Nuclear membrane lipid peroxidation products bind to nuclear macromolecules

Ascorbate-Fe2+-driven lipid peroxidation processes in isolated rat liver nuclei give rise to products that bind to DNA and total nuclear proteins. This has been demonstrated by integrating [3H]arachidonic acid into the nuclear membranes. Lipid peroxidation was estimated from the formation of 2-thiobarbituric acid chromophore, and from the relative distribution of 3H-peroxidation products between the lipidic fraction and the nonlipidic fraction of the nuclear suspensions during incubation. The amount of 3H-peroxidation products associated with DNA and total nuclear proteins increased about threefold, when compared to control experiments (no ascorbate-Fe2+), after 180 min of incubation. In contrast, the radioactivity associated with the histone fraction was observed to decrease during incubation. The positive correlation obtained between the formation of thiobarbituric acid chromophore and the association of radioactivity with DNA and nuclear proteins indicates that the binding processes were dependent on peroxidation of the nuclear membrane lipids.     

Studies on lipid peroxidation in rat liver nuclei and isolated nuclear membranes

Non-enzymatic and enzymatically-driven lipid peroxidation processes were studied in rat liver nuclei and isolated nuclear membranes, by evaluating the formation of thiobarbituric acid-chromophore, free malondialdehyde, lipofuscin-like pigments, and the degradation of polyunsaturated fatty acids of the nuclear membrane lipids. The results obtained show that: (1) both non-enzymatic and enzymatically driven lipid peroxidation processes are operative in cell nuclei and isolated nuclear membranes; (2) only for isolated nuclear membranes, a good qualitative and up to a great extent quantitative correlation between malondialdehyde and lipofuscin-like pigment formation was obtained; (3) there is a qualitative but not quantitative correlation between malondialdehyde formation and polyunsaturated fatty acid degradation; (4) lipid peroxidation processes in isolated nuclear membranes and intact nuclei have an essentially identical kinetic behaviour. No statistical differences in the relative increases in the concentrations of malondialdehyde and lipofuscin-like pigments or in the degradation of polyunsaturated fatty acids were obtained, when the two systems were compared, except in the presence of NADPH-ADP-Fe3+, which induced a significantly larger degradation of polyunsaturated fatty acids in isolated nuclear membranes than in intact nuclei, and (5) no malondialdehyde-DNA fluorescent adduct formation was observed in any of the experimental groups studied, as inferred from the characteristics of the fluorescent spectra of lipofuscin-like pigments extracted from incubated nuclear preparations.     

Altered lipid peroxidation and antioxidant potential in human uterine tumors.

A comparative study of lipid peroxidation and antioxidant potential has been made in human uterus and uterine tumor. Two types of uterine tumor used are: tumor (I), a fibroid which is the commonest benign solid tumor in uterus and tumor (II), an adenomyoma. Tumor microsomes are less susceptible to lipid peroxidation induced by both enzymic (NADPH-ADP-Fe3+ and xanthine-xanthine-oxidase) and non-enzymic (ascorbate-Fe2+) systems except in the case of tumor (II) microsomes when induced with xanthine-xanthine oxidase. Resistance of tumor microsomes to lipid peroxidation is associated with the low content of substrates in the form of polyunsaturated fatty acids (PUFAs), higher level of alpha-tocopherol, reduced glutathione and protein thiols and altered enzymic antioxidant potential (catalase and superoxide dismutase).     

Genetic polymorphism of the CYP1A1, CYP2E1, GSTM1 and GSTT1 genes and lung cancer susceptibility in a north Indian population

The present study investigates in a experimental system in vitro the relationship between the non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation in rat liver microsomes and nuclei. Chemiluminescence was measured as cpm/mg protein during 180 min at 37 degrees C. Approximately 50-55% of the fatty acids located in rat liver microsomes and nuclei are polyunsaturated with a prevalence of C18:2 n6 and C20:4 n6. The values of total light emission during the non-enzymatic and enzymatic lipid peroxidation were highest in microsomes than in nuclei. A significant decrease of C20:4 n6 and C22:6 n3 in rat liver microsomes and nuclei was observed during the lipid ascorbate-Fe2+-dependent peroxidation, whereas a significant decrease of C20:4 n6 in rat liver microsomes was observed during enzymatic lipid peroxidation. Over the time course studies, analysis of chemiluminescence in microsomes and nuclei demonstrated that the lipid peroxidation in the presence of ascorbate-Fe2+ reach a maximum at 50 and 30 min, respectively, whereas in the presence of NADPH it reachs a maximum at 20 min in both organelles. In liver microsomes and nuclei the peroxidizability index (pi) which indicates the degree of vulnerability to degradation of a selected membrane showed statistically significant differences between control versus ascorbate-Fe2+ when microsomes or nuclei were compared. Our results indicate that non-enzymatic (ascorbate-Fe2+) and enzymatic (NADPH) lipid peroxidation are operative in rat liver microsomes and nuclei but the sensitivities of both organelles to lipid peroxidation evidenced by chemiluminescence was greater in the presence of ascorbate-Fe2+ when compared with NADPH.     

The effect of ionic strength on the lipid peroxidation of porcine intestinal brush-border membrane vesicles

The effects of salt concentration gradient (inside to outside) on the lipid peroxidation of porcine intestinal brush-border membrane vesicles have been studied and several interesting features of the peroxidation have been elucidated. The addition of dithiothreitol and Fe2+ is far more effective in induction of the lipid peroxidation than any of the other metal ion species tested (Fe3+, Cu2+, Ni2+, Zn2+ and Cr3+). The peroxidation rate of the membrane vesicles induced by dithiothreitol plus Fe2+ was sensitive for the incubation temperature and was increased with increase of the temperature. Imposition of an inward salt concentration gradient on the membrane vesicles preloaded with 300 mM mannitol by addition of 100 mM chloride of K+, Na+, Li+, Rb+, NH4+ or choline to medium produces a very large reduction of the lipid peroxidation induced by dithiothreitol plus Fe2+. The membrane peroxidation is depressed more with the mannitol (300 mM)-preloaded vesicles than with the K2SO4 (100 mM)-preloaded vesicles when they are incubated in medium containing 20-100 mM of K2SO4. Addition of membrane-permeant anions such as SCN- and I-, but not addition of NO3-, to incubation medium has been found to decrease markedly the lipid peroxidation of the mannitol-preloaded vesicles. From these results it is suggested that the lipid peroxidation of the brush-border membranes by addition of dithiothreitol plus Fe2+ is sensitively changed with change in ionic strength.     

Is lipid peroxidation associated with DNA damage?

Specific binding to DNA of lipid peroxidation products was studied in rat hepatocytes labeled with [14C(U)]arachidonic acid after incubation at 37 degrees C either in the absence or in the presence of 200 microM FeSO4. The results obtained show that: (1) production of malondialdehyde-like thiobarbituric-reactive substances occurred in the absence of FeSO4 and was increased, albeit quite variably, by exposure to this pro-oxidant; (2) a low but appreciable binding of radioactivity to DNA and protein was constantly detected in 5 independent experiments; (3) there was no quantitative correlation between malondialdehyde formation and the amount of DNA-bound and protein-bound radioactivity, and any meaningful evidence of a GSH-depletion effect was absent. Taking into account the possible biosynthetic incorporation of radioactivity into DNA, the results of this study must be interpreted with caution, and solely as indicating that in the intact cell the covalent binding to DNA of reactive species generated by lipid peroxidation, if it occurs, should be minimal, corresponding in our experimental conditions approximately to 0.01 mumole of radioactive arachidonic acid per mole nucleotides.     

Binding of manganese in human and rat plasma

Albumin, transferrin and 'transmanganin' have all been proposed as the major Mn-binding ligand in plasma. The present investigations were initiated in order to resolve these discrepancies. Compared to other metals tested (109 Cd2+, 65Zn2+, 59Fe3+), 54Mn2+ bound poorly to purified albumin. The addition of exogenous albumin to plasma did not result in an increased 54Mn radioactivity associated with this protein. Also, incubation of 65Zn-albumin in the presence of excess Mn2+ (1 mM) did not result in the displacement of Zn from albumin or Mn binding. In contrast to these results, 54Mn was bound to purified transferrin, not as readily as Fe3+, but better than Zn2+ or Cd2+. Saturation of transferrin with Fe3+ (1.6 micrograms Fe/mg) prevented the binding of 54Mn indicating that Mn probably binds to Fe-binding sites on the protein. Polyacrylamide gel electrophoresis further demonstrated the association of 54Mn with transferrin rather than with albumin in both human and rat plasma. The amount of 54Mn radioactivity recovered with transferrin increased as incubation time was increased, probably due to oxidation of Mn2+ to Mn3+. Mn binding to transferrin reached a maximum within 5 and 12 h of incubation. About 50% of 54Mn migrated with transferrin, whereas only 5% was associated with albumin. A significant portion (20-55%) of the 54Mn radioactivity migrated with electrophoretically slow plasma components whose identity was not determined. Possibilities include alpha 2-macroglobulin, heavy gamma-globulins and/or heavy lipoproteins.     

The proteasomal system and HNE-modified proteins

Metabolic processes and environmental conditions cause the constant formation of oxidizing species over the lifetime of cells and organisms. This leads to a continuous oxidation of intracellular components, including lipids, DNA and proteins. During the extensively studied process of lipid peroxidation, several reactive low-molecular weight products are formed, including reactive aldehydes as 4-hydroxynonenal (HNE). These aldehydic lipid peroxidation products in turn are able to modify proteins. The degradation of oxidized and oxidatively modified proteins is an essential part of the oxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic and nuclear proteins is the proteasomal system. The proteasomal system by itself is a multicomponent system responsible for the degradation of the majority of intracellular proteins. It has been shown that some, mildly cross-linked, HNE-modified proteins are preferentially degraded by the proteasome, but extensive modification with this cross-linking aldehyde leads to the formation of protein aggregates, that can actually inhibit the proteasome. This review summarizes our knowledge of the interactions between lipid peroxidation products, proteins, and the proteasomal system.     

Lipid peroxidation in rat liver microsomes. II. Response of hydroperoxide formation to iron concentration

When rat liver microsomes were incubated with NADPH, the major products were hydroperoxides which increased with time indicating that endogenous iron content is able to promote lipid peroxidation. The addition of either 5 microM Fe2+ or Fe3+ ions strongly enhanced the hydroperoxide formation rate. However, due to the hydroperoxide breakdown, hydroperoxide concentration decreased with time in this case. Higher ferrous or ferric iron concentration did not change the situation much, in that both hydroperoxide breakdown and formation were similar to those when NADPH only was present in the incubation medium. After lipid peroxidation, analysis of fatty acids indicated that the highest amount of peroxidized PUFA occurred in the presence of 5 microM of either Fe2+ or Fe3+. This analysis also showed that after 8 min incubation with low iron concentration, PUFA depletion was about 77% of that observed after 20 min, whereas without any iron addition or in the presence of 30 microM of either Fe3+, PUFA decrease was only about 37% of that observed after 20 min. As far as the optimum Fe2+/Fe3+ ratio required to promote the initiation of microsomal lipid peroxidation in rat liver is concerned, the highest hydroperoxide formation was observed with a ratio ranging from 0.5 to 2. These results indicate that microsomal lipid peroxidation induced by endogenous iron is speeded up by the addition of low concentrations of either Fe2+ or Fe3+ ions, probably because free radicals generated by hydroperoxide breakdown catalyze the propagation process. In experimental conditions unfavourable to hydroperoxide breakdown the principal process is that of the initiation of lipid peroxidation.     

Oxygen radicals stimulate intracellular proteolysis and lipid peroxidation by independent mechanisms in erythrocytes

Exposure of red blood cells to oxygen radicals can induce hemoglobin damage and stimulate protein degradation, lipid peroxidation, and hemolysis. To determine if these events are linked, rabbit erythrocytes were incubated at 37 degrees C with various oxygen radical-generating systems and antioxidants. Protein degradation, measured by the production of free alanine, increased more than 11-fold in response to xanthine (X) + xanthine oxidase (XO). A similar increase in proteolysis occurred when the cells were incubated with acetaldehyde plus XO, with ascorbic acid plus iron (Asc + Fe), or with hydrogen peroxide (H2O2) alone. Upon addition of XO, increased proteolysis was evident within 5 min and was linear for up to 5 h. In contrast, lipid peroxidation, as shown by the production of malonyldialdehyde, conjugated dienes, or lipid hydroperoxides was observed only after 2 h of incubation with X + XO, acetaldehyde + XO, or H2O2. Ascorbate plus Fe2+ induced both protein degradation and lipid peroxidation; however, the addition of various antioxidants (urate, xanthine, glucose, or butylated hydroxytoluene) decreased lipid peroxidation without affecting proteolysis. Thus, these processes seem to occur by distinct mechanisms. Furthermore, at low concentrations of XO, protein degradation was clearly increased in the absence of detectable lipid peroxidation products. Hemolysis occurred only in a small number of cells (9%) and followed the appearance of lipid peroxidation products. Thus, an important response of red cells to oxygen radicals is rapid degradation of damaged cell proteins. Increased proteolysis seems to occur independently of membrane damage and to be a more sensitive indicator of cell exposure to oxygen radicals than is lipid peroxidation.     

Nicotinamide (vitamin B3) as an effective antioxidant against oxidative damage in rat brain mitochondria

Nicotinamide (vitamin B3) an endogenous metabolite, showed significant inhibition of oxidative damage induced by reactive oxygen species (ROS) generated by ascorbate-Fe2+ and photosensitization systems in rat brain mitochondria. It protected against both protein oxidation and lipid peroxidation, at millimolar concentrations. Inhibition was more pronounced against oxidation of proteins than peroxidation of lipids. Chemically related endogenous compounds, tryptophan and isonicotinic acid, showed comparable inhibitory properties. The protective effect observed, at biologically relevant concentrations, with nicotinamide was more than that of the endogenous antioxidants ascorbic acid and alpha-tocopherol. Hence our studies suggest that nicotinamide (vitamin B3) can be considered as a potent antioxidant capable of protecting the cellular membranes in brain, which is highly susceptible to prooxidants, against oxidative damage induced by ROS.     

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.     

Antioxidant properties of rimantadine in influenza virus infected mice and in some model systems

Influenza virus infection is associated with development of oxidative stress in lung and blood plasma, viz. increase of primary and secondary lipid peroxidation products. It was established that rimantadine treatment led to a decrease of the products of lipid peroxidation in tissues of mice experimentally infected with influenza virus A/Aichi/2/68 (H3N2). The effect is strongest in blood plasma (a decrease of about 50%) and weaker in the lung (about 20%). To elucidate the mechanism of this action of rimantadine, experiments were carried out with some model systems. The capability of rimantadine to scavenge superoxide radicals (scavenging properties) was studied in a system of xanthine-xanthine oxidase to generate superoxide. The amount of superoxide was measured spectrophotometrically by the NBT-test and chemiluminesce. Rimantadine does not show scavenging properties and its antioxidant effect observed in vivo, is not a result of its direct action on the processes of lipid peroxidation and/or interaction with antioxidant enzymes. The antioxidant properties of rimantadine were investigated by measurement of induced lipid peroxidation in a Fe2+ and (Fe2+ - EDTA) system with an egg liposomal suspension. Our findings with model systems do not prove an antioxidant or prooxidant effect of the drug on the processes of lipid peroxidation. Apparently, the observed antioxidant effect of rimantadine in vivo is not connected directly with free radical processes in the organism.     

Antiserum detection of reactive carbonyl species-modified DNA in human colonocytes

Polyunsaturated fats have been linked to occurrences of sporadic colon cancer. One possible cause may be degradation of polyunsaturated fats during cooking, resulting in multiple reactive carbonyl species (RCS) that can damage nuclear DNA and proteins, particularly in rapidly dividing colon crypt cells. This study describes a novel antiserum against RCS-modified DNA, with apparent order of reactivity to DNA modified with 4-hydroxy-trans-2-nonenal > glyoxal > acrolein > crotonaldehyde > malondialdehyde; some reactivity was also observed against conjugated Schiff base-type structures. Anti-(RCS-DNA) antiserum was successfully utilised to demonstrate formation of RCS-DNA in a human colon cell model, exposed to RCS insult derived from endogenous and exogenous lipid peroxidation sources. Further utilisation of the antiserum for immunohistochemical analysis confirmed RCS-modified DNA in crypt areas of 'normal' colon tissue. These results fully support a potential role for dietary lipid peroxidation products in the development of sporadic colon cancer.     

Biosynthesis of proteins by human gastric mucosa in vitro.

Incorporation of L-[U-14C]leucine and of D[U-14C]glucose into proteins of fresh human gastric mucosa in vitro was studied after incubation of homogenized tissue and of intact mucosal pieces. CsCl centrifugation was used to separate high-density mucus glycoproteins from other mucosal proteins, and the macromolecular nature of radioactive mucosal glycoprotein fractions was confirmed by SDS/polyacrylamide-gel electrophoresis and autoradiography of the polyacrylamide gels. In all experiments a substantial proportion of total incorporated radioactivity was associated with gastric-mucosal glycoprotein fractions (CsCl fraction L3), indicating their biosynthesis. Radioactivity of these fractions was shown to co-chromatograph with carbohydrates when fractionated either directly or after reduction and alkylation (1) Sephadex G-200 chromatography in the excluded fractions and (2) by DEAE-cellulose ion-exchange chromatography. On incubation of intact mucosa, the major portion of radioactivity associated with the glycoprotein fractions of both leucine- and glucose-labelled specimens was secreted into the mucosal media during the course of the experiment. It is suggested that biosynthesis of mucus in vivo by gastric mucosa may be associated with rapid secretion of the synthesized macromolecules into the lumen of the stomach and that investigations of the metabolic processes within the mucosa should consider the products of secretion of the tissue. Incorporation of L-[U-14C]leucine implies biosynthesis of the polypeptide components of the macromolecules.     

OxyR regulon controls lipid peroxidation-mediated oxidative stress in Escherichia coli

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. The oxyR gene product regulates the expression of enzymes and proteins that are needed for cellular protection against oxidative stress. Upon exposure to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the Escherichia coli oxyR overexpression mutant was much more resistant to lipid peroxidation-mediated cellular damage, when compared to the OxyR deletion mutant in regard to growth kinetics, viability, and DNA damage. The deletion of the OxyR gene in E. coli also resulted in increased susceptibility of superoxide dismutase to lipid peroxidation-mediated inactivation. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in free radical-induced cellular damage. Also, the oxyR regulon plays an important protective role in lipid peroxidation-mediated cellular damage.     

UVA-induced oxidative damage to rat liver nuclei: reduction of iron ions and the relationship between lipid peroxidation and DNA damage

Lipid peroxidation and DNA damage and the relationship between the two events were studied in rat liver nuclei irradiated with low dose UVA. Lipid peroxidation was measured as thiobarbituric acid-reactive substances (TBARS) by spectrophotometric method and as malondialdehyde-TBA adduct by HPLC, and DNA damage was measured as 8-hydroxy-deoxyguanosine (8-OH-dGu) and strand breakage (or loss of double-stranded DNA) by a fluorometric analysis of alkaline DNA unwinding method. The results show that UVA irradiation by itself increased nuclear lipid peroxidation but caused little or no DNA strand breakage or 8-OH-dGu. When 0.5 mM ferric (Fe+3) or ferrous (Fe+2) ions were added to the nuclei during UVA irradiation, lipid peroxidation and DNA damage, measured both as 8-OH-dGu and loss of double-stranded DNA, were strongly enhanced. Lipid peroxidation occurred concurrently with the appearance of 8-OH-dGu. Fe3+ ions were reduced to Fe2+ in this UVA/Fe2+/nuclei system. Lipid peroxidation and DNA damage were neither inhibited by scavengers of hydroxyl radical and singlet oxygen nor inhibited by superoxide dismutase and catalase. Inclusion of EDTA or chain-breaking antioxidants, butylated hydroxytoluene (BHT) and diphenylamine (an alkoxy radical scavenger), inhibited lipid peroxidation but not the level of 8-OH-dGu. BHT also did not inhibit the loss of double-stranded DNA in this system. This study demonstrates the reduction of exogenous Fe+3 by UVA when added to rat liver nuclei, and, as a result, oxidative damage is strongly enhanced. In addition, the results show that DNA damage is not a result of lipid peroxidation in this UVA/Fe2+/nuclei system.     

Identification of metabolic pathways of the lipid peroxidation product 4-hydroxynonenal by enterocytes of rat small intestine.

The cytotoxic lipid peroxidation product 4-hydroxynonenal (HNE1) is rapidly metabolized in enterocytes. The degradation of HNE and other aldehydic products of lipid peroxidation processes seems to be an antioxidative defense system. The metabolism of HNE was studied in suspensions of rat enterocytes at 37 degrees C, pH 7.4 and at initial HNE concentration of 100 microM. About 70% of the HNE were degraded within three minutes of incubation. Main products of HNE which were identified in enterocytes were the glutathione-HNE-1:1-conjugate, the hydroxynonenoic acid and the 1,4-dihydroxynonene. Furthermore, the formation of metabolites of the tricarboxylic acid cycle is suggested. The quantitative share of HNE binding to proteins was low with about 1% of total HNE consumption after three minutes of incubation.     

Oxalomalate, a competitive inhibitor of NADP+ -dependent isocitrate dehydrogenase, regulates lipid peroxidation-mediated apoptosis in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Recently, we demonstrated that the control of cytosolic redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic NADP⁺-dependent isocitrate dehydrogenase (IDPc) through to supply NADPH for antioxidant systems. The protective role of IDPc against lipid peroxidation-mediated apoptosis in U937 cells was investigated in control and cells pre-treated with oxlalomalate, a competitive inhibitor of IDPc. Upon exposure to 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the susceptibility to apoptosis was higher in oxalomalate-treated cells as compared to control cells. The results suggest that IDPc plays an important protective role in apoptosis of U937 cells induced by lipid peroxidation-mediated oxidative stress.     

Oxalomalate,a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase,enhances lipid peroxidation-mediated oxidative damage in U937 cells

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. Cytosolic NADP+-dependent isocitrate dehydrogenase (ICDH) in U937 cells produces NADPH, an essential reducing equivalent for the antioxidant system. The protective role of ICDH against lipid peroxidation-mediated oxidative damage in U937 cells was investigated in control cells pre-treated with oxalomalate, a competitive inhibitor of ICDH. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) to U937 cells, which induces lipid peroxidation in membranes, the viability was lower and the protein oxidation, lipid peroxidation, and oxidative DNA damage, reflected by an increase in 8-hydroxy-2'-deoxyguanosine, were higher in oxalomalate-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2',7'-dichlorodihydrofluorescin, as well as the significant decrease in the intracellular GSH level in oxalomalate-treated U937 cells upon exposure to AAPH. These results suggest that ICDH plays an important role as an antioxidant enzyme in cellular defense against lipid peroxidation-mediated oxidative damage through the removal of reactive oxygen species.