Glutathione-dependent Protection Research Articles

Sesquiterpene-evoked phytochemical toxicity in PC12 neuronal cells reveals a variable degree of oxidative stress and alpha-tocopherol and glutathione-dependent protection

Phytochemicals are often promoted generally as antioxidants and demonstrate variable levels of reactive oxygen species (ROS) sequestration in vitro, which attributes to their neuroprotective bioactivity. Sesquiterpenes from cannabis and essential oils may demonstrate bifunctional properties towards cellular oxidative stress, possessing pro-oxidant activities by generating ROS or scavenging ROS directly. Sesquiterpenes can also oxidize forming sesquiterpene oxides, however the relative contribution they make to the bioactivity or cytotoxicity of complex botanical extracts more generally is unclear, while selected cannabis-prevalent terpenes such as β-caryophyllene may also activate cannabinoid receptors as part of their biological activity. In the present study, we investigated selected sesquiterpenes β-caryophyllene and humulene and their oxidized forms (β-caryophyllene oxide and zerumbone, respectively) against established antioxidants (ascorbic acid, α-tocopherol, and glutathione) and in the presence of cannabinoid receptor 1 and cannabinoid receptor 2 antagonists, to gain a better understanding of the molecular and cellular mechanisms of neuroprotection versus neurotoxicity in semi-differentiated rat neuronal phaeochromocytoma (PC12) cells. Our results demonstrate that the sesquiterpenes β-caryophyllene, humulene and zerumbone possess concentration-dependent neurotoxic effects in PC12 cells. Both β-caryophyllene- and humulene-evoked toxicity was unaffected by CB1 or CB2 receptor antagonism, demonstrating this occurred independently of cannabinoid receptors. Both glutathione and α-tocopherol were variably able to alleviate the concentration-dependent loss of PC12 cell viability from exposure to β-caryophyllene, humulene and zerumbone. During 4-hour exposure to sesquiterpenes only modest increases in ROS levels were noted in PC12 cells, with glutathione co-incubation significantly inhibiting intracellular ROS production. However, significant increases in ROS levels in PC12 cells were demonstrated during 24-hour incubation with either antioxidants or sesquiterpenes individually, and with additive toxicity exhibited in combination. Overall, the results highlight a concentration-dependent profile of sesquiterpene neurotoxicity independent of cannabinoid receptors and dissociated from the formation of reactive oxygen species as a marker or correlate to the loss of cell viability.

Chrysotile inhibits glutathione-dependent protection against the onset of lipid peroxidation in rat lung microsomes.

The glutathione and vitamin E dependent protection of lipid peroxidation in an NADPH (0.4 mM) and chrysotile (500 micrograms/ml) containing system were investigated in vitro in rat lung microsomes. Addition of 1 mM glutathione to the above reaction system containing microsomes supplemented with vitamin E (1 nmol/mg protein) reduced lipid peroxidation. Similar protection by glutathione could be observed in normal unsupplemented microsomes though the degree of protection was less pronounced. Addition of free radical scavengers such as, superoxide dismutase (100 units/ml), catalase (150 units/ml), mannitol (1 mM) and beta-carotene (0.5 mM) to the reaction system showed an insignificant effect on lipid peroxidation. When the reaction was carried out in absence of glutathione, vitamin E content of peroxidizing microsomes decreased rapidly. In this system a concomitant increase in the activity of microsomal glutathione-S-transferase was observed which may serve as an alternative pathway to detoxify lipid peroxides. Addition of glutathione alone to the reaction system prevented both against the loss in vitamin E content and increase in the activity of glutathione-S-transferase. Supplementation of both vitamin E and glutathione was found to be effective in lowering glutathione-S-transferase activity to that of normal basal level. Our results suggest that chrysotile-mediated stimulation of NADPH-dependent lipid peroxidation may be due to hampering of glutathione-dependent protection which may ultimately exhaust membrane bound vitamin E. Our data further suggest that the lung tissue may have an inbuilt mechanism whereby glutathione-S-transferase may be triggered to cope with the excessive production of lipid peroxides.

Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation

Evidence that rat liver musomal glutathione transferase is responsible for the glutathione-dependent inhibition of lipid peroxidation in liver musomes has been obtained. Activation of the musomal glutathione transferase in musomes by cystamine renders this organelle even more resistant to lipid peroxidation in the presence of glutathione compared with untreated musomes. Upon examining the effect of seven glutathione analogues on lipid peroxidation, it was found that only those that serve as good substrates for the musomal glutathione transferase (Glutaryl- l-Cys-Gly and α- l-Glu- l-Cys-Gly) can inhibit lipid peroxidation. The lack of inhibition by the other five analogues (α d-Glu- l-Cys-Gly, γ- d-Glu- l-Cys-Gly, β- l-Asp- l-Cys-Gly, α- l-Asp- l-Cys-Gly and α- d-Asp- l-Cys-Gly) shows the specificity of the protection and rules out any non-enzymic component. Inhibitors of selenium- dependent glutathione peroxidase (mercaptosuccinate at 50 μM) and phospholipid hydroperoxide glutathione peroxidase (iodoacetate, 1 mM + glutathione, 0.5 mM) do not inhibit the glutathione-dependent protection of rat liver musomes against lipid peroxidation. Purified musomal glutathione transferase, NADPH-cytochrome P450 reductase and cytochrome P450 were reconstituted in musomal phospholipid vesicles by cholate dialysis. The resulting membranes contained functional enzymes and did display enzymic lipid peroxidation induced by 75 μM NADPH and 10 μM Fe-EDTA (2: 1). This model system was used to investigate whether musomal glutathione transferase could inhibit lipid peroxidation in a glutathione-dependent manner. The results show that 5 mM glutathione did inhibit lipid peroxidation when functional musomal glutathione transferase was included. This was not the case when the enzyme had been pre-inactivated with diethylpyrocarbonate. Furthermore, the protective effect of glutathione could be partly reversed by an inhibitor (100μM bromosulphophtalein) of the enzyme. Apparently, rat liver musomal glutathione transferase has the capacity to inhibit lipid peroxidation in a reconstituted system.

Hydrogen peroxide as a mediator of programmed cell death in the blastocyst

Abstract Previous work identified in blastocele fluid a soluble activity which killed cmbryonal carcinoma cells with trophectodermal potential but not those with embryonic potential [35]. From use of a malignant caricature of the late blastocyst, this toxic activity was postulated to be H20, [8]. The purpose of this paper was to determine if blastocele fluid also contained amounts of H 2O, capable of mediating the preferential killing of malignant pretrophectodermal cells (ECa 247). We not only observed that blastocele fluid is not toxic for these cells in the presence of catalase, but that malignant cells with embryonic potential (P19) that normally survive exposure to blastocele fluid become sensitive to it if their intracellular glutathione levels are lowered. Thus, it is concluded that the blastocyst contains amounts of H20, toxic to malignant pretrophectodermal cells and that glutathione-dependent mechanisms protect malignant inner cell mass cells with embryonic potential. Apparently, H202production and glutathione-dependent protection mechanisms are developmentally regulated in the inner cell mass. These results are discussed with regards to apoptosis and the regulation of tissue mass.

Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation

Evidence that rat liver microsomal glutathione transferase is responsible for glutathione-dependent protection against lipid peroxidation

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 μmol/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 μmol/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.

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

Ascorbate-Fe 3+-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 °C for 1 week diminished the activity. GSH could not be substituted by other sulfhydryl compounds tested. Deoxycholate (1 m m) and bromosulfophthalein (0.1 m m) inhibited GSH-dependent protection but did not inhibit microsomal GSH peroxidase activity. Iodoacetate (10 m m) inhibited GSH-dependent protection but did not inhibit microsomal GSH S-transferase. N-Ethylmaleimide (0.1 m m) and oxidized glutathione (10 m m) 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.

Decrease of the inhibition of lipid peroxidation by glutathione-dependent system in erythrocytes of non-insulin dependent diabetics.

The inhibition of lipid peroxidation of erythrocyte membranes by glutathione-dependent protection was studied in patients with non-insulin dependent diabetes mellitus. Incubation of red cells from diabetics with 1.5 mM t-butyl hydroperoxide resulted in a lipid peroxidation increase greater than that of normal controls. Glutathione-dependent and glutathione-independent protection against oxidative damage was examined using an artificial system, in which erythrocyte ghosts were incubated with t-butyl peroxide and dialysed hemolysate in the presence or the absence of 2 mM glutathione. The glutathione-dependent protection of hemolysate from diabetics was approximately 70% of that from normal controls. The results suggest that decrease in glutathione-dependent protection against lipid peroxidation, along with decrease in glutathione levels, increases oxidative damage in erythrocyte membranes taken from diabetic patients.

Glutathione, free radicals and chemotherapeutic agents: Mechanisms of free-radical induced toxicity and glutathione-dependent protection

Glutathione, free radicals and chemotherapeutic agents: Mechanisms of free-radical induced toxicity and glutathione-dependent protection

Glutathione-dependent protection against oxidative damage of the human red cell membrane

Glutathione (GSH) dependent protection against oxidative damage of human red cell membrane was examined. An artificial system was used in which chloroform/methanol-extracted red cell lipids, in the form of liposomes, were subjected to attack by a peroxidation system consisting of ascorbate-Fe3+. Human erythrocytes contained a nondialyzable factor, completely inactivated by heating in a boiling water bath for 3 min, which showed GSH-dependent inhibition against lipid peroxidation and was devoid of GSH peroxidase activity. On the other hand, GSH-S transferase, highly purified by affinity chromatography, had no inhibitory activity. These findings strongly indicate that the GSH- dependent protection against lipid peroxidation of human red cell membrane is mediated by one or more proteins other than GSH peroxidase and GSH-S transferase.

Influence of vitamin E and selenium on glutathione-dependent protection against microsomal lipid peroxidation

A GSH-dependent microsomal protein which inhibits lipid peroxidation has been described [R. F. Burk, Biochem. biophys. Acta 757, 21 (1983)]. Studies of its mechanism indicate that it scavenges free radicals. Vitamin E (α-tocopherol) and selenium are micronutrients which protect against lipid peroxidation. The effect of nutritional deficiencies of these substances on the GSH-dependent protection against rat liver microsomal lipid peroxidation was studied to determine whether GSH, selenium and α-tocopherol function through separate or shared mechanisms. In the ascorbate—iron microsomal lipid peroxidation system, there is a 1–3 min lag phase before lipid peroxidation begins. The length of the lag correlated well ( r = 0.87) with the microsomal α-tocopherol content as measured by high pressure liquid chromatography. Thus, the selenium-deficient microsomes, which had a shorter lag than controls, had a somewhat lower α-tocopherol content. The vitamin E-deficient microsomes, which had no detectable α-tocopherol, had the shortest lag, but a distinct lag was present. Addition of 0.1 mM GSH to control microsomes prolonged the lag by 270%. In selenium-deficient and vitamin E-deficient microsomes, which had shorter initial lags, GSH addition caused 345 and 280% increases respectively. This suggests that the function of the GSH-dependent protective mechanism is unimpaired in these deficiencies. Trypsin digestion of microsomes, which abolished the lag completely and destroyed the GSH-dependent protection, had no effect on microsomal α-tocopherol content, however. These experiments illustrate the importance of two defenses against microsomal lipid peroxidation: the GSH-dependent protein which is responsible for the existence of the lag, and α-tocopherol which affects the length of the lag. They suggest that these defenses function separately to prevent peroxidation of membrane polyunsaturated fatty acids. Selenium appears to affect microsomal α-tocopherol content but to have no other effect on the microsomal lipid peroxidation system.

Glutathione-dependent protection by rat liver microsomal protein against lipid peroxidation

GSH is an important cellular defense against oxidant injury. Its effect in the rat liver microsomal lipid peroxidation system has been examined. Incubation of fresh rat liver microsomes with ascorbic acid and ADP-chelated iron leads to the peroxidation of microsomal lipids (production of thiobarbituric acid-reactive substances and destruction of polyunsaturated fatty acids) following a 2 to 5 min lag. Addition of 0.1 mM GSH to the system lengthened the lag period by 5 to 15 min without affecting the rate or the extent of lipid peroxidation. GSH could not be replaced in prolonging the lag by cysteine, mercaptoethanol, dithiothreitol, propylthiouracil, or GSSG. The GSH effect on the lag was abolished by heating or trypsin digestion of the microsomes, indicating that microsomal protein is required for its expression. Progressively longer lags were observed as the GSH concentration was increased from 0.1 to 5 mM, but there was no evidence of GSH oxidation as a consequence of the protection against lipid peroxidation. GSH protected against heat inactivation of the microsomal protein responsible for the GSH effect. Experiments with an oxygen electrode revealed that the GSH protection did not alter the ratio of O 2 consumed to thiobarbituric acid-reactive substances produced. This implicated free radical scavenging as the mechanism of protection. These results indicate the existence of a GSH-dependent rat liver microsomal protein which scavenges free radical. This protein may be an important defense against free radical injury to the microsomal membrane.

Purification from pig liver of a protein which protects liposomes and biomembranes from peroxidative degradation and exhibits glutathione peroxidase activity on phosphatidylcholine hydroperoxides

The cell sap from pig liver contains a protein which protects phosphatidylcholine liposomes and biomembranes from peroxidative degradation in the presence of glutathione. The activity of this protein has been assayed by measuring the inhibition of aged phosphatidylcholine liposome peroxidation induced by the Fe 3+-triethylenetetramine complex. The peroxidation-inhibiting protein from pig liver has been purified 535-fold to homogeneity with overall recovery of activity of 12%. (NH 4) 2SO 4 precipitation, ion-exchange chromatography on DEAE-Sepharose CL-6B and CM23-cellulose, affinity chromatography on glutathionebromosulfophthalein-Sepharose and gel filtration on Sephadex G-50 were used. Gel filtration and SDS-polyacrylamide gel electrophoresis indicated a molecular weight of approximately 20000. The protein inhibited peroxidation by Fe 3+-triethylenetetramine following a 15 min preincubation of phosphatidylcholine liposomes in the presence of 5 mM glutathione or 2-mercapthoethanol. The pure protein exhibited glutathione peroxidase activity on hydroperoxide groups of phosphatidylcholine and on cumene and t-butyl hydroperoxides, with specific activities of 2.2, 3.8 and 0.9 μmol/min per mg protein, respectively. The protein appears to be distinct from the selenoenzyme glutathione peroxidase and from any known glutathione S-transferase. The peroxidation was studied also with fresh phosphatidylcholine liposomes and was induced in this case by Fe-ascorbate. To obtain protection by the peroxidation-inhibiting protein and glutathione, preincubation was not necessary, but α-tocopherol, incorporated in the liposomes in the molar ratio 1:250 to phosphatidylcholine, was required. Lipid peroxidation of rat liver mitoplasts and microsomes was blocked when these preparations were incubated in the perpxodizing mixture in the presence of peroxidation-inhibiting protein and glutathione. The protection from Fe 3+-triethylenetetramine-induced peroxidation is related apparently to reduction of hydroperoxide groups in polyunsaturated fatty acid residues of phospholipids and to inhibition of free radicals formation by chain branching. Protection from the Fe-ascorbate-induced peroxidation is apparently attributable to the same mechanism. However, the requirement of α-tocopherol for protection in the Fe-ascorbate-induced peroxidation suggests that the cooperation of a free-radical scavenger is necessary. It is probable that the glutathione peroxidase activity is involved also in the glutathione-dependent protection exhibited by the protein on lipid peroxidation of biomembranes.