Effect of ethanol and the catalase inhibitor aminotriazole on lipid peroxidation in the rat myocardium

The influence of chronic alcohol consumption and catalase inhibitor aminotriazole administration on the level of nonenzymatic lipid peroxidation has been studied in the rat myocardium. It was demonstrated that combined as well as separate treatment with ethanol or aminotriazole elevated the levels of chemiluminescence and enhanced the rate of accumulation of thiobarbituric acid-reactive products in the nuclei-free and total particulate fraction of the rat heart homogenate. The most pronounced effect was noted during combined application of ethanol and aminotriazole. The induction of chemiluminescence by ethanol was prevented by addition of natural (vitamin E, reduced glutathione) or artificial (dibunol) antioxidants into the incubation media. A putative role of the myocardial catalase-containing micro-peroxisomes in stimulation of the intracellular lipid peroxidation is discussed.     

Effect of clofibrate treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. A study was made of the effect of hypolipidemic drug clofibrate on the level of lipid peroxidation in homogenates and subcellular fractions of rat liver. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of clofibrate the levels of Fe/ADP-ascorbate-, as well as t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation were decreased in the whole and "post-nuclear" liver homogenates. Dilution of the homogenates prevented depressing effect of clofibrate on lipid peroxidation. 3. Clofibrate significantly decreased the level of the Bu'OOH-dependent lipid peroxidation, but did not affect the activity of the Fe/ADP-ascorbate-induced reaction in rat liver mitochondria and microsomes. 4. Peroxidative alteration of membrane lipids in vivo was evaluated by determining the extent of conjugated dienes formation (absorption at 233 nm). It was shown that clofibrate did not increase the level of ultraviolet absorption of lipids from rat liver subcellular fractions. 5. The data obtained indicate that cytosol from the clofibrate treated rat liver contains a factor(s) which prevents lipid peroxidation in the mitochondria and microsomes.     

Effect of chronic ethanol treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. The effect of chronic ethanol treatment on the level of lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that after chronic ethanol treatment the level of Fe/ADP-ascorbate-induced lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. Dilution of the homogenates prevented depressive effect of ethanol on lipid peroxidation. 3. Chronic ethanol treatment did not affect the intensity of the Fe/ADP-ascorbate-induced process in rat liver mitochondria and microsomes. 4. Peroxidative alteration of the liver lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). It was shown that ethanol did not increase the level of u.v. absorption of lipids from mitochondria and microsomes. Chronic alcohol treatment did not influence the steady-state concentration of malonic dialdehyde in the whole liver homogenate. 5. The data obtained indicate that cytosol from the ethanol treated rat liver contains a factor(s) which prevents Fe/ADP-ascorbate-dependent lipid peroxidation in biological membranes.     

Effect of chronic ethanol treatment on the t-butyl hydroperoxide-dependent lipid peroxidation in rat liver

1. The effect of chronic ethanol consumption on the level of the t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of ethanol the rate of lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. 3. Ethanol significantly decreased the intensity of lipid peroxidation in microsomes, but did not affect the Bu'OOH-dependent process in mitochondria. 4. The level of lipid peroxidation was reduced after incubation of the total particulate fraction (mitochondria plus microsomes) with the undialysed cytosol from ethanol-treated rat liver. Dialysis of the cytosol prevented depressive effect of ethanol treatment on lipid peroxidation. 5. Reduced glutathione (0.1-1.0 mM) was shown to decrease the rate of lipid peroxidation in rat liver microsomes, but did not affect its level in mitochondria. 6. Pyrazole injections to rats reduced and phenobarbital treatment increased the level of the Bu'OOH-dependent lipid peroxidation in liver microsomes. 7. The data obtained indicate that the Bu'OOH-dependent lipid peroxidation is not an appropriate marker of the ethanol-induced oxidative stress in rat liver cells.     

Effect of chronic ethanol treatment under partial catalase inhibition on the activity of enzymes related to peroxide metabolism in rat liver and heart

1. In order to test the hypothesis that the alcoholic cardiomyopathy under partial catalase inhibition is associated with the activation of lipid peroxidation in cardiomyocytes (Panchenko et al., Experientia 43, 580-581, 1987), the effects of ethanol and catalase inhibitor 3-amino-1,2,4-triazole (aminotriazole) on rat heart and liver content of reduced glutathione and on the activity of enzymes related to peroxide metabolism: catalase, superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase and glucose-6-phosphate dehydrogenase were investigated. 2. In accordance with the data obtained by Kino (J. molec, cell. Cardiol. 13, 5-12, 1981), when ethanol (36% of dietary calories) and aminotriazole were simultaneously administered an alcoholic cardiomyopathy developed while in the liver moderate fatty degeneration was revealed. 3. Chronic combined or separate administration of ethanol and aminotriazole was shown to increase glutathione concentration and glutathione-S-transferase activity in rat liver. In the groups of animals which received isocaloric carbohydrates in the diet instead of ethanol the liver glucose-6-phosphate dehydrogenase was increased. 4. Acute and chronic aminotriazole injections led to catalase inactivation and in the latter case also to inhibition of the liver superoxide dismutase and glutathione peroxidase activities. 5. Ethanol and aminotriazole treatment did not alter the glutathione level and the activity of all enzymes tested (except catalase) in rat myocardium.     

Role of monoaminoxidase in the intensification of peroxidation of lipids in mitochondria during experimental myocardial necrosis

The relationship between lipid peroxidation and rat heart mitochondrial monoamine oxidase activity was studied in experimental myocardial necrosis induced by adrenaline injection. It has been established that both the intensity of peroxidation and the activity of monoamine oxidase in mitochondria from adrenaline-injured rat myocardium were essentially increased. The preliminary administration of antioxidants (vitamin E and ionol) was shown to decrease both the intensity of lipid peroxidation and the activity of monoamine oxidase. It is suggested that intensification of lipid peroxidation which is considered to be the main pathogenic factor in ischemic myocardial injury depends on mitochondrial monoamine oxidase activity. Protective effects of antioxidants are realized by the action on two subsequent chains during the formation of active oxygen forms and destruction of lipid peroxidation products.     

Increase in cellular pool of low-molecular-weight iron during ethanol metabolism in rat hepatocyte cultures

Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (≤1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas α-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.     

Relationships between lipid peroxidation and anoxia tolerance in a range of species during post-anoxic reaeration

Peroxidation was studied in anoxically treated plant tissues and quantified as conjugated dienes/trienes in the total lipid fraction and as the production of thiobarbituric acid reactive substances (TBARS). Oxidative stress caused by re-exposure of plants to oxygen led to an increase of conjugated diene/triene formation in rhizomes of Iris germanica and roots of wheat ( Triticum aestivum L.) and oats ( Avena sativa L.), and after a long anoxic exposure (45 days) in the rhizomes of the very anoxia tolerant Iris pseudacorus . Second derivative (SD) spectrophotometry of the UV spectrum of lipid extracts confirmed the formation of dienes. However, determination of TBARS in Iris spp. showed no lipid peroxidation in the anoxia tolerant I. pseudacorus . In the rhizomes of the anoxia intolerant I. germanica , elevated levels of TBARS correlated positively with conjugated diene/triene formation. The results suggest that anoxic stress may induce qualitative changes in membrane lipids, as indicated by lipid peroxidation after restoration of aerobic conditions. The rate of lipid peroxidation correlated negatively with anoxic stress tolerance.     

Spin trapping of oxygen and carbon-centered free radicals in ischemic canine myocardium

Oxygen free radical injury has been postulated to occur during myocardial ischemia. We have used Electron Spin Resonance and Spin Trapping techniques to directly demonstrate the production of carbon-centered (R.) and oxygen-centered lipid radical (RO.) in ischemic canine heart. In addition, venous effluent from the ischemic region showed that conjugated dienes (lipid peroxidation products) increased with ischemic duration. Our results suggest that the formation of the oxygen-centered and carbon-centered lipid radical species during ischemia are a consequence of oxy-radical peroxidation of myocardial membrane lipids.     

On the role of rat liver cytosol in suppression of low-level chemiluminescence during nonenzymatic lipid peroxidation.

1. The effect of normal rat liver cytosol on the level of Fe/ADP-ascorbate-induced lipid peroxidation in the total particulate fraction (mitochondria plus microsomes) has been studied. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde (MDA) formation. 2. Dialysed cytosol significantly decreased the level of chemiluminescence, and to a much lesser extent, the rate of MDA production. 3. Gel filtration on a Sephadex G-200 column led to appearance of at least three cytosolic fractions which suppressed the low-level chemiluminescence. 4. The discovered components differed from each other by their molecular masses, kinetics of chemiluminescence inhibition and effects on intensity of MDA formation. 5. The putative functional role of antioxidative defence factors from rat liver cytosol is discussed.     

Loss of heart phospholipid arachidonic acid without phospholipid peroxidation in anaesthetized rats rewarmed after prolonged deep hypothermia

Hypothermia–rewarming of the heart results in contractile dysfunction under in vitro as well as in vivo conditions. Increase in reactive oxygen species (ROS), lipid peroxidation and calcium overload are proposed mechanisms. In the first protocol of this study, the effect of putative phospholipase and calcium channel modulator mepacrine during deep hypothermia (4 h 14 °C) plus rewarming was tested in an isolated perfused rat heart model previously reported not to involve increase in lipid peroxides. Contractile function was measured under isovolumetric conditions using an intra-ventricular balloon connected to a transducer and recording system. Mepacrine completely reversed hypothermia–rewarming induced contractile failure in this model (LV dP/dt_max: 3236 ± 517 vs. 1058 ± 185 mm Hg/s in untreated hearts). In the second part of the study, lipid peroxidation of the heart was examined in vivo in anesthetized rats subjected to 4 h of deep hypothermia followed by rewarming. In this model recovery of heart function judged by cardiac output is decreased whereas blood pressure and heart rate recover fully. Peroxy conjugated diene isomers of unsaturated fatty acids were measured in heart phospholipids. The composition of the non-esterified fatty acids and the phospholipid fatty acid pool was examined in order to reveal signs of membrane remodeling. The results demonstrated no significant changes in phospholipid peroxidation after rewarming (91.07 ± 5.23 vs. 88.63 ± 7.73 nmol/g dry wt. in control). There was significant relative reduction in the content of arachidonic acid in the phospholipid fraction (29.55 ± 1.65 vs. 24.76 ± 1.48%). There was marked decrease in non-esterified fatty acids in myocardial tissue (1992 ± 291 vs. 1069 ± 189 nmol/g dry wt.), but a significant relative increase in arachidonic acid (20:4) in this fraction (3.46 ± 0.42 vs. 4.99 ± 0.30%). In conclusion, rewarming from deep hypothermia is not associated with increased phospholipid peroxidation. There is, however, a significant remodeling of the phospholipid fraction of myocardial lipids in vivo probably as a result of receptor or calcium stimulated phospholipase activity. Calcium or calcium stimulated phospholipase activity could contribute to posthypothermic contractile dysfunction.     

Effects of hyperoxia and acrylonitrile on the phospholipase C isozyme protein levels in rat heart and brain

We previously showed that hyperoxia exerts oxidative stress on the rat cerebral cortex, and the protein levels of phospholipase C (PLC) -beta1 and -delta1, but not PLC-gamma1, were changed. Acrylonitrile (ACN) appears to induce astrocytomas through induction of oxidative stress on the rat brain selectively. This study compared hyperoxia or ACN treatments of rats with respect to lipid peroxidation and PLC levels in the heart and cerebral cortex. Treatment of rats with ACN promoted lipid peroxidation in the heart and cerebral cortex, the percent increase above control being greater in the cortex than heart. Hyperoxia did not cause significant increases in lipid peroxidation in the cerebral cortex or heart. In the ACN-treated cerebral cortex, significant increases in the PLC-beta1 and -delta1 in the cytosol, and PLC-gamma1 in the cytosolic and particulate fractions, and lysate were observed. In the rat heart, in which PLC-beta1 could not be detected, PLC-gamma1 and -delta1 were increased and decreased in the cytosolic and particulate fractions, respectively, by hyperoxia. In addition, the expression level of PLC-gamma1 was decreased in the lysate by the treatment. In the heart treated with ACN, there was no change in the level of PLC-gamma1, while PLC-delta1 was elevated in all fractions. These findings suggested that the expression levels of PLC isozymes are altered by hyperoxia and ACN, but there are apparent differences in these altered levels between the different levels of oxidative stress, and between the organs.     

Function of the membrane lipids of the heart sarcoplasmic network in izadrin myocarditis studied by using a pyrene probe

The fluorescent hydrophobic pyrene probe was employed to study the viscosity of membrane lipids of rat heart sarcoplasmic reticulum in isoproterenol myocarditis. During pyrene incorporation into the reticulum obtained from the affected myocardium, the increase in the microviscosity occurred at lower temperatures and more rapidly both in "bound" and "free" membrane lipids as compared with normal. The increase of the viscosity of the reticulum membranes in isoproterenol myocarditis was accompanied by a lowering of the activity of Ca, Mg-ATPase of the sarcoplasmic reticulum coupled with an elevation of the content of lipid peroxidation products.     

Hepatic oxidative stress and related defenses during treatment of mice with acetylsalicylic acid and other peroxisome proliferators

The peroxisome proliferators perfluorooctanoic acid (PFOA; 0.02% w/w), perfluorodecanoic acid (PFDA; 0.02%, w/w), nafenopin (0.125%, w/w), clofibrate (0.5%, w/w), and acetylsalicylic acid (ASA; 1%, w/w) were administered to male C57 BL/6 mice in their diet for two weeks. Parameters for Fe³⁺ ADP, NADPH or ascorbic acid-initiated lipid peroxidation in vitro were measured. Approximately a twofold increase in susceptibility to lipid peroxidation was obtained for all the peroxisome proliferators tested. Cotreatment of mice with the peroxisome proliferator ASA (1%, w/w) and a catalase inhibitor, 3-amino-1,2,4-triazole (AT; 0.4%, w/w) for 7 days resulted in little inhibition of peroxisome proliferation, an elevated level of H₂O₂ in vivo, and total inhibition of the increased susceptibility to lipid peroxidation in vitro. No increase in lipid peroxidation in vivo was observed. Certain antioxidant enzymes (DT-diaphorase, superoxide dismutase, glutathione transferase, glutathione peroxidase, and glutathione reductase) and components (ubiquinone and α-tocopherol) were also measured. The results showed that there was some induction of these antioxidant enzymes and components by ASA or aminotriazole, except for glutathione peroxidase and superoxide dismutase, which were inhibited. The possible involvement of oxidative stress in the carcinogenicity of peroxisome proliferators is discussed.     

Effect of alpha-tocopherol on peroxidative membrane damage caused by doxorubicin: an in vitro study in human erythrocytes

Level of lipid peroxidation in doxorubicin treated human erythrocytes was studied and compared with that of cells pretreated with alpha-tocopherol. Erythrocytes treated with alpha-tocopherol had reduced level of lipid peroxidation with concomitantly lowered membrane damage. The membrane damage was monitored by the levels of conjugated diene absorption, lipid hydroperoxides and lipid peroxides. alpha-tocopherol was not effective in inhibiting the conjugated diene formation, but the lipid hydroperoxides and the lipid peroxide levels were significantly decreased. Methemoglobin level was found to be increased in alpha-tocopherol pretreated cells, which protects the membrane from damage. Erythrocyte membrane lipids were found to be decreased during doxorubicin treatment and alpha-tocopherol significantly reduced the membrane lipid breakdown. Level of reduced glutathione was maintained in alpha-tocopherol pretreated cells. These results are discussed with reference to the antioxidant property of alpha-tocopherol.     

四种中草药抗白内障形成中晶状体脂类过氧化水平及脂类含量的变化

我们观察了中草药防治大鼠半乳糖性白内障形成中脂类含量的变化及脂类过氧化水平。结果表明,与正常晶状体相比,白内障晶状体中总脂类的含量明显降低,总胆固醇的含量及脂类过氧化水平明显升高,总脂类与总胆固醇之比明显下降。而同时分别用黄苓、石斛、菟丝子及玉蝴蝶四种中草药水煎剂灌胃的大鼠晶状体中,总胆类与总胆固醇的含量基本恢复至正常;脂类过氧化水平虽仍高于正常晶状体,但也明显低于白内障晶状体,表明脂类过氧化参与了白内障的形成,上述四种中草药具有抑制脂类过氧化的作用。     

Oxygen reduction and lipid peroxidation by iron chelates with special reference to ferric nitrilotriacetate

A certain iron chelate, ferric nitrilotriacetate (Fe3+-NTA) is nephrotoxic and also carcinogenic to the kidney in mice and rats, a distinguishing feature not shared by other iron chelates tested so far. Iron-promoted lipid peroxidation is thought to be responsible for the initial events. We examined its ability to initiate lipid peroxidation in vitro in comparison with that of other ferric chelates. Chelation of Fe2+ by nitrilotriacetate (NTA) enhanced the autoxidation of Fe2+. In the presence of Fe2+-NTA, lipid peroxidation occurred as measured by the formation of conjugated diene in detergent-dispersed linoleate micelles, and by the formation of thiobarbituric acid-reactive substances in the liposomes of rat liver microsomal lipids. Addition of ascorbic acid to Fe3+-NTA solution promoted dose-dependent consumption of dissolved oxygen, which indicates temporary reduction of iron. On reduction, Fe3+-NTA initiated lipid peroxidation both in the linoleate micelles and in the liposomes. Fe3+-NTA also initiated NADPH-dependent lipid peroxidation in rat liver microsomes. Although other chelators used (deferoxamine, EDTA, diethylenetriaminepentaacetic acid, ADP) enhanced autoxidation, reduction by ascorbic acid, or in vitro lipid peroxidation of linoleate micelles or liposomal lipids, NTA was the sole chelator that enhanced all the reactions.     

黑木耳多糖对抗离体心脏缺血/再灌注损伤的研究

目的:探讨黑木耳多糖(AAP)对离体大鼠心脏缺血/再灌注(I/R)损伤的防护作用及其机制。方法:健康雄性SD大鼠灌胃黑木耳多糖(50,100,200mg/(kg.d))4周后,采用离体心脏Langendorff灌流方法,全心停灌30min,复灌120min建立I/R模型。测定左心室动力学指标和再灌注各时间点冠脉流出液中乳酸脱氢酶(LDH)含量;实验结束测定心肌组织甲月赞(formazan)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活性的变化。结果:与单纯I/R组相比,AAP预处理明显提高心肌细胞的formazan含量,降低再灌注期间冠脉流出液中LDH含量,明显增强左室发展压、左心室内压最大上升速率和心率与发展压乘积的恢复,缓解冠脉流量的减少;高剂量AAP改善I/R心肌功能的作用要好于丹参预处理(4ml/(kg.d),gastricperfusion)组。中剂量AAP(100mg/(kg.d))预处理4周后明显抑制I/R心肌MDA的增加和SOD活性的减弱(P0.01),其效果要好于丹参阳性对照组。结论:在大鼠离体心脏灌流模型上,黑木耳多糖预处理具有抗心脏I/R损伤的作用,这种保护作用可能与其增加心肌SOD活性,减少脂质过氧化损伤有关。     

A possible mechanism for the peroxidation of lipids due to chronic ethanol ingestion.

The effects of chronic ethanol ingestion on NADPH-oxidase and on the NADPH-catalyzed peroxidation of lipids in rat liver microsomes have been studied. It was demonstrated that the rates of NADPH oxidation, of oxygen consumption, and of malondialdehyde formation increased significantly above control values after one month of ethanol ingestion. Further, the fatty acid composition of these microsomes revealed a decrease in arachidonate and in the C22 polyenes. Also, the energies of activation for the formation of malondialdehyde increased in the microsomes from the ethanol-treated animals. These results were interpreted to mean that ethanol ingestion had induced changes in the microsomal membranes such that additional or alternate, possibly abnormal, pathways for lipid peroxidation were functional. Finally, these data suggest a mechanism whereby chronic ethanol ingestion inhances the production of lipid peroxides via the microsomal-catalyzed oxidation of NADPH.     

Cholesteryl Esters Accumulate in the Heart in a Porcine Model of Ischemia and Reperfusion

Myocardial ischemia is associated with intracellular accumulation of lipids and increased depots of myocardial lipids are linked to decreased heart function. Despite investigations in cell culture and animal models, there is little data available on where in the heart the lipids accumulate after myocardial ischemia and which lipid species that accumulate. The aim of this study was to investigate derangements of lipid metabolism that are associated with myocardial ischemia in a porcine model of ischemia and reperfusion. The large pig heart enables the separation of the infarct area with irreversible injury from the area at risk with reversible injury and the unaffected control area. The surviving myocardium bordering the infarct is exposed to mild ischemia and is stressed, but remains viable. We found that cholesteryl esters accumulated in the infarct area as well as in the bordering myocardium. In addition, we found that expression of the low density lipoprotein receptor (LDLr) and the low density lipoprotein receptor-related protein 1 (LRP1) was up-regulated, suggesting that choleteryl ester uptake is mediated via these receptors. Furthermore, we found increased ceramide accumulation, inflammation and endoplasmatic reticulum (ER) stress in the infarcted area of the pig heart. In addition, we found increased levels of inflammation and ER stress in the myocardium bordering the infarct area. Our results indicate that lipid accumulation in the heart is one of the metabolic derangements remaining after ischemia, even in the myocardium bordering the infarct area. Normalizing lipid levels in the myocardium after ischemia would likely improve myocardial function and should therefore be considered as a target for treatment.