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.     

The influence of the acyl chain composition of cardiolipin on the stability of mitochondrial complexes; An unexpected effect of cardiolipin in α-ketoglutarate dehydrogenase and prohibitin complexes

The role of cardiolipin acyl chain composition in assembly/stabilization of mitochondrial complexes was investigated using three yeast deletion mutants (acb1Δ strain; taz1Δ strain; and acb1Δtaz1Δ strain). Deletion of the TAZ1 gene, involved in cardiolipin acyl chain remodeling, is known to increase the content of monolyso-cardiolipin (MLCL) at the expense of CL, and to decrease the unsaturation of the remaining CL. Deletion of the ACB1 gene encoding the acyl-CoA-binding protein, involved in fatty acid elongation, decreases the average length of the CL acyl chains. Furthermore, a TAZ1ACB1 double deletion mutant strain was used in this study which has both a decrease in the length of the CL acyl chains and an increase in MLCL. BN/SDS PAGE analysis revealed that cardiolipin is important for the prohibitin–m-AAA protease complex, the α-ketoglutarate dehydrogenase complex and respiratory chain supercomplexes. The results indicate that the decreased level of complexes in taz1Δ and acb1Δtaz1Δ mitochondria is due to a decreased content of CL or the presence of MLCL.     

Localization of lipid peroxidation products in liposomes after γ-irradiation

The distribution of lipid peroxidation products in liposomes after γ-irradiation at various doses was studied. Increases in thiobarbituric-acid-reactive substances, in the absorbance at 232 nm and in hydroperoxides were observed mainly in liposomal membranes after relatively low doses of irradiation, while carbonyl compounds were distributed both inside and outside the membranes. After higher doses of irradiation, however, the absorbance at 232 nm and the amount of hydroperoxides reached a maximal level in the membrane portion and then decreased when the decomposition products were released from the membranes. Under this condition, malondialdehyde and other carbonyl compounds were increased mainly in the medium of liposomal suspension. These results are discussed with reference to the lipid peroxidation process which is induced quantitatively by ionizing radiation.     

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.     

Electrochemical analysis of the effect of Ca on cardiolipin–cytochrome c interaction

Mitochondrial Ca²⁺ has been considered a trigger for the release of cytochrome c, which is a critical and early event in the induction of cell apoptosis, although the molecular mechanism underlying this effect is still not fully understood. Here we investigate the interaction between cytochrome c and cardiolipin and the effect of Ca²⁺ on this interaction using electrochemical methods. Experimental results revealed that modification of cardiolipin onto the surface of a pyrolytic graphite electrode could lead to a rapid direct electron transfer of cytochrome c through the electrostatic interaction between the protein and the cardiolipin. Addition of Ca²⁺ to the test solution containing cytochrome c could cause the decrease of the redox peaks of the protein, and the peaks could be recovered when Ca²⁺ was chelated by ethylenediaminetetraacetate. The cardiolipin–cytochrome c interaction and the Ca²⁺ effect were also investigated with the variation of the charges of lipids, buffer solutions, reaction time, and valencies of cations for comparison.     

Prooxidant action of aluminum Ion – Stimulation of iron-mediated lipid peroxidation by aluminum

Prooxidant nature of aluminum ion was analyzed in relation to iron coordination. Aluminum ion effectively enhanced the formation of thiobarbituric acid-reactive substances as a marker of lipid peroxidation of microsomes from rat liver under the acidic conditions, and this metal further attenuated the antioxidant action of flavonoids such as quercetin and baicalein under neutral conditions. Autooxidation of ferrous ion was markedly inhibited by aluminum ion. Aluminum can act as a prooxidant by stabilizing reduced iron the initiating species for lipid peroxidation, and by inhibiting the antioxidant action of flavonoid.     

Effect of succinate on mitochondrial lipid peroxidation. 1. Comparative studies on ferrous ion and ADP · Fe/NADPH-induced peroxidation

Lipid peroxidation in isolated rat liver mitochondria, mitoplast, and mitochondrial inner membrane fragments was induced either by ferrous ions, or in an NADPH-dependent process by complexing with adenine nucleotides (ADP or ATP) iron. The Fe²⁺-induced lipid peroxidation is nonenzymic when inner membrane fragments are used, while the differences in the inhibitory effect of Mn²⁺ ions and the stimulatory effect of the ionophore A-23187 in mitochondria and inner membrane fragments suggest an enzymic mechanism for ferrous ion-induced lipid peroxidation in intact mitochondria. Contrary to this the ADP/Fe/NADPH-dependent lipid peroxidation is an enzymic process both in mitochondria and inner membrane preparations. We have shown that cytochrome P₄₅₀ is involved in the ADP/Fe/NADPH-induced lipid peroxidation. Succinate, a known inhibitor of NADPH-dependent lipid peroxidation, inhibited the Fe²⁺-induced process also, and there was no difference in this effect when inner membrane preparations, mitochondria, or mitoplasts were used.     

Binding of doxorubicin to cardiolipin as compared to other anionic phospholipids—An evaluation of electrostatic effects

The binding of doxorubicin to large unilamellar vesicles consisting of cardiolipin or other anionic phospholipids was analyzed in terms of the local drug concentration at the membrane surface, according to the Gouy-Chapman theory. The analysis suggests strong positive binding cooperativity. Part of the drug binds in the uncharged form. The affinity for cardiolipin and other anionic phospholipids is comparable. A binding level of 0.5 doxorubicin per lipid-phosphorus is reached when the local concentration of free doxorubicin monomer-equivalents at the membrane surface is about 0.2–0.7 mM. This contrasts with earlier findings indicating a 300–1000 fold higher affinity for cardiolipin. The present analysis provides an explanation for this apparent discrepancy.     

Impairment of brain mitochondrial functions by β-hemolytic Group B Streptococcus. Effect of cardiolipin and phosphatidylcholine

Group B Streptococcus (GBS) causes severe infection in the central nervous system. In this study, brain mitochondrial function was investigated by simulating infection of isolated mitochondria with GBS, which resulted in loss of mitochondrial activity. The β-hemolysin expressing strains GBS-III-NEM316 and GBS-III-COH31, but not the gGBS-III-COH31 that does not express β-hemolysin, caused dissipation of preformed mitochondrial membrane potential (Δψ_m). This indicates that β-hemolysin is responsible for decreasing of the reducing power of mitochondria. GBS-III-COH31 interacted with mitochondria causing increase of oxygen consumption, due to uncoupling of respiration, blocking of ATP synthesis, and cytochrome c release outside mitochondria. Moreover, the mitochondrial systems contributing to the control of cellular Ca²⁺ uptake were lost. In spite of these alterations, mitochondrial phospholipid content and composition did not change significantly, as evaluated by MALDI-TOF mass spectrometry. However, exogenous cardiolipin (CL) and dipalmitoylphosphatidylcholine (DPPC) attenuated the uncoupling effect of GBS-III-COH31, although with different mechanisms. CL was effective only when fused to the inner mitochondrial membrane, probably reducing the extent of GBS-induced proton leakage. DPPC, which is not able to fuse with mitochondrial membranes, exerted its effect outside mitochondria, likely by shielding mitochondria against GBS β-hemolysin attack.     

Lipid peroxidation in chronic gastritis; any influence of Helicobacter pylori?

In an attempt to investigate the significance of lipid peroxidation in the pathogenesis of gastritis associated with or without Helicobacter pylori infection, malonodialdehyde (MDA) levels were measured by the thiobarbiturate assay in the gastric juice of 101 patients undergoing upper GI endoscopy and correlated with histopathological findings. Elevated MDA levels were found in all patients with gastritis compared with controls. MDA levels were significantly correlated with the extent of the mucosal inflammation and with disease activity in patients with reactive gastritis. In patients with H. pylori associated gastritis MDA levels were not correlated with disease activity but rather with the degree of atrophy. In this case, MDA levels were equal or even less than in patients with reactive gastritis. MDA levels were not affected by the history of consumption of PPIs, of H(2)-blockers or of NSAIDs over the last month before the endoscopy. It is concluded that lipid peroxidation is a mechanism involved in the pathogenesis of gastritis associated or not to H. pylori infection.     

Lipid peroxidation as the mechanism of modification of brain 5′-nucleotidase activity in vitro

The effect of lipid peroxidation on the Mg²⁺-independent and Mg²⁺-dependent activity of brain cell membrane 5-nucleotidase was determined and the affinity of the active sites of Mg²⁺-dependent enzyme for 5-AMP (substrate) and Mg²⁺ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 M ascorbate and 25 M FeCl₂ (resultant) for 10 min. The activity of 5-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg²⁺-independent 5-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 mol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg²⁺, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg²⁺-dependent 5-nucleotidase for 5-AMP tripled, as indicated by a significant decrease inK _m (K _m=95±2 M AMP for control;K _m=32±2 MAMP for peroxidized).V _max was significantly reduced from 3.35±0.16 in control to 1.70±.09 moles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg²⁺ significantly increased (K _m=6.17±0.37 mM Mg²⁺ for control;K _m=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg²⁺-dependent 5-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5-nucleotidase activity and the loss of Mg²⁺-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5-nucleotidase during hypoxia.     

Lipid peroxidation in microsomes of murine bone marrow after low-doseγ-irradiation

The principal aim of the study was to investigate the effect of low-dose-irradiation on lipid peroxidation (LPO) in murine bone marrow. To this end, the degree of LPO in suspensions of microsomes of murine bone marrow cells (BMC) was determined in terms of malondialdehyde (MDA) formation after whole-body or in vitro exposure to various doses of-radiation. These effects were compared to some extent with similar effects in liver and spleen preparations. As to the effect of-irradiation on LPO in BMC, the response depends on the dose level and on whether whole-body or in vitro exposures are involved. Whole-body irradiation did not result in an increase in LPO in BMC microsomes, even at such high doses as 15 Gy, although hepatic microsomes showed a marked increase. In contrast, in vitro irradiation of BMC microsomes with 0.1, 10 and 50 Gy brought about an increase in LPO. This increase was already significant (P <0.05) at 0.1 Gy following a post-irradiation incubation and substantial at 50 Gy, even without subsequent incubation. The results show that low doses of-irradiation are able to induce an elevation of LPO in murine BMC microsomes, but only after in vitro irradiation. In the case of whole-body irradiation cellular radical scavengers and other metabolic reactions may prevent a measurable increase in LPO. This is partly illustrated by the case of vitamin-E deficiency, where a substantial increase in LPO in BMC microsomes is observed even without-irradiation in comparison with euvitaminotic mice because normally occurring radicals are not scavenged sufficiently.On leave from the University of Rochester, School of Medicine and Dentistry, Rochester, NY 14678, USA     

Does mercury promote lipid peroxidation?

In order to explore the observed association among mercury, atherosclerosis, and coronary heart disease, the effects of mercury, copper, and iron on the peroxidation of low-density lipoprotein (LDL) and on the enzymatic activities of glutathione peroxidase and myeloperoxidase were investigated in vitro. On the basis of our nuclear magnetic resonance (NMR) experiments, we conclude that mercury does not promote the direct nonenzymatic peroxidation of LDL, like copper and iron. In our enzyme measurements, mercury inhibited slightly myeloperoxidase, although not significantly in presence of LDL. Instead, inorganic mercury, but not methylmercury chloride, inhibited glutathione peroxidase effectively and copper event at 10 μmol/L, below physiological concentrations, doubled the inhibition rate. Copper and iron had no direct effect on glutathione peroxidase, but they both seem to activate production of HOCl by myeloperoxidase. We conclude here that, first, mercury and methylmercury do not promote direct lipid peroxidation, but that, second, a simultaneous exposure to high inorganic mercury, copper, and iron and low selenium concentrations can lead to a condition in which mercury promotes lipid peroxidations. This mechanism provides a plausible molecular-level explanation for the observed association between high body mercury content and atherosclerosis.     

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

In the present study it was investigated if a-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°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 -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 -tocopherol (0.35 mol/mg protein) produce a 43.37% inhibition of the lipid peroxidation process evaluated as chemiluminiscence (total cpm originated in 160 min). The phospholipid species containing the highest amount of docosahexaenoic acid: phosphatidyletanolamine and phosphatidylserine were more affected than phosphatidylcholine during the lipid peroxidation process. Not all phospholipids, however, were equally protected after the addition of -tocopherol to the incubation medium. Phosphatidylcholine and phosphatidyletanolamine, were not protected by -tocopherol, the vitamin provides selective antioxidant protection only for phosphatidylserine. These results indicate that -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.     

Remedial effect of DL-α-lipoic acid against adriamycin induced testicular lipid peroxidation

Adriamycin (ADR), a cytotoxic antineoplastic drug is used in the treatment of various solid tumors. However, its efficacy continues to be challenged by significant toxicities including testicular toxicity. In the present study, the effect of lipoic acid, a "universal antioxidant" was investigated on ADR induced peroxidative damages in rat testis. Adult male albino rats of Wistar strain were administered ADR (1 mg/kg body weight, i.v.) once a week for 10 weeks. ADR injected rats showed a significant decline in the activities of enzymic antioxidants (catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase) and non-enzymic antioxidants (reduced glutathione, Vitamin A, Vitamin C and Vitamin E) with high malondialdehyde levels. The extent of testicular toxicity was evident from the decreased activities of testicular marker enzymes (sorbitol dehydrogenase and glucose-6-phosphate dehydrogenase). Treatment with lipoic acid (35 mg/kg body weight, i.p.) one day prior to ADR administration, maintained near normal activities of the enzymes and significantly reduced lipid peroxidation, thereby proving it to be an effective cytoprotectant.     

Relationship of tumor necrosis factor α expression with activation of sphingomyelinase and lipid peroxidation after removal of cholestatic factor

The goal of this work was to study the expression of tumor necrosis factor α (TNFα), sphingomyelin cycle activation, and lipid peroxidation (LPO) processes after the removal of a cholestatic factor in the liver subjected to different durations of cholestasis. Restored bile flow after a 9-day hepatic cholestasis normalized sphingomyelinase (SMase) activity and levels of TNFα and LPO products. The removal of a cholestatic factor after a 12-day cholestasis did not normalize the studied parameters: SMase activity and the levels of TNFα and LPO products remained much higher compared to control. A significant positive correlation between TNFα expression, SMase activity, and LPO rate has been revealed. The obtained data indicate that hepatocyte apoptosis after bile outflow restoration in late cholestasis can be due to the activation of the sphingomyelin cycle, LPO, and TNFα expression. The synergistic interaction can sharply increase the proapoptotic capacity of each of these factors since TNFα activates SMase and LPO, SMase activity depends on the LPO rate, while ceramide, an SMase-produced secondary messenger of apoptosis, can induce oxidative stress.     

How does the Bax-α1 targeting sequence interact with mitochondrial membranes? The role of cardiolipin

A key event in programmed cell death is the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. The first helix localized at the N-terminus of Bax (Bax-α1) can act here as an addressing sequence, which directs activated Bax towards the mitochondrial surface. Solid state NMR (nuclear magnetic resonance), CD (circular dichroism) and ATR (attenuated total reflection) spectroscopy were used to elucidate this recognition process of a mitochondrial membrane system by Bax-α1. Two potential target membranes were studied, with the outer mitochondrial membrane (OM) mimicked by neutral phospholipids, while mitochondrial contact sites (CS) contained additional anionic cardiolipin. ¹H and ³¹P magic angle spinning (MAS) NMR revealed Bax-α1 induced pronounced perturbations in the lipid headgroup region only in presence of cardiolipin. Bax-α1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free OM vesicles, thereby adopting β-sheet-like features, as confirmed by ATR. CD studies revealed, that the cardiolipin mediated electrostatic locking of Bax-α1 at the CS membrane surface promotes conformational changes into an α-helical state; a process which seems to be necessary to induce further conformational transition events in activated Bax which finally causes irreversible membrane permeabilization during the mitochondrial apoptosis.     

Enhanced lipid peroxidation and inflammation during heat exposure in rats of different ages: Role of α-tocopherol

To investigate early events possibly related to the development of heat shock, we examined whether inflammatory-(interleukin-6, tumor necrosis factor α and 15-keto-13,14-dihydro-PGF_2α) and peroxidative-(8-iso-PGF_2α and malondialdehyde) markers are altered during acute heat exposure and aging. We also studied the relationships between inflammatory and peroxidative markers in these settings. In order to prevent these reactions developed as a consequence of the conditions mentioned above, we tested the effects of α-tocopherol. Our results demonstrated that 15-keto-13,14-dihydro-PGF_2α and malondialdehyde in the liver were altered during acute heat exposure in the young and middle-aged rats and could be predicted by changes in the levels of circulatory cytokines. Regardless of age, the supplementation with α-tocopherol prevented changes in the plasma cytokine levels and 15-keto-13,14-dihydro-PGF_2α and malondialdehyde levels in the liver, during acute heat exposure. This study notably emphasized the ability of α-tocopherol to prevent different heat induced mechanisms, involved in induction of inflammatory or peroxidative reactions.     

Inhibition of xanthine oxidase — xanthine — iron mediated lipid peroxidation by eugenol in liposomes

The effect of eugenol on xanthine oxidase (XO) xanthine(X)-Fe⁺³-ADP mediated lipid peroxidation was studied in liver microsomal lipid liposomes. Eugenol inhibited the lipid peroxidation in a dose dependent manner as assessed by formation of thiobarbituric acid reactive substances. When tested for its effect on XO activity per se, (by measuring uric acid formation) eugenol inhibited the enzyme to an extent of 85% at 10 µm concentration and hence formation of O₂ also However, the concentration of eugenol required for XO inhibition was more in presence of metal chelators such as EDTA, EGTA and DETAPAC, but not in presence of deferoxamine, ADP and citrate. The antiperoxidative effect of eugenol was about 35 times more and inhibition of XO was about 5 times higher as compared to the effect of allopurinol. Eugenol did not scavenge O₂ generated by phenazine methosulfate and NAD but inhibited propagation of peroxidation catalyzed by Fe²⁺ EDTA and lipid hydroperoxide containing liposomes. Eugenol inhibits XO-X-Fe⁺³ ADP mediated peroxidation by inhibiting the XO activity per se in addition to quenching various radical species. (Mol Cell Biochem 166: 65-71, 1997)     

Effect of lipid peroxidation conditions on calcium-dependent activity of phosphodiesterase 3′,5′-cAMP in the rat brain

3′,5′-cAMP plays an important role as a second messenger molecule controlling multiple cellular processes in the brain. Its levels are decreased by phosphodiesterases (PDEs), responsible for hydrolysis of intracellular cAMP. A part of the PDE activity is dependent on the effect of calcium, mediated by its binding to calmodulin. During oxidative stress, precisely these changes in calcium concentration are responsible for cell damage. We have examined the effects of oxidative stress conditions on the activity of PDE in rat brain homogenates. We found a different influence of activated lipid peroxidation conditions (Fe²⁺ with ascorbate and increased temperature) on the calcium-dependent and calcium-independent PDE activity. The inhibition of Ca²⁺-dependent PDE was observed, while Ca²⁺-independent PDE was not influenced. We assume that it might be the impact of lipid peroxidation products or any mechanism activated by the higher temperature on the interaction of the Ca²⁺-dependent isoform of PDE with the complex calcium-calmodulin. Another explanation might be that the formation of the functioning calcium-calmodulin complex is impossible in these conditions.