Protective action of phospholipid hydroperoxide glutathione peroxidase against membrane-damaging lipid peroxidation. In situ reduction of phospholipid and cholesterol hydroperoxides

The general reactivity of membrane lipid hydroperoxides (LOOHs) with the selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX) has been investigated. When human erythrocyte ghosts (lipid content: 60 wt % phospholipid; 25 wt % cholesterol) were treated with GSH/PHGPX subsequent to rose bengal-sensitized photoperoxidation, iodometrically measured LOOHs were totally reduced to alcohols. Similar treatment with the classic glutathione peroxidase (GPX) produced no effect unless the peroxidized membranes were preincubated with phospholipase A2 (PLA2). However, under these conditions, no more than approximately 60% of the LOOH was reduced; introduction of PHGPX brought the reaction to completion. Thin layer chromatographic analyses revealed that the GPX-resistant (but PHGPX-reactive) LOOH was cholesterol hydroperoxide (ChOOH) consisting mainly of the 5 alpha (singlet oxygen-derived) product. Membrane ChOOHs were reduced by GSH/PHGPX to species that comigrated with borohydride reduction products (diols). Sensitive quantitation of PHGPX-catalyzed ChOOH reduction was accomplished by using [14C]cholesterol-labeled ghosts. Kinetic analyses indicated that the rate of ChOOH decay was approximately 1/6 that of phospholipid hydroperoxide decay. Photooxidized ghosts underwent a large burst of free radical-mediated lipid peroxidation when incubation with ascorbate/iron or xanthine/xanthine oxidase/iron. These reactions were only partially inhibited by PLA2/GSH/GPX treatment, but totally inhibited by GSH/PHGPX treatment, consistent with complete elimination of LOOHs in the latter case. These findings provide important clues as to how ChOOHs are detoxified in cells and add new insights into PHGPX's protective role.     

Enzymatic reduction of phospholipid and cholesterol hydroperoxides in artificial bilayers and lipoproteins

Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxidized liposomes underwent a burst of thiobarbituric acid-detectable lipid peroxidation which could be inhibited by prior GSH/PHGPX treatment, but not by GSH/GPX treatment. Additional experiments indicated that hydroperoxides of phosphatidylcholine, cholesterol and cholesteryl esters in low-density lipoprotein are also good substrates for PHGPX. An important role of PHGPX in cellular detoxification of a wide variety of LOOHs in membranes and internalized lipoproteins is suggested from these findings.     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.     

Hyperresistance to cholesterol hydroperoxide-induced peroxidative injury and apoptotic death in a tumor cell line that overexpresses glutathione peroxidase isotype-4.

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPX; GPX4) plays a key role in eukaryotic defense against potentially lethal peroxidative injury and also regulation of physiological peroxide tone. In this work we focused on the cytoprotective antiperoxidant effects of GPX4, using a breast tumor epithelial cell line that over-expresses the enzyme. Wild-type COH-BR1 cells, which exhibit little (if any) GPX4 activity, were transfected with a construct encoding the mitochondrion-targeted long (L) form of the enzyme. Several transfectant clones were selected which expressed relatively large amounts of GPX4, as determined by both Northern and Western analysis. Enzyme activity ranged from 15-fold to 190-fold greater than that of wild-type or null-transfected cells. The functional ramifications of GPX4 overexpression were tested by challenging cells with photochemically generated cholesterol hydroperoxides (ChOOHs) in liposomal form. Compared with vector controls, overexpressing clones were found to be substantially more resistant to ChOOH-induced killing, as determined by annexin-V (early apoptotic) and thiazolyl blue (mitochondrial dehydrogenase) reactivity. Concomitantly, the clones exhibited a striking hyper-resistance to free radical-mediated lipid peroxidation, as assessed by labeling cell membranes with [(14)C]cholesterol and measuring a family of radiolabeled oxidation products (ChOX). L-form GPX4's antiperoxidant and cytoprotective effects could reflect its ability to detoxify ChOOHs as they enter cells and/or cell-derived lipid hydroperoxides arising from ChOOH one-electron turnover.     

Lethal damage to murine L1210 cells by exogenous lipid hydroperoxides: protective role of glutathione-dependent selenoperoxidases

The effect of selenium deprivation on the viability of murine L1210 cells exposed to various exogenous lipid hydroperoxides has been investigated. Selenoperoxidase activities of cells grown for longer than 1 week in 1% serum with no added selenium [Se(-) cells] were less than 10% of the activities of selenium-satisfied controls [Se(+) cells] or selenium-repleted counterparts [Se(-/+) cells]. The enzymes measured were classical glutathione peroxidase (GPX) and phospholipid hydroperoxide glutathione peroxidase (PHGPX). Se(-) cells exhibited a compensatory increase in catalase activity. Dye exclusion and clonal survival assays indicated that Se(-) and Se(+) cells were relatively insensitive to photochemically generated phospholipid hydroperoxides in liposomal form. However, both cell types were sensitive to liposomal cholesterol hydroperoxides, e.g., 7-hydroperoxycholesterol (7-OOH), Se(-) being much more so (LD50 approximately 10 microM) than Se(+) (LD50 approximately 75 microM). By contrast, 7-hydroxycholesterol over a comparable concentration range was minimally toxic to Se(-) and Se(+) cells. Cell killing by 7-OOH was inhibited by desferrioxamine and by butylated hydroxytoluene, suggesting that iron-mediated free radical reactions are involved. The involvement of glutathione in cytoprotection was confirmed by showing that Se(+) cells were more sensitive to 7-OOH after treating with buthionine sulfoximine, an inhibitor of GSH synthesis. Cellular detoxification of 7-OOH is provisionally attributed to PHGPX rather than GPX, since 7-OOH and other cholesterol hydroperoxides were found to be good substrates for PHGPX in a cell free system, but were unreactive with GPX.     

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase

The reduction of membrane-bound hydroperoxides is a major factor acting against lipid peroxidation in living systems. This paper presents the characterization of the previously described 'peroxidation-inhibiting protein' as a 'phospholipid hydroperoxide glutathione peroxidase'. The enzyme is a monomer of 23 kDa (SDS-polyacrylamide gel electrophoresis). It contains one gatom Se/22 000 g protein. Se is in the selenol form, as indicated by the inactivation experiments in the presence of iodoacetate under reducing conditions. The glutathione peroxidase activity is essentially the same on different phospholipids enzymatically hydroperoxidized by the use of soybean lipoxidase (EC 1.13.11.12) in the presence of deoxycholate. The kinetic data are compatible with a tert-uni ping-pong mechanism, as in the case of the 'classical' glutathione peroxidase (EC 1.11.1.9). The second-order rate constants (K1) for the reaction of the enzyme with the hydroperoxide substrates indicate that, while H2O2 is reduced faster by the glutathione peroxidase, linoleic acid hydroperoxide is reduced faster by the present enzyme. Moreover, the phospholipid hydroperoxides are reduced only by the latter. The dramatic stimulation exerted by Triton X-100 on the reduction of the phospholipid hydroperoxides suggests that this enzyme has an 'interfacial' character. The similarity of amino acid composition, Se content and kinetic mechanism, relative to the difference in substrate specificity, indicates that the two enzymes 'classical' glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are in some way related. The latter is apparently specialized for lipophylic, interfacial substrates.     

The yeast glutaredoxins are active as glutathione peroxidases

The yeast Saccharomyces cerevisiae contains two glutaredoxins, encoded by GRX1 and GRX2, which are active as glutathione-dependent oxidoreductases. Our studies show that changes in the levels of glutaredoxins affect the resistance of yeast cells to oxidative stress induced by hydroperoxides. Elevating the gene dosage of GRX1 or GRX2 increases resistance to hydroperoxides including hydrogen peroxide, tert-butyl hydroperoxide and cumene hydroperoxide. The glutaredoxin-mediated resistance to hydroperoxides is dependent on the presence of an intact glutathione system, but does not require the activity of phospholipid hydroperoxide glutathione peroxidases (GPX1-3). Rather, the mechanism appears to be mediated via glutathione conjugation and removal from the cell because it is absent in strains lacking glutathione-S-transferases (GTT1, GTT2) or the GS-X pump (YCF1). We show that the yeast glutaredoxins can directly reduce hydroperoxides in a catalytic manner, using reducing power provided by NADPH, GSH, and glutathione reductase. With cumene hydroperoxide, high pressure liquid chromatography analysis confirmed the formation of the corresponding cumyl alcohol. We propose a model in which the glutathione peroxidase activity of glutaredoxins converts hydroperoxides to their corresponding alcohols; these can then be conjugated to GSH by glutathione-S-transferases and transported into the vacuole by Ycf1.     

GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis

Oxygen is necessary for aerobic metabolism but can cause the harmful oxidation of lipids and other macromolecules. Oxidation of cholesterol and phospholipids containing polyunsaturated fatty acyl chains can lead to lipid peroxidation, membrane damage, and cell death. Lipid hydroperoxides are key intermediates in the process of lipid peroxidation. The lipid hydroperoxidase glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid alcohols, and this process prevents the iron (Fe²⁺)‐dependent formation of toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to lipid peroxidation and can result in the induction of ferroptosis, an iron‐dependent, non‐apoptotic form of cell death. This review describes the formation of reactive lipid species, the function of GPX4 in preventing oxidative lipid damage, and the link between GPX4 dysfunction, lipid oxidation, and the induction of ferroptosis.     

Possible pathways of lipid peroxide reduction in the liver and their intracellular localization

Lipid peroxidase activity in rat liver was studied. Rat liver cytosolic fraction was found to be capable of reducing lipid hydroperoxides. On the contrary, no lipid hydroperoxide reduction was observed in microsomes. It was found that at least two proteins in rat liver cytosol are capable of reducing phospholipid hydroperoxides. One of them is precipitated by 33-55% at (NH4)2SO4 saturation and requires reduced glutathione (GSH) as a hydrogen donor, while the other one is precipitated by 55-80% at (NH4)2SO4 saturation and reduces phospholipid hydroperoxides in the presence of a unidentified low molecular weight cytosolic factor, but not GSH or NADPH.     

Different effects of Triton X-100, deoxycholate, and fatty acids on the kinetics of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase

The effects of Triton X-100, deoxycholate, and fatty acids were studied on the two steps of the ping-pong reaction catalyzed by Se-dependent glutathione peroxidases. The study was carried out by analyzing the single progression curves where the specific glutathione oxidation was monitored using glutathione reductase and NADPH. While the "classic" glutathione peroxidase was inhibited only by Triton, the newly discovered "phospholipid hydroperoxide glutathione peroxidase" was inhibited by deoxycholate and by unsaturated fatty acids. The kinetic analysis showed that in the case of glutathione peroxidase only the interaction of the lipophilic peroxidic substrate was hampered by Triton, indicating that the enzyme is not active at the interface. Phospholipid hydroperoxide glutathione peroxidase activity measured with linoleic acid hydroperoxide as substrate, on the other hand, was not stimulated by the Triton concentrations which have been shown to stimulate the activity on phospholipid hydroperoxides. Furthermore a slight inhibition was apparent at high Triton concentrations and the effect could be attributed to a surface dilution of the substrate. Deoxycholate and unsaturated fatty acids were not inhibitory on glutathione peroxidase but inhibited both steps of the peroxidic reaction of phospholipid hydroperoxide glutathione peroxidase, in the presence of either amphiphilic or hydrophilic substrates. This inhibition pattern suggests an interaction of anionic detergents with the active site of this enzyme. These results are in agreement with the different roles played by these peroxidases in the control of lipid peroxide concentrations in the cells. While glutathione peroxidase reduces the peroxides in the water phase (mainly hydrogen peroxide), the new peroxidase reduces the amphyphilic peroxides, possibly at the water-lipid interface.     

Phospholipid hydroperoxide glutathione peroxidase: specific activity in tissues of rats of different age and comparison with other glutathione peroxidases

The tissue distribution of phospholipid hydroperoxide glutathione peroxidase (PHGPX) was studied in rats of different ages. In the same samples the activities of Se-dependent glutathione peroxidase (GPX), and non-Se-dependent glutathione peroxidase (non Se-GPX) were also determined using specific substrates for each enzyme. Enzymatically generated phospholipid hydroperoxides were used as substrate for PHGPX, hydrogen peroxide for GPX, and cumene hydroperoxide for non-Se-GPX (after correction for the activity of GPX on this substrate). PHGPX specific activity in different organs is as follows: liver = kidney greater than heart = lung = brain greater than muscle. Furthermore, this activity is reasonably constant in different age groups, with a lower specific activity observed only in kidney and liver of young animals. GPX activity is expressed as follows: liver greater than kidney greater than heart greater than lung greater than brain = muscle, and substantial age-dependent differences have been observed (adult greater than old greater than young). Non-Se-GPX activity was present in significant amount only in liver greater than lung greater than heart and only in adult animals. These results suggest a tissue- and age-specific expression of different peroxidases.     

The putative glutathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase

A putative glutathione peroxidase gene (Swiss-Prot accession number Z 68200) of Plasmodium falciparum, the causative agent of tropical malaria, was expressed in Escherichia coli and purified to electrophoretic homogeneity. Like phospholipid hydroperoxide glutathione peroxidase of mammals, it proved to be monomeric. It was active with H(2)O(2) and organic hydroperoxides but, unlike phospholipid hydroperoxide glutathione peroxidase, not with phosphatidylcholine hydroperoxide. With glutathione peroxidases it shares the ping-pong mechanism with infinite V(max) and K(m) when analyzed with GSH as substrate. As a homologue with selenocysteine replaced by cysteine, its reactions with hydroperoxides and GSH are 3 orders of magnitude slower than those of the selenoperoxidases. Unexpectedly, the plasmodial enzyme proved to react faster with thioredoxins than with GSH and most efficiently with thioredoxin of P. falciparum (Swiss-Prot accession number 202664). It is therefore reclassified as thioredoxin peroxidase. With plasmodial thioredoxin, the enzyme also displays ping-pong kinetics, yet with a limiting K(m) of 10 microm and a k(1)' of 0.55 s(-)1. The apparent k(1)' for oxidation with cumene, t-butyl, and hydrogen peroxides are 2.0 x 10(4) m(-1) s(-1), 3.3 x 10(3) m(-1) s(-1), and 2.5 x 10(3) m (-1) s(-1), respectively. k(2)' for reduction by autologous thioredoxin is 5.4 x 10(4) m(-1) s(-1) (21.2 m(-1) s(-1) for GSH). The newly discovered enzymatic function of the plasmodial gene product suggests a reconsideration of its presumed role in parasitic antioxidant defense.     

Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases.

The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild-type S. cerevisiae and was diminished in extracts from gpx1 Delta, gpx2 Delta, and gpx3 Delta deletion mutants; PHGPx activity was almost absent in a gpx1 Delta/gpx2 Delta/gpx3 Delta triple mutant. Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1 Delta/gpx2 Delta/gpx3 Delta mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This sensitivity to 18:3 was more marked than sensitivity to H(2)O(2). Unlike H(2)O(2) toxicity, delayed toxicity of 18:3 toward gpx1 Delta/gpx2 Delta/gpx3 Delta cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with alpha-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress. This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.     

Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes

For a long time lipid peroxidation has only been considered a deleterious process leading to disruption of biomembranes and thus, to cellular dysfunction. However, when restricted to a certain cellular compartment and tightly regulated, lipid peroxidation may have beneficial effects. Early on during evolution of living organisms special lipid peroxidizing enzymes, called lipoxygenases, appeared and they have been conserved during phylogenesis of plants and animals. In fact, a diverse family of lipoxygenase isoforms has evolved starting from a putative ancient precursor. As with other enzymes, lipoxygenases are regulated on various levels of gene expression and there are endogenous antagonists controlling their cellular activity. Among the currently known mammalian lipoxygenase isoforms only 12/15-lipoxygenases are capable of directly oxygenating ester lipids even when they are bound to membranes and lipoproteins. Thus, these enzymes represent the pro-oxidative part in the cellular metabolism of complex hydroperoxy ester lipids. Its metabolic counterplayer, representing the antioxidative part, appears to be the phospholipid hydroperoxide glutathione peroxidase. This enzyme is unique among glutathione peroxidases because of its capability of reducing ester lipid hydroperoxides. Thus, 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase constitute a pair of antagonizing enzymes in the metabolism of hydroperoxy ester lipids, and a balanced regulation of the two proteins appears to be of major cell physiological importance. This review is aimed at summarizing the recent developments in the enzymology and molecular biology of 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase, with emphasis on cytokine-dependent regulation and their regulatory interplay.     

Lipid peroxidation and haemoglobin degradation in red blood cells exposed to t-butyl hydroperoxide. The relative roles of haem- and glutathione-dependent decomposition of t-butyl hydroperoxide and membrane lipid hydroperoxides in lipid peroxidation and haemolysis

Red cells exposed to t-butyl hydroperoxide undergo lipid peroxidation, haemoglobin degradation and hexose monophosphate-shunt stimulation. By using the lipid-soluble antioxidant 2,6-di-t-butyl-p-cresol, the relative contributions of t-butyl hydroperoxide and membrane lipid hydroperoxides to oxidative haemoglobin changes and hexose monophosphate-shunt stimulation were determined. About 90% of the haemoglobin changes and all of the hexose monophosphate-shunt stimulation were caused by t-butyl hydroperoxide. The remainder of the haemoglobin changes appeared to be due to reactions between haemoglobin and lipid hydroperoxides generated during membrane peroxidation. After exposure of red cells to t-butyl hydroperoxide, no lipid hydroperoxides were detected iodimetrically, whether or not glucose was present in the incubation. Concentrations of 2,6-di-t-butyl-p-cresol, which almost totally suppressed lipid peroxidation, significantly inhibited haemoglobin binding to the membrane but had no significant effect on hexose monophosphate shunt stimulation, suggesting that lipid hydroperoxides had been decomposed by a reaction with haem or haem-protein and not enzymically via glutathione peroxidase. The mechanisms of lipid peroxidation and haemoglobin oxidation and the protective role of glucose were also investigated. In time-course studies of red cells containing oxyhaemoglobin, methaemoglobin or carbonmono-oxyhaemoglobin incubated without glucose and exposed to t-butyl hydroperoxide, haemoglobin oxidation paralleled both lipid peroxidation and t-butyl hydroperoxide consumption. Lipid peroxidation ceased when all t-butyl hydroperoxide was consumed, indicating that it was not autocatalytic and was driven by initiation events followed by rapid propagation and termination of chain reactions and rapid non-enzymic decomposition of lipid hydroperoxides. Carbonmono-oxyhaemoglobin and oxyhaemoglobin were good promoters of peroxidation, whereas methaemoglobin relatively spared the membrane from peroxidation. The protective influence of glucose metabolism on the time course of t-butyl hydroperoxide-induced changes was greatest in carbonmono-oxyhaemoglobin-containing red cells followed in order by oxyhaemoglobin- and methaemoglobin-containing red cells. This is the reverse order of the reactivity of the hydroperoxide with haemoglobin, which is greatest with methaemoglobin. In studies exposing red cells to a wide range of t-butyl hydroperoxide concentrations, haemoglobin oxidation and lipid peroxidation did not occur until the cellular glutathione had been oxidized. The amount of lipid peroxidation per increment in added t-butyl hydroperoxide was greatest in red cells containing carbonmono-oxyhaemoglobin, followed in order by oxyhaemoglobin and methaemoglobin. Red cells containing oxyhaemoglobin and carbonmono-oxyhaemoglobin and exposed to increasing concentrations of t-butyl hydroperoxide became increasingly resistant to lipid peroxidation as methaemoglobin accumulated, supporting a relatively protective role for methaemoglobin. In the presence of glucose, higher levels of t-butyl hydroperoxide were required to induce lipid peroxidation and haemoglobin oxidation compared with incubations without glucose. Carbonmono-oxyhaemoglobin-containing red cells exposed to the highest levels of t-butyl hydroperoxide underwent haemolysis after a critical level of lipid peroxidation was reached. Inhibition of lipid peroxidation by 2,6-di-t-butyl-p-cresol below this critical level prevented haemolysis. Oxidative membrane damage appeared to be a more important determinant of haemolysis in vitro than haemoglobin degradation. The effects of various antioxidants and free-radical scavengers on lipid peroxidation in red cells or in ghosts plus methaemoglobin exposed to t-butyl hydroperoxide suggested that red-cell haemoglobin decomposed the hydroperoxide by a homolytic scission mechanism to t-butoxyl radicals.     

Rat liver cytosolic glutathione peroxidase: reactivity with linoleic acid hydroperoxide and cumene hydroperoxide.

The reactivity of rat liver glutathione (GSH) peroxidase with two hydroperoxides was determined using integrated rate equations. The bimolecular rate constant for the reaction of GSH peroxidase with linoleic acid hydroperoxide is approximately four times the rate constant with cumene hydroperoxide. The reactivity toward reduced glutathione is not altered by different hydroperoxides. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for lipid hydroperoxide in rat liver is approximated at 9.5 × 10^?5 min.     

The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress.

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human alpha-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (kcat/Km) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s-1 mM-1, respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s-1 mM-1, respectively. Immunotitration studies with human liver extracts showed that the antibodies against human alpha-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the alpha-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetraenoic acid, an important intermediate in the 5-lipoxygenase pathway.     

Hydroperoxide-metabolizing systems in rat liver.

1. Metabolism of added hydroperoxides was studied in hemoglobin-free perfused rat liver and in isolated rat hepatocytes as well as microsomal and mitochondrial fractions. 2. Perfused liver is capable of removing organic hydroperoxides [cumene and tert-butyl hydroperoxide] at rates up to 3--4 mumol X min-1 X gram liver-1. Concomitantly, there is a release of glutathione disulfide (GSSG) into the extracellular space in a relationship approx. linear with hydroperoxide infusion rates. About 30 nmol GSSG are released per mumol hydroperoxide added per min per gram liver. GSSG release is interpreted to indicate GSH peroxidase activity. 3. GSSG release is observed also with added H2O2. At rates of H2O2 infusion of about 1.5 mumol X min-1 X gram liver-1 a maximum of GSSG release is attained which, however, can be increased by inhibition of catalase with 3-amino-1,2,4-aminotriazole. 4. A contribution of the endoplasmic reticulum in addition to glutathione peroxidase in organic hydroperoxide removal is demonstrated (a) by comparison of perfused livers from untreated and phenobarbital-pretreated rats and (b) in isolated microsomal fractions, and a possible involvement of reactive iron species (e.g. cytochrome P-450-linked peroxidase activity) is discussed. 5. Hydroperoxide addition to microsomes leads to rapid and substantial lipid peroxidation as evidenced by formation of thiobarbituric-acid-reactive material (presumably malondialdehyde) and by O2 uptake. Like in other types of induction of lipid peroxidation, malondialdehyde/O2 ratios of 1/20 are observed. Cumene hydroperoxide (0.6 mM) gives rise to 4-fold higher rates of malondialdehyde formation than tert-butyl hydroperoxide (1 mM). Ethylenediamine tetraacetate does not inhibit this type of lipid peroxidation. 6. Lipid peroxidation in isolated hepatocytes upon hydroperoxide addition is much lower than in isolated microsomes or mitochondria, consistent with the presence of effective hydroperoxide-reducing systems. However, when NADPH is oxidized to the maximal extent as evidenced by dual-wavelength spectrophotometry, lipid peroxidation occurs at large amounts. 7. A dependence of hydroperoxide removal rates upon flux through the pentose phosphate pathway is suggested by a stimulatory effect of glucose in hepatocytes from fasted rats and by an increased rate of 14CO2 release from [1-14C]glucose during hydroperoxide metabolism in perfused liver.     

The selenoprotein GPX4 is essential for mouse development and protects from radiation and oxidative damage insults

Lipid peroxidation has been implicated in a variety of pathophysiological processes, including inflammation, atherogenesis, neurodegeneration, and the ageing process. Phospholipid hydroperoxide glutathione peroxidase (GPX4) is the only major antioxidant enzyme known to directly reduce phospholipid hydroperoxides within membranes and lipoproteins, acting in conjunction with alpha tocopherol (vitamin E) to inhibit lipid peroxidation. Here we describe the generation and characterization of GPX4-deficient mice by targeted disruption of the murine Gpx4 locus through homologous recombination in embryonic stem cells. Gpx4(-/-) embryos die in utero by midgestation (E7.5) and are associated with a lack of normal structural compartmentalization. Gpx4(+/-) mice display reduced levels of Gpx4 mRNA and protein in various tissues. Interestingly, cell lines derived from Gpx4(+/-) mice are markedly sensitive to inducers of oxidative stress, including gamma-irradiation, paraquat, tert-butylhydroperoxide, and hydrogen peroxide, as compared to cell lines derived from wild-type control littermates. Gpx4(+/-) mice also display reduced survival in response to gamma-irradiation. Our observations establish GPX4 as an essential antioxidant enzyme in mice and suggest that it performs broad functions as a component of the mammalian antioxidant network.