Cataract induction by products of lipid peroxidation

Liposome suspension prepared from the unsaturated phospholipids exposed to lipid peroxidation (LPO) induced posterior subcapsular cataracts after injection into the posterior vitreous of rabbit eyes. In the background of this model lies a type of lens opacity formed during retinal degeneration when toxic peroxide substances diffuse anteriorly through the vitreous body resulting in vitreous opacities and complicated cataracts. Saturated liposomes (prepared from beta-oleoyl-gamma-palmitoyl) L-alpha-lecithin) did not induce lens opacities, which is the evidence that a lipid peroxidation mechanism may be responsible for the posterior cataracts. Along with cataract formation accumulation of LPO fluorescent products in vitreous, aqueous humor and lens was observed. It was followed by a decreased level of reduced glutathione in the lens. The obtained results strongly support the hypothesis of LPO initial role in cataracts.     

Site-specific induction of lipid peroxidation by iron in charged micelles

Generation of hydroxyl radicals by the Fenton reaction resulted in lipid peroxidation of linoleic acid (LA) (H2O2-Fe2+-induced lipid peroxidation) in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles, but not in negatively charged sodium dodecyl sulfate (SDS) micelles. However, more OH radicals formed via the Fenton reaction were trapped by N-t-butyl-alpha-phenylnitrone (PBN) in SDS micelles than in TTAB micelles. When detergent-dispersed LA was contaminated with linoleic acid hydroperoxide (LOOH), lipid peroxidation was catalyzed by Fe2+ via reductive cleavage of LOOH (LOOH-Fe2+-induced lipid peroxidation), and Fe2+ was oxidized simultaneously in SDS micelles, even when H2O2 was not present. In contrast, LOOH-Fe2+-induced lipid peroxidation and simultaneous oxidation of Fe2+ were not observed in TTAB micelles. An ESR spectrum presumed to be due to an alkoxy radical trapped by PBN was also detected in SDS micelles, but not in TTAB micelles in the LOOH-Fe2+-induced lipid peroxidation system. The results are discussed in the light of the localization of iron, the unsaturated bonding moiety of LA, the OOH-group of LOOH, and the trapping site of PBN in different charged micelles.     

Cellular lipid peroxidation end-products induce apoptosis in human lens epithelial cells

Hydrogen peroxide (H(2)O(2)), an oxidant present in high concentrations in the aqueous humor of the elderly eyes, is known to impart toxicity to the lens---apoptosis being one of the toxic events. Since H(2)O(2) causes lipid peroxidation leading to the formation of reactive end-products, it is important to investigate whether the end-products of lipid peroxidation are involved in the oxidation-induced apoptosis in the lens. 4-Hydroxynonenal (HNE), a major cytotoxic end product of lipid peroxidation, has been shown to mediate oxidative stress-induced cell death in many cell types. It has been shown that HNE is cataractogenic in micromolar concentrations in vitro, however, the underlying mechanism is not yet clearly understood. In the present study we have demonstrated that H(2)O(2) and the lipid derived aldehydes, HNE and 4-hydroxyhexenal (HHE), can induce dose- and time-dependent loss of cell viability and a simultaneous increase in apoptosis involving activation of caspases such as caspase-1, -2, -3, and -8 in the cultured human lens epithelial cells. Interestingly, we observed that Z-VAD, a broad range inhibitor of caspases, conferred protection against H(2)O(2)- and HNE-induced apoptosis, suggesting the involvement of caspases in this apoptotic system. Using the cationic dye JC-1, early apoptotic changes were assessed following 5 h of HNE and H(2)O(2) insult. Though HNE exposure resulted in approximately 50% cells to undergo early apoptotic changes, no such changes were observed in H(2)O(2) treated cells during this period. Furthermore, apoptosis, as determined by quantifying the DNA fragmentation, was apparent at a much earlier time period by HNE as opposed to H(2)O(2). Taken together, the results demonstrate the apoptotic potential of the lipid peroxidation end-products and suggest that H(2)O(2)-induced apoptosis may be mediated by these end-products in the lens epithelium.     

Selective pacemaker interruption and disorders of atrial automaticity during induction of lipid peroxidation

The influence of lipid peroxidation (LPO) inductor H2O2 on spontaneous contractility and electrical activity of the right atrium was studied. LPO induction caused positive inotropic catecholamine-like effect, followed by brady-arrhythmia with the action of potential prolongation, blockade of electrical activity and atrial arrest. Electrical pacing during atrial arrest caused regular contractions of the same force as before electrical activity blockade. The results suggest that the arrest of spontaneous atrial activity under the influence of LPO inductor is due to the impairment of sinus node automaticity.     

Enzymatic lipid peroxidation

Biosynthesis of certain biologically active substances (prostaglandins, thromboxanes, prostacyclins and leukotrienes) in animal tissues occurs with participation of cyclooxygenases and lipoxygenases, enzymic systems of lipid peroxidation. In normal physiological and pathological processes the enzymic lipid peroxidation by microsomal dioxygenases is considerably more active than the nonenzymic one in the same membrane structures. The molecular structure of the products of the enzymic and nonenzymic peroxidation of lipids also differs essentially. An assumption is advanced that cytosol lipoxygenase may be an easily dissociating component of the cyclooxygenase multienzymic complex and its transition from the biomembrane to the cell cytoplasm is accompanied by changes in the enzyme conformation and chemical nature of the products resulted from polyenic lipids oxidation catalyzed by the enzyme.     

Enhanced lipid peroxidation is not necessary for induction of metallothionein-I by oxidative stress

A study has been made of factors which may influence the induction of metallothionein-I (MT-I) synthesis by the superoxide radical generating agent, paraquat (PQ). Hepatic concentrations of zinc (Zn) and MT-I increased in rats injected with PQ (40 mg/kg, s.c.) or fasting, but were greater in the former. Renal concentration of MT-I increased in fasted rats but not in PQ-treated rats. The data suggest that the increase in MT-I concentrations in PQ-treated rats is not caused by reduction in food intake. Administration of PQ increased hepatic concentrations of Zn, MT-I and thiobarbituric acid-reactive substances (TBA-RS), indicating the occurrence of lipid peroxidation. Treatment of rats with vitamin E (400 mg/kg, s.c.) on 4 successive days before injection of PQ prevented only the enhancement of lipid peroxidation. The data indicate that the induction of MT synthesis by PQ is not correlated with enhancement of lipid peroxidation. Similar results were obtained in the liver of rats subjected to the radical-generating conditions, such as fasting and exposure to carbon tetrachloride. Free radicals may induce MT synthesis by direct or indirect mechanisms.     

Transferrin-dependent lipid peroxidation

The potential for iron bound to transferrin to be released and promote the peroxidation of phospholipid liposomes was investigated using ADP as a low molecular weight chelator and Superoxide generated by the xanthine/ xanthine oxidase system as the reducing agent. Lipid peroxidation in this system was dependent upon transferrin as the source of iron; increasing the transferrin concentration resulted in increased rates of lipid peroxidation. Increasing the xanthine oxidase activity also caused increased rates of peroxidation. Catalase stimulated rates of peroxidation at all xanthine oxidase activities tested. Conditions resulting in the most rapid release of iron from transferrin (low pH, high ADP) did not promote the greatest rates of lipid peroxidation, indicating that at neutral pH, rates of lipid peroxidation may be limited by the availability of iron. It is concluded that transferrin is not a likely source of iron for catalysis of deleterious biological oxidations such as lipid peroxidation in vivo.     

Thiol-dependent lipid peroxidation

Initiation of lipid peroxidation in liposomes by cysteine, glutathione, or dithiothreitol required iron, and was not inhibited by superoxide dismutase. The absence of superoxide involvement in thiol autoxidation was confirmed by the inability of superoxide dismutase to inhibit thiol reduction of cytochrome c. Furthermore, the rate of cytochrome c reduction by thiols was not decreased under anaerobic conditions. We suggest that lipid peroxidation initiated by thiols and iron occurs via direct reduction of iron. Control of cellular thiol autoxidation, and reactions occurring as a consequence, such as lipid peroxidation, must therefore involve chelation of transition metals to control their redox reactions.     

Inhibition of lipid peroxidation

Lipid peroxy radicals (ROO-) were detected by electron spin resonance (ESR) at low temperature after formation by addition of H₂O₂ into a suspension of mice lymphocites. If lymphocytes were treated with selenomethionine (Se-Met) prior to addition of H₂O₂, ROO-formation was inhibited in a fashion that was dependent on Se-Met concentration. Formation of ROO- in the spleen of mice was induced by⁶⁰Co irradiation. Animals that were supplemented with Na₂SeO₃ prior to irradiation exhibited a lower ROO-concentration than that of nontreated animals. Based on our experiments, we have concluded that Se has an oxygen-free radical scavenging effect. This should be a protective effect against lipid peroxy radical cellular attack.     

The mechanism of NADPH-dependent lipid peroxidation. The propagation of lipid peroxidation.

NADPH-dependent lipid peroxidation occurs in two distinct sequential radical steps. The first step, initiation, is the ADP-perferryl ion-catalyzed formation of low levels of lipid hydroperoxides. The second step, propagation, is the iron-catalyzed breakdown of lipid hydroperoxides formed during initiation generating reactive intermediates and products characteristic of lipid peroxidation. Propagation results in the rapid formation of thiobarbituric acid-reactive material and lipid hydroperoxides. Propagation can be catalyzed by ethylenediamine tetraacetate-chelated ferrous ion, diethylenetriamine pentaacetic acid-chelated ferrous ion, or by ferric cytochrome P-450. However, cytochrome P-450 is destroyed during propagation.     

Iron induction of lipid peroxidation and effects on antioxidative enzyme activities in rice leaves

Lipid peroxidation in relation to toxicity of detached rice leavescaused by excess iron (FeSO₄) was investigated. ExcessFeSO₄, which was observed to induce toxicity, enhanced the contentoflipid peroxidation but not the content of H₂O₂.Superoxidedismutase activity was reduced by excess FeSO₄. Ascorbate peroxidaseand glutathione reductase activities were increased by excess FeSO₄.Free radical scavengers, such as mannitol and reduced glutathione, inhibitedexcess iron-induced toxicity and at the same time inhibited excessiron-enhancedlipid peroxidation, suggesting that lipid peroxidation enhanced by excess ironis mediated through free radicals.     

Crystalline lens "tumor immunity"

Three series of experiments on 72 rabbits have shown that lens tumor immunity is not related to the anaerobic nature of its energy metabolism and is not a consequence of lens capsule impermeability for chemical cancer genes. Ionizing radiation causes cataracts without malignancy of lens epithelial cells.     

Oxygen-dependent antagonism of lipid peroxidation

Measurements of the rates for formation of conjugated dienes, malonylaldehyde, and lipid hydroperoxides show that increasing the concentration of O2 from 0.11 mM to 0.35 mM or 0.69 mM can slow the rate of linoleic acid peroxidation in a xanthine oxidase/hypoxanthine system. This effect is seen at pH 7.0 but not 7.4 and depends on the presence of monounsaturated fatty acids (oleic, cis, or trans vaccenic acid). Oxygen antagonism of ascorbic acid-iron-EDTA mediated lipid peroxidation is similarly dependent on fatty acid mixtures and occurs at pH 5.0 and 6.0 but not 7.0. The efficiency of initiation of peroxidation in the xanthine oxidase system is unaffected by monounsaturated fatty acids and O2 concentration. Increasing the O2 concentration increases the rate of superoxide radical production, but there is no change in salicylate hydroxylation (e.g., OH. production) or ferrous ion concentration. Oxygen-mediated slower rates of lipid peroxidation are associated with either increased H2O2 production or, based on an indirect assay, singlet O2 production. Increased O2 concentrations increase the rate of azobisisobutyronitrile-initiated lipid peroxidation as expected but addition of exogenous superoxide radicals slows the rate. Under similar conditions superoxide reacts with fatty acids to produce singlet O2. Overall, the data suggest that O2-mediated antagonism occurs because of termination reactions between hydroperoxyl (HO2.) and organic radicals, and singlet O2 or H2O2 are products of these reactions.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe²⁺ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than α-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than α-tocopherol; (e) to be a weaker antiradical than α-tocopherol in the reduction of the stable radical DPPH^·. Resveratrol reduced Fe³⁺ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe³⁺, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like α-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Resveratrol inhibition of lipid peroxidation

To define the molecular mechanism(s) of resveratrol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process. Resveratrol proved (a) to inhibit more efficiently than either Trolox or ascorbate the Fe2+ catalyzed lipid hydroperoxide-dependent peroxidation of sonicated phosphatidylcholine liposomes; (b) to be less effective than Trolox in inhibiting lipid peroxidation initiated by the water soluble AAPH peroxyl radicals; (c) when exogenously added to liposomes, to be more potent than alpha-tocopherol and Trolox, in the inhibition of peroxidation initiated by the lipid soluble AMVN peroxyl radicals; (d) when incorporated within liposomes, to be a less potent chain-breaking antioxidant than alpha-tocopherol; (e) to be a weaker antiradical than alpha-tocopherol in the reduction of the stable radical DPPH*. Resveratrol reduced Fe3+ but its reduction rate was much slower than that observed in the presence of either ascorbate or Trolox. However, at the concentration inhibiting iron catalyzed lipid peroxidation, resveratrol did not significantly reduce Fe3+, contrary to ascorbate. In their complex, our data indicate that resveratrol inhibits lipid peroxidation mainly by scavenging lipid peroxyl radicals within the membrane, like alpha-tocopherol. Although it is less effective, its capacity of spontaneously entering the lipid environment confers on it great antioxidant potential.     

Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.     

3-Methylindole inhibits lipid peroxidation

The mechanism of pneumotoxicity of 3-methylindole has been postulated to occur via protein alkylation or lipid peroxidation. This report describes the effects of the addition of 3-methylindole to goat lung microsomes to evaluate the possibility that this xenobiotic may increase NADPH-supported lipid peroxidation. Concentrations of malondialdehyde were measured as an index of lipid peroxidation. Instead of a stimulation of lipid peroxidation by 3-methylindole, a complete inhibition of lipid peroxidation was produced by concentrations of 3-methylindole as low as 10 microM. The addition of 3-methylindole to actively peroxidizing microsomes (NADPH-supported) caused an immediate cessation of malondialdehyde production. These results demonstrate that 3-methylindole pneumotoxicity does not proceed by a mechanism of lipid peroxidation, but in fact, this molecule may act as an effective antioxidant to prevent lipid peroxidation in pulmonary tissue.     

Ferritin and superoxide-dependent lipid peroxidation

Ferritin was found to promote the peroxidation of phospholipid liposomes, as evidenced by malondialdehyde formation, when incubated with xanthine oxidase, xanthine, and ADP. Activity was inhibited by superoxide dismutase but markedly stimulated by the addition of catalase. Xanthine oxidase-dependent iron release from ferritin, measured spectrophotometrically using the ferrous iron chelator 2,2'-dipyridyl, was also inhibited by superoxide dismutase, suggesting that superoxide can mediate the reductive release of iron from ferritin. Potassium superoxide in crown ether also promoted superoxide dismutase-inhibitable release of iron from ferritin. Catalase had little effect on the rate of iron release from ferritin; thus hydrogen peroxide appears to inhibit lipid peroxidation by preventing the formation of an initiating species rather than by inhibiting iron release from ferritin. EPR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide was used to observe free radical production in this system. Addition of ferritin to the xanthine oxidase system resulted in loss of the superoxide spin trap adduct suggesting an interaction between superoxide and ferritin. The resultant spectrum was that of a hydroxyl radical spin trap adduct which was abolished by the addition of catalase. These data suggest that ferritin may function in vivo as a source of iron for promotion of superoxide-dependent lipid peroxidation. Stimulation of lipid peroxidation but inhibition of hydroxyl radical formation by catalase suggests that, in this system, initiation is not via an iron-catalyzed Haber-Weiss reaction.