Role of Fe(III) in Fe(II)citrate-mediated peroxidation of mitochondrial membrane lipids

In this report we study the effect of Fe(III) on lipid peroxidation induced by Fe(II)citrate in mitochondrial membranes, as assessed by the production of thiobarbituric acid-reactive substances and antimycin A-insensitive oxygen uptake. The presence of Fe(III) stimulates initiation of lipid peroxidation when low citrate:Fe(II) ratios are used ( 4:1). For a citrate:total iron ratio of 1:1 the maximal stimulation of lipid peroxidation by Fe(III) was observed when the Fe(II):Fe(III) ratio was in the range of 1:1 to 1:2. The lag phase that accompanies oxygen uptake was greatly diminished by increasing concentrations of Fe(III) when the citrate:total iron ratio was 1:1, but not when this ratio was higher. It is concluded that the increase of lipid peroxidation by Fe(III) is observed only when low citrate:Fe(II) ratios were used. Similar results were obtained using ATP as a ligand of iron. Monitoring the rate of spontaneous Fe(II) oxidation by measuring oxygen uptake in buffered medium, in the absence of mitochondria, Fe(III)-stimulated oxygen consumption was observed only when a low citrate:Fe(II) ratio was used. This result suggests that Fe(III) may facilitate the initiation and/or propagation of lipid peroxidation by increasing the rate of Fe(II)citrate-generated reactive oxygen species.     

H2O2 production and response to stress conditions by mitochondrial fractions from rat liver

Rat liver mitochondria, in different steps of the maturation process, were resolved by differential centrifugation at 1000g (M₁), 3000g (M₃), and 10,000g (M₁₀), and their characteristics determining susceptibility to stress conditions were investigated. Some parameters did not show gradual changes in the transition from M₁₀ to M₁ fraction because of the contamination of the M₁₀ fraction by microsomes and damaged mitochondria with relatively high lipid content. The highest and lowest rates of O₂ consumption and H₂O₂ production were exhibited by M₁ and M₁₀ fractions, respectively. Vitamin E and coenzyme Q levels were significantly higher in M₁₀ than in M₁ fraction, whereas whole antioxidant capacity was not significantly different. The degree of oxidative damage to lipids and proteins was higher in M₁ and not significantly different in M₃ and M₁₀ fractions. The order of susceptibility to both oxidative challenge and Ca²⁺-induced swelling was M₁ > M₃ > M₁₀. It seems that the Ca²⁺-induced swelling is due to permeabilization of oxidatively altered inner membrane and leads to discard mitochondria with high ROS production. If, as previous reports suggest, mitochondrial damage is initiating stimulus to mitochondrial biogenesis, the susceptibility of the M₁ mitochondria to stressful conditions could be important to regulate cellular ROS production. In fact, it should favor the substitution of the oldest ROS-overproducing mitochondria with neoformed mitochondria endowed with a smaller capacity to produce free radicals.     

Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxidation in rat liver mitochondria

Mitochondria are cellular organelles where the generation of reactive oxygen species may be high. They are, however, effectively protected by their high capacities of antioxidative systems, as enzymes and either water or lipid soluble low molecular weight antioxidants.These antioxidative defence systems can be effectively regenerated after or during an oxidative stress as long as the mitochondria are in an energized state. Energization of mitochondria mainly depends on the availability of suitable respiratory substrates which can provide hydrogen for the reduction of either the glutathione- or -tocopherol-system, since GSH is regenerated by glutathione reductase with the substrate NADPH and the -tocopheroxyl-radical likely by reduced coenzyme Q. It was shown that mitochondria do not undergo damages as long as they can keep a high energy state. The delicate balance between prooxidative/antioxidative activities can be shifted towards oxidation, if experimentally prooxidants were added. After exhaustion of the antioxidative defence systems damages of rnitochondrial functions become expressed followed by membrane injuries along with the oxidation and degradation of mitochondrial lipids and proteins leading finally to the total degradation of the mitoc hondria.Extramitochondrial antioxidants may assist the mitochondrial antioxidative defence systems in a complex way, whereby particularly ascorbic acid can act both as prooxidant and as antioxidant. (Mol Cell Biochem 174: 199–205, 1997)     

Lipid peroxidation in purified plasma membrane fractions of rat liver in relation to the hepatoxicity of carbon tetrachloride

Preparations of rat liver sinusoidal plasma membrane have been tested for their ability to metabolize the hepatotoxin carbon tetrachloride (CCl4) to reactive free radicals in vitro and compared in this respect with standard preparations of rat liver microsomes. The sinusoidal plasma membranes were relatively free of endoplasmic reticulum-associated activities such as the enzymes of the cytochrome P450 system and glucose-6-phosphatase. CCl4 metabolism was measured as (i) covalent binding of [14C]-CCl4 to membrane protein, (ii) electron spin resonance spin-trapping of CCl3. radicals and (iii) CCl4-induced lipid peroxidation. By all of these tests, purified sinusoidal plasma membranes were found unable to metabolize CCl4. The fatty acid composition of the plasma membranes was almost identical to that of the microsomal preparation and both membrane fractions exhibited similar rates of the lipid peroxidation that was stimulated non-enzymically by gamma-radiation or incubation with ascorbate and iron. The absence of CCl4-induced lipid peroxidation in the plasma membranes seems to be due, therefore, to an absence of CCl4 activation rather than an inherent resistance to lipid peroxidation. We conclude that damage to the hepatocyte plasma membrane during CCl4 intoxication is not due to a significant local activation of CCl4 to CCl3. within that membrane.     

Glycosaminoglycans reduce oxidative damage induced by copper (Cu+2), iron (Fe+2) and hydrogen peroxide (H2O2) in human fibroblast cultures

Acid glycosaminoglycans (GAGs) antioxidant activity was assessed in a fibroblast culture system by evaluating reduction of oxidative system-induced damage. Three different methods to induce oxidative stress in human skin fibroblast cultures were used. In the first protocol cells were treated with CuSO₄ plus ascorbate. In the second experiment fibroblasts were exposed to FeSO₄ plus ascorbate. In the third system H₂O₂ was utilised. The exposition of fibroblasts to each one of the three oxidant systems caused inhibition of cell growth and cell death, increase of lipid peroxidation evaluated by the analysis of malondialdehyde (MDA), decrease of reduced glutathione (GSH) and superoxide dismutase (SOD) levels, and rise of lactate dehydrogenase activity (LDH). The treatment with commercial GAGs at different doses showed beneficial effects in all oxidative models. Hyaluronic acid (HA) and chondroitin-4-sulphate (C4S) exhibited the highest protection. However, the cells exposed to CuSO₄ plus ascorbate and FeSO₄ plus ascorbate were better protected by GAGs compared to those exposed to H₂O₂. These outcomes confirm the antioxidant properties of GAGs and further support the hypothesis that these molecules may function as metal chelators. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

水分胁迫对甘蔗叶片线粒体膜流动性的影响及其与膜脂过氧化的关系

用荧光探剂ANS对抗旱性不同的甘蔗品种在水分胁迫下叶片线粒体膜流动性的变化进行的研究表明,水分胁迫降低了线粒体膜的流动性,抗旱性强的甘蔗品种Co 617和F.Y.79-9的下降幅度分别小于抗旱性弱的Co 740和M.T.77-208;水分胁迫下线粒体膜流动性的下降与膜脂过氧化产物丙二醛含量的增加有密切关系。外源自由基处理试验也表明,甘蔗叶片线粒体膜流动性的下降与膜脂过氧化作用有关。     

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H₂O₂-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.     

A mechanism of mitochondrial damage induced by tert-butyl hydroperoxide and microsomes in vitro

Isolated potato ( Solanum tuberosum L. cv. Dansyaku) tuber mitochondria showed a significant loss in respiratory activity when treated with tert -butyl hydroperoxide (BHP), especially in the presence of microsomes. The following alterations appeared in parallel with the gradual decrease in the respiratory activity: The outer membrane became leaky, probably due to peroxidation of phospholipids. The level of sulfhydryl (SH) groups in mitochondrial proteins decreased in contrast to non-protein SH groups. A considerable amount of phospholipids was degraded and lost. A mechanism of the mitochondrial damage induced by BHP and microsomes is discussed with respect to a significant role of free radicals which may be formed at the onset of senescence or physiological disorders.     

Tempol protection of spinal cord mitochondria from peroxynitrite-induced oxidative damage

Peroxynitrite (PN)-mediated mitochondrial dysfunction has been implicated in the secondary injury process after traumatic spinal cord injury (SCI). This study investigated the detrimental effects of the PN donor SIN-1 (3-morpholinosydnonimine) on isolated healthy spinal cord mitochondria and the protective effects of tempol, a catalytic scavenger of PN-derived radicals. A 5 min exposure of the mitochondria to SIN-1 caused a dose-dependent decrease in the respiratory control ratio (RCR) that was accompanied by significant increases in complex I-driven states II and IV respiration rates and decreases in states III and V. These impairments occurred together with an increase in mitochondrial protein 3-nitrotyrosine (3-NT), but not in lipid peroxidation (LP)-related 4-hydroxynonenal (4-HNE). Tempol significantly antagonized the respiratory effects of SIN-1 in parallel with an attenuation of 3-NT levels. These results show that the exogenous PN donor, SIN-1, rapidly causes mitochondrial oxidative damage and complex I dysfunction identical to traumatic spinal cord mitochondrial impairment and that this is mainly due to tyrosine nitration. Consistent with that, the protection of mitochondrial respiratory function by tempol is associated with a decrease in 3-NT levels in mitochondrial proteins also similar to the previously reported antioxidant actions of tempol in traumatically-injured spinal cord mitochondria.     

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.     

Deficit of mitochondria-derived ATP during oxidative stress impairs mouse MII oocyte spindles

Although the role of oxidative stress in maternal aging and infertility has been suggested, the underlying mechanisms are not fully understood. The present study is designed to determine the relationship between mitochondrial function and spindle stability in metaphase II （MII） oocytes under oxidative stress. MII mouse oocytes were treated with H2O2 in the presence or absence of permeability transition pores （PTPs） blockers cyclosporin A （CsA）. In addition, antioxidant N-acetylcysteine （NAC）, F0/F1 synthase inhibitor oligomycin A, the mitochondria uncoupler carbonyl cyanide 4-trifluoro- methoxyphenylhydrazone （FCCP） or thapsigargin plus 2.5 mM Ca^2＋ （Th＋2.5 mM Ca^2＋） were used in mechanistic studies. Morphologic analyses of oocyte spindles and chromosomes were performed and mitochondrial membrane potential （AWm）, cytoplasmic free calcium concentration （[Ca^2＋]c） and cytoplasmic ATP content within oocytes were also assayed. In a time- and H202 dose-dependent manner, disruption of meiotic spindles was found after oocytes were treated with H202, which was prevented by pre-treatment with NAC. Administration of H2O2 led to a dissipation of AWm, an increase in [Ca^2＋]c and a decrease in cytoplasmic ATP levels. These detrimental responses of oocytes to H2O2 treatment could be blocked by pre-incubation with CsA. Similar to H2O2, both oligomycin A and FCCP dissipated AWm, decreased cytoplasmic ATP contents and disassembled MII oocyte spindles, while high [Ca^2＋]c alone had no effects on spindle morphology. In conclusion, the decrease in mitochondria-derived ATP during oxidative stress may cause a disassembly of mouse MII oocyte spindles, presumably due to the opening of the mitochondrial PTPs.     

Action of the herbicides neburon and siduron on membrane permeabilities in potato tuber mitochondria

The herbicides neburon and siduron are uncouplers of oxidative phosphorylation in potato tuber (Solanum tuberosum L. cv. Bintje) mitochondria. Their effect on the ion permeabilities of the mitochondrial membrane was investigated using the acid-base pulse technique, swelling experiments and integrity tests. Both herbicides permeabilize the membrane to H⁺ ions. They have no action on the permeabilities of K⁺ and Fe(CN)^3?₆. The swelling observed with Ca²⁺ was better interpreted as an effect on membrane structure than as a true swelling. Diuron, a parent compound that does not uncouple oxidative phosphorylation, does not act on Ca²⁺-induced apparent swelling.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Decrease in beech (<Emphasis Type="Italic">Fagus sylvatica</Emphasis>) seed viability caused by temperature and humidity conditions as related to membrane damage and lipid composition

The aim of this study was to determine if the loss of germinability and viability of beech (Fagus sylvatica L.) seeds stored at different variants of temperature (4, 20, and 30 °C) and relative humidity (RH: 45 and 75 %) is associated with a loss of membrane integrity and changes in lipid composition. Beech seeds stored for 9 weeks gradually lost viability at a rate dependent on temperature and humidity. The harmful effect of temperature increased with growing humidity. The loss of seed viability was strongly correlated with an increase in membrane permeability and with production of lipid hydroxyperoxides (LHPO), which was regarded as an indicator of peroxidation of unsaturated fatty acids. The condition of membranes was assessed on the basis of their permeability and the state of lipid components: phospholipids and fatty acids. During seed storage we observed a decline in concentration of individual phospholipids and fatty acids, proportional to the loss of seeds viability. We also detected a decrease in concentrations of α-tocopherol and sterols, which play an important role in protection of membranes against the harmful influence of the environment. Our results show that the germinability of beech seeds declines rapidly at temperature above 0 °C and growing humidity. This is due mainly to the loss of membrane integrity, caused by peroxidation of unsaturated fatty acids.     

谷氨酰胺对心肌细胞线粒体膜PTP开放干预作用的实验研究

目的:探讨谷氨酰胺(Gln)对过度训练状态下心肌线粒体膜通透性转换孔(PTP)开放的干预作用及其可能机制。方法:30只SD大鼠随机分为3组(n=10):对照组(CG组)、过度训练组(OG组)和补充Gln+过度训练组(GOG组)。采用分光光度法检测大鼠心肌线粒体PTP开放程度,电化学法检测心肌丙二醛(MDA)、还原型谷胱苷肽(GSH)含量和磷脂酶A2(PLA2)活性。结果:OG组与GOG组比较,吸光度(A0)显著下降(P<0.05),吸光度变化(△A)值显著降低(P<0.05);荧光剂罗丹明123(Rh123)的荧光强度(F0)显著增强(P<0.05),Rh123荧光强度变化(△F)值明显降低(P<0.05)。与GOG组比较,线粒体GSH含量显著降低(P<0.05),PLA2活性显著增加(P<0.05);MDA含量显著升高(P<0.05)。结论:过度训练可导致心肌细胞线粒体PTP开放增加,过度训练状态下线粒体活性氧生成增多,PLA2活性增加及GSH的含量下降,补充外源性的Gln对这些变化有显著的干预作用。     

Hydrogen peroxide is involved in the sclerotial differentiation of filamentous phytopathogenic fungi

Aims: The purpose of this study was to investigate the role of H₂O₂ and the related oxidative stress markers catalase (CAT) and lipid peroxidation in the sclerotial differentiation of the phytopathogenic filamentous fungi Sclerotium rolfsii, Sclerotinia minor, Sclerotinia sclerotiorum and Rhizoctonia solani. Methods and Results: Using the H₂O₂‐specific scopoletin fluorometric assay and the CAT‐dependent H₂O₂ consumption assays, it was found that the production rate of intra/extracellular H₂O₂ and CAT levels in the sclerotiogenic fungi were significantly higher and lower, respectively, than those of their nondifferentiating counterpart strains. They peaked in the transition between the undifferentiated and the differentiated state of the sclerotiogenic strains, suggesting both a cell proliferative and differentiative role. In addition, the indirect indicator of oxidative stress, lipid peroxidation, was substantially decreased in the nondifferentiating strains. Conclusions: These findings suggest that the differentiative role of H₂O₂ is expressed via induction of higher oxidative stress in the sclerotiogenic filamentous phytopathogenic fungi. Significance and Impact of the Study: This study shows that the direct marker of oxidative stress H₂O₂ is involved in the sclerotial differentiation of the phytopathogenic filamentous fungi S. rolfsii, S. minor, S. sclerotiorum and R. solani, which could have potential biotechnological implications in terms of developing antifungal strategies by regulating intracellular H₂O₂ levels.     

Effect of endothelin-1 induced ischemia on peroxidative damage and membrane properties in rat striatum synaptosomes

Synaptosomes obtained from rat striata lesioned by central injection of endothelin-1 (ET-1) were analyzed for the levels of lipid peroxidation products, the susceptibility to lipid peroxidation, the phospholipid and free fatty acid composition and the activity of Na⁺,K⁺-ATPase one hour after ET-1 treatment. The intrastriatal injection of ET-1 promoted an increase of endogenous thiobarbituric reactive substances (TBARS), as index of free radical mediated lipid damage, and a greater susceptibility to iron/ascorbate-induced lipid peroxidation. The pattern of free fatty acids showed a significant decrease of arachidonic and docosahexaenoic acid consequent to ET-1 treatment. The analysis of lipid composition showed a significant loss of phospholipids: among phospholipid species, sphingomyelin and phosphatidylethanolamine plasmalogen were particularly reduced by ET-1 treatment. The activity of membrane-bound Na⁺,K⁺-ATPase was also significantly reduced in synaptosomes obtained from ET-1 lesioned striata. Taken together these results indicate a significant modification of synaptosomal membrane of ET-1 treated rat striata, possibly due to a free radical mediated damage.     

Proteomic characterization of oxidative dysfunction in human umbilical vein endothelial cells (HUVEC) induced by exposure to oxidized LDL

The oxidative modification of low-density lipoprotein (LDL) and subsequent alteration of endothelial cell function are generally accepted as an important early event in the pathogenesis of atherosclerosis. To understand the mechanism by which oxidized LDL (oxLDL) causes dysfunction in endothelial cells, human umbilical vein endothelial cells (HUVEC) were exposed to oxLDL at a concentration that induces cellular dysfunction, and proteomic analysis was carried out, together with the analysis of cellular lipid peroxidation products. Time-dependent accumulation of 7-ketocholesterol and the progression of oxidative modification of peroxiredoxin 2 were observed, together with the suppression of cell proliferation. Proteomic analysis using two-dimensional gel electrophoresis (2-D gel) revealed that nucleophosmin, stathmin, and nucleolin were differentially expressed after exposure to oxLDL. Both 2-D gel and western blot analyses revealed that (1) nucleophosmin was dephosphorylated in a time-dependent manner; (2) stathmin was transiently phosphorylated at 6 h, and the unphosphorylated form was continuously down-regulated; and (3) nucleolin was identified as a 20-kDa fragment and a 76-kDa form, which were down-regulated. These observations suggest that the exposure of HUVEC to oxLDL results in the suppression of cell proliferation, which is ascribed to protein modification and/or altered expression of nucleophosmin, stathmin, and nucleolin under these oxidative stress conditions.