Raymond F. Burk

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 O2 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 radicals. This protein may be an important defense against free radical injury to the microsomal membrane.

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, Biochim. biophys. Acta 757, 21 (1983)]. Studies of its mechanism indicate that it scavenges free radicals. Vitamin E (alpha-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 alpha-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 alpha-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 alpha-tocopherol content. The vitamin E-deficient microsomes, which had no detectable alpha-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 alpha-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 alpha-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 alpha-tocopherol content but to have no other effect on the microsomal lipid peroxidation system.

Ethane production and liver necrosis in rats after administration of drugs and other chemicals

Lipid peroxidation and liver necrosis due to a number of drugs and chemicals were studied. The agents were administered to control, vitamin E-deficient, and selenium-deficient rats. Vitamin E deficiency was documented by a low serum tocopherol level (0.34 mg/100 ml) and selenium deficiency by a specific activity of the selenoenzyme glutathione peroxidase in 105,000g liver supernatant which was approximately 1% of the control value. Glutathione S-transferase specific activity, measured with 1-chloro-2,4-dinitrobenzene as substrate, was doubled by selenium deficiency. Ethane production was measured as the index of in vivo lipid peroxidation. Hepatic necrosis was assessed histologically and by measuring serum glutamic-pyruvic transaminase. When no agents were administered, vitamin E-deficient rats produced more ethane than did control or selenium-deficient rats. Phenobarbital did not cause an increase in ethane production. CCl4 (60 μl/100 g) and BrCCl3 (5 μl/100 g) caused large ethane production and moderately severe liver necrosis. The vitamin E-deficient and selenium-deficient groups given BrCCl3 produced more ethane than the controls but had no more severe hepatic necrosis than the controls. Thioacetamide (5 mmol/kg) and acetaminophen (3 g/kg) caused moderate to severe liver necrosis but only small ethane production. Vitamin E deficiency potentiated acetaminophen hepatotoxicity and selenium deficiency inhibited it. Iodipamide (1.5 mmol/kg) caused very large ethane production and death within hours in vitamin E-deficient rats. At a dose of 2 mmol/kg it caused severe liver necrosis in control rats but only small ethane production. This dose caused no liver necrosis in selenium-deficient rats. Acetylhydrazine (30 mg/kg) caused moderate ethane production in all groups but no liver necrosis. A single dose of ethanol (0.68 ml/100 g) led to small production of ethane in all groups. This study shows that many agents are capable of causing in vivo lipid peroxidation, but that lipid peroxidation does not always correlate with liver necrosis. Selenium deficiency is not a simple glutathione peroxidase deficiency state, as hepatic glutathione S-transferase is increased by it and it protects against acetaminophen and iodipamide hepatotoxicity.

Selenoprotein P associates with endothelial cells in rat tissues

Abstract Selenoprotein P is an extracellular heparin-binding protein that has been implicated in protecting the liver against oxidant injury. Its location in liver, kidney, and brain was determined by conventional immunohistochemistry and confocal microscopy using a polyclonal antiserum. Selenoprotein P is associated with endothelial cells in the liver and is more abundant in central regions than in portal regions. It is also present in kidney glomeruli associated with capillary endothelial cells. Staining of selenoprotein P in the brain is also confined to vascular endothelial cells. The heparin-binding properties of selenoprotein P could be the basis for its binding to tissue. Its localization to the vicinity of endothelial cells is potentially relevant to its oxidant defense function.

Effect of oxygen tension on the generation of alkanes and malondialdehyde by peroxidizing rat liver microsomes

The alkanes, ethane and pentane, are often used as indices of lipid peroxidation. Because it has been indicated that O2 tension can affect the yield of these compounds, a systematic study of this was carried out. Rat liver microsomes were peroxidized using an iron-ascorbate system. The incubations were carried out in sealed flasks at 37 degrees under N2 and various concentrations of O2 up to 100%. Ethane and pentane production were measured by gas chromatography, and malondialdehyde was measured by the thiobarbituric acid reaction. Microsomal fatty acids were measured by gas chromatography. Polyunsaturated fatty acids were lost during lipid peroxidation. There was no loss of saturated or monounsaturated fatty acids. Loss of polyunsaturated fatty acids correlated with O2 tension in the flask. Half-maximal losses of docosahexaenoic acid, arachidonic acid, and linoleic acid occurred at 3, 5, and 35% O2 respectively. Malondialdehyde formation reflected polyunsaturated fatty acid loss at all O2 concentrations. Alkane formation reflected polyunsaturated fatty acid loss below 5% O2 but not above it. The ratio of alkane formed to precursor polyunsaturated fatty acid lost decreased progressively as O2 concentration was increased above 5%. For example, the molar yield of pentane formed per precursor polyunsaturated fatty acid lost was 0.3% at 5% O2 but only 0.003% at 100% O2. This indicates that quantitation of lipid peroxidation using alkane formation requires consideration of O2 tension at the site of alkane formation.

Effect of selenium deficiency on the disposition of plasma glutathione

Selenium deficiency causes increased hepatic synthesis and release of GSH into the blood. The purpose of this study was to examine the effect of selenium deficiency on the disposition of plasma glutathione. Plasma glutathione concentration was 40 +/- 3.4 nmol GSH equivalents/ml in selenium-deficient rats and 17 +/- 5.4 nmol GSH equivalents/ml in control rats. The half-life and systemic clearance of plasma glutathione were found to be the same in selenium-deficient and control rats (t1/2 = 3.4 +/- 0.7 min). Because selenium-deficient plasma glutathione concentration was twice that of control, the determination that selenium deficiency did not affect glutathione plasma systemic clearance indicated that the flux of glutathione through the plasma was doubled by selenium deficiency. It has been proposed that the kidney is responsible for the removal of a major fraction of plasma glutathione. In these studies, renal clearance accounted for 24% of plasma systemic glutathione clearance in controls and 44% in selenium-deficient rats. This indicates that a significant amount of glutathione is metabolized at extrarenal sites, especially in control animals. More than half of the increased plasma glutathione produced in selenium deficiency was removed by the kidney. Thus, selenium deficiency results in a doubling of cysteine transport in the form of glutathione from the liver to the periphery as well as a doubling of plasma glutathione concentration.

Protection by gsh against lipid peroxidation induced by ascorbate and iron in rat liver microsomes

Abstract GSH is considered to be a potent inhibitor of 1ipid peroxidation, but the mechanisms by which it carries out this function are not clear. GSH-dependent factors which inhibit 1ipid peroxidation in the NADPH and in the ascorbate-iron microsomal 1ipid peroxidation systems have been demonstrated in rat liver 105,000 g supernatant (1,2). This communication describes a GSH-dependent factor in the microsomal fraction of rat liver which inhibits ascorbate and iron-induced microsomal 1ipid peroxidation.

Toxicity studies in isolated hepatocytes from selenium-deficient rats and vitamin E-deficient rats.

Isolated hepatocytes from selenium-deficient, vitamin E-deficient, and control rats were treated with cumene hydroperoxide (CuOOH), phorone (diisopropylene acetone), acetaminophen, and diquat. The effect of these chemicals on cell viability, glutathione synthesis and release, and lipid peroxidation as measured by thiobarbituric acid (TBA)-reactive substances was determined during a 4-hr incubation in a complete medium under 95% O2:5% CO2 at 37 degrees C. CuOOH-treated (0.5 mM) selenium-deficient and vitamin E-deficient hepatocytes lost viability sooner than control hepatocytes. Thus, loss of selenium or vitamin E from the hepatocyte resulted in a cell more susceptible to damage by CuOOH. Phorone treatment (1.65 mM) resulted in depletion of intracellular glutathione in all three groups to approximately 20% of that in untreated hepatocytes. Cell viability and TBA-reactive substances were the same in treated and untreated hepatocytes. Thus, lowering of intracellular glutathione did not result in the spontaneous loss of cell viability or increased lipid peroxidation in selenium-deficient or in vitamin E-deficient hepatocytes. Acetaminophen appeared to be less toxic to selenium-deficient hepatocytes than to controls. This finding is in agreement with whole animal studies reported previously showing that selenium deficiency protects rats against acetaminophen hepatotoxicity. A potential explanation of this result is stimulation of glutathione synthesis by selenium deficiency. Severely vitamin E-deficient hepatocytes were protected from cell death by 12.5 and 25.0 mM acetaminophen, apparently by its antioxidant properties, while 50.0 mM acetaminophen was toxic to them. At all concentrations used, acetaminophen decreased the TBA-reactive substances present in the hepatocyte suspensions. Diquat (0.1 mM) caused more rapid cell death and higher levels of TBA-reactive substances in selenium-deficient hepatocytes than in control hepatocytes. Diquat toxicity in selenium-deficient isolated hepatocytes was not as severe as its toxicity in selenium-deficient whole animals, however.

Effect of inhibition of γ-glutamyltranspeptidase by AT-125 (Acivicin) on glutathione and cysteine levels in rat brain and plasma

SummaryAT-125 (Acivicin) is an inhibitor of γ-glutamyltranspeptidase (γ-GTP) which initiates glutathione catabolism to cysteine. We measured plasma and brain glutathione and cysteine in rats treated with AT-125. Six h after AT-125 treatment, plasma glutathione had increased 6-fold and plasma cysteine had fallen significantly. Brain cysteine fell after 24 h of AT-125 treatment, and brain glutathione had also decreased 18%. AT-125 pretreatment inhibited brain uptake of 35S when it was given as 35S-GSH but had no effect when it was given as 35S-cysteine. These results suggest that plasma glutathione is catabolized by γ-GTP, and cysteine derived from it is taken up by the brain. N-acetylcysteine was administered to AT-125 treated rats in an attempt to supply cysteine to the brain in the face of γ-GTP inhibition. N-acetylcysteine supported brain glutathione levels, suggesting that it can serve as a source of cysteine under these conditions.

Non-selenium-dependent glutathione peroxidase activity in rat lung: Association with lung glutathione S-transferase activity and the effects of hyperoxia

To determine if non-selenium-dependent glutathione peroxidase (Non-Se GSH-Px) activity is present in rat lung, we fractionated rat lung soluble fractions from rats fed a selenium-deficient or control diet and measured glutathione peroxidase activity with both cumene hydroperoxide and hydrogen peroxide as substrates. We also measured glutathione S-transferase (GSH S-transferase) activity in the fractions with 1-chloro-2,4-dinitrobenzene as substrate. Non-Se GSH-Px activity was present (about 34% of total GSH-Px activity), and the peak present in the gel filtration chromatogram coeluted with the GSH S-transferase peak. We then measured GSH S-transferase activity in lung-soluble fractions from rats exposed to room air or 85% O2 for 5 days. Lung GSH S-transferase activity was increased in the oxygen-exposed animals when compared to the air-exposed controls. The increase in GSH S-transferase activity could represent the induction of lung non-Se GSH-Px activity.