Metabolism of 4-hydroxynonenal by rat Kupffer cells

Kupffer cells are known to participate in the early events of liver injury involving lipid peroxidation. 4-Hydroxy-2,3-(E)-nonenal (4-HNE), a major aldehydic product of lipid peroxidation, has been shown to modulate numerous cellular systems and is implicated in the pathogenesis of chemically induced liver damage. The purpose of this study was to characterize the metabolic ability of Kupffer cells to detoxify 4-HNE through oxidative (aldehyde dehydrogenase; ALDH), reductive (alcohol dehydrogenase; ADH), and conjugative (glutathione S-transferase; GST) pathways. Aldehyde dehydrogenase and GST activity was observed, while ADH activity was not detectable in isolated Kupffer cells. Additionally, immunoblots demonstrated that Kupffer cells contain ALDH 1 and ALDH 2 isoforms as well as GST A4-4, P1-1, Ya, and Yb. The cytotoxicity of 4-HNE on Kupffer cells was assessed and the TD50 value of 32.5+/-2.2 microM for 4-HNE was determined. HPLC measurement of 4-HNE metabolism using suspensions of Kupffer cells incubated with 25 microLM 4-HNE indicated a loss of 4-HNE over the 30-min time period. Subsequent production of 4-hydroxy-2-nonenoic acid (HNA) suggested the involvement of the ALDH enzyme system and formation of the 4-HNE-glutathione conjugate implicated GST-mediated catalysis. The basal level of glutathione in Kupffer cells (1.33+/-0.3 nmol of glutathione per 10(6) cells) decreased significantly during incubation with 4-HNE concurrent with formation of the 4-HNE-glutathione conjugate. These data demonstrate that oxidative and conjugative pathways are primarily responsible for the metabolism of 4-HNE in Kupffer cells. However, this cell type is characterized by a relatively low capacity to metabolize 4-HNE in comparison to other liver cell types. Collectively, these data suggest that Kupffer cells are potentially vulnerable to the increased concentrations of 4-HNE occurring during oxidative stress.     

Ability of different hepatoma cells to metabolize 4-hydroxynonenal

4-Hydroxynonenal (4-HNE), produced during the oxidative lipid breakdown of biological membranes, modulates various biochemical processes in normal liver and in hepatoma cells. It is very probable that the effects of 4-HNE are related to the quantity formed in the cells and the cells' ability to metabolize it. Aldehyde catabolism takes place within the cells through oxidative and reductive enzymes, and through conjugation with intracellular glutathione. In this paper, the various enzymatic pathways involved in the metabolism of 4-HNE were studied in normal hepatocytes and in hepatoma cells. The hepatocyte pathway undergoes a complex variety of change during neoplastic transformation. In hepatoma cells, generally, 4-HNE metabolism was due mainly to aldehyde dehydrogenases, whereas in normal hepatocytes 4-HNE metabolism was mainly due to alcohol dehydrogenase and glutathione-S-transferase. The increase in oxidative enzymes compared to normal tissue was not the same in all types of hepatoma: in HTC hepatoma cells, the enzyme levels were considerably higher; in AH-130 hepatoma cells of Yoshida, they were lower in subcellular particles and similar in the cytosol. Indeed, consumption of externally-added 4-HNE in hepatoma cells was proportional to their content of 4-HNE metabolizing enzymes.     

Molecular mechanisms of ALDH3A1-mediated cellular protection against 4-hydroxy-2-nonenal

Evidence suggests that aldehydic molecules generated during lipid peroxidation (LPO) are causally involved in most pathophysiological processes associated with oxidative stress. 4-Hydroxy-2-nonenal (4-HNE), the LPO-derived product, is believed to be responsible for much of the cytotoxicity. To counteract the adverse effects of this aldehyde, many tissues have evolved cellular defense mechanisms, which include the aldehyde dehydrogenases (ALDHs). Our laboratory has previously characterized the tissue distribution and metabolic functions of ALDHs, including ALDH3A1, and demonstrated that these enzymes may play a significant role in protecting cells against 4-HNE. To further characterize the role of ALDH3A1 in the oxidative stress response, a rabbit corneal keratocyte cell line (TRK43) was stably transfected to overexpress human ALDH3A1. These cells were studied after treatment with 4-HNE to determine their abilities to: (a) maintain cell viability, (b) metabolize 4-HNE and its glutathione conjugate, (c) prevent 4-HNE-protein adduct formation, (d) prevent apoptosis, (e) maintain glutathione homeostasis, and (f) preserve proteasome function. The results demonstrated a protective role for ALDH3A1 against 4-HNE. Cell viability assays, morphological evaluations, and Western blot analyses of 4-HNE-adducted proteins revealed that ALDH3A1 expression protected cells from the adverse effects of 4-HNE. Based on the present results, it is apparent that ALDH3A1 provides exceptional protection from the adverse effects of pathophysiological concentrations of 4-HNE such as may occur during periods of oxidative stress.     

Formation of 4-hydroxynonenal from cardiolipin oxidation: Intramolecular peroxyl radical addition and decomposition

We report herein that oxidation of a mitochondria-specific phospholipid tetralinoleoyl cardiolipin (L(4)CL) by cytochrome c and H(2)O(2) leads to the formation of 4-hydroxy-2-nonenal (4-HNE) via a novel chemical mechanism that involves cross-chain peroxyl radical addition and decomposition. As one of the most bioactive lipid electrophiles, 4-HNE possesses diverse biological activities ranging from modulation of multiple signal transduction pathways to the induction of intrinsic apoptosis. However, where and how 4-HNE is formed in vivo are much less understood. Recently a novel chemical mechanism has been proposed that involves intermolecular dimerization of fatty acids by peroxyl bond formation; but the biological relevance of this mechanism is unknown because a majority of the fatty acids are esterified in phospholipids in the cellular membrane. We hypothesize that oxidation of cardiolipins, especially L(4)CL, may lead to the formation of 4-HNE via this novel mechanism. We employed L(4)CL and dilinoleoylphosphatidylcholine (DLPC) as model compounds to test this hypothesis. Indeed, in experiments designed to assess the intramolecular mechanism, more 4-HNE is formed from L(4)CL and DLPC oxidation than 1-palmitoyl-2-linoleoylphosphatydylcholine. The key products and intermediates that are consistent with this proposed mechanism of 4-HNE formation have been identified using liquid chromatography-mass spectrometry. Identical products from cardiolipin oxidation were identified in vivo in rat liver tissue after carbon tetrachloride treatment. Our studies provide the first evidence in vitro and in vivo for the formation 4-HNE from cardiolipin oxidation via cross-chain peroxyl radical addition and decomposition, which may have implications in apoptosis and other biological activities of 4-HNE.     

Aldehydes from n-6 fatty acid peroxidation. Effects on aminophospholipids

4-Hydroxy-nonenal (4-HNE) is a major by-product of n-6 fatty acid peroxidation. It has been described to covalently bind biomolecules expressing primary amine, especially the Lys residues in proteins. Low-density lipoproteins (LDL) are well-described macromolecules to be modified by 4-HNE, making them available to scavenger receptors on macrophages. Those macrophages then become foam cells and play an active role in atherogenesis. This paper reports on the covalent binding of 4-HNE to phosphatidylethanolamine (PE), a major aminophospholipid in biological membranes. In contrast, phosphatidylserine (PS) is virtually not modified by 4-HNE. One stable adduct, the Michael adduct PE/4-HNE is a poor substrate of secreted phospholipase A(2) and is not cleaved by phospholipase D. Plasmalogen PE, an important subclass of PE, is covalently modified by 4-HNE as well, but appears to be further degraded on its sn-1 position, the alkenyl chain, which might alter the antioxidant potential of the molecule. An aldehyde homologous to 4-HNE has been characterized as a breakdown product of 12-hydroperoxyeicosatetraenoic acid (12-HpETE) and named 4-hydroxy-2E,6Z-dodecadienal (4-HDDE). This compound as well as 4-HNE was detected in human plasma. Finally, 4-HDDE appears almost 3-fold more active than 4-HNE to make covalent adducts with PE. We conclude that 4-HNE and 4-HDDE are two biologically relevant markers of n-6 fatty acid peroxidation that may alter the phospholipid-dependent cell signaling.     

Revealing mechanisms of selective,concentration-dependent potentials of 4-hydroxy-2-nonenal to induce apoptosis in cancer cells through inactivation of membrane-associated catalase

Tumor cells generate extracellular superoxide anions and are protected against superoxide anion-mediated intercellular apoptosis-inducing signaling by the expression of membrane-associated catalase. 4-Hydroxy-2-nonenal (4-HNE), a versatile second messenger generated during lipid peroxidation, has been shown to induce apoptosis selectively in malignant cells. The findings described in this paper reveal the strong, concentration-dependent potential of 4-HNE to specifically inactivate extracellular catalase of tumor cells both indirectly and directly and to consequently trigger apoptosis in malignant cells through superoxide anion-mediated intercellular apoptosis-inducing signaling. Namely, 4-HNE caused apoptosis selectively in NOX1-expressing tumor cells through inactivation of their membrane-associated catalase, thus reactivating subsequent intercellular signaling through the NO/peroxynitrite and HOCl pathways, followed by the mitochondrial pathway of apoptosis. Concentrations of 4-HNE of 1.2 µM and higher directly inactivated membrane-associated catalase of tumor cells, whereas at lower concentrations, 4-HNE triggered a complex amplificatory pathway based on initial singlet oxygen formation through H₂O₂ and peroxynitrite interaction. Singlet-oxygen-dependent activation of the FAS receptor and caspase-8 increased superoxide anion generation by NOX1 and amplification of singlet oxygen generation, which allowed singlet-oxygen-dependent inactivation of catalase. 4-HNE and singlet oxygen cooperate in complex autoamplificatory loops during this process. The finding of these novel anticancer pathways may be useful for understanding the role of 4-HNE in the control of malignant cells and for the optimization of ROS-dependent therapeutic approaches including antioxidant treatments.     

Regulation of autophagy,mitochondrial dynamics,and cellular bioenergetics by 4-hydroxynonenal in primary neurons

The production of reactive species contributes to the age-dependent accumulation of dysfunctional mitochondria and protein aggregates, all of which are associated with neurodegeneration. A putative mediator of these effects is the lipid peroxidation product 4-hydroxynonenal (4-HNE), which has been shown to inhibit mitochondrial function, and accumulate in the postmortem brains of patients with neurodegenerative diseases. This deterioration in mitochondrial quality could be due to direct effects on mitochondrial proteins, or through perturbation of the macroautophagy/autophagy pathway, which plays an essential role in removing damaged mitochondria. Here, we use a click chemistry-based approach to demonstrate that alkyne-4-HNE can adduct to specific mitochondrial and autophagy-related proteins. Furthermore, we found that at lower concentrations (5–10 μM), 4-HNE activates autophagy, whereas at higher concentrations (15 μM), autophagic flux is inhibited, correlating with the modification of key autophagy proteins at higher concentrations of alkyne-4-HNE. Increasing concentrations of 4-HNE also cause mitochondrial dysfunction by targeting complex V (the ATP synthase) in the electron transport chain, and induce significant changes in mitochondrial fission and fusion protein levels, which results in alterations to mitochondrial network length. Finally, inhibition of autophagy initiation using 3-methyladenine (3MA) also results in a significant decrease in mitochondrial function and network length. These data show that both the mitochondria and autophagy are critical targets of 4-HNE, and that the proteins targeted by 4-HNE may change based on its concentration, persistently driving cellular dysfunction.     

Enantioselective metabolism of trans-4-hydroxy-2-nonenal by brain mitochondria

Trans-4-hydroxy-2-nonenal (HNE) is a product of lipid peroxidation with many cellular effects. HNE possesses a stereogenic center at the C4 carbon that influences the metabolism and alkylation targets of HNE. We tested the hypothesis that rat brain mitochondria metabolize HNE in an enantioselective manner after exposure to racemic HNE. The study of HNE chirality, however, is hindered by the lack of facile methods to chromatographically resolve (R)-HNE and (S)-HNE. We used a chiral hydrazine, (S)-carbidopa, as a derivatization reagent to form diastereomers with (R)-HNE and (S)-HNE that were separated by reverse-phase HPLC. After exposure to racemic HNE, rat brain mitochondria metabolized HNE enantioselectively with a higher rate of (R)-HNE metabolism. By using the purified enantiomers of HNE, we found that this enantioselective metabolism of HNE was the result of higher rates of enzymatic oxidation of (R)-HNE by aldehyde dehydrogenases compared to (S)-HNE. Conjugation of HNE to glutathione was a minor metabolic pathway and was not enantioselective. These studies demonstrate that the chirality of HNE affects its mitochondrial metabolism and potentially other processes in the central nervous system.     

Role of DDAH-1 in lipid peroxidation product-mediated inhibition of endothelial NO generation

Altered nitric oxide (NO) biosynthesis is thought to play a role in the initiation and progression of atherosclerosis and may contribute to increased risk seen in other cardiovascular diseases. It is hypothesized that altered NO bioavailability may result from an increase in endogenous NO synthase (NOS) inhibitors, asymmetric dimethly araginine (ADMA), and N(G)-monomethyl arginine, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). Lipid hydroperoxides and their degradation products are generated during inflammation and oxidative stress and have been implicated in the pathogenesis of cardiovascular disorders. Here, we show that the lipid hydroperoxide degradation product 4-hydroxy-2-nonenal (4-HNE) causes a dose-dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4-HNE (50 microM) on endothelial NO production were partially reversed with L-Arg supplementation (1 mM). Overexpression of human DDAH-1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4-HNE (50 microM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.     

Transfection of mGSTA4 in HL-60 cells protects against 4-hydroxynonenal-induced apoptosis by inhibiting JNK-mediated signaling

The mammalian alpha-class glutathione S-transferase (GST) isozymes mGSTA4-4, rGSTA4-4, and hGSTA4-4 are known to utilize 4-hydroxynonenal (4HNE) as a preferred substrate. During the present studies, we have examined the effect of transfecting human myeloid HL-60 cells with mGSTA4, on 4-HNE-induced apoptosis and the associated signaling mechanisms. Results of these studies show that treatment of the wild-type or vector-only-transfected HL-60 cells with 20 microM 4-HNE caused apoptosis within 2 h. The cells transfected with mGSTA4 did not undergo apoptosis under these conditions even after 4 h. In the wild-type and vector-transfected cells, apoptosis was preceded by JNK activation and c-Jun phosphorylation within 30 min, and an increase in AP-1 binding within 2 h of treatment with 20 microM 4-HNE. In mGSTA4-transfected cells, JNK activation and c-Jun phosphorylation were observed after 1 h, and increased AP-1 binding was observed after 8 h under these conditions. In the control cells, 20 microM 4-HNE caused caspase 3 activation and poly(ADP-ribose) polymerase cleavage within 2 h, while in mGSTA4-transfected cells, a lesser degree of these effects was observed even after 8 h. Transfection with mGSTA4 also provided protection to the cells from 4-HNE and doxorubicin cytotoxicity (1.6- and 2.6-fold, respectively). These results show that 4-HNE mediates apoptosis through its effects on JNK and caspase 3, and that 4-HNE metabolizing GST isozyme(s) may be important in the regulation of this pathway of oxidative-stress-induced apoptosis.     

4-Hydroxy-2-nonenal induces chronic obstructive pulmonary disease-like histopathologic changes in mice

The α,β-unsaturated aldehyde 4-hydroxy-2-nonenal (4-HNE) is an endogenous product of oxidative stress that is found at increased levels in the lungs of patients with chronic obstructive pulmonary disease (COPD) and animal models of this lung disorder. In the present study, levels of 4-HNE adducts were increased in two different mouse models of COPD. Challenging lungs with 4-HNE enlarged the airspace and induced goblet cell metaplasia of the airways in mice, two characteristics of COPD. 4-HNE induced the accumulation of inflammatory cells expressing high levels of MMP-2 and MMP-9. Our results indicate that 4-HNE production during oxidative stress is a key pathway in the pathogenesis of COPD.     

4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation

Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.     

Relationship of electrophilic stress to aging

This review begins with the premise that an organism's life span is determined by the balance between two countervailing forces: (i) the sum of destabilizing effects and (ii) the sum of protective longevity-assurance processes. Against this backdrop, the role of electrophiles is discussed, both as destabilizing factors and as signals that induce protective responses. Because most biological macromolecules contain nucleophilic centers, electrophiles are particularly reactive and toxic in a biological context. The majority of cellular electrophiles are generated from polyunsaturated fatty acids by a peroxidation chain reaction that is readily triggered by oxygen-centered radicals, but propagates without further input of reactive oxygen species(ROS). Thus, the formation of lipid-derived electrophiles such as 4-hydroxynon-2-enal (4-HNE) is proposed to be relatively insensitive to the level of initiating ROS, but to depend mainly on the availability of peroxidation-susceptible fatty acids. This is consistent with numerous observations that life span is inversely correlated to membrane peroxidizability, and with the hypothesis that 4-HNE may constitute the mechanistic link between high susceptibility of membrane lipids to peroxidation and shortened life span. Experimental interventions that directly alter membrane composition (and thus their peroxidizability) or modulate 4-HNE levels have the expected effects on life span, establishing that the connection is not only correlative but causal. Specific molecular mechanisms are considered, by which 4-HNE could (i) destabilize biological systems via nontargeted reactions with cellular macromolecules and (ii) modulate signaling pathways that control longevity-assurance mechanisms.     

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal,a key molecule in stress mediated signaling

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.     

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H(2)O(2) and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.     

Increased 4-Hydroxynonenal Formation Contributes to Obesity-Related Lipolytic Activation in Adipocytes

Oxidative stress in adipose tissue plays an etiological role in a variety of obesity-related metabolic disorders. We previously reported that increased adipose tissue 4-hydroxynonenal (4-HNE) contents contributed to obesity-related plasma adiponectin decline in mice. In the present study, we investigated the effects of intracellular 4-HNE accumulation on lipolytic response in adipocytes/adipose tissues and underlying mechanisms. In both fully-differentiated 3T3-L1 and primary adipocytes, a 5-hour 4-HNE exposure elevated lipolytic reaction in a dose-dependent manner at both basal and isoproterenol-stimulated conditions, evidenced by significantly increased glycerol and fatty acids releases. This conclusion was corroborated by the comparable observations when the minced human visceral adipose tissues were used. Mechanistic investigations revealed that 4-HNE-stimulated lipolytic activation is multifactorial. 4-HNE exposure quickly increased intracellular cyclic AMP (cAMP) level, which was concomitant with increased phosphorylations of protein kinase A (PKA) and its direct downstream target, hormone sensitive lipase (HSL). Pre-incubation with H89, a potent PKA inhibitor, prevented 4-HNE stimulated glycerol release, suggesting that enhanced lipolytic action in response to 4-HNE increase is mediated mainly by cAMP/PKA signal pathway in adipocytes. In addition to activating cAMP/PKA/HSL pathway, 4-HNE exposure also suppresses AMP-activated protein kinase (AMPK), a suppressive pathway for lipolysis, measured by both Western blotting for phosphorylated form of AMPK and ELISA for enzyme activity. Furthermore, 5-Aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside (AICAR), a pharmacological AMPK activator, alleviated 4-HNE-induced lipolysis, suggesting that AMPK suppression also contributes to 4-HNE elicited lipolytic response. In conclusion, our findings indicate that increased intracellular 4-HNE accumulation in adipocytes/adipose tissues contributes to obesity-related lipolytic activation.     

Effects of mGST A4 transfection on 4-hydroxynonenal-mediated apoptosis and differentiation of K562 human erythroleukemia cells

Cellular levels of downstream products of membrane lipid oxidation appear to regulate differentiation in K562 human erythroleukemia cells. 4-Hydroxynonenal (4-HNE) is a diffusible and relatively stable product of peroxidation of arachidonic and linoleic acids, cellular levels of which are regulated through metabolism to glutathione (GSH) conjugate by glutathione S-transferases (GSTs). A group of immunologically related alpha-class mammalian GSTs expressed in mice (mGST A4-4), rat (rGST A4-4), human (hGST A5.8), and other species, as well as the more distantly related human hGST A4-4, preferentially utilize 4-HNE as a substrate and are suggested to be major determinants of intracellular levels of 4-HNE. Present studies were designed to examine the effects of 4-HNE on K562 cells and to study the effect of transfection of mGSTA4-4 in these cells. Exposure of K562 cells to 20 microM 4-HNE for 2 h resulted in a rapid erythroid differentiation of K562 cells, as well as apoptosis evidenced by characteristic DNA laddering. Stable transfection of cells with mGST A4-4 resulted in a fivefold increase in GST-specific activity toward 4-HNE compared with wild-type or vector-only transfected cells. The mGST A4-4-transfected cells were resistant to the cytotoxic, apoptotic, and differentiating effects of 4-HNE. The mGST A4 transfection also conferred resistance to direct oxidative stress (IC(50) of H(2)O(2) 22, 23, and 35 microM for wild-type, vector-transfected, and mGST A4-transfected cells, respectively). mGST A4-4-transfected cells also showed a higher rate of proliferation compared with wild-type or vector-transfected K562 cells (doubling time 22.1 +/- 0.7, 31 +/- 1.2, and 29 +/- 0.6 h, respectively). Cellular 4-HNE levels determined by mass spectrometry were lower in mGST A4-4-transfected cells compared to cells transfected with vector alone (5.9 pmol/5 x 10(7) cells and 62.9 pmol/5 x 10(7) cells, respectively). Our studies show that 4-HNE can induce erythroid differentiation in K562 cells and that overexpression of mGST A4 suppresses 4-HNE levels and inhibits erythroid differentiation and apoptosis.     

Characterization of 4-hydroxy-2-nonenal metabolism in stellate cell lines derived from normal and cirrhotic rat liver

During oxidative stress, reactive aldehydes, including trans-4-hydroxy-2-nonenal (4-HNE), are generated by peroxidation of membrane lipids and purportedly stimulate hepatic stellate cells to produce excessive extracellular matrix, including type I collagen. An important question concerning the ability of 4-HNE to modulate collagen production by stellate cells is the potential of these specialized cells to detoxify 4-HNE. The objective of the present study was to characterize the ability of stellate cell lines, derived from normal (NFSC) and cirrhotic (CFSC) rat livers, to metabolize 4-HNE by oxidative, reductive and conjugative pathways. These two stellate cell lines were noted to have differing susceptibilities to the cytotoxic effect of 4-HNE. Treatment of both stellate cell lines with a range of 4-HNE doses demonstrated that the concentration which was cytotoxic to 50% of CFSC (TD(50)) was 25% greater than that for NFSC (967.57+/-9.26 nmol/10(6) cells vs. 769.90+/-5.32 nmol/10(6) cells respectively). The capacity of these cell lines to metabolizes 4-HNE was determined by incubating them in suspension with 50 microM 4-HNE (10 nmol/10(6) cell); 4-HNE elimination and metabolite formation were quantified over a 20 min time course. Both stellate cell lines rapidly metabolized 4-HNE, with the CFSC line eliminating 4-HNE at a rate that was approx. 2-fold greater than the NFSC line. The rate of 4-HNE metabolism attributable to glutathione S-transferase (GST) was similar in both cell lines, though differential cell specific expressions of GST isoforms GSTP1-1 and GSTA4-4 were observed. The greater rate of 4-HNE elimination by CFSC was attributable to its aldehyde dehydrogenase (ALDH) activity which accounted for approx. 50% of 4-HNE metabolism in CFSC but was insignificant in NFSC. Neither cell line had detectable alcohol dehydrogenase activity or protein levels. Measurement of cellular GSH concentrations revealed that NFSC contain approx. 2-fold greater concentrations of GSH when compared to CFSC and that following 4-HNE treatment, GSH levels were rapidly depleted from both cell lines. Concomitant with 4-HNE mediated GSH depletion, a corresponding increase in the 4-HNE-glutathione adduct formation was observed with the NFSC line forming greater amounts of the glutathione adduct than did the CFSC line. Taken together, these data demonstrate that both stellate cell lines have the capacity to metabolize 4-HNE but that CFSC have a greater rate of metabolism which is attributable to their greater ALDH activity, suggesting that the stellate cells isolated from cirrhotic liver may be differentially responsive to the biologic effects of 4-HNE.