Regulation of 4-hydroxynonenal-mediated signaling by glutathione S-transferases

4-Hydroxynonenal (HNE), one of the major end products of lipid peroxidation, has been shown to be involved in signal transduction and available evidence suggests that it can affect cell cycle events in a concentration-dependent manner. Glutathione S-transferases (GSTs) can modulate the intracellular concentrations of HNE by affecting its generation during lipid peroxidation by reducing hydroperoxides and also by converting it into a glutathione conjugate. We have recently demonstrated that overexpression of the Alpha class GSTs in cells leads to lower steady-state levels of HNE, and these cells acquire resistance to apoptosis induced by lipid peroxidation-causing agents such as H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics, suggesting that signaling for apoptosis by these agents is transduced through HNE. Cells with the capacity to exclude HNE from the intracellular environment at a faster rate are relatively more resistant to apoptosis caused by H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics as well as by HNE, suggesting that HNE may be a common denominator in mechanisms of apoptosis caused by oxidative stress. We have also shown that transfection of adherent cells with HNE-metabolizing GSTs leads to transformation of these cells due to depletion of HNE. These recent studies from our laboratories, which strongly suggest that HNE is a key signaling molecule and that GSTs, being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.     

Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth

Products of lipid peroxidation such as 4-hydroxy-trans-2-nonenal (HNE) trigger multiple signaling cascades that variably affect cell growth, differentiation, and apoptosis. Because glutathiolation is a significant metabolic fate of these aldehydes, we tested the possibility that the bioactivity of HNE depends upon its conjugation with glutathione. Addition of HNE or the cell-permeable esters of glutathionyl-4-hydroxynonenal (GS-HNE) or glutathionyl-1,4-dihydroxynonene (GS-DHN) to cultures of rat aortic smooth muscle cells stimulated protein kinase C, NF-kappaB, and AP-1, and increased cell growth. The mitogenic effects of HNE, but not GS-HNE or GS-DHN, were abolished by glutathione depletion. Pharmacological inhibition or antisense ablation of aldose reductase (which catalyzes the reduction of GS-HNE to GS-DHN) prevented protein kinase C, NF-kappaB, and AP-1 stimulation and the increase in cell growth caused by HNE and GS-HNE, but not GS-DHN. The growth stimulating effect of GS-DHN was enhanced in cells treated with antibodies directed against the glutathione conjugate transporters RLIP76 (Ral-binding protein) or the multidrug resistance protein-2. Overexpression of RLIP76 abolished the mitogenic effects of HNE and its glutathione conjugates, whereas ablation of RLIP76 using RNA interference promoted the mitogenic effects. Collectively, our findings suggest that the mitogenic effects of HNE are mediated by its glutathione conjugate, which has to be reduced by aldose reductase to stimulate cell growth. These results raise the possibility that the glutathione conjugates of lipid peroxidation products are novel mediators of cell signaling and growth.     

4-hydroxynonenal as a bioactive marker of pathophysiological processes

The review is focused on the currently major aspect of 4-hydroxynonenal (HNE) research--studies that combine biological activities of the aldehyde together with the methods of its identification in cells and tissues. Because there were some excellent reviews on HNE published in recent years, starting in 1990 and 1991 with supreme reviews done by Hermann Esterbauer, who discovered the aldehyde, and colleagues from the Institute of Biochemistry in Graz, this article pays most of attention to the most recent articles, published in the last 15 months. Additionally, an overview on the relevance of HNE is given with respect to the research and publication trends in the period of 10 years (1993-2002) according to the data in the Current Contents and Medline data bases. It is obvious that HNE started in 1993 as a "toxic product of lipid peroxidation" and "second toxic messenger of free radicals", to become in 2002 a reliable marker of oxidative stress, a possible causative agent of several diseases (such as Alzheimer's disease), growth modulating factor and a signaling molecule. Novel analytical methods developed suitable pathways for HNE to become a clinically applicable marker of lipid peroxidation on one side and on the other a standardized parameter of food quality control. As it is also present physiologically in various cells and tissues, it is likely that HNE will soon become one of the most attractive factors for those who search for a small and reactive molecular link between genomics and proteomics.     

4-Hydroxy-2,3-nonenal as a signal for cell function and differentiation.

4-Hydroxy-2,3-nonenal (HNE) is a biologically active aldehydic end product of oxidative decomposition of omega-3 and omega-6 polyunsaturated fatty acids of membrane phospholipids, a process referred to as lipid peroxidation. HNE has been detected in several experimental and clinical conditions in which oxidative stress has been reported to occur and several authors have suggested that HNE and related 4-hydroxy-2,3-alkenals (HAKs) of different chain length may act not only as toxic and mutagenic mediators of oxidative stress-related injury but also as biological signals in normal and pathological conditions. In this paper we will review the literature supporting the concept that HNE and HAKs may act as signal molecules able to modulate biological events such as chemotaxis, signal transduction, gene expression, cell proliferation and cell differentiation.     

Post-translational Modification of Serine/Threonine Kinase LKB1 via Adduction of the Reactive Lipid Species 4-Hydroxy-trans-2-nonenal (HNE) at Lysine Residue 97 Directly Inhibits Kinase Activity

Oxidative stress is pathogenic in a variety of diseases, but the mechanism by which cellular signaling is affected by oxidative species has yet to be fully characterized. Lipid peroxidation, a secondary process that occurs during instances of free radical production, may play an important role in modulating cellular signaling under conditions of oxidative stress. 4-Hydroxy-trans-2-nonenal (HNE) is an electrophilic aldehyde produced during lipid peroxidation that forms covalent adducts on proteins, altering their activity and function. One such target, LKB1, has been reported to be inhibited by HNE adduction. We tested the hypothesis that HNE inhibits LKB1 activity through adduct formation on a specific reactive residue of the protein. To elucidate the mechanism of the inhibitory effect, HEK293T cells expressing LKB1 were treated with HNE (10 μm for 1 h) and assayed for HNE-LKB1 adduct formation and changes in LKB1 kinase activity. HNE treatment resulted in the formation of HNE-LKB1 adducts and decreased LKB1 kinase activity by 31 ± 9% (S.E.) but had no effect on the association of LKB1 with its adaptor proteins sterile-20-related adaptor and mouse protein 25. Mutation of LKB1 lysine residue 97 reduced HNE adduct formation and attenuated the effect of HNE on LKB1 activity. Taken together, our results suggest that adduction of LKB1 Lys-97 mediates the inhibitory effect of HNE.     

Effect of 4-hydroxynonenal on antioxidant capacity and apoptosis induction in Jurkat T cells

4-Hydroxynonenal (HNE) is one of the major end products of lipid peroxidation and may have either physiological or pathological significance regulating cell proliferation. We studied some biochemical effects of HNE, at various concentrations (0.1-100 μM), on Jurkat T cells incubated thereafter for 24, 48 and 72 h. HNE at low concentrations significantly enhanced the proliferation index, whereas at higher concentrations progressively blocked cell proliferation. Caspase 3 activity increased significantly at HNE concentrations between 1 and 10 μM and decreased at higher concentrations. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) increased progressively with HNE concentrations, particularly GSH-Px. Glucose-6-phosphate dehydrogenase (G6PDH) showed a different pattern, increasing at low HNE (1-5 μM) concentrations and rapidly declined thereafter. These results show that HNE may induce growth inhibition of Jurkat T cells and regulate the activity of typical antioxidant enzymes. Furthermore, the protective effect of doubling the foetal calf serum still points out the risk that cultured cells undergo oxidative stress during incubation.     

4-Hydroxy-2-nonenal is a powerful endogenous inhibitor of endothelial response

There is increasing evidence that lipid peroxidation is involved in many of the pathophysiologies associated with cardiovascular diseases, such as atherosclerosis and the long-term complications of diabetes. Among the products which originate from the peroxidation of cellular membrane lipids, 4-hydroxy-2-nonenal (HNE) is believed to be largely responsible for the cytopathological effects observed during oxidative stress in vivo. Here we found that HNE dramatically inhibited the expression of adhesion molecules induced by inflammatory stimuli in human aortic endothelial cells. The inhibition was found to be accompanied by a significant reduction of NF-kappaB activation followed by nuclear localization. This and the observation that the HNE treatment of the cells resulted in a rapid reduction of intracellular glutathione levels suggest that redox regulation of NF-kappaB may be involved in the modulation of the endothelial response by reactive aldehydes.     

Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP₊-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Impact of 4‐hydroxynonenal on matrix metalloproteinase‐9 regulation in lipopolysaccharide‐stimulated RAW 264.7 cells

Tissue degradation and leukocyte extravasation suggest proteolytic destruction of the extracellular matrix (ECM) during severe malaria. Matrix metalloproteinases (MMPs) play an established role in ECM turnover, and increased MMP‐9 protein abundance is correlated with malarial infection. The malaria pigment hemozoin (Hz) is a heme detoxification biomineral that is produced during infection and associated with biologically active lipid peroxidation products such as 4‐hydroxynonenal (HNE) adsorbed to its surface. Hz has innate immunomodulatory activity, and many of its effects can be reproduced by exogenously added HNE. Hz phagocytosis enhances MMP‐9 expression in monocytes; thus, this study was designed to examine the ability of HNE to alter MMP‐9 regulation in activated cells of macrophage lineage. Data show that treatment of lipopolysaccharide‐stimulated RAW 264.7 cells with HNE increased MMP‐9 secretion and activity. HNE treatment abolished the cognate tissue inhibitor of metalloproteinase‐1 protein levels, further decreasing MMP‐9 regulation. Phosphorylation of both p38 mitogen‐activated protein kinase (MAPK) and c‐Jun NH2‐terminal kinase was induced by HNE, but only p38 MAPK inhibition lessened MMP‐9 secretion. These results demonstrate the in vitro ability of HNE to cause MMP‐9 dysregulation in an activated cell model. The findings may extend to myriad pathologies associated with lipid peroxidation and elevated MMP‐9 levels leading to tissue damage. Copyright © 2015 John Wiley & Sons, Ltd.     

Hydroxynonenal-generated crosslinking fluorophore accumulation in Alzheimer disease reveals a dichotomy of protein turnover

Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently bind amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated in intimate association with the pathological lesions of Alzheimer disease (AD), suggesting that oxidative stress is a major component of AD pathogenesis. Some HNE-protein products result in protein crosslinking through a fluorescent compound similar to lipofuscin, linking lipid peroxidation and the lipofuscin accumulation that commonly occurs in post-mitotic cells such as neurons. In this study, brain tissue from AD and control patients was examined by immunocytochemistry and immunoelectron microscopy for evidence of HNE-crosslinking modifications of the type that should accumulate in the lipofuscin pathway. Strong labeling of granulovacuolar degeneration (GVD) and Hirano bodies was noted but lipofuscin did not contain this specific HNE-fluorophore. These findings directly implicate lipid crosslinking peroxidation products as accumulating not in the lesions or the lipofuscin pathways, but instead in a distinct pathway, GVD, that accumulates cytosolic proteins.     

Stimulation of HeLa cell growth by physiological concentrations of 4-hydroxynonenal

The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimualting effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin–HNE conjugate. HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 μM), which was primarily cytotoxic and a physiological range (below 10 μM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.     

Involvement of PI3K/PKG/ERK1/2 signaling pathways in cortical neurons to trigger protection by cotreatment of acetyl-L-carnitine and alpha-lipoic acid against HNE-mediated oxidative stress and neurotoxicity: implications for Alzheimer's disease

Oxidative stress has been shown to underlie neuropathological aspects of Alzheimer's disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of lipid peroxidation of unsaturated lipids. HNE-induced oxidative toxicity is a well-described model of oxidative stress-induced neurodegeneration. GSH plays a key role in antioxidant defense, and HNE exposure causes an initial depletion of GSH that leads to gradual toxic accumulation of reactive oxygen species. In the current study, we investigated whether pretreatment of cortical neurons with acetyl-L-carnitine (ALCAR) and alpha-lipoic acid (LA) plays a protective role in cortical neuronal cells against HNE-mediated oxidative stress and neurotoxicity. Decreased cell survival of neurons treated with HNE correlated with increased protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (HNE) accumulation. Pretreatment of primary cortical neuronal cultures with ALCAR and LA significantly attenuated HNE-induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose-dependent manner. Additionally, pretreatment of ALCAR and LA also led to elevated cellular GSH and heat shock protein (HSP) levels compared to untreated control cells. We have also determined that pretreatment of neurons with ALCAR and LA leads to the activation of phosphoinositol-3 kinase (PI3K), PKG, and ERK1/2 pathways, which play essential roles in neuronal cell survival. Thus, this study demonstrates a cross talk among the PI3K, PKG, and ERK1/2 pathways in cortical neuronal cultures that contributes to ALCAR and LA-mediated prosurvival signaling mechanisms. This evidence supports the pharmacological potential of cotreatment of ALCAR and LA in the management of neurodegenerative disorders associated with HNE-induced oxidative stress and neurotoxicity, including AD.     

Reactive aldehydes – second messengers of free radicals in diabetes mellitus

Abstract

Elevated levels of pro-oxidants and various markers of oxidative tissue damage were found in diabetic patients, indicating involvement of oxidative stress in the pathogenesis of diabetes mellitus (DM). On one side, physiological levels of reactive oxygen species (ROS) play an important role in redox signaling of various cells, while on the other, excessive ROS production can jeopardize the integrity and physiological functions of cellular macromolecules, in particular proteins, thus contributing to the pathogenesis of DM. Reactive aldehydes, especially 4-hydroxynonenal (HNE), are considered as second messengers of free radicals that act both as signaling molecules and as cytotoxic products of lipid peroxidation causing long-lasting biological consequences, in particular by covalent modification of macromolecules. Accordingly, the HNE and related reactive aldehydes may play important roles in the pathophysiology of DM, both in the development of the disease and in its progression and complications due to the following: (i) exposure of cells to supraphysiological levels of 4-hydroxyalkenals, (ii) persistent and sustained generation of 4-hydroxyalkenals that progressively affect vulnerable cells that lack an efficient bioactive aldehyde neutralization system, (iii) altered redox signaling influenced by reactive aldehydes, in particular by HNE, and (iv) induction of extracellular generation of similar aldehydes under secondary pathological conditions, such as low-grade inflammation.     

Vascular smooth muscle cell activation and growth by 4-hydroxynonenal

The present study examines the signal transduction mechanism that is involved in the growth of vascular smooth muscle cells exposed to 4-hydroxynonenal (HNE) in vitro. This aldehyde component of oxidized low-density lipoprotein has been identified in atherosclerotic lesion. Exposure to HNE caused ERK, JNK, and p38 MAP kinase activation as well as the induction of c-fos and c-jun gene expression. AP-1 activity was also significantly induced by HNE treatment. These intracellular activities appear to be the mechanism of HNE-caused mitogenesis. Indeed, HNE induced vascular smooth muscle cell proliferation as determened by Alamar-Blue assay and stimulated DNA synthesis as determined by bromodeoxyuridine incorporation. These observations are consistent with a role of lipid peroxidation products in vascular smooth muscle cell growth in atherogenesis.     

Stimulation of Epstein-Barr virus-infected human B cell growth by physiological concentrations of 4-hydroxynonenal

We had previously shown that cyclosporin A (CsA) directly promoted the immortalization of Epstein-Barr virus (EBV)-infected human B cells (EBV-B cells) via an oxidative stress mechanism. 4-Hydroxynonenal (HNE) is a reactive end-product of lipid peroxidation. We hypothesized that HNE may mediate a direct oxidative stress-promoting effect of CsA on EBV-B cells. HNE-protein adducts in CsA-treated EBV-B cell extracts were assayed immunochemically using a Slot-Blot method. Cell proliferation was assayed by [(3)H]-thymidine incorporation. EBV oncogene latent membrane protein-1 (LMP1) expression was assayed by using PE-conjugated anti-LMP1 antibody in flow cytometry. We found that CsA at 500 ng ml(-1) and 1000 ng ml(-1) significantly increased the level of HNE-protein adducts in EBV-B cells over the control (arbitrary units +/- SE) by 251.3 +/- 7.5 to 361.3 +/- 9.7 and 342.7 +/- 10.7, respectively (p < 0.05, n = 3). EBV-B cells treated with a physiological concentration of HNE (1 microM) for 0.5 and 1 h and cultured for 2 and 4 weeks showed significantly increased [(3)H]-thymidine incorporation. EBV-B cells treated with HNE (1 microM) for 1 h and subsequently cultured for 2 and 4 weeks had a significantly higher ( > 2.0 times) LMP1-positive cell population over the control. In conclusion, in accordance with our previous findings, we show that CsA treatment of EBV-B cells results in increased production of the lipid peroxidation reactive end-product HNE that directly promotes EBV-B cell proliferation and LMP1 expression. This observation provides evidence for further understanding the mechanism of CsA-induced oxidative stress on EBV-related post-transplant lymphoproliferative disorder (PTLD).     

4-Hydroxynonenal induces oxidative stress and death of cultured spinal cord neurons

Primary spinal cord trauma can trigger a cascade of secondary processes leading to delayed and amplified injury to spinal cord neurons. Release of fatty acids, in particular arachidonic acid, from cell membranes is believed to contribute significantly to these events. Mechanisms of fatty acid-induced injury to spinal cord neurons may include lipid peroxidation. One of the major biologically active products of arachidonic acid peroxidation is 4-hydroxynonenal (HNE). The levels of HNE-protein conjugates in cultured spinal cord neurons increased in a dose-dependent manner after a 24-h exposure to arachidonic acid. To study cellular effects of HNE, spinal cord neurons were treated with different doses of HNE, and cellular oxidative stress, intracellular calcium, and cell viability were determined. A 3-h exposure to 10 microM HNE caused approximately 80% increase in oxidative stress and 30% elevation of intracellular calcium. Exposure of spinal cord neurons to HNE caused a dramatic loss of cellular viability, indicated by a dose-dependent decrease in MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium, inner salt] conversion. The cytotoxic effect of HNE was diminished by pretreating neurons with ebselen or N-acetylcysteine. These data support the hypothesis that formation of HNE may be responsible, at least in part, for the cytotoxic effects of membrane-released arachidonic acid to spinal cord neurons.     

4-Hydroxy-2-nonenal: a product and mediator of oxidative stress

The onset of lipid peroxidation within cellular membranes is associated with changes in their physiochemical properties and with the impairment of enzymatic functions located in the membrane environment. There is increasing evidence that aldehydic molecules generated endogenously during the process of lipid peroidation are causally involved in most of the pathophysiological effects associated with oxidative stress in cells and tissues. 4-Hydroxy-2-nonenal (HNE), among them, is believed to be largely responsible for cytopathological effects observed during oxidative stree in vivo and has achieved the status of one of the best recognized and most studied of the cytotoxic products of lipid peroxidation. In the present review, I provide a comprehensive summary of HNE, as the product and mediator or oxidative stress.     

Inhibition of antioxidants and hyperthermia enhance bleomycin-induced cytotoxicity and lipid peroxidation in Chinese hamster ovary cells.

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. Heat enhances the cytotoxic effect of certain anticancer agents such as bleomycin, but the mechanisms involved in cell killing are currently unknown. Bleomycin generates reactive oxygen species. It is likely that hyperthermia itself also increases oxidative stress in cells. We evaluate whether oxidative stress has a role in the mechanism of cell death caused by bleomycin and heat in Chinese hamster ovary cells. Heat (41 to 44 degrees C) increased cytotoxicity of bleomycin, evaluated by clonogenic cell survival. Decreased levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus enhancing cytotoxic responses to heat and to oxidant-generating drugs. We determine the involvement of four major cellular antioxidant defenses, superoxide dismutase (SOD), the glutathione redox cycle (GSH cycle), catalase, and glutathione S-transferase (GST), in cellular sensitivity to bleomycin, alone or combined with hyperthermia. These cellular defenses were inhibited by diethyldithiocarbamate, l-buthionine sulfoximine, aminotriazole, and ethacrynic acid, respectively. We show that levels of antioxidants (SOD, GSH cycle, and GST) affect cellular cytotoxic responses to bleomycin, at normal and elevated temperatures (41 to 44 degrees C), suggesting the involvement of oxidative stress. Bleomycin and iron caused oxidative damage to membrane lipids in intact cells, at 37 and 43 degrees C. Lipid peroxidation was evaluated by fluorescence detection of thiobarbituric acid-reactive products. There was an increase in damage to membrane lipids when the antioxidant defenses, SOD and catalase, were inhibited. The differing effects of antioxidant inhibitors on bleomycin-induced cytotoxicity and membrane lipid damage suggest that different mechanisms are involved in these two processes. However, free radicals appear to be involved in both cases. The marked sensitization of cells by diethyldithiocarbamate, to both bleomycin-induced cytotoxicity and lipid peroxidation, suggests that superoxide could be involved in both of these processes.     

Glutathione Cycle Impairment Mediates Aβ-induced Cell Toxicity

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP⁺-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.