Vitamin E dependent reduced glutathione inhibition of rat liver microsomal lipid peroxidation

Effects of reduced glutathione (GSH) were investigated on $\text{in}$$\text{vitro}$ lipid peroxidation of hepatic microsomes obtained from Long-Evans Hooded rats fed chemically defined, purified diets containing adequate or documented deficiencies of vitamin E (E), selenium (Se) or both. Glutathione inhibited lipid peroxidation mediated by both NADPH-dependent enzymatic and ascorbate-dependent non-enzymatic systems. The inhibitory effect of GSH was observed in microsomes obtained from E supplemented groups whereas it had no effect on microsomes from E deficient animals. Selenium status had no effect on GSH inhibition. Glutathione was found to be specific for the E dependent inhibition of lipid peroxidation and could not be substituted by other sulfhydryl compounds tested. Also, GSH did not inhibit non-enzymatic lipid peroxidation of heat-denatured microsomes from either E-supplemented groups or any of the other dietary regimens.     

Rat lung microsomal lipid peroxidation: effects of vitamin E and reduced glutathione

Lung microsomal membranes that contain the redox active components associated with the mixed-function oxidase system can be peroxidized in vitro. To investigate the characteristics of rat lung microsomal lipid peroxidation, we performed experiments using a variety of peroxidation initiators and microsomes obtained from normal and vitamin E-deficient rats. We found that lung microsomes obtained from normal rats are peroxidized much less than liver microsomes obtained from the same animals. Only initiation systems using very high concentrations of ferrous iron produced any significant peroxidation of normal rat lung microsomes. Lung microsomes obtained from vitamin E-deficient rats were found to be much more susceptible to peroxidation. Glutathione (GSH) was effective in inhibiting peroxidation when lung microsomes from normal rats were peroxidized. GSH was not effective in decreasing peroxidation when microsomes from vitamin E-deficient rats were peroxidized in the same system. We conclude that both GSH and vitamin E protect lung microsomal membranes from peroxidation. Glutathione protection appears to be related to the presence of a sulfhydryl group.     

Rabbit liver microsomal lipid peroxidation. The effect of lipid on the rate of peroxidation

Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.     

Enzymic lipid peroxidation in the microsomal fraction of rat brain

Abstract: An enzymic lipid peroxidation system has been demonstrated in the microsomal fraction of rat brain and the requirements and optimal conditions for assay determined. The involvement of NADPH-cytochrome c reductase was demonstrated in vesicles reconstituted with lipids extracted from the brain microsomal fraction. Further characterization of the system made use of substances shown to inhibit the liver microsomal system. α-Tocopherol was shown to be an effective inhibitor of lipid peroxidation in the brain microsomal system, whereas Na₂SO₃ had no effect, which is indicative that free radical transfer occurs only in the hydrophobic regions. Neither superoxide dismutase nor catalase inhibited lipid peroxidation. The implications of an NADPH-cytochrome c reductase-dependent lipid peroxidation system that is not linked to a drug hydroxylation system and appears to differ from the liver microsomal system in a number of other ways are discussed.     

Growth-related lipid peroxidation in tumour microsomal membranes and mitochondria.

Microsomes and mitochondria isolated from Morris hepatomas 3924A (fast-growing) and 44 (slow-growing) and Ehrlich ascites tumour cells exhibit a NADPH-dependent peroxidation of endogenous lipids lower than that of the corresponding fractions from rat liver. Moreover, the O2- and ascorbate-dependent lipid peroxidations are decreased in microsomes from the two Morris hepatomas. The peroxidative activity appears to be inversely related to the growth rate of the tumours. It is suggested that the low susceptibility of tumour membranes to peroxidative agents may be a factor responsible for the high mitotic activity of this tissue.     

Antioxidant effect of caeruloplasmin on microsomal lipid peroxidation

Microsomal NADPH-dependent lipid peroxidation catalyzed by ADP-Fe³⁺ was inhibited by the addition of caeruloplasmin. The antioxidant effect of caeruloplasmin was independent of the superoxide anion (O^?₂ scavenging activity. Since caeruloplasmin enhanced the function of ADP-Fe³⁺ acting as electron acceptor for microsomal electron transport system, the antioxidant effect of caeruloplasmin is considered to depend on the ferroxidase activity.     

The effect of lipid peroxidation on the calcium-accumulating ability of the microsomal fraction isolated from chicken breast muscle.

The effect of lipid peroxidation on the Ca2+-accumulating and Ca2+-retaining abilities of the microsomal fraction from chicken breast muscle was investigated. At 25 degrees C, enzymic lipid peroxidation did not seriously affect either of these abilities unless ascorbic acid was present, when both were diminished. At 37 degrees C, Ca2+-concentrating ability was decreased further by the effects of heat damage to the membrane. Membrane lipid peroxidation did not affect microsomal adenosine triphosphatase activity unless the microsomal fraction was subsequently washed with albumin. This effect of albumin is possibly due to removal of lipid-breakdown products. Addition of soya-bean phospholipids to the peroxidized vesicles washed with albumin restored adenosine triphosphatase activity, demonstrating a non-specific phospholipid requirement.     

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.     

The inhibitory effect of succinate on radiation-enhanced mitochondrial lipid peroxidation

The degree of mitochondrial ADP/Fe/NADPH-induced lipid peroxidation was increased up to the fourth day after 9.0 Gy whole body gamma-irradiation. The lipid peroxidation inhibiting effect of succinate added to isolated mitochondria was diminished as a consequence of irradiation. The succinate, administered in vivo prior to irradiation, decreased the amount of malondialdehyde production and protected the succinate dehydrogenase enzyme against inactivation. The mean survival of succinate-pretreated animals was much longer than that of controls. The role of mitochondrial lipid peroxidation in the pathogenesis of radiation injury is discussed.     

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.     

Radioprotective effect of 2-mercaptopropionylglycine on radiation-induced microsomal lipid peroxidation

Radiation-induced microsomal lipid peroxidation was measured following gamma-irradiation (66-399 Gy) in the presence or absence of 2-mercaptopropionylglycine (MPG) at low and high concentrations (0.03 and 0.1 mg/ml). At high concentrations of MPG, enhancement of lipid peroxidation was observed instead of reduction. Following gamma-irradiation at 266.4 Gy, lipid peroxidation increased with increasing concentrations of MPG. The spontaneous lipid peroxidation also increased with increasing concentrations of MPG. When MPG and EDTA were added together, radioprotection was observed even at high concentrations of MPG after different doses of radiation. An increase in radioprotection with increasing concentrations of MPG in the presence of EDTA at 266.4 Gy was also observed. The exogenous supply of Fe2+ during irradiation was found to eliminate the 'threshold dose' which has to be delivered before MPG shows an 'enhancement effect'. A possible mechanism for the enhancement of radiation-induced lipid peroxidation is proposed.     

The mechanism of liver microsomal lipid peroxidation

In the presence of Fe³⁺ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1,3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. These results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe³⁺ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe²⁺ by oxygen.     

The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis

The testis is a remarkably active metabolic organ; hence it is suitable not only for studies of lipid metabolism in the organ itself but also for the study of lipid peroxidation processes in general. The content of fatty acids in testis is high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. Studies were carried out to evaluate the effect of alpha-tocopherol in vitro on ascorbate-Fe(++) lipid peroxidation of rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition were used as an index of the oxidative destruction of lipids. Special attention was paid to the changes produced on the highly PUFA [C20:4 n6] and [C22:5 n6]. Lipid peroxidation of testis microsomes or mitochondria induced a significant decrease of both fatty acids. Total chemiluminescence was similar in both kinds of organelles when the peroxidized without (control) and with ascorbate-Fe(++) (peroxidized) groups were compared. Arachidonic acid was protected more efficiently than docosapentaenoic acid at all alpha-tocopherol concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbate-Fe(++). The maximal percentage of inhibition in both organelles was approximately 70%; corresponding to an alpha-tocopherol concentration between 1 and 0.25 mM. IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.144 mM) than mitochondria (0.078 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6]+[C22:5 n6] in microsomes that in mitochondria. It is proposed that the vulnerability to lipid peroxidation of rat testis microsomes and mitochondria is different because of the different proportion of PUFA in these organelles The peroxidizability index (PI) was positively correlated with the level of long chain fatty acids. The results demonstrated the protective effect of alpha-tocopherol on lipid peroxidation in microsomes and mitochondria from rat testis.     

Differential effect of L-thyroxine on phospholipid biosynthesis in mitochondria and microsomal fraction.

1. The action of L-thyroxine on the incorporation of radioactive choline or CDP-choline into phosphatidylcholine in vitro was explored in liver and brain microsomal fraction and mitochondria obtained from young adult rats. 2. In liver mitochondria isolated from animals treated with L-thyroxine (40 mg/kg body wt. during 6 days), the incorporation of both radioactive precursors into phosphatidylcholine was significantly decreased compared with normal controls, whereas in the total homogenate and in the microsomal fraction the incorporation was similar in the experimental and control groups. In subcellular fractions isolated from brain, the incorporation of precursors was similar in L-thyroxine-treated and normal animals. 3. Liver mitochondria isolated from normal animals incubated in vitro with CDP-choline, in the presence of different concentrations of L-thyroxine, showed also a marked decrease in the incorporation of label into phosphatidylcholine, whereas no significant changes were found in the total homogenate and in the microsomal fraction compared with control experiments. 4. The differential effect of L-thyroxine on the incorporation of radioactive precursors into phosphatidylcholine of isolated liver subcellular fractions gives further support to the hypothesis that liver mitochondria can independently synthesize part of their own phospholipids. 5. Possible mechanisms of the action of the hormone at the mitochondrial level are discussed.     

The mechanism of liver microsomal lipid peroxidation.

In the presence of Fe-3+ and complexing anions, the peroxidation of unsaturated liver microsomal lipid in both intact microsomes and in a model system containing extracted microsomal lipid can be promoted by either NADPH and NADPH : cytochrome c reductase or by xanthine and xanthine oxidase. Erythrocuprein effectively inhibits the activity promoted by xanthine and xanthine oxidase but produces much less inhibition of NADPH-dependent peroxidation. The singlet-oxygen trapping agent, 1, 3-diphenylisobenzofuran, had no effect on NADPH-dependent peroxidation but strongly inhibited the peroxidation promoted by xanthine and xanthine oxidase. NADPH-dependent lipid peroxidation was also shown to be unaffected by hydroxyl radical scavengers.. The addition of catalase had no effect on NADPH-dependent lipid peroxidation, but it significantly increased the rate of malondialdehyde formation in the reaction promoted by xanthine and xanthine oxidase. The results demonstrate that NADPH-dependent lipid peroxidation is promoted by a reaction mechanism which does not involve either superoxide, singlet oxygen, HOOH, or the hydroxyl radical. It is concluded that NADPH-dependent lipid peroxidation is initiated by the reduction of Fe-3+ followed by the decomposition of hydroperoxides to generate alkoxyl radicals. The initiation reaction may involve some form of the perferryl ion or other metal ion species generated during oxidation of Fe-2+ by oxygen.     

Nitrofuran inhibition of microsomal lipid peroxidation

Two nitrofuran compounds, nifurtimox and nitrofurantoin, inhibited in a concentration-dependent manner the NADPH-, iron-induced lipid peroxidation in rat liver microsomes, as shown by the decreased rate of MDA accumulation. Other nitro compounds (benznidazole and chloramphenicol) were relatively inactive. Nifurtimox inhibition affected polyenoic fatty acids and cytochrome P-450 degradation that follows lipid peroxidation. The ascorbate- or tert-butyl hydroperoxide-dependent lipid peroxidations were much less inhibited than the NADPH-dependent one. Nifurtimox and nitrofurantoin, but not benznidazole and chloramphenicol, strongly stimulated the microsomal NADPH-oxidase activity, thus supporting electron diversion, as the main cause of the inhibition of peroxidation initiation.     

Glutathione disulfide enhances the reduced glutathione inhibition of lipid peroxidation in rat liver microsomes

Experiments were undertaken to examine the effects of reduced (GSH) and oxidized (GSSG) glutathione on lipid peroxidation of rat liver microsomes. Dependence on microsomal alpha-tocopherol was shown for the GSH inhibition of lipid peroxidation. However, when GSH (5 mM) and GSSG (2.5 mM) were combined in the assay system, inhibition of lipid peroxidation was enhanced markedly over that with GSH alone in microsomes containing alpha-tocopherol. Surprisingly, the synergistic inhibitory effect of GSH and GSSG was also observed for microsomes that were deficient in alpha-tocopherol. These data suggest that there may be more than one factor responsible for the glutathione-dependent inhibition of lipid peroxidation. The first is dependent upon microsomal alpha-tocopherol and likely requires GSH for alpha-tocopherol regeneration from the alpha-tocopheroxyl radical during lipid peroxidation. The second factor appears to be independent of alpha-tocopherol and may involve the reduction of lipid hydroperoxides to their corresponding alcohols. One, or possibly both, of these factors may be activated by GSSG through thiol/disulfide exchange with a protein sulfhydryl moiety.     

Chemically-induced glutathione depletion and lipid peroxidation

Malondialdehyde (MDA) formation in mouse liver homogenates was measured in the presence of various glutathione depletors (5 mmol/l). After a lag phase of 90 min, the MDA formation increased from 1.25 nmol/mg protein to 14.5 nmol/mg in the presence of diethyl maleate (DEM), to 10.5 with diethyl fumarate (DEF) and to 4 with cyclohexenon by 150 min. It remained at 1.25 nmol/mg with phorone and in the control. On the other hand, glutathione (GSH) dropped from 55 nmol/mg to 50 nmol/mg in the control to, < 1 with DEM, to 46 with DEF, to 3 with cyclohexenon and to 7 with phorone. The data show that the potency to deplete GSH is not related to MDA production in this system. DEM stimulated in vitro ethane evolution in a concentration-dependent manner and was strongly inhibited by SKF 525A. From type I binding spectra to microsomal pigments the following spectroscopic binding constants were determined: 2.5 mmol/l for phorone, 1.2 mmol/l for cyclohexenon, 0.5 mmol/l for DEM and 0.3 mmol/l for DEF. In isolated mouse liver microsomes NADPH-cytochrome P-450 reductase and NADH-cytochrome b₅ reductase activity were unaffected by the presence of DEM, whereas ethoxycoumarin dealkylation was inhibited. Following in vivo pretreatment, hepatic microsomal electron flow as determined in vitro was augmented in the presence of depleting as well as non-depleting agents, accompanied by a shift from O₂⁻ to H₂O₂ production. It is concluded that it is not the absence of GSH which causes lipid peroxidation after chemically-induced GSH depletion but rather the interaction of the chemicals with the microsomal monoxygenase system.     

Effects of lipid peroxidation on adrenal microsomal monooxygenases

Incubation of guinea pig adrenal microsomes with 10^?6 M ferrous (Fe²⁺) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe²⁺ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe²⁺ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[α]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.     

Effect of antioxidants on adriamycin-induced microsomal lipid peroxidation

Adriamycin (25 μM) stimulated NADPH-dependent microsomal lipid peroxidation about fourfold over control values. The tested antioxidants, zinc, superoxide dismutase, vitamin E, and desferrioxamine (Desferal) inhibited Adriamycin-enhanced lipid peroxidation to varying degrees. Others antioxidants, e.g., glutathione, catalase, and selenium, were found to have no effects. Our in vitro studies suggest that adriamycin effect is mediated by a complex oxyradical cascade involving superoxide, hydroxyl radical, and small amounts of iron.