Induced Membrane Lipid Peroxidation Research Articles

Alterations in Glutathione Reductase, Superoxide Dismutase and Lipid Peroxidation of Tadpoles (Xenopus laevis) Exposed to Bonny Light Crude Oil and its Fractions

We determined the effects of sub-lethal levels of Bonny Light crude oil (WC) and its water soluble (WSF) and insoluble (WIF) fractions on malondialdehyde (MDA) and stress enzymes in tadpoles (Xenopus laevis) following two and 4 weeks exposure at different concentrations. We observed that the treatment of tadpoles with WC, WSF, or WIF decreased the weight of tadpoles, increased MDA levels, and increased superoxide dismutase (SOD) and glutathione reductase (GR) at lower concentrations of exposure and decreased the enzymes at higher doses of exposure. We found that the WC had a lesser negative effect on the parameters of tadpoles compared to the WSF. Longer exposure of tadpoles to WC, WSF, or WIF even at lower concentrations resulted in a negative effect on MDA, SOD, and GR activities The study shows that sub-lethal contaminations with WC, WSF, or WIF induces membrane lipid peroxidation and reduce the ability of tadpoles to produce SOD and GR which may have metabolic costs.

Protection against amyloid beta-peptide (1–42)-induced loss of phospholipid asymmetry in synaptosomal membranes by tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester: Implications for Alzheimer's disease

Amyloid-beta (1-42) [Abeta (1-42)] deposition in the brain is a hallmark of Alzheimer's disease (AD) and has been shown to induce apoptosis and disrupt cellular ion homeostasis. Abeta (1-42) induces membrane lipid peroxidation, and 4-hydroxynonenal (HNE) and 2-propenal (acrolein) are the two reactive products of lipid peroxidation, which structurally modify proteins by covalent interaction and inhibit enzyme function. Phosphatidylserine (PS), an aminophospholipid, is sequestered in the inner leaflet of the plasma membrane in nonstimulated cells. An early signal of synaptosomal apoptosis is the loss of phospholipid asymmetry and the appearance of phosphatidylserine in the outer leaflet of the membrane. The ATP-requiring enzyme, flippase, maintains phospholipid asymmetry of PS. Here, we have investigated the inactivation of the transmembrane enzyme aminophospholipid-translocase (or flippase) by Abeta (1-42). Flippase activity depends on a critical cysteine residue, a putative site of covalent modification by the Abeta (1-42)-induced lipid peroxidation products, HNE or acrolein. The present study is aimed to investigate the protective effects of tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester (FAEE) on Abeta (1-42) induced modulation in phospholipid asymmetry in the synaptosomal membranes. Pretreatment of synaptosomes with D609 and FAEE significantly protected Abeta (1-42)-induced loss of phospholipid asymmetry in synaptosomal membranes. Our results suggest that D609 and FAEE exert protective effects against Abeta (1-42) induced apoptosis. The increase in intracellular Ca(2+) might not be the sole cause for the loss of flippase activity. Rather, other mechanisms that could modulate the function of flippase might be important in the modulation of phospholipid asymmetry. The results of this study are discussed with relevance to neuronal loss in the AD brain.

Neuronal and glial calcium signaling in Alzheimer’s disease

Cognitive impairment and emotional disturbances in Alzheimer’s disease (AD) result from the degeneration of synapses and death of neurons in the limbic system and associated regions of the cerebral cortex. An alteration in the proteolytic processing of the amyloid precursor protein (APP) results in increased production and accumulation of amyloid β-peptide (Aβ) in the brain. Aβ has been shown to cause synaptic dysfunction and can render neurons vulnerable to excitotoxicity and apoptosis by a mechanism involving disruption of cellular calcium homeostasis. By inducing membrane lipid peroxidation and generation of the aldehyde 4-hydroxynonenal, Aβ impairs the function of membrane ion-motive ATPases and glucose and glutamate transporters, and can enhance calcium influx through voltage-dependent and ligand-gated calcium channels. Reduced levels of a secreted form of APP which normally regulates synaptic plasticity and cell survival may also promote disruption of synaptic calcium homeostasis in AD. Some cases of inherited AD are caused by mutations in presenilins 1 and 2 which perturb endoplasmic reticulum (ER) calcium homeostasis such that greater amounts of calcium are released upon stimulation, possibly as the result of alterations in IP 3 and ryanodine receptor channels, Ca 2+-ATPases and the ER stress protein Herp. Abnormalities in calcium regulation in astrocytes, oligodendrocytes, and microglia have also been documented in studies of experimental models of AD, suggesting contributions of these alterations to neuronal dysfunction and cell death in AD. Collectively, the available data show that perturbed cellular calcium homeostasis plays a prominent role in the pathogenesis of AD, suggesting potential benefits of preventative and therapeutic strategies that stabilize cellular calcium homeostasis.

Hypochlorous acid-induced oxidative damage of human red blood cells: effects of tert-butyl hydroperoxide and nitrite on the HOCl reaction with erythrocytes

Hypochlorous acid, one of the most powerful biological oxidants, is believed to be important in the pathogenesis of some diseases. The purpose of this study was to further characterise the membrane and intracellular events which resulted in HOCl-induced oxidative impairments and haemolysis of human erythrocytes and interaction of different oxidative agents, which accumulated during respiratory burst, in the process of RBS oxidation. The sequence of cellular events after red blood cell exposure to HOCl: cell morphological transformations, oxidation of cellular constituents, enzyme modifications, and haemolysis have been evaluated. It was shown that HOCl-treated cells underwent colloid-osmotic haemolysis, preceded by rapid morphological transformations and membrane structural transitions. The activation energy of the process of haemolysis (after removal of the excess of oxidative agent) was estimated to be 146±22 kJ/mol at temperatures above the break point of Arrhenius plot (31–32 °C). This value corresponds to the activation energy of the process of protein denaturation. Modification of erythrocytes by HOCl inhibited membrane acetylcholinesterase (uncompetitive type of inhibition), depleted intracellular glutathione, activated intracellular glutathione peroxidase, but did not induce membrane lipid peroxidation. The presence of other oxidants, nitrite or tert-butyl hydroperoxide ( t-BHP), promoted the oxidative damage induced by HOCl and led to new oxidative reactions.

Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: A comparative study

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.

Perturbed endoplasmic reticulum function, synaptic apoptosis and the pathogenesis of Alzheimer's disease.

Endoplasmic reticulum (ER) appears to be a focal point for alterations that result in neuronal dysfunction and death in Alzheimer's disease (AD). Aberrant proteolytic processing and/or trafficking of the beta-amyloid precursor protein (APP) in ER may promote neuronal degeneration by increasing the levels of the neurotoxic forms of beta-amyloid (A beta) and by decreasing the levels of the neuroprotective secreted form of APP (sAPP alpha). Some cases of AD are caused by mutations in the genes encoding presenilin 1 (PS1). When expressed in cultured neuronal cells and transgenic mice, PS1 mutations cause abnormalities in ER calcium homoeostasis, enhancing the calcium responses to stimuli that activate IP3- and ryanodine-sensitive ER calcium pools. Two major consequences of this disrupted ER calcium regulation are altered proteolytic processing of APP and increased vulnerability of neurons to apoptosis and excitotoxicity. The impact of PS1 mutations and aberrant APP processing is particularly great in synaptic terminals. Perturbed synaptic calcium homoeostasis promotes activation of apoptotic cascades involving production of Par-4 (prostate apoptosis response-4), mitochondrial dysfunction and caspase activation. A beta 42 (the 42-amino-acid form of A beta) induces membrane lipid peroxidation in synapses and dendrites resulting in impairment of membrane ion-motive ATPases and glucose and glutamate transporters. This disrupts synaptic ion and energy homoeostasis thereby promoting synaptic degeneration. In contrast, sAPP alpha activates signalling pathways that protect synapses against excitotoxicity and apoptosis. In the more common sporadic forms of AD, the initiating causes of the neurodegenerative cascade are less well defined, but probably involve increased levels of oxidative stress and impaired energy metabolism. Such alterations have been shown to disrupt neuronal calcium homoeostasis in experimental models, and may therefore feed into the same neurodegenerative cascade initiated by mutations in presenilins and APP. Perturbed synaptic ER calcium homoeostasis and consequent alterations in APP processing appear to be pivotal events in both sporadic and familial forms of AD.

Increased doxorubicin uptake and toxicity in multicellular tumour spheroids treated with DC electrical fields.

Electrochemotherapy (ECT) is a new approach to the treatment of tumours. In the present study, multicellular prostate tumour spheroids were treated with non-lethal direct current (DC) electrical fields, and uptake and toxicity of doxorubicin were investigated. An electrical field with a field strength of 500 Vm−1 applied for a duration of 90 s resulted in neither reversible nor irreversible membrane breakdown as revealed by fluid phase uptake studies of the membrane impermeant tracer Lucifer yellow. However, treated spheroids showed an increased uptake of doxorubicin and, consequently, an increased toxicity following electrical field exposure. The electrical field raised intracellular reactive oxygen species (ROS) as revealed using 2′,7′-dichlorofluorescein diacetate (H2DCFDA) as an indicator. ROS induced membrane lipid peroxidation since the lipid peroxidation end products malondialdehyde (MDA) and 4-hydroxy-2-(E)-nonenal (4-HNE) were detected after electrical field treatment. Moreover, lipid peroxidation decreased the lipid diffusion coefficient D from 4.2 × 10−10 cm2 s−1 to 2.7 × 10−10 cm2 s−1 in the control and treated sample, respectively, as revealed by fluorescence recovery after photobleaching (FRAP) experiments. The field effects could be mimicked by incubating spheroids with 100 nM hydrogen peroxide and were inhibited by the radical scavengers dehydroascorbate (DHA) and α-tocopherol (vitamin E), indicating that the increased uptake of doxorubicin after electrical field treatment is owing to lipid peroxidation and decreased membrane lipid mobility. Treatment of tumours with low intensity electrical fields may be useful to improve the cytotoxic capacity of anthracyclines. © 1999 Cancer Research Campaign

Effects of amyloid precursor protein derivatives andoxidative stress on basal forebrain cholinergic systems inALZHEIMERS disease

The dysfunction and degeneration of cholinergic neuronal circuits in the brain is aprominent feature of Alzheimers disease. Increasing data suggest that age-related oxidative stresscontributes to degenerative changes in basal forebrain cholinergic systems. Experimental studieshave shown that oxidative stress, and membrane lipid peroxidation in particular, can disruptmuscarinic cholinergic signaling by impairing coupling of receptors to GTP-binding proteins.Altered proteolytic processing of the β-amyloid precursor protein (APP) may contributeto impaired cholinergic signaling and neuronal degeneration in at least two ways. First, levels ofcytotoxic forms of amyloid β-peptide (Aβ) are increased ; Aβdamages and kills neurons by inducing membrane lipid peroxidation resulting in impairment ofion-motive ATPases, and glucose and glutamate transporters, thereby rendering neuronsvulnerable to excitotoxicity. The latter actions of Aβ may be mediated by4-hydroxynonenal, an aldehydic product of membrane lipid peroxidation that covalently modifiesand inactivates the various transporter proteins. Subtoxic levels of Aβ can also suppresscholine acetyltransferase levels, and may thereby promote dysfunction of intact cholinergiccircuits. A second way in which altered APP processing may endanger cholinergic neurons is byreducing levels of a secreted form of APP which has been shown to modulate neuronalexcitability, and to protect neurons against excitotoxic, metabolic and oxidative insults. Mutationsin presenilin genes, which are causally linked to many cases of early-onset inherited Alzheimersdisease, may increase vulnerability of cholinergic neurons to apoptosis. The underlying mechanismappears to involve perturbed calcium regulation in the endoplasmic reticulum, which promotesloss of cellular calcium homeostasis, mitochondrial dysfunction and oxyradical production.Knowledge of the cellular and molecular underpinnings of dysfunction and degeneration ofcholinergic circuits is leading to the development of novel preventative and therapeuticapproaches for Alzheimers disease and related disorders.

Oxidative stress of vanadium-mediated oxygen free radical generation stimulated by aluminium on human erythrocytes.

It has been suggested that aluminium stimulates vanadium-mediated superoxide radical generation. The oxidative stress of generated superoxide radicals on antioxidant enzyme activity, oxidation of NADH and NADPH, membrane lipid peroxidation and osmotic fragility in human red blood cells (RBC) was investigated. RBC were incubated with varying concentrations of vanadium and aluminium ions at 37 degrees C for 2 h. RBC incubated with vanadium ions showed significantly increased superoxide dismutase and catalase activities, and oxidized NADH and NADPH concentrations compared with control RBC preparations. Erythrocyte lipid peroxidation was assessed by measuring thiobarbituric acid reactivity. RBC incubated with elevated levels of vanadium showed significantly increased membrane lipid peroxidation when compared with control RBC; it increased further on addition of aluminium. A significant positive correlation was observed between the extent of vanadium induced membrane lipid peroxidation and the osmotic fragility of treated RBC. In the presence of vanadium, aluminium stimulates superoxide dismutase and catalase activities. NADH and NADPH oxidation and membrane lipid peroxidation, as well as increasing osmotic fragility of human erythrocytes. The stimulatory effect of aluminium was dependent on concentration. These results may have implications for the mechanism of toxicity of aluminium and vanadium in haemodialysis patients.

17?-Estradiol attenuates oxidative impairment of synaptic Na+/K+-ATPase activity, glucose transport, and glutamate transport induced by amyloid ?-peptide and iron

Synapse loss, deposits of amyloid beta-peptide (Abeta), impaired energy metabolism, and cognitive deficits are defining features of Alzheimer's disease (AD). Estrogen replacement therapy reduces the risk of developing AD in postmenopausal women. Because synapses are likely sites for initiation of neurodegenerative cascades in AD, we tested the hypothesis that estrogens act directly on synapses to suppress oxidative impairment of membrane transport systems. Exposure of rat cortical synaptosomes to Abeta25-35 (Abeta) and FeSO4 induced membrane lipid peroxidation and impaired the function of the plasma membrane Na+/K+-ATPase, glutamate transporter, and glucose transporter. Pretreatment of synaptosomes with 17beta-estradiol or estriol largely prevented impairment of Na+/K+-ATPase activity, glutamate transport, and glucose transport; other steroids were relatively ineffective. 17Beta-estradiol suppressed membrane lipid peroxidation induced by Abeta and FeSO4, but did not prevent impairment of membrane transport systems by 4-hydroxynonenal (a toxic lipid peroxidation product), suggesting that an antioxidant property of 17beta-estradiol was responsible for its protective effects. By suppressing membrane lipid peroxidation in synaptic membranes, estrogens may prevent impairment of transport systems that maintain ion homeostasis and energy metabolism, and thereby forestall excitotoxic synaptic degeneration and neuronal loss in disorders such as AD and ischemic stroke.

Aβ25–35 induces rapid lysis of red blood cells: contrast with Aβ1–42 and examination of underlying mechanisms

Amyloid β-peptide (Aβ) is produced by many different cell types and circulates in blood and cerebrospinal fluid in a soluble form. In Alzheimer's disease (AD), Aβ forms insoluble fibrillar aggregates that accumulate in association with cells of the brain parenchyma and vasculature. Both full-length Aβ (Aβ1–40/42) and the Aβ25–35 fragment can damage and kill neurons by a mechanism that may involve oxidative stress and disruption of calcium homeostasis. Circulating blood cells are exposed to soluble Aβ1–40/42 and may also be exposed to Aβ aggregates associated with the luminal surfaces of cerebral microvessels. We therefore examined the effects of Aβ25–35 and Aβ1–42 on human red blood cells (RBCs) and report that Aβ25–35, in contrast to Aβ1–42, induces rapid (10–60 min) lysis of RBCs. The mechanism of RBC lysis by Aβ25–35 involved ion channel formation and calcium influx, but did not involve oxidative stress because antioxidants did not prevent cell lysis. In contrast, Aβ1–42 induced a delayed (4–24 h) damage to RBCs which was attenuated by antioxidants. The damaging effects of both Aβ25–35 and Aβ1–42 towards RBCs were completely prevented by Congo red indicating a requirement for peptide fibril formation. Aβ1–42 induced membrane lipid peroxidation in RBC, and basal levels of lipid peroxidation in RBCs from AD patients were significantly greater than in age-matched controls, suggesting a possible role for Aβ1–42 in previously reported alterations in RBCs from AD patients.

Impairment of Glucose and Glutamate Transport and Induction of Mitochondrial Oxidative Stress and Dysfunction in Synaptosomes by Amyloid β‐Peptide: Role of the Lipid Peroxidation Product 4‐Hydroxynonenal

Deposits of amyloid beta-peptide (A beta), reduced glucose uptake into brain cells, oxidative damage to cellular proteins and lipids, and excitotoxic mechanisms have all been suggested to play roles in the neurodegenerative process in Alzheimer's disease. Synapse loss is closely correlated with cognitive impairments in Alzheimer's disease, suggesting that the synapse may be the site at which degenerative mechanisms are initiated and propagated. We report that A beta causes oxyradical-mediated impairment of glucose transport, glutamate transport, and mitochondrial function in rat neocortical synaptosomes. A beta induced membrane lipid peroxidation in synaptosomes that occurred within 1 h of exposure; significant decreases in glucose transport occurred within 1 h of exposure to A beta and decreased further with time. The lipid peroxidation product 4-hydroxynonenal conjugated to synaptosomal proteins and impaired glucose transport; several antioxidants prevented A beta-induced impairment of glucose transport, indicating that lipid peroxidation was causally linked to this adverse action of A beta. FeSO4 (an initiator of lipid peroxidation), A beta, and 4-hydroxynonenal each induced accumulation of mitochondrial reactive oxygen species, caused concentration-dependent decreases in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, and reduced cellular ATP levels significantly. A beta also impaired glutamate transport, an effect blocked by antioxidants. These data suggest that A beta induces membrane lipid peroxidation, which results in impairment of the function of membrane glucose and glutamate transporters, altered mitochondrial function, and a deficit in ATP levels; 4-hydroxynonenal appears to be a mediator of these actions of A beta. These data suggest that oxidative stress occurring at synapses may contribute to the reduced glucose uptake and synaptic degeneration that occurs in Alzheimer's disease patients. They further suggest a sequence of events whereby oxidative stress promotes excitotoxic synaptic degeneration and neuronal cell death in a variety of different neurodegenerative disorders.

Diclofenac Sodium and Mefenamic Acid: Potent Inducers of the Membrane Permeability Transition in Renal Cortex Mitochondria

The ability of nonsteroidal anti-inflammatory drugs (NSAIDs) to induce Ca2+-mediated/cyclosporin A-sensitive mitochondrial membrane permeability transition (MMPT) was evaluated by monitoring swelling of isolated rat renal cortex mitochondria in the presence of 20 μmCaCl2. Dipyrone and paracetamol did not induce MMPT, while piroxicam and acetylsalicylic acid (and its metabolite salicylate) were poor inducers. In contrast, diclofenac sodium and mefenamic acid were potent triggering agents, inducing MMPT at 2 μm, a concentration below those previously shown to uncouple and/or inhibit oxidative phosphorylation. When compared to salicylate, a classical uncoupler and inducer of MMPT, the potency of diclofenac sodium and mefenamic acid was about 50-fold greater. Swelling was completely prevented by EGTA, cyclosporin A, or MgCl2, and only partially by ADP or dithiothreitol. Under the same experimental conditions as for the swelling assays, the drugs depressed the membrane potential of mitochondria, an effect prevented by cyclosporin A and restored by EGTA. Also, the drugs did not induce membrane lipid peroxidation or changes in GSSG levels, but led to a small decrease in protein thiol content, as well as to a substantial decrease in the NADPH levels of mitochondria. Hence, membrane depolarization and pyridine nucleotide oxidation seem to be involved in MMPT induction by these NSAIDs. The potency in eliciting the process, like the uncoupling activity, seems to be influenced by the lipophilic character of the molecules.

Lipid Peroxidation Products in Tomato Fruit (Lycopersicon esculentum) during Storage after Exposure to Hormic Doses of UV Light

Fresh fruit and vegetables are highly perishable because of their active metabolism during the postharvest phase. Previous studies showed that hormic dose of UV cause a delay in the senescence of tomato fruit by about 7 days. The objective of this study was to elucidate whether UV acts on the cell membrane in producing the phenomenon of delayed senescence, since it is known that UV radiation can provoke photooxidation of membrane lipids. Membrane lipid peroxidation was studied in tomato fruit (Lycopersicon esculentum Mill cv. Trust) treated by hormic UV dose, and was followed by assaying products of lipid oxidation during the storage period. We observed the production of lipofuscin-like compounds, malondialdehyde, aldehydes, pentane, ethane, and hydrogen peroxide within few days of the treatment. An increase in the efflux of electrolytes (total, potassium, and calcium) was also observed. An immediate increase in the level of these products of oxidation supports the hypothesis that UV radiation induces membrane lipid peroxidation. However, beyond 5 to 7 days after treatment, the production of oxidation products and electrolyte leakage were lower than the control fruits. Thereafter, the level of products of lipid oxidation associated with senescence was higher in control fruits than in treated ones. Results suggest that the initial oxidation stress by the exposure to UV led to biochemical reactions inducing the production of stress compounds, such as polyamines, which are non specific antioxidants. Consequently, a delay in the senescence was observed.

Effects of Ferulic Acid on Fertile and Asthenozoospermic Infertile Human Sperm Motility, Viability, Lipid Peroxidation, and Cyclic Nucleotides

The capacity of human sperm fertilization principally depends on sperm motility and membrane integrity. Reactive oxygen species, such as superoxide anion and hydrogen peroxide, are known to impair sperm motility and membrane integrity by inducing membrane lipid peroxidation (LPO). Ferulic acid (FA), an effective constituent in various medicinal herbs, has recently been shown to scavenge oxygen free radicals and increase the intracellular cAMP and cGMP. The aim of this study is to investigate the effects of FA on human sperm motility, viability, lipid peroxidation, and cyclic nucleotides in fertile and asthenozoospermic infertile individuals in vitro. The sperm samples were obtained from 10 fertile volunteers and 10 asthenozoospermic infertile patients. Washed spermatozoa were incubated at 37°C in Ham's F-10 medium with 0, 0.1, 0.2, 0.4, 0.8, or 1.6 mM of FA. Samples were analyzed for viability, determined by eosin-Y dye exclusion method at 0, 1, 2, 3, 5, and 6 h of incubation; motility, determined by the trans-membrane migration method within 2 h of incubation; LPO, determined by thiobarbituric acid (TBA) method at 3 h of incubation and the intracellular cAMP and cGMP, determined, respectively, by 3H-cAMP and 125I-cGMP radioimmunoassay at 3 h of incubation. The results showed: in both fertile and infertile spermatozoa, the viability, trans-membrane migration ratio (TMMR) and the levels of intracellular cAMP and cGMP in FA-treated spermatozoa were significantly higher than those of spermatozoa in control groups, while TBA-reactive substances contents in treated spermatozoa were significantly lower than those in control spermatozoa. The effects of FA on these processes were concentration dependent. These data suggested that FA is beneficial to sperm viability and motility in both fertile and infertile individuals, and that reduction of lipid peroxidative damage to sperm membranes and increase of intracellular cAMP and cGMP may be involved in these benefits. It is possible that FA may be used for cure of asthenozoospermic infertility.Copyright © 1997 Elsevier Science Inc.

EFFECT OF LIPID PEROXIDATION ON THE NMDA RECEPTOR CPP BINDING SITE DURING CEREBRAL HYPOXIA IN NEWBORN PIGLETS. ▴ 2229

Previous studies have shown that hypoxia induces membrane lipid peroxidation and alters the N-methyl-D-aspartate (NMDA) receptor in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia-induced modification of the 3-(2-carboxypiperazin-4-yl)-1-phosphonic acid (CPP) binding site of the NMDA receptor is correlated with hypoxia-induced lipid peroxidation in brain cell membranes. Studies were performed in 18 anesthetized, ventilated piglets divided into 2 groups - 8 normoxic and 10 exposed to decreased FiO2 at different concentrations and durations to achieve various degrees of hypoxia. P2 membrane fractions were prepared and 3H-CPP binding was performed at concentrations ranging from 2.5 to 1500 nM at 23°C for 40 mins. Conjugated dienes (CDs) were measured as an index of lipid peroxidation.[ATP] and [phosphocreatine] (PCr) were measured biochemically to document tissue hypoxia. In the normoxic group the [CD] was 0.0 nmol/g brain. Bmax(receptor number) 343±80 fmole/mg protein and Kd (dissociation constant) 130±46 nM. The animals exposed to decreased FiO2 concentrations demonstrated [CDs] (nmol/g brain), Bmax (fmole/mg protein) and Kd (nM) concentrations as follows: (3.5, 142, 80), (25, 193, 118), (49, 195, 80), (56, 261, 119), (70, 326, 112), (98, 97, 47), (112, 61, 65), (117, 166, 156), (123, 59, 28) and (202, 50, 58). The results show that as the [CDs] increase both the receptor Bmax (r=0.7) and Kd (r=0.6) decrease in a linear fashion. The data demonstrate that during hypoxia progressive NMDA receptor modification occurs as lipid peroxidation increases. We speculate that the hypoxia-induced progressive modification of the NMDA recognition site may be due either to an alteration of brain cell membrane structure secondary to conjugated diene formation or to a direct modification of the receptor by hypoxia-induced free radicals. (Funded by NIH-HD-20337, MOD #6-FY94-0135, UCPR 506-93)

Contribution of plasma membrane and endoplasmic reticulum Ca 2+-ATPases to the synaptosomal [Ca 2+] i increase during oxidative stress

In the present study we analyzed the effect of ascorbate (0.8 mM)/Fe 2+ (2.5 μM)-induced membrane lipid peroxidation on the levels of intracellular free calcium, [Ca 2+] i, and on the possible mechanisms involved in the perturbation of intracellular calcium homeostasis during oxidative stress. For this purpose, the influence of the ascorbate/iron oxidant system on the plasma membrane and endoplasmic reticulum Ca 2+-dependent ATPases of brain cortical synaptosomes was studied. In addition, the influence of the peroxidative process on the uptake of calcium ( 45Ca 2+) and on the Na +/Ca 2+ exchange activity at the plasma membrane was evaluated. After ascorbate/Fe 2+-induced membrane lipid peroxidation of the order of 18.05 ± 4.20 nmol TBARS/mg protein, an increase in [Ca 2+] i occured, under basal or depolarizing conditions (30 mM KCI), which was dependent on the extracellular calcium concentration. Thus, for 1 and 3 mM extracellular calcium concentration, an increase of the resting [Ca 2+] i values of 19.8% and 33.7% was observed, while after the K +-depolarization the enhancement of the [Ca 2+] i was 18.4% and 29.5%, respectively. The Na +/Ca 2+ exchange activity and the time-dependent influx of 45 Ca 2+, observed in basal conditions and after the 30 mM K +-depolarization, were not affected under the peroxidative conditions. The Ca 2+-dependent ATPase activity of the synaptosomal plasma membrane was significantly depressed following peroxidation of membrane lipids, decreasing the V max by 48.1%, without significant changes in the affinity of the enzyme for calcium ( K m for Ca 2+ was 0.54 ± 0.04 μM in control conditions and 0.56 ± 0.034 μM in peroxidized conditions). The Ca 2+-ATPase activity of the endoplasmic reticulum was also affected during ascorbate/iron-induced oxidative stress; thus, an inhibition of 45.2% was observed 5 min after adding ATP. These data suggest that the increase in synaptosomal [Ca 2+] i due to oxidative stress may result from the inhibition of the plasma membrane and the endoplasmic reticulum membrane Ca 2+-ATPase activities, probably as a result of the alteration of the lipid environment required for the maximal activity of these membrane enzymes. The consequent increase in [Ca 2+] i may be responsible for the injury of the nervous tissue observed during several pathological conditions in which free radical generation seems to be involved.

Effect of elevated glucose concentrations on cellular lipid peroxidation and growth of cultured human kidney proximal tubule cells.

This study has examined whether elevated glucose can induce lipid peroxidation and contribute to the inhibition of cell growth in human kidney proximal tubule(HPT) cells. HPT cells were cultured in media containing glucose concentrations of 8 mM (control), 25 mM, and 50 mM. Lipid peroxidation was assessed by the thiobarbituric acid reactivity and cell growth was assessed by 3H-thymidine uptake. Results show decreased (59%, p < 0.01) growth of HPT cells cultured in 50 mM glucose. Cells cultured in 50 mM mannitol did not show any growth inhibition, suggesting that the decreased cell growth associated with glucose is not due to osmolarity changes. There was an increase (108%, p < 0.02) in lipid peroxidation in cells cultured with high levels of glucose (50 mM) compared with controls and cells cultured with 50 mM mannitol. To examine if membrane lipid peroxidation or malondialdehyde (MDA, an end product of lipid peroxidation) has any role in the inhibition of cell growth, we examined the effect of tertiary butylhydroperoxide (TBH, known to cause lipid peroxidation and generate MDA) on the growth of HPT cells. TBH decreased cell growth (49, 17 and 3% of controls at 0.1, 0.25, and 0.5 mumole TBH/ml medium). Similarly, a marked reduction in the growth was observed with exogenous MDA (72, 69 and 34% of controls at 0.1, 0.25, and 0.5 mumole MDA/ml medium). This suggests that elevated glucose can induce membrane lipid peroxidation and accumulation of MDA, which in turn can inhibit cellular growth and contribute to the altered structure and function of HPT cells in diabetes.

Possible role of nitric oxide on fertile and asthenozoospermic infertile human sperm functions.

The capacity of human sperm fertilization is principally dependent on sperm motility and membrane integrity. Oxygen-derived free radicals, such as superoxide anion, are known to impair sperm motility and membrane integrity by inducing membrane lipid peroxidation (LPO). Nitric oxide (NO), a biologically active free radical, has recently been shown to inactivate superoxide and increase intracellular guanosine-3', 5'-cyclic monophosphate (cGMP). The aim of this study is to investigate the effects of NO on human sperm motility, viability, lipid peroxidation and cGMP in fertile and asthenozoospermic infertile individuals in vitro. Semen samples were obtained from 10 fertile volunteers and 10 asthenozoospermic infertile patients. Washed spermatozoa were incubated at 37 degrees C in Ham's F-10 medium with 0, 25, 50, 100, 200, or 400nM sodium nitroprusside (SNP, Na2 [Fe(CN) 5NO] 2H2O), a nitric oxide releaser. Samples were analyzed for viability, determined by eosin-Y dye exclusion method at 0, 1, 2, 3, 5 and 6 h of incubation; motility, determined by the trans-membrane migration method within 2 h of incubation; LPO determined by malondialdehyde (MDA)-thiobarbituric acid method at 3 h of incubation; and the intracellular cGMP, determined by 125I-cGMP radioimmunoassay at 3 h of incubation. The results showed: in both fertile and infertile samples, viability, trans-membrane migration ratio and the levels of intracellular cGMP in 25-100nM SNP-treated spermatozoa were significantly higher than those in control groups, while MDA contents in treated groups were significantly lower than those in controls. However, when concentrations of SNP increased to 200-400nM, the opposite effects were exhibited. The effects of SNP on these processes were biphasic within 25-400nM. The most effective concentration was 100nM. These data suggested that NO is beneficial to sperm viability and motility in both fertile and infertile individuals, and that reduction of lipid peroxidative damage to sperm membranes and increase of intracellular cGMP may be involved in these benefits.

Membrane lipid peroxidation induces changes in γ-[ 3H]aminobutyric acid transport and calcium uptake by synaptosomes

In the present study, we analyze the effect of Fe 2+/ascorbate-induced lipid peroxidation on Ca 2+-dependent and Ca 2+-independent release and on the uptake of γ-[ 3H]aminobutyric acid (GABA) by sheep brain synaptosomes. In addition, we study the effect of lipid peroxidation on the levels of cytosolic calcium and on the uptake of calcium ( 45Ca 2+). After membrane lipid peroxidation, a decrease in the uptake of GABA is observed. After ascorbate/Fe 2+-induced membrane lipid peroxidation, a significant decrease in [ 3H]GABA release in response to K +-depolarization occurs, in the absence and in the presence of Ca 2+. The influx of 45Ca 2+ induced by K +-depolarization is significantly depressed under peroxidative conditions, while basal calcium uptake is inhibited to a much lesser degree. The levels of free ionic calcium [Ca 2+] i, as determined by the fluorescent dye Indo-1, are increased after synaptosomes were submitted to the ascorbate/Fe 2+ oxidative stress. It is concluded that membrane lipid peroxidation induces a decrease in Ca 2+-dependent and Ca 2+-independent efflux of accumulated [ 3H]GABA in response to elevated K + pulses (60 mM) and in the depolarization-induced calcium influx, while free ionic calcium levels increase. The Ca 2+-dependent efflux is interpreted to reflect stimulus-secretion coupling process and the Ca 2+-independent efflux may reflect membrane transport processes. Thus, the results suggest a possible relationship between a reduced calcium movement across the membrane, the decrease in neurotransmitters uptake and release and oxidative stress.