Microsomal glutathione-dependent protection against lipid peroxidation acts through a factor other than glutathione peroxidase and glutathione S-transferase in rat liver

Ascorbate-Fe3+-induced and NADPH-induced lipid peroxidation of rat liver microsomes were inhibited by glutathione (GSH). This inhibition was due to microsomal GSH-dependent factor. This factor was heat labile, and storage of microsomes at 4 degrees C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 mM) and bromosulfophthalein (0.1 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 mM) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 mM) and oxidized glutathione (10 mM) inhibited GSH-dependent protection but activated microsomal GSH S-transferase activity. These results indicate the existence of a heat-labile, microsomal GSH-dependent protective factor against lipid peroxidation that acts through a factor other than GSH-peroxidase and GSH S-transferase.     

Interplay between lipoic acid and glutathione in the protection against microsomal lipid peroxidation

Reduced glutathione (GSH) delays microsomal lipid peroxidation via the reduction of vitamin E radicals, which is catalyzed by a free radical reductase (Haenen, G.R.M.M. et al. (1987) Arch. Biochem. Biophys. 259, 449-456). Lipoic acid exerts its therapeutic effect in pathologies in which free radicals are involved. We investigated the interplay between lipoic acid and glutathione in microsomal Fe2+ (10 microM)/ascorbate (0.2 mM)-induced lipid peroxidation. Neither reduced nor oxidized lipoic acid (0.5 mM) displayed protection against microsomal lipid peroxidation, measured as thiobarbituric acid-reactive material. Reduced lipoic acid even had a pro-oxidant activity, which is probably due to reduction of Fe3+. Notably, protection against lipid peroxidation was afforded by the combination of oxidized glutathione (GSSG) and reduced lipoic acid. It is shown that this effect can be ascribed completely to reduction of GSSG to GSH by reduced lipoic acid. This may provide a rationale for the therapeutic effectiveness of lipoic acid.     

Protective effect of green tea against lipid peroxidation in the rat liver, blood serum and the brain

This paper reports data on the effect of green tea on the lipid peroxidation products formation and parameters of antioxidative system of the liver, blood serum and central nervous tissue of healthy young rats drinking green tea for five weeks. The rats were permitted free access to solubilized extract of green tea. Bioactive ingredients of green tea extract caused in the liver an increase in the activity of glutathione peroxidase and glutathione reductase and in the content of reduced glutathione as well as marked decrease in lipid hydroperoxides (LOOH), 4-hydroksynonenal (4-HNE) and malondialdehyde (MDA). The concentration of vitamin A increased by about 40%. Minor changes in the measured parameters were observed in the blood serum. GSH content increased slightly, whereas the index of the total antioxidant status increased significantly. In contrast, the lipid peroxidation products, particularly MDA was significantly diminished. In the central nervous tissue the activity of superoxide dismutase and glutathione peroxidase decreased while the activity od glutathione reductase and catalase increased after drinking green tea. Moreover the level of LOOH, 4-HNE and MDA significantly decreased. The use of green tea extract appeared to be beneficial to rats in reducing lipid peroxidation products. These results support and substantiate traditional consumption of green tea as protection against lipid peroxidation in the liver, blood serum, and central nervous tissue.     

The isolation, characterisation and kinetics of glutathione-S-transferase from human platelets

Glutathione S-transferase activity from human platelets was purified to homogeneity by affinity chromatography. The purified enzyme was found to be the acidic form and its molecular and catalytic properties were identical to acidic glutathione S-transferases purified from other human tissues. The purified platelet enzyme had no peroxidase activity and did not protect microsomes against peroxidation.     

Isolation and characterization of an anionic glutathione S-transferase from rat liver cytosol

An anionic glutathione S-transferase representing approximately 20% of the total glutathione S-transferase protein and 10% of the total transferase activity toward 1-chloro 2,4-dinitrobenzene has been purified to homogeneity from the 105,000 x g supernatant of rat liver homogenate. The SDS gel electrophoretic data on subunit composition revealed that the anionic isozyme is composed of two subunits with an identical Mr of 26,000. The Km values for 1-chloro 2,4-dinitrobenzene and reduced glutathione were determined to be 0.94 mM and 0.23 mM respectively. A significant amount of glutathione peroxidase activity toward cumene hydroperoxide is associated with the new isozyme.     

Activity of rat liver microsomal glutathione transferase toward products of lipid peroxidation and studies of the effect of inhibitors on glutathione-dependent protection against lipid peroxidation

Rat liver microsomal glutathione transferase displays glutathione peroxidase activity with linoleic acid hydroperoxide, linoleic acid ethyl ester hydroperoxide, and dilinoleoyl phosphatidylcholine hydroperoxide, with rates of 0.2, 0.3, and 0.3 mumol/min/mg, respectively. The activities are increased between three- and fourfold when the enzyme is activated with N-ethylmaleimide. Microsomal glutathione transferase can also conjugate 4-hydroxynon-2-enal with a specific activity of 0.5 mumol/min/mg. These findings show that the enzyme can remove harmful products of lipid peroxidation and thereby possibly protect intracellular membranes against oxidative stress. A set of glutathione transferase inhibitors (rose bengal, tributyltin acetate, S-hexylglutathione, indomethacin, cibacron blue, and bromosulfophtalein) which abolish the glutathione-dependent protection against lipid peroxidation in liver microsomes have been characterized. These inhibitors were found to be effective in the micromolar range and could prove valuable in studying the factor responsible for glutathione-dependent protection against lipid peroxidation.     

Effects of piperine on antioxidant pathways in tissues from normal and streptozotocin-induced diabetic rats

Using diabetes mellitus as a model of oxidative damage, this study investigated whether subacute treatment (10 mg/kg/day, intraperitoneally for 14 days) with the compound piperine would protect against diabetes-induced oxidative stress in 30-day streptozotocin-induced diabetic Sprague-Dawley rats. Liver, kidney, brain, and heart were assayed for degree of lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively) content, and activities of the free-radical detoxifying enzymes catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase. Piperine treatment of normal rats enhanced hepatic GSSG concentration by 100% and decreased renal GSH concentration by 35% and renal glutathione reductase activity by 25% when compared to normal controls. All tissues from diabetic animals exhibited disturbances in antioxidant defense when compared with normal controls. Treatment with piperine reversed the diabetic effects on GSSG concentration in brain, on renal glutathione peroxidase and superoxide dismutase activities, and on cardiac glutathione reductase activity and lipid peroxidation. Piperine treatment did not reverse the effects of diabetes on hepatic GSH concentrations, lipid peroxidation, or glutathione peroxidase or catalase activities; on renal superoxide dismutase activity; or on cardiac glutathione peroxidase or catalase activities. These data indicate that subacute treatment with piperine for 14 days is only partially effective as an antioxidant therapy in diabetes.     

Selenium-independent glutathione peroxidase activity associated with glutathione S-transferase from the housefly, Musca domestica

1. A glutathione S-transferase having Se-independent glutathione peroxidase activity was isolated from 100,000 g supernatant from housefly homogenate. 2. The specific activity of the partially purified Se-independent glutathione peroxidase was 1776 nmol NADPH oxidized/min/mg protein, representing an 87-fold purification. 3. The Mr of this enzyme was estimated to be 37,000 and 26,000 by gel filtration chromatography and gel electrophoresis, respectively. 4. Selenium-dependent glutathione peroxidase activity could not be detected in this same supernatant. 5. Se-independent glutathione peroxidase activity should be considered in future studies of the insect antioxidant defense system.     

Antioxidative effects of Cinnamomi cassiae and Rhodiola rosea extracts in liver of diabetic mice

Both Cinnamomi cassiae and Rhodiola rosea extracts are used as anti-diabetic folk medicines. Recently, increased oxidative stress was shown to play an important role in the etiology and pathogenesis of diabetes mellitus and its complications. This study was designed to examine the effects of Cinnamomi cassiae and Rhodiola rosea extracts on blood glucose, lipid peroxidation, the level of reduced glutathione and its related enzymes (glutathione reductase, glutathione S-transferase), and the activity of the antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) in the liver of db/db mice. Diabetic C57BL/Ks db/db mice were used as experimental models. Mice were divided into control (n=10), Cinnamomi cassiae (200 mg/kg/day, n=10), and Rhodiola rosea (200 mg/kg/day, n=10) treated groups for 12 weeks of treatment. These type II diabetic mice were used to investigate the effects of Cinnamomi cassiae and Rhodiola rosea on blood glucose, reduced glutathione, glutathione reductase, glutathione S-transferase, glutathione peroxidase, lipid peroxidation, catalase and superoxide dismutase. Cinnamomi cassiae and Rhodiola rosea extracts significantly decreased on blood glucose, increased levels of reduced glutathione and the activities of glutathione reductase, glutathione S-transferase, glutathione peroxidase, catalase and superoxide dismutase in the liver. Extract treatment also significantly decreased lipid peroxidation. Cinnamomi cassiae and Rhodiola rosea extracts may be effective for correcting hyperglycemia and preventing diabetic complications.     

Cytosolic glutathione peroxidase from liver of pacu (Piaractus mesopotamicus), a hypoxia-tolerant fish of the Pantanal

Pacu (Piaractus mesopotamicus Holmberg, 1887, Characiformes) dwells in waters of Pantanal, in which it has adapted for alternate concentrations of dissolved oxygen. Intracellular antioxidant protection should be vital for such an adaptation. Accordingly, we found that cytosol from liver of pacu has the highest antioxidant glutathione peroxidase activity so far reported for fish and murine species. To clarify whether this activity was due to a selenium independent glutathione S-transferase or to a glutathione peroxidase, we purified it and studied its kinetics. The substrates cumene hydroperoxide and hydrogen peroxide were promptly reduced by the enzyme, but peroxidized phosphatidylcholine had to undergo previous fatty acid removal with phospholipase A(2). Augmenting concentrations (from 2 to 6 mM) of reduced glutathione activated the pure enzyme. Curves of velocity versus different micromolar concentrations of hydrogen peroxide in the presence of 2, 4 or 8 mM reduced glutathione indicated that at least 2.5 mM reduced glutathione should be available in vivo for an efficient continuous destruction of micromolar concentrations of hydrogen peroxide by this peroxidase. Molecular exclusion HPLC and SDS-polyacrylamide gel electrophoresis indicated that the purified peroxidase is a homotetramer. Data from internal sequences showed selenocysteine in its primary structure and that the enzyme was a homologue of the type-1 glutathione peroxidase found in rat, bull, trout, flounder and zebra fish. Altogether, our data establish that in liver cells of pacu, a hypoxia-tolerant fish from South America, there are high levels of a cytosolic GPX-1 capable of quenching hydrogen peroxide and fatty acid peroxides, providing an effective antioxidant action.     

Effects of phenol compounds, glutathione analogues and a diuretic drug on glutathione S-transferase, glutathione reductase and glutathione peroxidase from canine erythrocytes.

1. Phenol compounds (ellagic acid, quercetin and purpurogallin), glutathione analogues (S-hexylglutathione and S-octylglutathione) and a diuretic drug (ethacrynic acid) were compared for their inhibitory effects on glutathione S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GSH-Px) in the canine erythrocytes. 2. All these compounds inhibited GST activity; quercetin was found to be the most potent inhibitor. 3. Ellagic acid, purpurogallin, quercetin and ethacrynic acid inhibited GR activity; S-hexylglutathione and S-octylglutathione had no effect on GR and GSH-Px activities. 4. Quercetin and purpurogallin inhibited GST non-competitively toward glutathione, whereas ellagic acid showed a competitive inhibition. Ellagic acid and purpurogallin inhibited GR non-competitively toward oxidized glutathione.     

Gastroprotective and Anti-oxidative Properties of Ascorbic Acid on Indomethacin-induced Gastric Injuries in Rats

Reactive oxygen species (ROS) have been implicated in the etiology of indomethacin-induced gastric mucosal damage. This study investigated ascorbic acid (vitamin C)'s protective effects against oxidative gastric mucosal damage induced by indomethacin. Ascorbic acid is a powerful antioxidant because it can donate a hydrogen atom and form a relatively stable ascorbyl free radical. We have investigated alterations in the levels of myeloperoxidase, antioxidant system enzymes (glutathione S-transferase, superoxide dismutase, glutathione reductase, catalase, glutathione peroxidase), lipid peroxidation and glutathione, as markers for ulceration process following oral administration of ascorbic acid, famotidine, lansoprazole, and ranitidine in rats with indomethacin-induced ulcers. In the present study, we found that (1) ascorbic acid, famotidine, lansoprazole and ranitidine reduced the development of indomethacin-induced gastric damages; (2) the administration of indomethacin caused a significant decrease in the levels of superoxide dismutase, glutathione peroxidase, glutathione S-transferase and glutathione, and an increase in the lipid peroxidation level; (3) the administration of ascorbic acid reversed the trend, inducing a significant increase of these enzymes' levels and a reduction in lipid peroxidation level in tissues; and (4) catalase, glutathione reductase and myeloperoxidase activities, increased by indomethacin, were found to be lower in the ascorbic acid, famotidine, lansoprazole and ranitidine-treated groups. The results indicate that the gastroprotective properties of ascorbic acid could be related to its positive effects on the antioxidant system and myeloperoxidase activity in indomethacin-induced gastric ulcers in rats.     

Effect of aldehydes on polyamine metabolism. III. Inhibition of S-adenosyl methionine decarboxylase (SAMD) by CCl4 and by aldehydes produced during lipid peroxidation

In isolated rat liver cells in which lipid peroxidation is stimulated by CCl4, a strong inhibition of S-adenosylmethionine decarboxylase (SAMD) activity occurs. Some purified aldehydes, which are produced during lipid peroxidation, are able to inhibit SAMD activity in Yoshida hepatoma cells. The most active aldehyde is hydroxypentenal (HPE). It inhibits by 50% SAMD activity at 0.5 mM concentration in entire hepatoma cells, or in hepatoma cell sap, and at 0.1 mM concentration in partially purified hepatoma cell sap fractions.     

Effects of glutathione on the alpha-tocopherol-dependent inhibition of nuclear lipid peroxidation

Endogenous alpha-tocopherol levels in isolated rat liver nuclei were determined to be 0.045 mol% (mol alpha-tocopherol per mol phospholipid x 100). This value corresponds to 970 polyunsaturated fatty acid (PUFA) moieties to one molecule of alpha-tocopherol in the nuclear membrane. Isolated nuclei, when incubated with various concentrations of exogenous alpha-tocopherol, took up only a small percent of initial levels of alpha-tocopherol present in the incubation media. Exogenous alpha-tocopherol, when incorporated in isolated nuclei above a threshold value of 0.085 mol%, effectively inhibited NADPH-induced lipid peroxidation. The addition of 1 mM glutathione lowered the threshold levels of alpha-tocopherol needed to inhibit lipid peroxidation to about 0.040 mol%. We suggest the data indicate a glutathione-dependent enhancement of the ability of alpha-tocopherol to inhibit nuclear lipid peroxidation.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. Effects of the hexose monophosphate shunt as mediated by glutathione and ascorbate.

Lipid peroxidation and haemoglobin degradation were the two extremes of a spectrum of oxidative damage in red cells exposed to t-butyl hydroperoxide. The exact position in this spectrum depended on the availability of glucose and the ligand state of haemoglobin. In red cells containing oxy- or carbonmono-oxy-haemoglobin, hexose monophosphate-shunt activity was mainly responsible for metabolism of t-butyl hydroperoxide; haem groups were the main scavengers in red cells containing methaemoglobin. Glutathione, via glutathione peroxidase, accounted for nearly all of the hydroperoxide metabolizing activity of the hexose monophosphate shunt. Glucose protection against lipid peroxidation was almost entirely mediated by glutathione, whereas glucose protection of haemoglobin was only partly mediated by glutathione. Physiological concentrations of intracellular or extracellular ascorbate had no effect on consumption of t-butyl hydroperoxide or oxidation of haemoglobin. Ascorbate was mainly involved in scavenging chain-propagating species involved in lipid peroxidation. The protective effect of intracellular ascorbate against lipid peroxidation was about 100% glucose-dependent and about 50% glutathione-dependent. Extracellular ascorbate functioned largely without a requirement for glucose metabolism, although some synergistic effects between extracellular ascorbate and glutathione were observed. Lipid peroxidation was not dependent on the rate or completion of t-butyl hydroperoxide consumption but rather on the route of consumption. Lipid peroxidation appears to depend on the balance between the presence of initiators of lipid peroxidation (oxyhaemoglobin and low concentrations of methaemoglobin) and terminators of lipid peroxidation (glutathione, ascorbate, high concentrations of methaemoglobin).     

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.     

The effect of 2,4-dichlorophenol and pentachlorophenol on antioxidant system in the leaves of Phalaris arudinacea

The purpose of this work was to evaluate the effect of 2,4-dichlorophenol (2,4-DCP) and pentachlorophenol (PCP) on the activity of antioxidative system and lipid peroxidation in the leaves of reed canary grass (Phalaris arudinacea). The activity of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), glutathione reductase (GR) and glutathione S-transferase (GST) as well as the content of glutathione, ascorbate and phenolic compounds were determined. An induced-increase in the APX, CAT, GPX and GR activities was stronger for PCP, while a significant increase in the GST activity was noted only for 2,4-DCP. Both compounds increased the content of phenolic compounds, oxidized and reduced glutathione as well as the content of ascorbic acid. PCP induced stronger increase in lipid peroxidation than 2,4-DCP. The observed changes revealed that chlorophenols induce oxidative stress and oxidative damage in the leaves of reed canary grass.     

Nickel-mediated inhibition in the glutathione-dependent protection against lipid peroxidation

Glutathione protects liver microsomes against the rapid onset of lipid peroxidation via a sulfhydryl dependent heat labile factor known as free radical reductase. The administration of nickel to mice resulted in an inhibition in the activity of free radical reductase, and enhanced lipid peroxidation and the activity of glutathione S-transferase in a dose dependent manner. The pretreatment of cyclam, a known specific chelator of nickel restored free radical reductase and glutathione S-transferase activities and alleviated nickel mediated enhancement of lipid peroxidation. Our results indicate that nickel-mediated inhibition in free radical reductase activity and activation of glutathione S-transferase may be due to the interaction of nickel with sensitive-SH groups located on these proteins.     

Phospholipid hydroperoxide glutathione peroxidase of rat testis. Gonadotropin dependence and immunocytochemical identification.

A high glutathione peroxidase activity toward phospholipid hydroperoxides is present in rat testis. The attribution of this activity to the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) was supported by cross-reactivity with antibodies raised against pig heart PHGPX which had been purified and characterized. Rat testis PHGPX is partially cytosolic and partially linked to nuclei and mitochondria. The soluble and organelle-bound enzymes appear identical by Western blot analysis. PHGPX, but neither selenium-dependent nor non-selenium-dependent glutathione peroxidase activity, is expressed in testes only after puberty, disappears after hypophysectomy, and is partially restored by gonadotropin treatment. Specific immunostaining of testes by antiserum against PHGPX appears as a fine granular brown pattern localized throughout the cytoplasm in more immature cells but is confined to the peripheral part of the cytoplasm, the nuclear membrane, and mitochondria in maturating spermatogenic cells. As expected, immunostaining of spermatogenic cells in hypophysectomized animals was negative, but gonadotropin treatment only marginally increased the immunoreactivity. The expression of PHGPX in testes is consistent with the previously described specific requirement for selenium for synthesis of a 15-20-kDa selenoprotein which is related to the production of functional spermatozoa.     

Photoperiodic changes of the glutathione system of the brain under acute hypoxia]

The effect of acute hypoxic hypobaric hypoxia on the content of reduced glutathione and the activity of glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and glutathione S-transferase, as well as 5'-nucleotidase in homogenates of juvenile male rats under conditions of varying photoperiodic duration: natural conditions of illumination, continuous illumination and continuous darkness were studied. Photoperiodic changes were revealed in the glutathione system of the control animals: the activity of glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase reduces under constant light, while the activity of glutathione peroxidase and glutathione S-transferase increases under conditions of constant darkness. The greatest inhibitory effect on the state of the glutathione system is brought about by constant light in case of acute hypoxia: the content of reduced glutathione decreases along with a sharp drop of the activity of glutathione S-transferase and glucose-6-phosphate dehydrogenase, observed against the background of decreased glutathione reductase activity. Permanent dark conditions eliminate partially or completely the negative effect of acute hypoxia on the glutathione system of the brain. The obtained results are indicator of a possibility of protecting role of melatonin in case of acute hypoxia.