Factors in rat liver cytosol, inhibiting chemiluminescence during nonenzymatic lipid peroxidation]

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 the chemiluminescence technique and by malonic dialdehyde (MDA) production. Dialyzed cytosol significantly decreased the level of chemiluminescence and, to a much lesser extent, the rate of MDA production. Gel filtration on a Sephadex G-200 column led to the appearance of at least three cytosolic fractions which suppressed the low-level chemiluminescence. These fractions differed from one another by their molecular masses, kinetics of chemiluminescence inhibition and effects on the intensity of MDA production. The putative functional role of antioxidative defence factors from rat liver cytosol is discussed.     

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 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 dibenzo[a,c]cyclooctene lignans isolated from Fructus schizandrae on lipid peroxidation and anti-oxidative enzyme activity.

The effect of nine dibenzo[a,c]cyclooctene lignans isolated from Fructus schizandrae on in vitro and in vivo lipid peroxidation of liver microsomes as well as on anti-oxidative enzyme activities were studied. Seven of the nine lignans (1 mM) were shown to inhibit Vit C/NADPH induced lipid peroxidation (malondialdehyde (MDA) formation) of rat liver microsomes. Of these compounds, schisanhenol (Sal), S(-)schizandrin C (S(-)sin C) and S(-)schizandrin B (S(-)sin B) were shown to be more potent than Vit E at the same concentration. Sal and Sin B were able to inhibit gossypol-induced superoxide anion generation in rat liver microsomes. In addition, oral administration of Sal and Sin B markedly reduced liver MDA formation induced by ethanol, 15 ml/kg in mice, and increased superoxide dismutase and catalase activities in rat liver cytosol. The data of this paper are in favor of the conclusion that some lignans, like Sal, have strong anti-oxidant activity. The mechanisms of anti-oxidant activity of the lignans were discussed.     

Protective effects of lazaroids against oxygen-free radicals induced lysosomal damage

Lipid peroxidation of membranes by oxygen free radicals has been implicated in various disease states. Different antioxidants and iron chelators have been used to reduce lipid peroxidation. Lazaroids have been used for the acute treatment of central nervous system disorders such as trauma and ischemia wherein lipid peroxidative processes take place.In this study we evaluated the effect of lazaroids (U-785 18F and U-74389F) on the release of acid phosphatase activity and formation of malondialdehyde (MDA) in rat liver lyosomes subjected to exogenously generated oxygen free radicals. There was a significant increase in the acid phosphatase release and MDA formation in the presence of oxygen free radicals. This was prevented by both the lazaroids. In a separate study the effect of lazaroid U-74389F was seen on the zymosan-stimulated polymorphonuclear (PMN) leukocyte-derived chemiluminescence. The PMN leukocyte chemiluminescent activity was attenuated by the lazaroid in a dose-dependent manner. These studies suggest that lazaroids may inhibit lipid peroxidation and stabilize the membrane.     

Distinct temporal relation among oxygen uptake, malondialdehyde formation, and low-level chemiluminescence during microsomal lipid peroxidation

An oxystat system was employed in conjunction with a single-photon counting apparatus for simultaneous monitoring of oxygen uptake, oxidative decomposition of membrane lipids, and occurrence of electronically excited species during microsomal lipid peroxidation. During NADPH/ADP-iron-promoted lipid peroxidation at a steady state oxygen partial pressure (pO2) of 30 mm Hg, complex time relationships among oxygen uptake, malondialdehyde (MDA) formation, and low-level chemiluminescence were observed. While the first two parameters occurred nearly simultaneously, low-level chemiluminescence occurred with a significant delay. A decrease of the steady state pO2 to 3 mm Hg led to significant increases of the lag phases of all three parameters and a further enhancement of the time displacement of low-level chemiluminescence in relation to oxygen uptake and MDA formation. At a pO2 of 0.5 mm Hg, the lowest pO2 maintained during this study, no low-level chemiluminescence was observed while oxygen uptake and MDA formation were still detected. In contrast, during NADPH/CCl4-promoted lipid peroxidation at a pO2 of 0.5 mm Hg a sudden drastic rise of low-level chemiluminescence accompanying oxygen uptake and MDA formation was observed. At pO2 between 0.5 and 3 mm Hg all three parameters occurred nearly concomitantly during the entire incubation. At pO2 levels above 3 mm Hg all three parameters showed principally the same behavior. However, the respective maxima of low-level chemiluminescence were reached with some delay. The present observations support the assumption that the decomposition of membrane lipid peroxyl radicals to MDA and the formation of electronically excited species proceed via different pathways. The time displacement between oxygen uptake and MDA formation, on the one hand, and low-level chemiluminescence, on the other hand, depends on the type of initiating radical system and on the steady state pO2 level. It is suggested that the differences are due to distinct subsets (chemical or spatial) of secondary peroxyl radicals in the membrane.     

A high involvement of O2- possibly generated in inner membranes for iron-induced microsomal lipid peroxidation.

The lipid peroxidation of and the O2- generation by rat liver microsomes in the presence of NADPH or both NADPH and Fe3+ were determined by thiobarbituric acid-reacting substance formation and by chemiluminescence intensities with a cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]pyrazin-3-one(MCLA), as a chemiluminescence probe. Judging from the experiments with various inhibitors on the O2- generation and the lipid peroxidation, O2- generated, at intramembranous site, by cytochrome P-450 system is considered to be highly involved in the iron-induced lipid peroxidation.     

Effect of chronic ethanol, catalase inhibitor 3-amino-1,2,4-triazole and clofibrate treatment on lipid peroxidation in rat myocardium

1. The effect of chronic alcohol consumption, catalase inhibitor 3-amino-1,2,4-triazole (amino-triazole) and peroxisome proliferator clofibrate on the level of Fe/ADP-ascorbate-induced lipid peroxidation has been studied in the rat myocardium. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. Combined us well as separate treatment with ethanol (36% of dietary calories) and aminotriazole caused elevation of the rate of lipid peroxidation in the nuclear-free homogenate or total particulate fraction of the rat heart. The most pronounced effect was noted during combined application of ethanol and aminotriazole. 3. Prolonged clofibrate treatment significantly increased the level of nonenzymatic lipid peroxidation in the rat myocardium. 4. Peroxidative alteration of the myocardial lipids in vivo was evaluated by measurement of conjugated dienes (absorbance at 233 nm). Separate ethanol, aminotriazole or clofibrate treatment did not affect the level of u.v. absorption of lipids from the total particulate fraction. However, when ethanol and aminotriazole were administered simultaneously an increase of conjugated diene formation was observed. 5. The data obtained confirm the hypothesis that ethanol or clofibrate-induced activation of the myocardial lipid peroxidation may be due to the increase of hydrogen peroxide-generating capacity of the heart microperoxisomes.     

Sex-related differences in cadmium-induced lipid peroxidation in the rat

Male and female rats were dosed once a day for 2 days injection with 1.5 mg of Cd/kg as CdCl₂. 24 hr after administration of cadmium, lipid peroxidation determined by estimation of malondialdehyde (MDA) was greatly increased in male rat liver, but was not in female rats. Cadmium in a larger dose of 4.5 mg/kg, subcutaneous single injection, significantly increased content of MDA in female rat liver. These results suggest that sex-related differences exist in the ability of cadmium to induce MDA formation in rat liver, although administration of cadmium causes the enhancement of MDA formation in both male and female rats. The reason why sex-related differences exist in lipid peroxidation of rat liver is discussed.     

The role of selenium in the regulation of lipid peroxidation in biological membranes and glutathione peroxidase activity

The antioxidative effect of selenium cannot be exclusively due to the functioning of the selenium-dependent glutathione peroxidase mechanism of utilization of various hydroperoxides. This hypothesis is based on the following experimental evidence. Selenium ions are readily incorporated into animal organs and tissues immediately after injection (2 hours) as well as into cell organelles and cytosol where they inhibit lipid peroxidation. The activity of glutathione peroxidase (EC 1.1.1.19) in rat liver and guinea pig cytosol is thereby unchanged but increases drastically after 12 hours reaching a maximum an the 3rd-4th day. The effectiveness of lipid peroxidation inhibition does not increase under these conditions. Although the glutathione peroxidase activity is absent in the nuclei and microsomes, exogenous selenium inhibits lipid peroxidation in these organelles. The activity of the rat liver cytosolic enzyme markedly exceeds that of its guinea pig counterpart. However, lipid peroxidation in guinea pig liver occurs less intensively than that in rat liver cytosol.     

Assay of liver cytosol lipoxygenase by differential pulse polarography

Lipoxygenase activity assayed by differential pulse polarography was found to be more sensitive than that determined by the increase in absorption at 234 nm. The lipoxygenase activity level in the liver cytosol of rats fed oxidized palm oil was significantly higher than that of the control animals fed either saline or fresh palm oil. The effects of flavonoids on the inhibition of lipoxygenase activity level in liver cytosol of rats were in the decreasing order quercetin greater than myricetin greater than morin greater than phloretin. The observed free malonaldehyde (MDA) in liver cytosol of rats determined by high-performance liquid chromatography using malonaldehyde-dinitro-phenylhydrazine complex was 23, 25, and 51.2% for rats fed saline, fresh palm oil, and oxidized palm oil, respectively. A linear relationship between the lipoxygenase activity and the free liver cytosol MDA was shown. The assay of lipoxygenase by differential pulse polarography provides a simple, sensitive, and quantitative method for the study of liver lipid peroxidation.     

Glutathione and lipid peroxide levels in rat liver following administration of methapyrilene and analogs

The possibility was examined that the induction of tumors in rat liver by feeding methapyrilene, which is not mutagenic, is related to effects on glutathione levels and lipid peroxidation. Fischer 344 rats were given single-dose and multiple-dose treatments with the anti-histamine methapyrilene (MP), which is carcinogenic in rats, and with two non-carcinogenic analogs, methafurylene (MF) and thenyldiamine (TD) and the effects on malonaldehyde (MDA) formation and glutathione (GSH) levels in the liver were investigated. After a single dose, MDA levels were increased at 6 h by MF and TD and at 24 h by MP. MDA levels returned to normal after 30 h with MP and MF, but not with TD. Levels of MDA (and other TBA-reactive products) after four daily treatments were most elevated by TD, less elevated by MP, and were lowered by MF. Forty-two hours following treatment with both MP and MF, MDA levels had returned to normal, but in TD-treated animals MDA remained high. GSH levels were highest after MF and MP, and remained high at 42 h, but TD induced only a small increase. There appears to be increased lipid peroxidation in the liver as a result of treatment of rats with MP, MF and TD. The greater response induced by TD, as well as the increased liver GSH levels after repeated administration of all three drugs indicate that lipid peroxidation in rat liver is not a particular effect related to the liver carcinogen methapyrilene.     

运动性内源自由基对大鼠肝线粒体的影响

采用大鼠耗竭游泳作为动物运动模型,用戊巴比妥酸(TBA)法测定脂质过氧化水平,薄层色谱—定磷法测定心磷脂含量,细胞色素C还原法测定细胞色素C氧化酶活性。结果如下:耗竭运动时,肝线粒体脂质过氧化水平升高24％;心磷脂含量下降21％;细胞色素C氧化酶活性下降25％。上述结果表明:耗竭运动时,机体内源自由基的产生是运动损伤和整体疲劳的原因之一。     

Paracetamol-stimulated lipid peroxidation in isolated rat and mouse hepatocytes

Treatment of isolated hepatocytes from 3-methylcholanthrene induced rats with 1 mM paracetamol has been found to greatly decrease cellular reduced glutathione (GSH) content and to promote lipid peroxidation, evaluated as malonaldehyde (MDA) production and conjugated diene absorbance. A similar dosing of hepatocytes from phenobarbital-induced or normal rats is ineffective in that respect. On the other hand, the aspecific stimulation of the cytochrome P-450-mediated paracetamol activation due to acetone addition further increases GSH depletion as well as MDA production.Isolated hepatocytes with basal low GSH content are also more susceptible to paracetamol-induced lipid peroxidation, indicating that the rate of the drug metabolism and the cellular GSH content are critical factors in the determination of such peroxidative attack.In isolated mouse liver cells paracetamol does not require preliminary cytochrome P-450 induction to stimulate MDA formation, even at concentrations ineffective in rat cells.However, 5 mM paracetamol, despite a great depletion of cellular GSH content, does not promote MDA formation either in the rat or in the mouse hepatocytes. This effect may be due to the ability of paracetamol to scavenge lipid peroxides under defined conditions, as tested in various lipid peroxidizing systems.Membrane leakage of lactate dehydrogenase (LDH) is evident in paracetamol treated cells undergoing lipid peroxidation, but not when MDA formation is inhibited by high doses of the drug or by addition of antioxidants such as α-tocopherol and diphenylphenylenediamine (DPPD).Nevertheless in these conditions the covalent binding of activated paracetamol metabolites is not affected, suggesting that lipid peroxidation might play a role in the pathogenesis of liver damage following paracetamol overdose.     

Genetic polymorphism of the CYP1A1, CYP2E1, GSTM1 and GSTT1 genes and lung cancer susceptibility in a north Indian population

The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH.     

Cytosolic factors which affect microsomal lipid peroxidation in lung and liver

Studies were carried out to determine the effects of lung and liver cytosol on pulmonary and hepatic mierosomal lipid peroxidation, to determine the cytosolic concentrations of various substances which affect lipid peroxidation, and to determine which of these substances is responsible for the effects of the cytosol on lipid peroxidation. Lung cytosol inhibits both enzymatic (NADPH-induced) and nonenzymatic (Fe²⁺-induced) lung microsomal lipid peroxidation. In contrast, liver cytosol stimulates lipid peroxidation in hepatic microsomes during incubation alone, enhances Fe²⁺-stimulated lipid peroxidation, and has no effect on the NADPH-induced response. Substances which are known to be involved in inhibition of lipid peroxidation, including glutathione, glutathione reductase, glutathione peroxidase, and superoxide dismutase, are found in greater concentrations in liver cytosol than in lung cytosol. However, ascorbate is found in approximately equal concentrations in pulmonary and hepatic cytosol. Most of the effects of the cytosol on lipid peroxidation seem to be due to ascorbate and glutathione. For example, ascorbate, in concentrations found in lung cytosol, inhibits lung microsomal lipid peroxidation to about the same extent as the cytosol. The effects of liver cytosol on hepatic microsomal lipid peroxidation can be duplicated by concentrations of ascorbate and glutathione normally found in the cytosol; i.e., ascorbate stimulates and glutathione inhibits lipid peroxidation with the net effect being similar to that of liver cytosol. The results indicate that ascorbate has opposite effects on pulmonary and hepatic microsomal lipid peroxidation and suggest that ascorbate plays a major role in protecting pulmonary tissue against the harmful effects of lipid peroxidation.     

Sensitivity of mitochondria isolated from liver and kidney of rat and bovine to lipid peroxidation: A comparative study of light emission and fatty acid profiles

Much work has been carried out on non-enzymatic–induced lipid peroxidation of mitochondria obtained from different tissues of monogastric animals, but little information is available about this process in poligastric animals. Studies were carried out to determine the sensitivity of mitochondria isolated from liver and kidney of rat and bovine to lipid peroxidation (ascorbate-Fe²⁺ dependent) by comparison of light emission and fatty acid profiles. Mitochondria from both species were susceptible to lipid peroxidation. Measurements of chemiluminescence indicate that the lipid peroxidation process was more effective in mitochondria from rat liver than in the organelle obtained from bovine, whereas changes were not observed in mitochondria from rat and bovine kidney. The fatty acid composition of total lipids isolated from liver and kidney mitochondria of both species was substantially modified when subjected to non-enzymatic lipid peroxidation with a decrease of arachidonic and docosahexaenoic acids. The polyunsaturated fatty acid (PUFA) composition was higher in mitochondria obtained from rat liver (43.11± 4.16) than in bovine (15.78 ± 0.76). As a consequence, the unsaturation index (UI), was higher in mitochondria of rat liver than in bovine. Nevertheless, the PUFA composition of kidney mitochondria from both species was similar; therefore, statistically significant differences in the UI were not observed. The results suggest that mainly the PUFAs present in hepatic and kidney mitochondria were sensitive to oxidative damage. The lipid peroxidation process was more effective in rat liver mitochondria than in bovine. (Mol Cell Biochem xxx: 77–82, 2005) Member of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)     

Non-enzymatic lipid peroxidation of microsomes and mitochondria isolated from liver and heart of pigeon and rat

Studies were carried out to determine the level of ascorbate-Fe2+ dependent lipid peroxidation of mitochondria and microsomes isolated from liver and heart of rat and pigeon. Measurements of chemiluminescence indicate that the lipid peroxidation process was more effective in mitochondria and microsomes from rat liver than in the same organelles obtained from pigeon. In both mitochondria and microsomes from liver of both species a significant decrease of arachidonic acid was observed during peroxidation. The rate C18:2 n6/C20:4 n6 was 4.5 times higher in pigeon than in rat liver. This observation can explain the differences noted when light emission and unsaturation index of both species were analysed. A significant decrease of C18:2 n6 and C20:4 n6 in pigeon liver mitochondria was observed when compared with native organelles whereas in pigeon liver microsomes only C20:4 n6 diminished. In rat liver mitochondria only arachidonic acid C20:4 n6 showed a significant decrease whereas in rat liver microsomes C20:4 n6 and C22:6 n3 decreased significantly. However changes were not observed in the fatty acid profile of mitochondria and microsomes isolated from pigeon heart. In the heart under our peroxidation conditions the fatty acid profile does not appear to be responsible for the different susceptibility to the lipid peroxidation process. The lack of a relationship between fatty acid unsaturation and sensitivity to peroxidation observed in heart suggest that other factor/s may be involved in the protection to lipid peroxidation in microsomes and mitochondria isolated from heart.     

Arachidonic acid hydroperoxide stimulates lipid peroxidation in rat liver nuclei and chromatin fractions

Arachidonic acid, the most abundant polyunsaturated fatty acid in rat liver nuclei phospholipids is a major target of free radical attack, which induces lipid peroxidation. The non-enzymatic lipid peroxidation process in intact rat liver nuclei and in several chromatin fractions indicated that the most sensitive fatty acid for peroxidation is arachidonic acid C20:4 n-6. In this study, the effect of different amounts of arachidonic acid hydroperoxide on the lipid peroxidation of rat liver nuclei and chromatin fractions was studied; rat liver nuclei and chromatin fractions deprived of exogenous added hydroperoxide were utilized as control. The addition of arachidonic acid hydroperoxide to liver nuclei produces a marked increase in light emission that was hydroperoxide concentration dependent. The maximal peak of chemiluminescence displayed by the different chromatin fractions analyzed was observed between 20 and 80 min of incubation. The highest value of light emission was displayed by the high-density chromatin fractions, the 27.5 K fraction showed intermediate values of light emission, whereas the lowest density fraction produced very low chemiluminescence. A high correlation between arachidonic acid hydroperoxide concentration and chemiluminescence in the different chromatin fractions was observed. AC is Members of Carrera del Investigador Científico, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.