Radioprotective effect of 2-mercaptopropionyl glycine on radiation-induced lipid peroxidation and enzyme release in erythrocytes

gamma-Irradiation of erythrocyte suspensions resulted in lipid peroxidation and enzyme (acetylcholinesterase, AChE) release. The presence of 2-mercaptopropionyl glycine (MPG) during irradiation decreased lipid peroxidation and enzyme (acetylcholinesterase, AChE) release. The presence of 2-mercaptopropionyl glycine (MPG) during irradiation decreased lipid peroxidation and from erythrocytes of high and low concentrations was observed at 480 and 320 Gy, respectively. This implied that the extent of enzyme release is likely to be masked when only a single dose of radiation is used, unless it happens to be an optimum dose. MPG mediated inhibition of lipid peroxidation and enzyme release indicated that lipid peroxidation may induce the breakdown of the phosphatidylinositol/enzyme interaction. Further, the enzyme damage was assigned to the direct and indirect effects of radiation on the enzyme in situ.     

Loss of latent activity of liver microsomal membrane enzymes evoked by lipid peroxidation. Studies of nucleoside diphosphatase, glucose-6-phosphatase, and UDP glucuronyltransferase

The effects of lipid peroxidation on latent microsomal enzyme activities were examined in NADPH-reduced microsomes from phenobarbital-pretreated male rats. Lipid peroxidation, stimulated by iron or carbon tetrachloride, was assayed as malondialdehyde formation. Independent of the stimulating agent of lipid peroxidation, latency of microsomal nucleoside diphosphatase activity remained unaffected up to microsomal peroxidation equivalent to the formation of about 12 nmol malondialdehyde/mg microsomal protein. However, above this threshold a close correlation was found between lipid peroxidation and loss of latent enzyme activity. The loss of latency evoked by lipid peroxidation was comparable to the loss of latency attainable by disrupting the microsomal membrane by detergent. Loss of latent enzyme activity produced by lipid peroxidation was also observed for microsomal glucose-6-phosphatase and UDPglucuronyltransferase. In contrast to nucleoside diphosphatase, however, both enzymes were inactivated by lipid peroxidation, as indicated by pronounced decreases of their activities in detergent-treated microsomes. According to the respective optimal oxygen partial pressure (po2) for lipid peroxidation, the iron-mediated effects on enzyme activities were maximal at a po2 of 80 mmHg and the one mediated by carbon tetrachloride at a po2 of 5 mmHg. Under anaerobic conditions no alterations of enzyme activities were detected. These results demonstrate that loss of microsomal latency only occurs when peroxidation of the microsomal membrane has reached a certain extent, and that beyond this threshold lipid peroxidation leads to severe disintegration of the microsomal membrane resulting in a loss of its selective permeability, a damage which should be of pathological consequences for the liver cell. Because of its resistance against lipid peroxidation nucleoside diphosphatase is a well-suited intrinsic microsomal parameter to estimate this effect of lipid peroxidation on the microsomal membrane.     

Sensitivity of ATPase-ADPase activities from synaptic plasma membranes of rat forebrain to lipid peroxidation in vitro and the protective effect of vitamin E

The in vitro effects of membrane lipid peroxidation on ATPase-ADPase activities in synaptic plasma membranes from rat forebrain were investigated. Treatment of synaptic plasma membranes with an oxidant generating system (H₂O₂/Fe²⁺/ascorbate) resulted in lipid peroxidation and inhibition of the enzyme activity. Besides, trolox as a water soluble vitamin E analogue totally prevented lipid peroxidation and the inhibition of enzyme activity. These results demonstrate the susceptibility of ATPase-ADPase activities of synaptic plasma membranes to free radicals and suggest that the protective effect against lipid peroxidation by trolox prevents the inhibition of enzyme activity. Thus, inhibition of ATPase-ADPase activities of synaptic plasma membranes in cerebral oxidative stress probably is related to lipid peroxidation in the brain.     

Glutathione transferases in the tapeworm Moniezia expansa.

Four forms of GSH transferase were resolved from Moniezia expansa cytosol by GSH-Sepharose affinity chromatography and chromatofocusing in the range pH 6-4, and the presence of isoenzymes was further suggested by analytical isoelectric focusing. The four GSH transferase forms in the cestode showed no clear biochemical relationship to any one mammalian GSH transferase family. The N-terminal of the major GSH transferase form showed sequence homology with the Mu and Alpha family GSH transferases. The major GSH transferase appeared to bind a number of commercially available anthelmintics but did not appear to conjugate the compounds with GSH. The major GSH transferase efficiently conjugated members of the trans-alk-2-enal and trans,trans-alka-2,4-dienal series, established secondary products of lipid peroxidation.     

Dehydrogenase conversion to oxidase and lipid peroxidation in brain after carbon monoxide poisoning.

The conversion of xanthine dehydrogenase to xanthine oxidase and lipid peroxidation were measured in brain from carbon monoxide- (CO) poisoned rats. Sulfhydryl-irreversible xanthine oxidase increased from a control level of 15% to a peak of 36% over the 90 min after CO poisoning, while the conjugated diene level doubled. Reversible xanthine oxidase was 3-6% of the total enzyme activity over this span of time but increased to 31% between 90 and 120 min after poisoning. Overall, reversible and irreversible xanthine oxidase represented 66% of total enzyme activity at 120 min after poisoning. Rats depleted of this enzyme by a tungsten diet and those treated with allopurinol before CO poisoning to inhibit enzyme activity exhibited no lipid peroxidation. Treatment immediately after poisoning with superoxide dismutase or deferoxamine inhibited lipid peroxidation but had no effect on irreversible oxidase formation. Biochemical changes only occurred after removal from CO, and changes could be delayed for hours by continuous exposure to 1,000 ppm CO. These results are consistent with the view that CO-mediated brain injury is a type of postischemic reperfusion phenomenon and indicate that xanthine oxidase-derived reactive oxygen species are responsible for lipid peroxidation.     

Effect of 3-trifluoromethyl-alpha-ethylbenzhydrol (flumecinol) on lipid peroxidation in early human placenta

A study was made of the lipid peroxidation in the microsomal fraction of human placenta tissue in the presence of flumecinol (Zixoryn, Gedeon Richter Pharmaceutical Works, Budapest, Hungary). The tissue samples were obtained from placenta in early pregnancy and also from placenta at term. Up to a concentration of 1 mumolar, flumecinol (3-trifluoromethyl-alpha-ethyl-benzhydrol), as an enzyme inductor, significantly enhanced the rate of lipid peroxidation but above 1 molar it scarcely influenced it. The in vitro study proved the enzyme-inducing effect of flumecinol on lipid peroxidation in early human placenta.     

Subcellular localization of adrenal cortical glutathione peroxidase and protective role of the mitochondrial enzyme against lipid peroxidative damage

In view of the physiological importance of adrenocortical lipid peroxidation, we have carried out subcellular fractionation to determine the location of glutathione peroxidase, an enzyme which protects against lipid peroxidation. Glutathione peroxidase is present in both cytosolic (92%) and mitochondrial (8%) fractions. The small activity in mitochondria is not due to contamination by the cytosolic activity as evidenced by several rigorous approaches. The mitochondrial enzyme is located in the matrix and appears to be effective in protection from NADPH-dependent lipid peroxidative damage of cytochrome P-450 and succinic dehydrogenase, which are located exclusively in the inner membrane.     

Inhibition of phospholipid hydrolysis by phospholipase A2 in microsomal and mitochondrial membranes subjected to lipid peroxidation

The rate of phospholipid hydrolysis in rat liver microsomal and mitochondrial membranes catalyzed by phospholipase A2 was shown to decrease after ascorbate + Fe2+-induced lipid peroxidation. The degree of inhibition was linearly dependent on the amount of lipid peroxidation products (malonyl dialdehyde) accumulated in the membrane. The decreased phospholipid hydrolysis rate in membranes after lipid peroxidation was registered using phospholipases A2 from two sources: porcine pancreas and bee venom. It was established that the inhibitory action of phospholipid peroxidation products was not linked with a direct effect on the enzyme and was not caused by depletion of phospholipase reaction substrates (as a result of lipid peroxidation). A possible role of lateral separation of oxidized and non-oxidized lipid phases in the mechanisms of inhibition of phospholipid hydrolysis by phospholipase A2 is discussed.     

Antioxidant enzyme activities and lipid peroxidation in the freshwater cladoceran Daphnia magna exposed to redox cycling compounds

Contaminant-related changes in antioxidative processes in the freshwater crustacea Daphnia magna exposed to model redox cycling contaminant were assessed. Activities of key antioxidant enzymes including catalase, superoxide dismutase, glutathione peroxidase and glutathione S-transferases and levels of lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS) and lipofucsin pigment content were determined in D. magna juveniles after being exposed to sublethal levels of menadione, paraquat, endosulfan, cadmium and copper for 48 h. Results denoted different patterns of antioxidant enzyme responses, suggesting that different toxicants may induce different antioxidant/prooxidant responses depending on their ability to produce reactive oxygen species and antioxidant enzymes to detoxify them. Low responses of antioxidant enzyme activities for menadione and endosulfan, associated with increasing levels of lipid peroxidation and enhanced levels of antioxidant enzyme activities for paraquat, seemed to prevent lipid peroxidation, whereas high levels of both antioxidant enzyme activities and lipid peroxidation were found for copper. For cadmium, low antioxidant enzyme responses coupled with negligible increases in lipid peroxidation indicated low potential for cadmium to alter the antioxidant/prooxidant status in Daphnia. Among the studied enzymes, total glutathione peroxidase, catalase and glutathione S-transferase appeared to be the most responsive biomarkers of oxidative stress.     

Purification and characterisation of a novel cysteine conjugate beta-lyase from the tapeworm Moniezia expansa

The paper presents the first report of the purification of an invertebrate cysteine conjugate beta-lyase (CCBL). CCBL activity was shown to predominate within the cytosolic fraction of tissue from the tapeworm Moniezia expansa. The monomeric cytosolic enzyme was isolated with a M(r) of 72 kDa and co-purified with transaminase activity towards L-aspartate. The substrate profile for M. expansa CCBL is different from that of mammalian CCBLs. Exploiting the differences in mammalian and parasite substrate profiles will facilitate the development of helminth targeted conjugates which will not be activated by host (mammalian) CCBLs.     

Effect of cigarette smoke on lipid peroxidation,antioxidant enzymes and NMDA receptor subunits 2A and 2B concentration in rat hippocampus

The effect of cigarette smoke on lipid peroxidation and antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and on the concentration of N-methyl-d-aspartate receptor (NMDAR) subunits 2A and 2B in the hippocampus of Sprague-Dawley rats exposed to cigarette smoke for 2h/day for a period of 4 weeks was determined. It was observed that NMDAR 2A and 2B concentrations in the hippocampus were enhanced in the case of animals exposed to cigarette smoke, whereas lipid peroxidation and antioxidant enzyme activities did not show any change as compared to control animals. The results of our study suggest that cigarette smoke induces NMDAR 2A and 2B expression in the hippocampus, and that this is not due to an increased lipid peroxidation, because cigarette smoke has no effect on lipid peroxidation and antioxidant enzyme activities in the hippocampus.     

Cytochrome P450 2E1 dependent catalytic activity and lipid peroxidation in rat blood lymphocytes

To investigate the similarities in the catalytic activity of blood lymphocyte P450 2E1 in blood lymphocyte with the liver isoenzyme, NADPH dependent lipid peroxidation and activity of N-nitrosodimethyamine demethylase (NDMA-d) was studied in rat blood lymphocytes. Blood lymphocytes were found to catalyse NADPH dependent (basal) lipid peroxidation and demethylation of N-nitrosodimethylamine (NDMA). Pretreatment with ethanol or pyrazole or acetone resulted in significant increase in the NADPH dependent lipid peroxidation and the activity of NDMA-d in blood lymphocytes and liver microsomes. In vitro addition of CCl(4) to the blood lymphocytes isolated from control or ethanol pretreated rats resulted in an increase in the NADPH dependent lipid peroxidation. Significant inhibition of the basal and CCl(4) supported NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocytes isolated from control or ethanol pretreated rats by dimethyl formamide or dimethyl sulfoxide or hexane, solvents known to inhibit P450 2E1 catalysed reactions in liver and anti- P450 2E1, have indicated the role of P450 2E1 in the NADPH dependent lipid peroxidation in rat blood lymphocytes. The data indicating similarities in the NADPH dependent lipid peroxidation and NDMA-d activity in blood lymphocyte with the liver microsome have provided evidence that blood lymphocyte P450 2E1 could be used as a surrogate to monitor and predict hepatic levels of the enzyme.     

Regional effects of ageing on Na+,K(+)-ATPase activity in rat brain and correlation with multiple unit action potentials and lipid peroxidation.

Effects of ageing on Na+,K(+)-ATPase activity in crude synaptosomal fractions from the rat brain parietal cortex, hippocampus, striatum and thalamus has been studied. From 12 months to 24 months, a progressive decline in enzyme activity in the parietal cortex, hippocampus and striatum was found which correlated with increase in lipid peroxidation in the three brain regions. In the thalamus, ageing did not affect the enzyme activity and lipid peroxidation. Age-related decline in multiple unit action potentials was also observed in two brain regions, viz. hippocampus and parietal cortex. Statistical correlations calculated by Pearson's correlation coefficient showed that decline in Na+,K(+)-ATPase activity correlated to decline in multiple unit action potentials. There was rise in lipid peroxidation also and the data indicate that age-related changes in lipid peroxidation and Na+,K(+)-ATPase activity contribute to the deterioration of electrophysiological activity.     

Lipid peroxidation inactivates rat liver microsomal glycerol-3-phosphate acyl transferase. Effect of iron and copper salts and carbon tetrachloride

Lipid peroxidation is known to affect the activity of several enzymes including microsomal enzymes such as glucose-6-phosphatase; but its effect on the enzymes of lipid biosynthesis has not been investigated. Glycerol-3-phosphate acyltransferase (GPAT) represents the first committed step and probably the rate limiting step in glycerolipid synthesis and thus may be a good candidate for study. Rat liver microsomal GPAT was assayed after preincubating the microsomes under conditions known to induce peroxidation. In 30 min, 10 microM Fe2+ can diminish the activity by as much as 80%. The inactivating effect can be blocked to different extents by several antioxidants, while ascorbic acid enhances it. These effects, along with the concomitant measurement of lipid peroxidation, indicate that microsomal GPAT activity is inactivated by lipid peroxidation in a sensitive and rapid fashion. This is further confirmed by the inactivating effect of carbon tetrachloride, which is known to induce lipid peroxidation in microsomes. Fe3+ also inactivates the enzyme, but at a higher concentration. Copper salts inactivate GPAT by a mechanism apparently different from that of iron. The mechanism might involve a direct sulfhydryl modification by copper and lipid peroxidation apparently different from that induced by iron. It is suggested that the inactivation of GPAT by lipid peroxidation could accelerate the process of membrane disintegration caused by lipid peroxidation in pathological conditions involving free radical-mediated tissue injury.     

The effect of paraquat on the peroxide metabolism enzymes in erythrocytes of freshwater fish species

The toxic effects of 10 ppm paraquat in vivo on the enzymes superoxide dismutase (SOD), catalase (C), peroxidase (P), glutathione peroxidase (GSH-Px) and on lipid peroxidation (LP) were estimated in erythrocytes of the carp, the tench and the crucian carp. Paraquat caused activity enhancement of the peroxide metabolism enzymes and increase of the lipid peroxidation in the carp and the crucian carp. The enzyme activities and lipid peroxidation were dependent on the species and on the length of the exposure to paraquat.     

Changes of CYP1A1, GST, and ALDH3 enzymes in hepatoma cell lines undergoing enhanced lipid peroxidation

Hepatoma cells show alterations in the response to oxidative stress (decreased lipid peroxidation) and in xenobiotic metabolism enzymes (decreased P450, increased GST and ALDH3). This study examined the effect of lipid peroxidation on the expression of the above enzymes in two rat hepatoma cell lines (MH(1)C(1) and 7777). To induce oxidative stress, cells were exposed to arachidonic acid (to increase lipid peroxidation substrate) and/or to beta-naphthoflavone (to increase CYP450), and treated with one dose of iron/histidine. The cells, that were still viable after the challenge, were refed with the culture medium and CYP1A1, GST, and ALDH3 enzymes monitored for 1, 6, 12, and 24 h. Treatments that increased markers indicative of lipid peroxidation are associated with a decrease in enzyme activities, which was permanent for CYP1A1 and transient for the other enzymes. We speculate from these data that aldehydic byproducts of lipid peroxidation may be responsible for these effects. Thus, restoration of lipid peroxidation in hepatoma cells seems to induce a rapid adaptation to oxidative stress, which is achieved by a simultaneous decrease of reactive oxygen species production and an increase in the two main enzymes involved in the removal of the aldehydic products of lipid peroxidation.     

Amyloid β-Peptides Increase Annular and Bulk Fluidity and Induce Lipid Peroxidation in Brain Synaptic Plasma Membranes

Abstract: Amyloid β-peptides (Aβ) may alter the neuronal membrane lipid environment by changing fluidity and inducing free radical lipid peroxidation. The effects of Aβ_1–40 and Aβ_25–35 on the fluidity of lipids adjacent to proteins (annular fluidity), bulk lipid fluidity, and lipid peroxidation were determined in rat synaptic plasma membranes (SPM). A fluorescent method based on radiationless energy transfer from tryptophan of SPM proteins to pyrene and pyrene monomer-eximer formation was used to determine SPM annular fluidity and bulk fluidity, respectively. Lipid peroxidation was determined by the thiobarbituric acid assay. Annular fluidity and bulk fluidity of SPM were increased significantly ( p ≤ 0.02) by Aβ_1–40. Similar effects on fluidity were observed for Aβ_25–35 ( p ≤ 0.002). Increased fluidity was associated with lipid peroxidation. Both Aβ peptides significantly increased ( p ≤ 0.006) the amount of malondialdehyde in SPM. The addition of a water-soluble analogue of vitamin E (Trolox) inhibited effects of Aβ on lipid peroxidation and fluidity in SPM. The fluidizing action of Aβ peptides on SPM may be due to the induction of lipid peroxidation by those peptides. Aβ-induced changes in neuronal function, such as ion flux and enzyme activity, that have been reported previously may result from the combined effects of lipid peroxidation and increased membrane fluidity.     

Cu-Zn-superoxide dismutase activity in Moniezia expansa: inhibition by pyrimidine derivatives

Copper-zinc, cyanide-sensitive superoxide dismutase (Cu-Zn-SOD) was detected in homogenates of Moniezia expansa. The enzyme was purified by a sequence of multiple differential centrifugations, ammonium sulphate precipitation, ion-exchange and G-75 Sephadex column chromatography. The final enzyme preparation had a specific activity of 623.00 +/- 9.97U per mg protein and, after isolation, a single-staining band on acrylamide-SDS gels was detected which coincided with enzyme activity. The inhibitory activities of several benzimidazoles and several novel pyrimidine derivatives were determined on purified extracts of the M. expansa Cu-Zn-SOD. The results indicated that the percentage inhibition of Cu-Zn-SOD by some pyrimidine derivatives (6-amino-1, 3-dimethyl-5-nitroso-uracil, 6-amino-5-methyl-5-nitroso-uracil and 5-amino-uracil) was markedly higher than inhibition with the benzimidazoles.     

Triacontanol inhibits both enzymatic and nonenzymatic lipid peroxidation

The effect of the plant growth regulator, triacontanol (TRIA) on lipid peroxidation was studied in three different systems: (i) isolated chloroplasts of spinach (Spinacea oleracea L.) leaves; (ii) egg lecithin liposomes; and (iii) soybean lipoxygenase (LOX) system. The nonenzymatic lipid peroxidation in isolated chloroplasts and egg lecithin liposomes was measured as the amount of thiobarbituric acid reactive substances (TBARS) formed. Inhibition of Fe2+ and/or light-induced lipid peroxidation by TRIA was observed in both isolated chloroplasts and egg lecithin liposomes. The kinetics of soybean lipoxygenase-1 (LOX-1) was studied using linoleic acid as the substrate. The enzyme was competitively inhibited by TRIA. The Ki for TRIA inhibition of the enzyme was estimated to be 3.2-5.0 microM according to different methods of estimation. TRIA has been known to exhibit anti-inflammatory action in animals and this anti-inflammatory effect of TRIA might be mediated through inhibition of lipid peroxidation. Since LOX inhibitors have been extensively used as therapeutic agents, TRIA, being a natural compound has been suggested to be an effective anti-inflammatory drug.     

Effect of succinate on mitochondrial lipid peroxidation. 2. The protective effect of succinate against functional and structural changes induced by lipid peroxidation

The damaging effects of ADP/Fe/NADPH-induced lipid peroxidation were studied on the enzymes and membranes of rat liver mitochondria. Succinate, an inhibitor of mitochondrial lipid peroxidation, prevented or delayed most of the damage caused by the peroxidation on different mitochondrial structures and functions. There were marked abnormalities on the electrophoretic pattern of mitochondrial proteins during the course of lipid peroxidation. The disappearance of particular polypeptide bands and the accumulation of high-molecular-weight aggregates could be observed. Succinate was found to delay these effects. As a consequence of lipid peroxidation the succinate oxidase activity of mitochondria was decreased. The succinate dehydrogenase enzyme and the component(s) of the respiratory chain were inactivated. Succinate prevented the inactivation of succinate dehydrogenase but did not protect the other components of terminal oxidation chain. From the matrix enzymes the glutamate dehydrogenase retained its full activity but the NADP-linked isocitrate dehydrogenase was inactivated. The mitochondrial membranes became permeable to large protein molecules. Succinate prevented the inactivation of isocitrate dehydrogenase and delayed the release of protein molecules from mitochondria.