The role of superoxide and singlet oxygen in lipid peroxidation promoted by xanthine oxidase

The peroxidative oxidation of extracted rat liver microsomal lipid, assayed as malondialdehyde production, can be promoted by milk xanthine oxidase in the presence of 0.2 mM FeCl₃ and 0.1 mM EDTA. The reaction is inhibited by the superoxide dismutase activity of erythrocuprein. The reaction is also inhibited by 1,3-diphenylisobenzofuran, which reacts with singlet oxygen to yield dibenzoylbenzene. During inhibition of the lipid peroxidation reaction by 1,3-diphenylisobenzofuran, o-dibenzoylbenzene was produced. The rate of superoxide production by xanthine oxidase was not affected by 1,3-diphenylisobenzofuran. Lipid peroxidation promoted by ascorbic acid is not inhibited by either erythrocuprein or 1,3-diphenylisobenzofuran. Therefore it is suggested that the peroxidative oxidation of unsaturated lipid promoted by xanthine oxidase involves the formation of singlet oxygen from superoxide, and the singlet oxygen reacts with the lipid to form fatty acid hydroperoxides.     

Free radical and freezing injury to cell membranes of winter wheat

The symptoms of injury in microsomal membranes isolated from crowns of seedlings of Triticum aestivum , L. cultivar Fredrick after a lethal freeze-thaw stress included an increased lipid phase transition temperature, loss of lipid phosphate (lipid-P), and increased free fatty acid levels. However, minimal changes in fatty acid saturation were observed, suggesting minimal amounts of lipid peroxidation. All of these injury symptoms, including the lack of lipid peroxidation, were simulated in vitro by treatment of isolated membranes with oxygen free radicals, generated from either xanthine oxidase (EC 1.1.3.22) or paraquat (l,r-dimethyl-4,4'-bipyridinium dichloride). Further evidence indicating a relationship between free radicals and freezing injury comes from the observation that both protoplasts and microsomal membranes isolated from wheat seedlings, that had been acclimated to induce freezing tolerance, also had increased tolerance of oxygen free radicals, and contained higher lipid-soluble antioxidant levels, than those from non-acclimated seedlings. Lipid-soluble antioxidants accumulated in the crown tissue of the wheat seedling during the acclimation period. Freezing stress accelerated the formation of oxygen free radicals. Membranes isolated from crowns after a freeze–thaw stress tended to produce higher levels of superoxide as shown by the reduction of Tiron (1,2-dihydroxy-l,3-benzenedisulfonic acid). In protoplasts, increased superoxide production coincided with lethal freezing injury. These results are discussed in terms of the possible involvement of oxygen free radicals in mediating aspects of freezing injury to cell membranes.     

Effect of oxidants on small intestinal brush border membranes and colonic apical membranes--a comparative study

This study compares composition of the rat small intestinal brush border membranes (BBM) and colonic apical membranes (CAM) and their susceptibility to in vitro exposure to various oxidants. Differences were observed between BBM and CAM in their lipid composition, sugar content, alkaline phosphatase (ALP) activity and cholesterol/phospholipid ratio. BBM and CAM were exposed to superoxide generated by xanthine+xanthine oxidase (X-XO) or peroxides such as tertiary butyl hydroperoxide (tBuOOH) and hydrogen peroxide (H(2)O(2)) and alterations in ALP activity, peroxidation parameters and membrane lipids were analyzed. Exposure of BBM and CAM to superoxide resulted in decrease in ALP activity and increase in peroxidation parameters such as protein carbonyl content, malondialdehyde and conjugated diene. Superoxide exposure also resulted in lipid alterations specifically in certain phospholipids. These alterations were prevented either by superoxide dismutase or by allopurinol. Peroxides did not have any significant effect. These results suggest that both BBM and CAM are susceptible to superoxide, which can bring about peroxidation and degradation of membrane lipids specifically, certain phospholipids.     

Effect of a zinc-deficient diet on lipid peroxidation in liver and tumor subcellular membranes

The effect of a zinc-deficient diet on lipid peroxidation in liver and tumor mitochondrial and microsomal membrane preparations from BALB/c mice was investigated. Mitochondrial and microsomal membranes from both tissues displayed increased rates of in vitro peroxidation, both enzymatic and nonenzymatic. Measurement of in vivo peroxidation, using diene conjugation as an index of measurement revealed slight increases in tissues from zinc-deficient animals that were not statistically significant. Serum lipoperoxides analyzed from all three groups revealed no significant differences. The results point to an alteration in the peroxidation potential of mitochondrial and microsomal membranes due to zinc deficiency which may be related to an alteration in fatty acid composition.     

Superoxide dismutase content and microsomal lipid composition of tumours with different growth rates

The content of cytosolic superoxide dismutase has been determined in Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and in ascites tumour cells (Novikoff hepatoma and Ehrlich-Lettré). The enzyme is decreased in all the tumours examined. The lowest amounts were found in the tumours with the fastest growth rates. Measurements of the lipid composition and fluidity of microsomal membranes isolated from Morris hepatomas show that also these parameters are changed in relation to the growth rate. The lipid to protein ratio and the degree of fatty acid unsaturation decrease gradually from rat liver to hepatoma 44 and 3924A microsomes. The different lipid composition is reflected also by differences in the physical environment of the bilayer, as indicated by data obtained with spin-labeled fatty acids. It is proposed that the changes in the membrane lipid composition and organization are consequent to the decrease in the protective effect of cytosolic superoxide dismutase against the O2- induced lipid peroxidation.     

Disordered functioning of the superoxide radical-superoxide dismutase system in rat liver ischemia

The rate of O2 radical generation in microsomal membranes (VO2), the activity of cytosol superoxide dismutase (Cu, ZnSOD) and mitochondrial superoxide dismutase (MnSOD), and the activity of xanthine oxidizing system (XO) after a two-hour ischemia following a 24-hour reoxygenation of the rat liver were investigated. The high value of VO2, as compared to Cu, ZnSOD activity, may result in regulation disorders in O2-SOD system during ischemia. During reoxygenation, xanthine oxidizing system in combination with lowered Cu, ZnSOD activity may substantially contribute to the disturbance.     

Lipid peroxidation increases the molecular order of microsomal membranes

The effect of enzymatic lipid peroxidation on the molecular order of microsomal membranes was evaluated by ESR spectroscopy using the spin probes 5-, 12-, and 16-doxyl-stearic acid. Rat liver microsomal membranes were peroxidized by the NADPH-dependent reaction in the presence of the chelate ADP-Fe3+. Peroxidation resulted in a preferential depletion of polyenoic fatty acids and an increase in the percentage composition of shorter fatty acyl chains. There was no change in the cholesterol/phospholipid ratio of the peroxidized microsomes. The molecular order of both control and peroxidized membranes decreased toward the central region of the bilayer, and the order parameter (S) of each probe was temperature dependent. Peroxidation of the microsomal membrane lipids resulted in an increase in the order parameter determined with the three stearic acid spin probes. Of the three probes, 12-doxylstearic acid was the most sensitive to the changes in membrane organization caused by peroxidation. These data indicate that ESR spectroscopy is a sensitive method of detecting changes in membrane order accompanying peroxidation of membrane lipids.     

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.     

Mitochondrial lipid peroxidation is influenced by dietary factors in early colon carcinogenesis

Oxidative damage to mitochondrial proteins, lipids, and DNA seem to influence the promotion and progression of tumors. High-fat diets and diets high in iron decrease manganese superoxide dismutase activity, a mitochondrial antioxidant, in colon mucosa. Lipid peroxidation products are low in microsomal preparations from colonic mucosa even under peroxide-inducing conditions. However, damage specific to mitochondrial membranes is unknown. This study was designed to investigate dietary lipid and iron effects on fatty acid incorporation and lipid peroxide formation in mitochondrial membranes of colonic mucosa. Male Fischer rats were fed high-fat diets containing either corn oil or menhaden oil with an iron level of either 35 or 535 mg/kg diet. Animals were given two injections of the colon carcinogen, azoxymethane, or saline. Colon tissue was collected 1 and 6 weeks after injections. Mitochondrial and microsomal fractions were prepared for fatty acid analysis and quantitation of lipid peroxidation products. Results showed that lipid composition of both subcellular fractions were influenced by diet. Fatty acid composition of mitochondria differed from microsomes, but overall saturation remained constant. Peroxidation products in mitochondrial membranes were significantly greater than in microsomal membranes. Dietary treatment significantly affected mitochondrial peroxidation in carcinogen-treated animals. Therefore, mitochondria from colon mucosa are more susceptible to peroxidation than are microsomes, dietary factors influence the degree of peroxidation, and the resulting damage may be important in early colon carcinogenesis.     

Superoxide-dependent lipid peroxidation and vitamin E content of microsomes from hepatomas with different growth rates

Lipid peroxidation of microsomal membranes isolated from rat liver, and Morris hepatomas 9618A (slow-growing) and 3924A (fast-growing) was induced by superoxide radicals generated by the action of xanthine oxidase on xanthine. The peroxidation, measured as malondialdehyde and lipid hydroperoxide formation, was optimized with regard to iron concentration and chelation of iron by ADP. In such conditions hepatoma microsomes catalyze lower rates of lipid peroxidation than the normal counterpart. However, while microsomes from hepatoma 3924A show a marked decrease in both the malondialdehyde and hydroperoxide production rates, microsomes from hepatoma 9618A differ moderately from the control, mainly in the long-term production of hydroperoxides. It is also reported here that the 9618A microsomes partially lack cytochrome P-450 (about 40% deficiency), but they have a fatty acid composition similar to that of control. No differences were found in the content of vitamin E between normal and hepatoma 3924A microsomes. Moreover, induction of vitamin E deficiency in hepatoma 3924A microsomes does not influence the rate of either malondialdehyde or lipid hydroperoxide production. On the basis of these results and previous data on the lipid composition of hepatoma 3924A microsomes it is proposed that the high resistance to superoxide-dependent lipid peroxidation of hepatoma 3924A microsomes is related to the low substrate availability rather than the content of membrane antioxidants; and a limitation only in the propagation phase characterizes the hepatoma 9618A microsomal lipid peroxidation and would be due to the partial deficiency of the endogenous propagating agent, cytochrome P-450.     

Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins

The effects of all-zinc metallothionein (Zn-metallothionein) and predominantly cadmium metallothionein (Cd/Zn-metallothionein) on free radical lipid peroxidation have been investigated, using erythrocyte ghosts as the test system. When treated with xanthine and xanthine oxidase, Zn-metallothionein and Cd/Zn-metallothionein underwent thiolate group oxidation and metal ion release that was catalase-inhibitable, but superoxide dismutase-non-inhibitable. Similar treatment in the presence of ghosts and added Fe(III) resulted in metallothioneen oxidation that was significantly inhibited by superoxide dismutase. Ghosts incubated with xanthine/xanthine oxidase/Fe(III) underwent H₂O₂- and O₂⁻-dependent lipid peroxidation, as measured by thiobarbituric acid reactivity. Neither type of metallothionein had any effect on xanthine oxidase activity, but both strongly inhibited lipid peroxidation when added to the membranes concurrently with xanthine/xanthine oxidase/iron. This inhibition was far greater and more sustained than that caused by dithiothreitol at a concentration equivalent to that of metallothionein thiolate. Significant protection was also afforded when ghosts plus Cd/Zn-metallothionein or Zn/metallothionein were preincubated with H₂O₂ and Fe(III), and then subjected to vigorous peroxidation by the addition of xanthine and xanthine oxidase. These results could be mimicked by using Cd(II) or Zn(II) alone. Previous studies suggested that Zn(II) inhibits xanthine/xanthine oxidase/iron-driven lipid peroxidation in ghosts by interfering with iron binding and redox cycling. Therefore, the primary determinant of metallothionein proteciion appears to be metal release and subsequent uptake by the membranes. These results have important implications concerning the antioxidant role of metallothionein, a protein known to be induced by various prooxidant conditions.     

The mechanism of liver microsomal lipid peroxidation

In the presence of Fe³⁺ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1,3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. These results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe³⁺ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe²⁺ by oxygen.     

Superoxide-dependent redox cycling of citrate-Fe3+: evidence for a superoxide dismutaselike activity

Citrate-Fe3+, reportedly a physiological chelate, exhibits superoxide dismutaselike activity, as evidenced by the inhibition of xanthine oxidase-dependent cytochrome c reduction; the dismutation of xanthine oxidase-generated superoxide to hydrogen peroxide and oxygen, and the enhanced disproportionation of potassium superoxide. The catalytic activity of citrate-Fe3+ corresponds, on a molar basis, to 0.03% of that of copper- and zinc-containing superoxide dismutase. Although weak, this activity enables citrate-Fe3+ to inhibit superoxide and ADP-Fe3+ -dependent peroxidation of extracted microsomal lipids. Also, the dismutase activity of citrate-Fe3+ interferes with its ability to promote lipid peroxidation. It is proposed that chelation of Fe3+ by citrate may represent a protective mechanism against the deleterious consequences of superoxide generation.     

The effect of dietary polyenylphosphatidylcholine on microsomal delta-6-desaturase activity, fatty acid composition, and microviscosity in rat liver under oxidative stress

Polyenylphosphatidylcholine is a choline-glycerophospholipid containing up to 80% of total fatty acids as linoleic acid and may be an important factor in ensuring normal functioning of cell membranes. We tested the effect of a polyenylphosphatidylcholine-supplemented diet and compared it with both a trilinolein-supplemented and a laboratory chow diet on the fatty acid composition, microviscosity, and delta-6-desaturase activity of liver microsomal membranes of 12-month-old rats, in the absence or presence of oxidative stress induced by adriamycin. Polyenylphosphatidylcholine- and trilinolein-supplemented diets showed a similar increase in linoleic acid content and delta-6-desaturase activity in liver microsomes, indicating that low amounts of linoleic acid are able to partially restore the enzyme activity in old rats, independent of the source of linoleic acid. After adriamycin treatment, delta-6-desaturase activity increased in polyenylphosphatidylcholine and trilinolein groups, indicating a protective mechanism against the damage induced by polyunsaturated fatty acid peroxidation. The measurement of malondialdehyde production showed a protective effect on adriamycin-induced lipid peroxidation by polyenylphosphatidylcholine supplementation only. Microsomal membrane microviscosity did not change independent of diet and adriamycin treatment, suggesting that the response of microsomes to lipid peroxidation might be the maintenance of a given membrane order. Administration of polyenylphosphatidylcholine can prevent or minimize the liver damage induced by adriamycin treatment.     

Regulation of γ-Aminobutyric Acid/Barbiturate Receptor-Gated Chloride Ion Flux in Brain Vesicles by Phospholipase A2: Possible Role of Oxygen Radicals

Preincubation of brain membranes with phospholipase A2 (PLA2) has been shown previously to affect the binding characteristics of various recognition sites associated with the gamma-aminobutyric acid (GABA) receptor complex. In the present study, we have investigated the effects of PLA2 (from Naja naja siamensis venom) on the functional activity of the GABA receptor/chloride ion channel. PLA2 (0.001-0.02 U/mg protein) preincubation decreased pentobarbital-induced 36Cl- efflux and muscimol-induced 36Cl- uptake in rat cerebral cortical synaptoneurosomes. The effect of PLA2 was prevented by EGTA and two nonselective PLA2 inhibitors, mepacrine and bromophenacyl bromide. The removal of free fatty acids by addition of bovine serum albumin both prevented and reversed the effect of PLA2. Products of the catalytic activity of PLA2, such as the unsaturated free fatty acids, arachidonic and oleic acids, mimicked the effect of PLA2. However, the saturated fatty acid, palmitic acid, and lysophosphatidyl choline had no effect on pentobarbital-induced 36Cl- efflux. Because unsaturated free fatty acids are highly susceptible to peroxidation by oxygen radicals, the role of oxygen radicals was investigated. Xanthine plus xanthine oxidase, a superoxide radical generating system, mimicked the effect of PLA2, whereas the superoxide radical scavenger, superoxide dismutase, diminished the effects of PLA2 and arachidonic acid on pentobarbital-induced 36Cl- efflux. Similarly, the effect of PLA2 was also inhibited by methanol (1 mM), a scavenger of the hydroxyl radical, and by catalase. These data indicate that exogenously added PLA2 induces alterations in membrane phospholipids, possibly promoting the generation of oxygen radicals and fatty acid peroxides which can ultimately modulate GABA/barbiturate receptor function in brain.     

Inhibition of rat liver microsomal lipid peroxidation by N-acyldehydroalanines: an in vitro comparative study

Captodative substituted olefins are radical scavengers which react with free radicals to form stabilized radical adducts. One of those compounds, N-(paramethoxyphenylacetyl)dehydroalanine (AD-5), may react and scavenge both superoxide anion (O-2) and alk-oxyl radicals (RO.), and in this way prevent the appearance of their mediated biological effects. Nitrofurantoin and tert-butyl hydroperoxide were used as model compounds to stimulate free radical production and their mediated lipid peroxidation in rat liver microsomes. In addition, lipid peroxidation was also initiated by exposure of rat liver microsomal suspensions to ionizing radiation (gamma rays). The microsomal lipid peroxidation induced by these chemicals and physical agents was inhibited by the addition of AD-5. These effects were dose-dependent in a millimolar range of concentration. In addition, AD-5 has no effect on microsomal electron transport, showing that NADPH-cytochrome P450 reductase activity was not modified. These data, together with the comparisons of the effects of AD-5 and some antioxidant molecules such as superoxide dismutase, uric acid, and mannitol, support the conclusion that inhibition of lipid peroxidation by AD-5 is the result of its free radical scavenger activity. In addition, the inhibitory effect of AD-5 on microsomal lipid peroxidation was dependent of the nature of the free radical species involved in the initiation of the process, suggesting that O-2 is scavenged more efficiently than RO.     

Synthesis of acylated SOD derivatives which bind to the biomembrane lipid surface and dismutate extracellular superoxide radicals

Involvement of oxygen radicals in the pathogenesis of various inflammatory diseases has been the focus of recent attention. Since lipid peroxidation of cell membranes is postulated to be one of the major reasons for radical-induced tissue injury, inhibition of oxygen toxicity at or near plasma membranes is important. To metabolize extracellular superoxide radicals effectively at or near cell membranes, we synthesized amphipathic superoxide dismutase (SOD) derivatives (AC-SOD) by covalently linking hydrophobic fatty acids with different chain lengths, such as caprylic acid, capric acid, lauric acid and myristic acid, to the lysyl amino groups of the enzyme. When incubated with erythrocytes or polymorphonuclear leukocytes (PMNs), AC-SOD, but not SOD, bound to plasma membranes of these cells. When topically instilled to the eye, AC-SOD also bound to corneal epithelial cell surface. Upon activation by phorbolmyristyl acetate, extracellular cytochrome c was rapidly reduced by PMNs which were pretreated with SOD. In contrast, PMNs preincubated with AC-SOD failed to catalyze the reduction of cytochrome c under the same experimental conditions. These results suggested that AC-SOD bound to cell membranes and effectively dismutated superoxide radicals at or on the outer surface of plasma membranes.     

Lipid peroxidation by gamma-radiation in Salmonella typhi membranes: the effect of ascorbic acid

Lipid peroxidation in isolated membranes of Salmonella typhi occurred following gamma irradiation. The presence of the sensitizer, ascorbic acid, during irradiation resulted in a stimulation in the level of peroxidation. This enhancement of lipid damage could be prevented by the addition of the free radical scavenger sodium formate. Catalase and superoxide dismutase appeared less effective in this respect. Peroxidation in an unsaturated fatty acid and a phospholipid was also affected by gamma radiation and ascorbic acid in a manner similar to that observed in the cell membranes. The results suggest that hydroxyl radicals may be the major species responsible for the increased peroxidation effected by the sensitizer.     

The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

Interaction of rat liver microsomes containing saturated or unsaturated fatty acids with fatty acid binding protein: Peroxidation effect

In the studies described here rat liver microsomes containing labeled palmitic, stearic, oleic or linoleic acids were incubated with fatty acid binding protein (FABP) and the rate of removal of¹⁴C-labeled fatty acids from the membrane by the soluble protein was measured using a model system. More unsaturated than saturated fatty acids were removed from native liver microsomes incubated with similar amounts of FABP. Thein vitro peroxidation of microsomal membranes mediated by ascorbate-Fe⁺⁺, modified its fatty acid composition with a considerable decrease of the peroxidizability index. These changes in the microsomes facilitated the removal of oleic and linoeic acids by FABP, but the removal of palmitic and stearic acids was not modified. This effect is proposed to result from a perturbation of membrane structure following peroxidation with release of free fatty acids from susceptible domains.Abbreviations BSA bovine serum albumin - FABP fatty acid binding protein