gy). Using electrospray ionization mass spectrometry we found that the major classes of intestine phospholipids—phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol—included clusters with highly oxidizable molecular species containing docosahexaenoic fatty acid. Molecular species of cardiolipin were represented by only two major less oxidizable individual molecular species—tetralinoleoylcardiolipin and trilinoleoyl-mono-oleoylcardiolipin. Selective and robust oxidation of two anionic phospholipids—cardiolipin in mitochondria and phosphatidylserine outside of mitochondria—was observed 24 h after γ-irradiation. MS analysis detected several TBI-induced molecular species of oxidized cardiolipin: (C 18:2) 3(C 18:2–OOH), (C 18:2) 2(C 18:2–OOH) 2, (C 18:2) 1(C 18:2–OOH) 3, and (C 18:2–OOH) 4. The major molecular species involved in TBI-triggered peroxidation of phosphatidylserine included C 18:0/C 22:6–OOH, C 18:0/C 22:5–OOH, and C 18:0/C 22:4–OOH. More abundant phospholipids—phosphatidylcholine and phosphatidylethanolamine—did not reveal any oxidative stress responses despite the presence of highly oxidizable docosahexaenoic fatty acid residues in their molecular species. A marked activation of caspases 3/7 that was detected in the intestine of γ-irradiated mice indicates the involvement of apoptotic cell death in the TBI injury. Given that oxidized molecular species of cardiolipin and phosphatidylserine accumulate during apoptosis of different cells in vitro we speculate that cardiolipin and phosphatidylserine oxidation products may be useful as potential biomarkers of γ-irradiation-induced intestinal apoptosis in vivo and may represent a promising target for the discovery of new radioprotectors and radiosensitizers.

Cytochrome c release is required for phosphatidylserine peroxidation during fas‐triggered apoptosis in lung epithelial A549 cells

Oxidation of phosphatidylserine (PtdSer) has been shown to play a pivotal role in signaling during cell apoptosis and subsequent recognition of apoptotic cells by phagocytes. However, the redox catalytic mechanisms involved in selective PtdSer oxidation during apoptosis remain poorly understood. Here we employed anti-Fas antibody CH-11-treated A549 cells as a physiologically relevant model to investigate the involvement of PtdSer oxidation and its potential mechanism during apoptosis. We demonstrated that ligation of CH-11 with its cognate receptor initiated execution of apoptotic program in interferon gamma-pretreated A549 cells as evidenced by activation of caspase and DNA fragmentation. A significant increase of cytochrome c (cyt c) content in the cytosol as early as 2 h after CH-11 exposure was detected indicating that Fas-induced apoptosis in A549 cells proceeds via extrinsic type II pathway and includes mitochondrial signaling. PtdSer was selectively oxidized 3 h after anti-Fas triggering while two more abundant phospholipids--phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn)--and the major intracellular antioxidant, glutathione, remained nonoxidized. A pan-caspase inhibitor, z-VAD, fully blocked cyt c release and oxidation of PtdSer in Fas-treated A549 cells. On the other hand, z-DQMD, a caspase-3 inhibitor, completely inhibited caspase-3 activity but did not fully block caspase-8 activation and release of cyt c. Importantly, z-DQMD failed to protect PtdSer from oxidation. In addition, in a model system, we demonstrated that peroxidase activity of cyt c was greatly enhanced in the presence of dioleoylphosphatidylserine containing liposomes by monitoring oxidation of 2',7'-dichlorodihydrofluorescein to 2',7'-dichlorofluorescein. We further showed that peroxidase activity of cyt c catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine using a newly developed HPLC assay. MS analysis of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine revealed that in addition to its mono- and dihydroperoxides, several different PtdSer oxidation products can be formed. Overall, we concluded that cyt c acts as a catalyst of PtdSer oxidation during Fas-triggered A549 cell apoptosis.

Peroxidation and externalization of phosphatidylserine associated with release of cytochrome c from mitochondria.

Production of reactive oxygen species (ROS) during apoptosis is associated with peroxidation of phospholipids particularly of phosphatidylserine (PS). The mechanism(s) underlying preferential PS oxidation are not well understood. We hypothesized that cytochrome c (cyt c) released from mitochondria into cytosol acts as a catalyst that utilizes ROS generated by disrupted mitochondrial electron transport for PS oxidation. Selectivity of PS oxidation is achieved via specific interactions of positively charged cyt c with negatively charged PS. To test the hypothesis we employed temporary transfection of Jurkat cells with a pro-apoptotic peptide, DP1, a conjugate consisting of a protein transduction domain, PTD-5, and an antimicrobial domain, KLA [(KLAKLAK)2], known to selectively disrupt mitochondria. We report that treatment of Jurkat cells with DP1 yielded rapid and effective release of cyt c from mitochondria and its accumulation in cytosol accompanied by production of H2O2. Remarkably, this resulted in selective peroxidation of PS while more abundant phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) remained nonoxidized. Neither PTD-5 alone nor KLA alone exerted any effect on PS peroxidation. Redox catalytic involvement of cyt c in PS oxidation was further supported by our data demonstrating that: (i) specific interactions of cyt c with PS resulted in the formation of EPR-detectable protein-centered tyrosyl radicals of cyt c upon its interaction with H2O2 in the presence of PS-containing liposomes, and (ii) integration of cyt c into cytochrome c null (Cyt c -/-) cells or HL-60 cells specifically stimulates PS oxidation in the presence of H2O2 or t-BuOOH, respectively. We further demonstrated that DP1 elicited externalization of PS on the surface of Jurkat cells and enhanced their recognition and phagocytosis by J774A.1 macrophages. Our results are compatible with the hypothesis that catalysis of selective PS oxidation during apoptosis by cytosolic cyt c is important for PS-dependent signaling pathways such as PS externalization and recognition by macrophage receptors.

14] Peroxidation of phosphatidylserine in mechanisms of apoptotic signaling

Oxidative stress is a frequent trigger of apoptosis in a variety of cells and is also thought to be involved as a component of the common pathway in the execution of apoptosis. While effects of oxidative stress on protein components of apoptotic machinery, such as caspases, have been well characterized, information on selective oxidation of specific classes of phospholipids in live cells is limited. The direct measurement of oxidized lipid products is problematic, as cells possess a very effective system for remodeling and repairing oxidatively modified phospholipids that interferes with their accurate measurement. The authors have developed a sensitive, specific, and reliable procedure for the assessment of oxidative stress in different classes of membrane phospholipids in intact live cells based on their metabolic acylation with an oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid (cis-PnA), as a reporter molecule. Basic procedures and some applications of the technique are described in the chapter. The model of cis-PnA-labeled cells offers a unique model system for quantitative studies of oxidative stress and selective oxidation of specific classes of phospholipids under normal physiological conditions, as well as during cell injury and apoptotic death.

Selective inhibition of the non-enzymatic lipid peroxidation of phosphatidylserine in rod outer segments by alpha-tocopherol.

In the present study it was investigated if alpha-tocopherol shows protection against in vitro lipid peroxidation of phospholipids located in rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe2+ system, at 37 degrees C during 160 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of alpha-tocopherol. The fatty acid composition of total lipids isolated from rod outer segment membranes was substantially modified when subjected to non-enzymatic lipid peroxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3). The incorporation of alpha-tocopherol (0.35 micromol/mg protein) produce a 43.37% inhibition of the lipid peroxidation process evaluated as chemiluminescence (total cpm originated in 160 min). The phospholipid species containing the highest amount of docosahexaenoic acid: phosphatidylethanolamine and phosphatidylserine were more affected than phosphatidylcholine during the lipid peroxidation process. Not all phospholipids, however, were equally protected after the addition of alpha-tocopherol to the incubation medium. Phosphatidylcholine and phosphatidylethanolamine, were not protected by alpha-tocopherol, the vitamin provides selective antioxidant protection only for phosphatidylserine. These results indicate that alpha-tocopherol may act as antioxidant protecting rod outer segment membranes from deleterious effect by a selective mechanism that diminishes the loss of docosahexaenoic acid from phosphatidylserine.

Paraquat-induced phosphatidylserine oxidation and apoptosis are independent of activation of PLA2.

Paraquat is a pneumotoxin that causes lung injury by enhancing oxidative stress; however, the cellular responses to these redox events are undefined. We previously showed that paraquat produced selective peroxidation of phosphatidylserine that preceded apoptosis in 32D cells. We now report that the phospholipase A2 (PLA2) inhibitor quinacrine can attenuate phosphatidylserine oxidation and also block paraquat-induced apoptosis. Therefore, we investigated the potential for PLA2 to mediate apoptosis after paraquat. We found that, in contrast to quinacrine, the PLA2 inhibitors manoalide, aristolochic acid, and arachidonyl trifluoromethylketone failed to prevent paraquat-induced apoptosis. Moreover, no evidence of PLA2 activation was observed within 7 h after paraquat exposure. Finally, quinacrine failed to inhibit basal and 4-bromo-A-23187-induced release of [3H]arachidonic acid at concentrations that protected paraquat-induced apoptosis. We conclude that paraquat-induced phosphatidylserine oxidation and apoptosis occurred in the absence of PLA2 activation and that quinacrine protected phosphatidylserine and cell viability after paraquat in a PLA2-independent manner.

Nitric oxide protects cardiomyocytes against tert-butyl hydroperoxide-induced formation of alkoxyl and peroxyl radicals and peroxidation of phosphatidylserine.

We studied protective effects of nitric oxide against tert-butyl hydroperoxide-induced oxidative damage to cardiac myocytes. Two distinct free radicals species--alkoxyl radicals associated with non-heme iron catalytic sites and myoglobin protein-centered peroxyl radicals--were found in low-temperature EPR spectra of cardiac myocytes exposed to t-BuOOH. The t-BuOOH-induced radical formation was accompanied by site-specific oxidative stress in membrane phospholipids (peroxidation of phosphatidylserine) assayed by fluorescence HPLC after metabolic labeling of cell phospholipids with oxidation-sensitive cis-parinaric acid. An NO-donor, (Z)-1-[N-(3-ammonio-propyl)-N-(n-propyl) amino]-diazen-1-ium-1,2-diolate], protected cardiac myocytes against tert-butyl hydroperoxide-induced: (i) formation of non-protein- and protein-centered free radical species and (ii) concomitant peroxidation of phosphatidylserine. Thus nitric oxide can act as an effective antioxidant in live cardiomyocytes.