Peroxidative Changes in Brain Cortical Fatty Acids and Phospholipids, as Characterized During Fe2+- and Ascorbic Acid-Stimulated Lipid Peroxidation In Vitro

Abstract: The occurrence of peroxidative damage, as distinguished from anaerobic damage, to brain fatty acids and phospholipids was characterized in vitro. Fe²⁺ and ascorbic acid were used to stimulate peroxidation in cortical homogenates from rat brain incubated with or without oxygen. Lipid peroxidation was established in samples incubated with oxygen by increased diene conjugation, accumulation of thiobarbituric acid-reactive material (TBAR) and of lipid-soluble fluorescent products. No peroxidation occurred in samples incubated in the absence of oxygen (100% N₂). Lipid peroxidation was characterized by a selective loss of arachidonic acid and docosahexaenoic acid and by degradation of ethanolamine phosphoglyceride, while choline phosphoglyceride did not change. During the course of peroxidation there were parallel increases in products of lipid peroxidation concomitant with the decrease in polyenoic fatty acids. The maximal changes in diene conjugation and TBAR occurred earlier than the maximal changes in fluorescent material and fatty acids. It is concluded that measurements of changes in brain fatty acid and phospholipid composition may be a useful tool to establishment of whether peroxidative damage is important in vivo in situations with a critically reduced oxygen supply. Estimation of lipid-soluble fluorescence in vivo may also be useful, since it is considered to reflect the accumulation of stable end products of peroxidation.     

Ultraviolet-treated lipoproteins as a model system for the study of the biological effects of lipid peroxides on cultured cell. I. Chemical modifications of ultraviolet-treated low-density lipoproteins

A new experimental model system constituted by ultraviolet-treated low-density lipoproteins (LDL) has been designed in order to investigate the biological effects of lipid peroxides entering the cell through the endocytotic pathway. This paper reports the chemical modifications of the lipid components and apolipoproteins of the ultraviolet-treated LDL. Human LDL were submitted to short ultraviolet radiations (254 nm, 0.5 mW/cm2, for variable periods of time) and compared to LDL peroxidized by iron. The lipid peroxidation was monitored by following the formation of the peroxidation products (conjugated dienes, thiobarbituric acid-reactive substances (TBARS) and fluorescent lipid-soluble products) and the change of the composition in polyunsaturated fatty acids, carotenes and vitamin E. Several parameters of the apo B-100 structure were investigated: molecular size (by SDS-PAGE) and TNBS-reactive amino groups (chemical determination by trinitrobenzene sulfonic acid). The most important feature was the absence of major modification of apo B-100 in ultraviolet-treated LDL: the molecular weight and the content in TNBS-reactive amino groups of apo B-100 were not modified. In contrast, iron-treated LDL exhibited a loss of the apo B-100 band and a decrease in the number of TNBS-reactive amino group. Both ultraviolet radiations and iron ions induced a significant decrease in the content of polyunsaturated fatty acids, carotenes and vitamin E together with a large formation of lipid peroxidation products. However, the time-course of the formation of conjugated dienes, TBARS and fluorescent lipid-soluble products was quite different using the two oxidative systems. These results demonstrate that ultraviolet radiations induced a strong peroxidation of the lipid content of LDL and no (or only minor) changes in the apolipoprotein moiety whereas iron-catalyzed peroxidation resulted in the formation fo lipid peroxidation products as well as apo B alterations.     

The antioxidative property of green tea against iron-induced oxidative stress in rat brain

The antioxidative property of green tea against iron-induced oxidative stress was investigated in the rat brain both in vivo and in vivo. Incubation of brain homogenates at 37 degrees C for 4 hours in vitro increased the formation of Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Auto-oxidation (without exogenous iron) of brain homogenates was inhibited by green tea extract in a concentration-dependent manner. Moreover, incubation with iron (1 microM) elevated lipid peroxidation of brain homogenates after 4-hour incubation at 37 degrees C. Co-incubation with green tea extract dose-dependently inhibited the iron-induced elevation in lipid peroxidation. For the in vivo studies: ferrous citrate (iron, 4.2 nmoles) was infused intranigrally and induced degeneration of the nigrostriatal dopaminergic system of rat brain. An increase in lipid peroxidation in substantia nigra as well as a decrease in dopamine content in striatum was observed seven days after the iron infusion. Intranigral infusion of green tea extract alone did not increase, and in some cases, even decreased lipid peroxidation in substantia nigra. Co-infusion of green tea extract prevented oxidative injury induced by iron. Both iron-induced elevation in lipid peroxidation in substantia nigra and iron-induced decrease in dopamine content in striatum were suppressed. Oral administration of green tea extract for two weeks did not prevent the iron-induced oxidative injury in nigrostriatal dopaminergic system. Our results suggest that intranigral infusion of green tea extract appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of green tea extract protects the nigrostriatal dopaminergic system from the iron-induced oxidative injury.     

Ferric(III) ions inhibits copper(II)/hydrogen peroxide-catalyzing lipid peroxidation in human erythrocyte membranes.

1. Effect of ferric ions (Fe3+) on the lipid peroxidation catalyzed by copper ions (Cu2+) and hydrogen peroxide (H2O2) was studied in human erythrocyte membranes. 2. The formation of thiobarbituric acid-reactive products elicited by CuCl2/H2O2 was inhibited by FeCl3 in a concentration-dependent manner; 0.25 mM FeCl3 were enough to cause 50% inhibition of the formation of peroxides. 3. The inhibitory effect of FeCl3 is not due to competition against Cu2+. 4. FeCl3 inhibited the initiation, but did not inhibit the propagation of Cu2+/H2O2-catalyzing lipid peroxidation. 5. In the heat- or trypsin-treated erythrocyte membranes, FeCl3 had no inhibitory effect on Cu2+/H2O2-catalyzing lipid peroxidation. 6. Sodium azide, an inhibitor of catalase, had no effect on the inhibitory effect of FeCl3. 7. These results suggest that a protein factor(s), which is not catalase, is involved in the inhibition of Cu2+/H2O2-catalyzing lipid peroxidation by Fe3+.     

Carboxyfullerene prevents iron-induced oxidative stress in rat brain

Carboxyfullerene, a water-soluble carboxylic acid derivative of a fullerene, was investigated as a protective agent against iron-induced oxidative stress in the nigrostriatal dopaminergic system of anesthetized rats. Intranigral infusion of exclusive carboxyfullerene did not increase lipid peroxidation in substantia nigra or deplete dopamine content in striatum. Infusion of ferrous citrate (iron II) induced degeneration of the nigrostriatal dopaminergic system. An increase in lipid peroxidation in substantia nigra as well as decreases in K+-evoked dopamine overflow and dopamine content in striatum were observed 7 days after the infusion. Co-infusion of carboxyfullerene prevented iron-induced oxidative injury. Furthermore, tyrosine hydroxylase-immunoreactive staining showed that carboxyfullerene inhibited the iron-induced loss of the dopaminergic nerve terminals in striatum. The antioxidative action of carboxyfullerene was verified by in vitro studies. Incubation of brain homogenates increased the formation of the Schiff base fluorescent products of malonaldehyde, an indicator of lipid peroxidation. Both autooxidation (without exogenous iron) and iron-induced elevation of lipid peroxidation of brain homogenates were suppressed by carboxyfullerene in a dose-dependent manner. Our results suggest that intranigral infusion of carboxyfullerene appears to be nontoxic to the nigrostriatal dopaminergic system. Furthermore, the potent antioxidative action of carboxyfullerene protects the nigrostriatal dopaminergic system from iron-induced oxidative injury.     

Melatonin and pinoline prevent aluminium-induced lipid peroxidation in rat synaptosomes.

The serum concentrations of aluminum, a metal potentially involved in the pathogenesis of Alzheimer's disease, increase with age. Also, intense and prolonged exposure to aluminum may result in dementia. Melatonin and pinoline are two well known antioxidants that efficiently reduce lipid peroxidation due to oxidative stress. Herein, we investigated the effects of melatonin and pinoline in preventing aluminum promotion of lipid peroxidation when the metal was combined with FeCl3 and ascorbic acid in rat synaptosomal membranes. Lipid peroxidation was estimated by quantifying malondialdehyde (MDA) and 4-hydroxyalkenal (4-HDA) concentrations in the membrane suspension. Under the experimental conditions used herein, the addition of aluminum (0.0001 to 1 mmol/L) enhanced MDA + 4-HDA formation in the synaptosomes. Melatonin and pinoline reduced, in a concentration-dependent manner, lipid peroxidation due to aluminum, FeCl3 and ascorbic acid in the synaptosomal membranes. These results suggest that the indoleamine melatonin and the beta-carboline pinoline may potentially act as neuroprotectant agents in the therapy of those diseases with elevated aluminum concentrations in the tissues.     

NADH-dependent reductive stress and ferritin-bound iron in allyl alcohol-induced lipid peroxidation in vivo: the protective effect of vitamin E.

The role of iron in allyl alcohol-induced lipid peroxidation and hepatic necrosis was investigated in male NMRI mice in vivo. Ferrous sulfate (0.36 mmol/kg) or a low dose of ally alcohol (0.6 mmol/kg) itself caused only minor lipid peroxidation and injury to the liver within 1 h. When FeSO4 was administered before allyl alcohol, lipid peroxidation and liver injury were potentiated 50-100-fold. Pretreatment with DL-tocopherol acetate 5 h before allyl alcohol protected dose-dependently against allyl alcohol-induced lipid peroxidation and liver injury in vivo. Products of allyl alcohol metabolism, i.e. NADH and acrolein, both mobilized trace amounts of iron from ferritin in vitro. Catalytic concentrations of FMN greatly facilitated the NADH-induced reductive release of ferritin-bound iron. NADH effectively reduced ferric iron in solution. Consequently, a mixture of NADH and Fe3+ or NADH and ferritin induced lipid peroxidation in mouse liver microsomes in vitro. Our results suggest that the reductive stress (excessive NADH formation) during allyl alcohol metabolism can release ferrous iron from ferritin and can reduce chelated ferric iron. These findings provide a rationale for the strict iron-dependency of allyl alcohol-induced lipid peroxidation and hepatotoxicity in mice in vivo and document iron mobilization and reduction as one of several essential steps in the pathogenesis.     

Age pigments and free radicals: fluorescent lipid complexes formed by iron- and copper-containing proteins

Haem and non-haem iron-containing proteins stimulate lipid peroxidation with the formation of fluorescent lipid complexes. This process requires the presence of lipid hydroperoxides which release ferrozine-reactive iron from haem-containing proteins. Stimulation of lipid peroxidation by the released iron is inhibited by the iron chelator desferrioxamine. Copper ions, although more stimulatory towards fluorescent lipid complex formation than iron ions, do not stimulate lipid peroxidation when tightly bound at the active centre of proteins, but are reactive when loosely bound to albumin and histidine.     

Endotoxin increases hepatic susceptibility to lipid peroxidation: a possible role of iron

The purpose of this study was to investigate the possible mechanism by which endotoxin enhances peroxidative damage to membrane lipids. Male B6C3 mice were treated with endotoxin intraperitoneally 0 or 20 mg/kg body weight for 24 h. Freshly prepared liver homogenate was incubated with either 1-5 mM of reduced glutathione (GSH), glucose, H(2)O(2), ascorbic acid (AA), FeSO(4), FeCl(3), EDTA, FeCl(3) plus AA, AA plus EDTA or EDTA plus FeCl(3) in phosphate-buffered saline (PBS), pH 7.0, or PBS, at 37 degrees C for 60 min. The levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), were significantly higher in the liver of endotoxin-treated mice, and the values were markedly increased following incubation. Compared to PBS, incubation with H(2)O(2), FeCl(3), FeSO(4), and AA, but not glucose, significantly enhanced TBAR formation. The greatest increase of TBAR was found when AA and FeCl(3) were added together. On the other hand, EDTA and GSH inhibited the formation of TBAR during incubation. When added before AA, EDTA completely inhibited the peroxidative effect of AA or FeSO4, and when added subsequent to AA, EDTA partially prevented the adverse effect of AA. The results obtained suggest that ionic iron plays an important role in initiating endotoxin-induced peroxidative damage to membrane lipids, and that AA may be involved in releasing iron from its protein complex and/or maintaining ionic iron in a reduced or catalytic state.     

Lipid peroxidation induced “in vivo” by iron-carbohydrate complex in the rat brain cortex

In view of the emerging role of metals and particularly iron in the pathogenesis of several ischemic or degenerative CNS diseases, via a lipid peroxidative process, a model of slow iron-induced peroxidative damage in the rat brain cortex has been carried out. Iron-carbohydrate complexes were injected in the right brain cortex, and biochemical assays were performed on ipsilateral and contralateral samples two hours or seven days after injection. Iron-sacchararate caused a significant increase in the ipsilateral cortex in TBARS, conjugated dienes and fluorescent substances seven days after injection, whereas no biochemical alteration was observed two hours after treatment. In order to prevent or to limit lipid peroxidation, some drugs known for chelating and/or scavening activity were administered to iron-injected rats. DL--tocopherol, methylprednisolone, D-penicillamine significantly decreased the value of fluorescent products formed by iron-saccharate, whereas desferrioxamine was not effective.     

Ethanol- and Fe(+2)-induced membrane lipid oxidation is not additive in developing chick brains

In order to study the effects of exogenous EtOH and/or Fe(+2) on membrane lipid peroxidation, exogenous EtOH, FeCl(2), FeCl(2) & EtOH, NaCl and NaCl & EtOH were injected into fertile chicken eggs. Controls were either shams or injected with saline. These injections were made at 0 days or 0-2 days of development and tissue removed at stage 37 (11 days of development). Embryonic exposure to exogenous EtOH and/or Fe(+2) promoted decreased brain mass, decreased levels of brain membrane polyunsaturated fatty acids, elevated levels of brain lipid hydroperoxides, and elevated levels of Fe(+2) within embryonic brain and liver. These alterations were more severe in triple-injected embryos (E0-2/E11) as compared to single-injected embryos (E0/E11). While exogenous treatments of either EtOH and/or FeCl(2) promoted increased levels of endogenous brain Fe(+2), the effects were not additive. These observations are consistent with the hypothesis that embryonic exposure to exogenous EtOH and/or Fe(+2) promotes brain membrane lipid peroxidation.     

Activation of endogenous lipid peroxidation in the brain in oxidation stress caused by administration of iron and its prevention by vitamin E

Looking for an appropriate model of accelerated aging in vivo we investigated the content of endogenous products of lipid peroxidation (LP) in the rat brain after single or 4 day-lasting intramuscular injection of complex-bind iron (ferum Hausman, 50 mg/kg body weight) like promoter of LP. We found that the single administration of this iron complex fails to induce endogenous LP; after 4 day-application of iron we observed significant increase in content of primary (lipid peroxides) and final (fluorescent) products of LP. Iron-promoted activation of endogenous LP could be abolished by animal pretreatment with the natural antioxidant alpha-tocopherol. The calcium antagonist nifedipine didn't affect the content of endogenous LP products neither alone nor in combination with alpha-tocopherol.     

Stimulation and inhibition of iron-dependent lipid peroxidation by desferrioxamine

Peroxidation of rat brain synaptosomes was assessed by the formation of thiobarbituric acid reactive products in either 50 mM potassium phosphate buffer (pH 7.4) or pH adjusted saline. In phosphate, addition of Fe2+ resulted in a dose-related increase in lipid peroxidation. In saline, stimulation of lipid peroxidation by Fe2+ was maximal at 30 uM, and was less at concentrations of 100 uM and above. Whereas desferrioxamine caused a dose-related inhibition of iron-dependent lipid peroxidation in phosphate, it stimulated lipid peroxidation with Fe2+ by as much as 7-fold in saline. The effects of desferrioxamine depended upon the oxidation state of iron, and the concentration of desferrioxamine and lipid. The results suggest that lipid and desferrioxamine compete for available iron. The data are consistent with the hypothesis that either phosphate or desferrioxamine may stimulate iron-dependent lipid peroxidation under certain circumstances by favoring formation of Fe2+/Fe3+ ratios.     

In vitro lipid peroxidation of human serum catalyzed by cupric ion: antioxidant rather than prooxidant role of ascorbate.

The dual role of ascorbate as an antioxidant and a prooxidant has been clearly documented in the literature. Ascorbate acts as an antioxidant by protecting human serum from lipid peroxidation induced by azo dye-generated free radicals. On the other hand, ascorbate is readily oxidized in the presence of transition metal ions, (especially cupric ion) and accelerates lipid peroxidation in tissue homogenates by producing free radicals. Interestingly, we observed an antioxidant rather than an expected prooxidant role of ascorbate when human serum supplemented with 1.2mmol/L ascorbate underwent lipid peroxidations initiated by 2mmol/L copper sulfate. The antioxidant role of ascorbate was confirmed by studying the conventional thiobarbituric acid reactive substances (TBARS) as well as by observing the protective effect of ascorbate on the copper-induced peroxidation of unsaturated and polyunsaturated fatty acids. The antioxidation protection provided by ascorbate was comparable to that of equimolar alpha-tocopherol when incubated for 24h. However, lipid peroxidation products were lower in serum supplemented with alpha-tocopherol after 48h of incubation. This effect may be attributed to the binding of copper by plpha-tocopherol after serum proteins, thus preventing direct interaction between cupric ions and ascorbate. This proposed mechanism is based on the observation that the concentration of ascorbate decreased more slowly in serum than in phosphate buffer at physiological pH. Our results also indicate an outstanding anti-oxidant property of human serum due to the chelation of transition metal ions (even at high concentrations) by various serum proteins.     

Vanadium exposure enhances lipid peroxidation in the kidney of rats and mice

Vanadium (V) as sodium orthovanadate induces an increase in lipid peroxidation in the kidneys after a single subcutaneous or intraperitoneal injection to rats or mice. The rate of malondialdehyde (MDA) formation, an index of lipid peroxidation, by kidney homogenates increased by more than 100% 1 h after injection. Chronic exposure of rats to vanadium sulfate, initially through maternal milk and later in the drinking water, resulted after 10 weeks in a significant increase in MDA formation by kidney but not by other tissues. In both acute and chronic studies in rats and mice, no significant increase in lipid peroxidation by V treatment was detected in brain, heart, lung, spleen, or liver. In mice, administration of ascorbate prior to acute exposure to V diminished both toxicity, i.e., respiratory depression and limb paralysis, and the formation of MDA in kidney.     

Lipid peroxidation in the rats striatum during stress after cortisol administration

Lipid peroxidation in the rat striatum under stress after cortisole injection was investigated. Three days cortisole injections (25 mg/kg every day) do not affect the level of lipid peroxidation products 6 days after termination of the hormone injection. However, in these periods, cortisole injected rats had a more significant response of lipid peroxidation to stress than the control animals (decrease of intermediate products and increase of Shift bases). Thus, the hormone injection induced a long-term changes in so important a regulatory system of the organisms as the lipid peroxidation, causing sensitization of its response to stress.     

Effects of a direct injection of liposoluble iron into rat striatum. Importance of the rate of iron delivery to cells

For a better understanding of the role of iron imbalance in neuropathology, a liposoluble iron complex (ferric hydroxyquinoline, FHQ) was injected into striatum of rats. The effects of two modalities of iron injections on brain damage, hydroxyl radical ( •OH) production (assessed by the salicylate method coupled to microdialysis) and tissue reactive iron level (evaluated ex vivo by the propensity of the injected structure for lipid peroxidation) were examined. Rapid injection of FHQ (10 nmoles of 5 mM FHQ pH 3 solution over 1-min period) but not that of corresponding vehicle led to extensive damage associated with increased tissue free iron level in the injected region. Conversely, neither lesion nor free iron accumulation was observed after slow FHQ injection (10 nmoles of a 100 μM FHQ pH 7 solution over 1-h period) as compared to corresponding vehicle injection. Production of •OH was induced by slow FHQ injection but not by rapid FHQ injection, probably as a result of in vivo abolition of iron-induced •OH formation by acid pH. Indeed, rapid injection of FAC pH 7 (ferric ammonium citrate, 5 mM in saline) was associated with •OH formation whereas rapid injection of FAC pH 3 did not. Our results identify the rate of iron delivery to cells as an important determinant of iron toxicity and do not support a major role for extracellular •OH in damage associated with intracerebral iron injection.     

Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide.

Endothelial cells, macrophages, neutrophils, and neuronal cells generate superoxide (O2-) and nitric oxide (.NO) which can combine to form peroxynitrite anion (ONOO-). Peroxynitrite, known to oxidize sulfhydryls and to yield products indicative of hydroxyl radical (.OH) reaction with deoxyribose and dimethyl sulfoxide, is shown herein to induce membrane lipid peroxidation. Peroxynitrite addition to soybean phosphatidylcholine liposomes resulted in malondialdehyde and conjugated diene formation, as well as oxygen consumption. Lipid peroxidation was greater at acidic and neutral pH, with no significant lipid peroxidation occurring above pH 9.5. Addition of ferrous (Fe+2) or ferric (Fe+3) iron did not enhance lipid peroxide formation over that attributable to peroxynitrite alone. Diethylenetetraminepentacetic acid (DTPA) or iron removal from solutions by ion-exchange chromatography decreased conjugated diene formation by 25-50%. Iron did not play an essential role in initiating lipid peroxidation, since DTPA and iron depletion of reaction systems were only partially inhibitory. In contrast, desferrioxamine had an even greater concentration-dependent inhibitory effect, completely abolishing lipid peroxidation at 200 microM. The strong inhibitory effect of desferrioxamine on lipid peroxidation was due to direct reaction with peroxynitrous acid in addition to iron chelation. We conclude that the conjugate acid of peroxynitrite, peroxynitrous acid (ONOOH), and/or its decomposition products, i.e., .OH and nitrogen dioxide (.NO2), initiate lipid peroxidation without the requirement of iron. These observations demonstrate a potential mechanism contributing to O2-(-)and .NO-mediated cytotoxicity.     

Transient Formation of Superoxide Radicals in Polyunsaturated Fatty Acid-Induced Brain Swelling

The involvement of superoxide free radicals and lipid peroxidation in brain swelling induced by free fatty acids has been studied in brain slices and homogenates. The polyunsaturated fatty acids linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4), and docosahexaenoic acid (22:6) caused brain swelling concomitant with increases in superoxide and membrane lipid peroxidation. Palmitic acid (16:0) and oleic acid (18:1) had no such effect. Furthermore, superoxide formation was stimulated by NADPH and scavenged by the addition of exogenous superoxide dismutase in cortical slice homogenates. These in vitro data support the hypothesis that both superoxide radicals and lipid peroxidation are involved in the mechanism of polyunsaturated fatty acid-induced brain edema.