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.     

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.     

Analysis of lipid peroxidation mechanisms in human spermatozoa

The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal* and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe²⁺-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe²⁺-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation. © 1993 Wiley-Liss, Inc.     

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.     

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.     

Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs

The age-related changes in the activities of antioxidant enzymes of mitochondrial and cytosolic fractions were measured in different regions of the central nervous system (CNS) in 10 and 32 months old guinea pigs. In old animals, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced (p < 0.05) in all the regions of CNS studied but catalase (CAT) declined significantly only in the cerebral cortex, hypothalamus and cerebellum. Glutathione reductase (GRd) activity declined in cerebral cortex and hypothalamus in the cytosolic fractions and only in cerebellum in the mitochondrial fraction. It is concluded that age-related decline in the activities of antioxidant enzymes is both region and enzyme specific. The endogenous lipid peroxide was found to be significantly higher (p < 0.05) in the 32 month old animals whereas, lipid peroxidation after incubating the tissue homogenate in air was found to be lower (p < 0.05). The in vitro mitochondrial lipid peroxidation decreased with age. The results indicate that accumulation of lipid peroxides takes place with ageing but the susceptibility of lipid peroxidation decreases in the older animals.     

Study on lipid peroxidation potential in different tissues induced by ascorbate-Fe2+: Possible factors involved in their differential susceptibility

Susceptibility of four major rat tissues to oxidative damage in terms of lipid peroxidation induced by in vitro by ascorbate-Fe²⁺ in homogenates and mitochondria has been examined. Lipid peroxidation, as assessed by thiobarbituric acid reactive substances (TBARS) and conjugated dienes was maximum in brain followed by liver, kidney and heart. However, the time course of lipid peroxidation showed different patterns in tissues examined. The higher susceptibilities of brain and liver can be explained by substrate availability and to a lesser extent the level of antioxidants. The differences observed in the tissues studied may reflect their susceptibility to degenerative diseases and xenobiotic toxicity which are considered as a result of oxidative damage to membranes.     

Sensitivity to in vitro lipid peroxidation in liver and brain of aged rats.

Lipid peroxidation in rat liver and brain has been studied to see if it increases with old age. No significant differences in the level of endogenous, nonstimulated lipid peroxidation (TBA-RS) is found between 9 month-old (mature adults) and 28 month-old animals in liver or cerebral cortex. Liver homogenates subjected in vitro to an oxidative stress (ascorbate-Fe++), show a clearly slower peroxidation rate in old than in young animals. On the other hand, the in vitro peroxidation rate of cerebral homogenates was similar in young and old animals. The in vitro peroxidation rate was much higher in brain than in liver tissue. These results do not support the view that old rats liver and brain are more susceptible to free radical oxidative damage than those of young ones.     

Nadph-initiated cytochrome P450-dependent free iron-independent microsomal lipid peroxidation: Specific prevention by ascorbic acid

In this paper we demonstrate that ascorbic acid specifically prevents NADPH-initiated cytochrome P450 (P450)-mediated microsomal lipid peroxidation in the absence of free iron. Lipid peroxidation has been evidenced by the formations of conjugated dienes, lipid hydroperoxide and malondialdehyde. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, glutathione, -tocopherol, uric acid, thiourea, mannitol, histidine, -carotene and probucol are ineffective to prevent the NADPH-initiated P450-mediated free iron-independent microsomal lipid peroxidation. Using a reconstituted system comprised of purified NADPH-P450 reductase, P450 and isolated microsomal lipid or pure L--phosphatidylcholine diarachidoyl, a mechanism has been proposed for the iron-independent microsomal lipid peroxidation and its prevention by ascorbic acid. It is proposed that the perferryl moiety P450 Fe³⁺. O₂ initiates lipid peroxidation by abstracting methylene hydrogen from polyunsaturated lipid to form lipid radical, which then combines with oxygen to produce the chain propagating peroxyl radical for subsequent formation of lipid peroxides. Apparently, ascorbic acid prevents initiation of lipid peroxidation by interacting with P450 Fe³⁺. O₂. (Mol Cell Biochem 166: 35-44, 1997)     

NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation induced skin injury

Oxidative stress and lipid peroxidation are major causes of skin injury induced by ultraviolet (UV) irradiation. Ferroptosis is a form of regulated necrosis driven by iron-dependent peroxidation of phospholipids and contributes to kinds of tissue injuries. However, it remains unclear whether the accumulation of lipid peroxides in UV irradiation-induced skin injury could lead to ferroptosis. We generated UV irradiation-induced skin injury mice model to examine the accumulation of the lipid peroxides and iron. Lipid peroxides 4-HNE, the oxidative enzyme COX2, the oxidative DNA damage biomarker 8-OHdG, and the iron level were increased in UV irradiation-induced skin. The accumulation of iron and lipid peroxidation was also observed in UVB-irradiated epidermal keratinocytes without actual ongoing ferroptotic cell death. Ferroptosis was triggered in UV-irradiated keratinocytes stimulated with ferric ammonium citrate (FAC) to mimic the iron overload. Although GPX4 protected UVB-injured keratinocytes against ferroptotic cell death resulted from dysregulation of iron metabolism and the subsequent increase of lipid ROS, keratinocytes enduring constant UVB treatment were markedly sensitized to ferroptosis. Nicotinamide mononucleotide (NMN) which is a direct and potent NAD⁺ precursor supplement, rescued the imbalanced NAD⁺/NADH ratio, recruited the production of GSH and promoted resistance to lipid peroxidation in a GPX4-dependent manner. Taken together, our data suggest that NMN recruits GSH to enhance GPX4-mediated ferroptosis defense in UV irradiation-induced skin injury and inhibits oxidative skin damage. NMN or ferroptosis inhibitor might become promising therapeutic approaches for treating oxidative stress-induced skin diseases or disorders.     

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.     

Importance of two iron-reducing systems in lipid peroxidation of rat brain: implications for oxygen toxicity in the central nervous system

The mechanism of lipid peroxidation in the central nervous system has been studied using oxygen-flushed rat brain homogenates at pH 7.4. Brain lipid peroxidation was monitored by chemiluminescence and by determination of thiobarbituric acid-reactive substances. Less involvement of O2-., H2O2 and probably .OH in the initiation of lipid peroxidation was indicated. Deferroxamine was an extremely potent inhibitor of lipid peroxidation, suggesting that lipid peroxidation was catalyzed by endogenous iron. Brain tissues were shown to contain at least two iron-reducing systems for promotion of lipid peroxidation. One was ascorbate-dependent and the other NADPH-dependent. The former was much more potent than the latter with respect to iron-reducing activity.     

Chloditan-induced changes in lipid peroxidation in the adrenals of dogs and guinea pigs

The effect of adrenocorticolytic drug chloditan (1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane;) on the content and the rate of lipid peroxidation in dog and guinea pig adrenals was studied. In adrenals of dogs and guinea the amount of malondialdehyde (MDA) was increased. In vitro spontaneous lipid peroxidation was delayed in adrenal homogenate of DDD-treated dogs. Spontaneous lipid peroxidation in adrenal homogenate was not decreased by feeding DDD in the guinea pig. DDD consumption did not affect on lipid peroxidation promoted by ascorbic acid and ferrous iron. The accumulation of MDA in dog and guinea pig adrenal homogenates was inhibited by NADPH.     

Hyperoxia results in transient oxidative stress and an adaptive response by antioxidant enzymes in goldfish tissues

The effects of hyperoxia on the status of antioxidant defenses and markers of oxidative damage were evaluated in goldfish tissues. The levels of lipid peroxides, thiobarbituric acid reactive substances, carbonyl proteins and the activities of some antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of goldfish, Carassius auratus L., over a time course of 3-12 h of hyperoxia exposure followed by 12 or 36 h of normoxic recovery. Exposure to high oxygen resulted in an accumulation of protein carbonyls in tissues throughout hyperoxia and recovery whereas lipid peroxides and thiobarbituric acid reactive substances accumulated transiently under short-term hyperoxia stress (3-6 h) but were then strongly reduced. This suggests that hyperoxia stimulated an enhancement of defenses against lipid peroxidation or mechanisms for enhancing the catabolism of peroxidation products. The activities of principal antioxidant enzymes, superoxide dismutase and catalase, were not altered under hyperoxia but catalase increased during normoxic recovery; activities may rise in anticipation of further hyperoxic excursions. In most tissues, the activities of glutathione-utilizing enzymes (glutathione peroxidase, glutathione-S-transferase, glutathione reductase) as well as glucose-6-phosphate dehydrogenase, were not affected under hyperoxia but increased sharply during normoxic recovery. Correlations between some enzyme activities and oxidative stress markers were found, for example, an inverse correlation was seen between levels of thiobarbituric acid reactive substances and glutathione-S-transferase activity in liver and catalase and glucose-6-phosphate dehydrogenase in kidney. The results suggest that liver glutathione-S-transferase plays an important role in detoxifying end products of lipid peroxidation accumulated under hyperoxia stress.     

Effect of reactive oxygen species on the erythrocyte calcium-pump function in protein-energy malnutrition

The presence of detectagle amounts of non-heme iron in erythrocyte ghost membranes have been postulated to lead to the initiation of membrane lipid peroxidation and the attendant perturbation of membrane functions. We have investigated the presence of non-heme iron and endogenous products of lipid peroxidation in erythrocyte membranes of normal and kwashiorkor (KWA) subjects and assessed the susceptibility of the membranes to exogenously generated reactive oxygen species. The modulation of the basal and calmodulin-stimulated calcium-pumping activity of these membranes by reactive oxygen species was also assessed. The results show the presence of significant amounts of non-heme iron and endogenous free radical reaction products in the red cell membranes of KWA subjects compared with that of normal children. Estimation of the extent of lipid peroxidation in the presence of exogenously generated reactive oxygen species further revealed that erythrocyte ghost membranes of KWA subjects are more susceptible to oxidative stress than those of normal individuals. Although both the basal and calmodulin-stimulated activities of the membrane-bound Ca²⁺-pump enzyme in normal and KWA subjects were inhibited by oxygen-free radicals, the erythrocyte enzyme in KWA subjects showed higher susceptibility to inhibition by oxygen free radicals than that of normal individuals. We propose that the reduced erythrocyte calcium-pump function in KWA is not unconnected with excessive generation of reactive oxygen species.Abbreviations PMSF phenylmethylsulfonylfuloride - TLCK N--p-tosyl-l-lysine chloromethylketone - EGTA ethyleneglycol-bis (B-aminoethylether) N,N-tetraacetic acid - EDTA ethylene diamine tetraacetic acid - ATP Adenosine 5-triphosphate - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - Tris-HCl Tris (hydroxymethyl) aminomethane-hydrochloride - SDS Sodium dodecyl sulphate - TBAR thiobarbituric acid-reactive products TBA, thiobarbituric acid - TCA trichloroacetic acid     

Lipid peroxidation as a source of oxidative damage in Helicobacter pylori: protective roles of peroxiredoxins

Oxidative stress conditions lead to enzymatic and non-enzymatic unsaturated fatty acid-initiated lipid peroxidation reactions. One exacerbating product is lipid hydroperoxide (LOOH) which itself promotes formation of several additional peroxyl radicals. Helicobacter pylori mutant strains with disruptions in genes encoding the peroxiredoxins, alkyl hydroperoxide reductase (ahpC) and the bacterioferritin comigratory protein (bcp), were more sensitive than the parent strain to oxidizing agents. These mutant strains were particularly sensitive, compared to the wild type, to killing by the unsaturated fatty acid linolenic acid but were not sensitive to the saturated fatty acid palmitic acid. A double mutant strain (ahpC bcp) accumulated more than 3-fold more lipid peroxides than the parent strain, indicating these peroxiredoxins together play a role in detoxifying lipid peroxides. The level of free iron accumulation, a signature of oxidative stress damage, was correlated specifically to organic peroxide-mediated stress by both in vivo and in vitro approaches. Free iron accumulation and concomitant destruction of [Fe-S] cluster-containing proteins (hydrogenase and aconitase) was correlated to damage mediated by exogenous t-butyl peroxide, or separately to intracellular accumulation of lipid peroxides in mutant strains. A major macromolecular target of accumulating lipid peroxides in H. pylori is DNA, as mutant analysis approaches combined with quantitative DNA fragmentation studies and specific DNA damage assessment (i.e. 8-oxoguanine formation) were used to demonstrate that such damage was especially associated with ahpC and ahpC bcp strains.     

Effect of Long-Term Normobaric Hyperoxia on Oxidative Stress in Mitochondria of the Guinea Pig Brain

Normobaric hyperoxia (NBO) is applied for treatment of various clinical conditions related to hypoxia, but it can potentially also induce generation of reactive oxygen species, causing cellular damage. In this study, we examined the effects of 60 h NBO treatment on lipid and protein oxidative damage and activity of superoxide dismutase (Mn-SOD) in brain mitochondria of guinea pigs. Despite significant stimulation of Mn-SOD expression and activity the NBO treatment resulted in accumulation of markers of oxidative lesions, including lipid peroxidation (conjugated dienes, thiobarbituric acid reactive substances) and protein modification (bityrosines, adducts with lipid peroxidation products, oxidized thiols). When inhaled O₂ was enriched with oxygen cation, O₂^•+, the Mn-SOD expression and activity were stimulated to similar extend, but lipid peroxidation and protein oxidation were prevented. These results suggest that long-term NBO treatment causes oxidative stress, but enrichment of inhaled oxygen by oxygen cation can protect the brain again adverse effects of hyperoxia.     

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.     

Indices of oxidative stress. 2. Lipid peroxides

Two methods of the determination of lipid peroxidation products have been compared which are based on Fe(II) oxidation by them at acid pH values in the presence of xylenol orange which binds Fe(III) have been compared. The first method uses cumene hydropeoxide as an internal standard. In the second one, lipid peroxides are previously reduced by triphenylphosphine and these substances content is measured as a difference of the production of complexes with xylenol orange and iron ions in the control (with reduction) and experimental sample (without reduction). The optimization of measurement conditions is described. The levels of lipid peroxides in goldfish tissues assayed simultaneously by two methods were similar. The method with cumene hydroperoxide needs less amounts of biological material; moreover, there is no necessity in a calibration curve. Effects of hyperoxia on lipid peroxide levels in goldfish tissues were studied with the cumene method. Within the first hours of hyperoxia this index increased 13-times in the liver and 2-times in the brain and muscle. The further exposure rebounded this parameter to the initial level. Levels of lipid peroxides positively correlated with levels of end products of lipid peroxidation (thiobarbiturate acid reactive substances) in the goldfish tissues. The method of quantification of lipid peroxides with cumene is recommended for wide using in biological investigations.