2-nonenal--(trans)-isomer and 4-hydroxy-2-nonenal

Reactive oxygen species (ROS) are elevated in the heart in response to hemodynamic and metabolic stress and promote hypertrophic signaling. ROS also mediate the formation of lipid peroxidation-derived aldehydes that may promote myocardial hypertrophy. One lipid peroxidation by-product, 4-hydroxy-trans-2-nonenal (HNE), is a reactive aldehyde that covalently modifies proteins thereby altering their function. HNE adducts directly inhibit the activity of LKB1, a serine/threonine kinase involved in regulating cellular growth in part through its interaction with the AMP-activated protein kinase (AMPK), but whether this drives myocardial growth is unclear. We tested the hypothesis that HNE promotes myocardial protein synthesis and if this effect is associated with impaired LKB1-AMPK signaling. In adult rat ventricular cardiomyocytes, exposure to HNE (10 μM for 1h) caused HNE-LKB1 adduct formation and inhibited LKB1 activity. HNE inhibited the downstream kinase AMPK, increased hypertrophic mTOR-p70S6K-RPS6 signaling, and stimulated protein synthesis by 27.1 ± 3.5%. HNE also stimulated Erk1/2 signaling, which contributed to RPS6 activation but was not required for HNE-stimulated protein synthesis. HNE-stimulated RPS6 phosphorylation was completely blocked using the mTOR inhibitor rapamycin. To evaluate if LKB1 inhibition by itself could promote the hypertrophic signaling changes observed with HNE, LKB1 was depleted in adult rat ventricular myocytes using siRNA. LKB1 knockdown did not replicate the effect of HNE on hypertrophic signaling or affect HNE-stimulated RPS6 phosphorylation. Thus, in adult cardiac myocytes HNE stimulates protein synthesis by activation of mTORC1-p70S6K-RPS6 signaling most likely mediated by direct inhibition of AMPK. Because HNE in the myocardium is commonly increased by stimuli that cause pathologic hypertrophy, these findings suggest that therapies that prevent activation of mTORC1-p70S6K-RPS6 signaling may be of therapeutic value.

Topics: Aldehydes; AMP-Activated Protein Kinases; Animals; Cells, Cultured; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Hypertrophy, Left Ventricular; Lipid Peroxidation; Male; Mechanistic Target of Rapamycin Complex 1; Multiprotein Complexes; Myocardium; Myocytes, Cardiac; Phosphorylation; Protein Biosynthesis; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Glutathione S-transferase activity has been shown to be associated with the microsomal fraction of Taenia solium. Electron microscopy and subcellular enzyme markers indicate the purity of the microsomal fraction that contains the glutathione S-transferase activity. T. solium microsomes were solubilized under conditions used to solubilize integral microsomal proteins. This procedure proved necessary to obtain enzymatic activity. To characterize this parasite enzyme activity, several substrates and inhibitors were used. The optimum activity for microsomal glutathione S-transferase was found to be pH 6.6, with a specific enzyme activity of 0.9, 0.1, 0.067, 0.03, and 0.05 micromol min(-1) mg(-1) with the substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 2,4-hexadienal, and trans-2-nonenal, respectively. No activity of glutathione peroxidase was observed. T. solium microsomes had an appKm (GSH)=0.161 microM, appKm (CDNB)=14.5 microM, and appVmax of 0.15 and 27.9 micromol min(-1) mg(-1) for GSH and CDNB, respectively. T. solium microsomes were inhibited by several glutathione S-transferase enzyme inhibitors, and it was possible to establish a simple inhibition system as well as corresponding Ki's for each inhibitor. These results indicate that the T. solium microsomal glutathione S-transferase is different from the parasite cytoplasmic enzymes that catalyze similar reactions.

Topics: Aldehydes; Alkadienes; Animals; Dinitrochlorobenzene; Enzyme Inhibitors; Enzyme Stability; Glutathione Peroxidase; Glutathione Transferase; Hydrogen-Ion Concentration; Kinetics; Microsomes; Nitrobenzenes; Taenia solium

Transient activation of fibroblasts or fibroblast-like cells to proliferate and to produce elevated quantities of extracellular matrix is essential to fibrosis. This activation is regulated by several cytokines produced by various inflammation-associated cells. Among these, transforming growth factor beta1 (TGFbeta1) is considered of major importance. Many studies have shown that lipid peroxidation play a key role in the initiation and progression of fibrosis in different organs. In fact, 4-hydroxy-2,3-nonenal (HNE), the major aldehydic product of lipid peroxidation, is able to induce TGFbeta1 expression and synthesis, and activation of activator protein-1 (AP-1) transcription factor. In this study, using the murine macrophage line J774-A1, we show that these effects are strictly related to the chemical structure of HNE, since neither 2-nonenal nor nonanal are biologically active to the same extent. Moreover, we demonstrate that HNE can indeed contribute to the onset of fibrosis by stimulating AP-1 binding to DNA and consequently inducing TGFbeta1 expression, since thiol-group reagents, such as N-ethylmaleimide and 4-(chloro-mercuri)-benzenesulfonic acid, that down-modulate HNE entrance and localisation inside the cell, prevent both phenomena. The possibility to control fibrogenic cytokine levels by means of antioxidant or dietetic treatments opens new potential pharmacological and nutritional horizons in the treatment of many chronic diseases characterised by excessive fibrosis.

Topics: Aldehydes; Animals; Cell Line; DNA; Electrophoretic Mobility Shift Assay; Fibrosis; Gene Expression; Lipid Peroxidation; Macrophages; Mice; Proteins; Structure-Activity Relationship; Transcription Factor AP-1

Elevated levels of 4-hydroxy-trans-2-nonenal (HNE) are implicated in the pathogenesis of numerous neurodegenerative disorders. Although well-characterized in the periphery, the mechanisms of detoxification of HNE in the CNS are unclear. HNE is oxidized to a non-toxic metabolite in the rat cerebral cortex by mitochondrial aldehyde dehydrogenases (ALDHs). Two possible ALDH enzymes which might oxidize HNE in CNS mitochondria are ALDH2 and succinic semialdehyde dehydrogenase (SSADH/ALDH5A). It was previously established that hepatic ALDH2 can oxidize HNE. In this work, we tested the hypothesis that SSADH oxidizes HNE. SSADH is critical in the detoxification of the GABA metabolite, succinic semialdehyde (SSA). Recombinant rat SSADH oxidized HNE and other alpha,beta-unsaturated aldehydes. Inhibition and competition studies in rat brain mitochondria showed that SSADH was the predominant oxidizing enzyme for HNE but only contributed a portion of the total oxidizing activity in liver mitochondria. In vivo administration of diethyldithiocarbamate (DEDC) effectively inhibited (86%) ALDH2 activity but not HNE oxidation in liver mitochondria. The data suggest that a relationship between the detoxification of SSA and the neurotoxic aldehyde HNE exists in the CNS. Furthermore, these studies show that multiple hepatic aldehyde dehydrogenases are able to oxidize HNE.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Aldehyde Oxidoreductases; Aldehydes; Animals; Benomyl; Brain Chemistry; Ditiocarb; Enzyme Inhibitors; gamma-Aminobutyric Acid; Male; Mitochondria; Mitochondria, Liver; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Succinate-Semialdehyde Dehydrogenase

The effects of three lipid peroxidation end-products, 4-hydroxynonenal (HNE), 2-nonenal (NE) and nonanal, on phosphoinositide-specific phospholipase C (PL-C) activity were studied in HL-60 cells. Enzymatic activity was determined by measuring the amounts of inositol-P3 (Ins-P3) produced by the cells incubated at 37 degrees C in the presence of the various compounds. HNE was shown to activate PL-C at concentrations of between 10(-8) and 10(-6) M; 10(-9) and 10(-8) M of NE also strongly stimulated PL-C. In contrast, nonanal failed to modify enzymatic activity. The concentrations of HNE and NE active on PL-C showed good correspondence with those that have been reported to be chemotactic towards rat neutrophils. The pretreatment of cells with 1 microM pertussis toxin completely prevented the increase of Ins-P3 production induced by HNE and NE. Maximal PL-C stimulation was produced by 10 nM NE; the degree of inositol-P3 production induced by the simultaneous addition of an equimolar dose of HNE was not significantly different from the activity value induced by NE alone, suggesting a possible competition between the two compounds. The data indicate that both HNE and NE share a common mechanism of action which, as with other better-known chemoattractants, involves PL-C activation through a G regulatory protein.

Topics: Aldehydes; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Activation; HL-60 Cells; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Lipid Peroxidation; Pertussis Toxin; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

The promyelocytic cell line HL-60 has been used as an in vitro model to study the mechanism of action of two chemotactic aldehydes, 2-nonenal and 4-hydroxynonenal. Increasing aldehyde concentrations have been added to undifferentiated and DMSO-differentiated cells incubated at 37 degrees C and their effect on phosphoinositide-specific phospholipase C has been analysed by using a specific inositol-1,4,5-tris-phosphate assay system. Concentrations of 2-nonenal between 10(-9) and 10(-7) M significantly increased the enzymatic-activity in DMSO-differentiated HL-60 cells, while 10(-9) and 10(-8) M concentrations were active in the undifferentiated cells. 4-Hydroxynonenal was able to activate phospholipase C both in undifferentiated and DMSO-differentiated cells at concentrations ranging from 10(-8) to 10(-6) M. The concentrations of both compounds active on phospholipase C displayed a good correspondence with those which had been reported to be chemotactic towards rat neutrophils. In the case of 4-hydroxynonenal, the present results confirm its ability to activate phospholipase C, which we had previously shown in isolated neutrophil plasma membranes. The comparison of the effects of 2-nonenal and 4-hydroxynonenal on chemotaxis and phospholipase C activation suggests a common mechanism of action for both aldehydes, for which the presence of the double bond seems to be required.

Topics: Acid Phosphatase; Aldehydes; Animals; Cell Differentiation; Cell Membrane; Chemotaxis; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Enzyme Activation; Free Radical Scavengers; HL-60 Cells; Humans; Inositol 1,4,5-Trisphosphate; Ligands; Lipid Peroxidation; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphatidylinositol Diacylglycerol-Lyase; Rats; Type C Phospholipases

Lipid peroxidation produces several toxic carbonyls, including biologically active aldehydes. In previous studies, we demonstrated that 4-hydroxynonenal (HNE), one of the major products of lipoperoxidation, inhibited growth and c-myc expression in K562 and HL-60 human leukemic cells. In this study, we compared the HNE effects with those of 4-hydroxyoctenal (HOE), 4-hydroxyundecenal (HUE; different lengths of the lipophilic tail), and the analogous aldehydes 2-trans-nonanal (lacking the OH group) and nonenal (lacking the OH group and the trans CC double bond), on HL-60 cell proliferation and c-myc expression. HUE and HOE inhibited growth and c-myc expression in a dose-dependent fashion, with an effectiveness comparable with that of HNE, whereas 2-nonenal and nonanal did not affect these parameters. Our results showed that different aldehydes produced from lipid peroxidation may contribute to growth inhibition by c-myc downregulation and that the molecular features involved seem to be the hydroxy group and the trans CC double bond.

Topics: Aldehydes; Blotting, Northern; Cell Division; Cell Line; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA; Time Factors

To indicate the extent of lipid peroxidation induced by oxidative stress, by measuring aldehyde end products in biological samples.. A highly specific gas chromatography and mass spectrometry (GC/MS) method was used to measure plasma concentrations of aliphatic aldehydes within the first week of life in 13 premature infants who subsequently developed chronic lung disease (CLD) and 11 infants without CLD (non-CLD). The oxime-tert-butyldimethylsilyl derivatives of aldehydes were analysed using 2,2,6,6-d4-cyclohexanone as the internal standard.. All of the aldehydes measured were raised in those infants with CLD compared with non-CLD infants. Plasma concentrations of heptanal, 2-nonenal, and 4-hydroxynonenal (HNE) were significantly increased in CLD infants on the day of birth, while the differences in all aldehydes between the two groups were not significant at 4-6 days of age. Logistic regression analysis showed that the increase in these three aldehydes within the first 24 hours of life independently showed significant associations with the development of CLD. In particular, an HNE concentration of > or = 200 nM on day 0 was the best predictor for the early detection of CLD (odds ratio = 32.0), followed by a 2-nonenal concentration of > or = 150 nM (odds ratio = 16.0).. These findings suggest that lipid peroxidation may have a role in the pathogenesis of neonatal CLD.

Topics: Aldehydes; Antioxidants; Bilirubin; Female; Gas Chromatography-Mass Spectrometry; Humans; Infant, Newborn; Infant, Premature; Lipid Peroxidation; Lung Diseases, Obstructive; Male; Predictive Value of Tests; Prognosis; Regression Analysis; Respiration, Artificial; Respiratory Distress Syndrome, Newborn; Statistics, Nonparametric; Vitamin E

Inhibition of conceptal biosynthesis of all-trans-retinoic acid (t-RA) by aldehydes generated from lipid peroxidation was investigated. Oxidative conversion of all-trans-retinal (t-RAL, 18 microM) to t-RA catalyzed by rat conceptal cytosol (RCC) was sensitive to inhibition by trans-2-nonenal (tNE), nonyl aldehyde (NA), 4-hydroxy-2-nonenal (4HNE), and hexanal. With an initial molar ratio of aldehyde/t-RAL of 2:1, tNE, NA, and 4HNE caused 70, 65, and 40% reductions of t-RA synthesis, respectively. Hexanal reduced generation of t-RA by approximately 50% as the ratio of aldehyde/t-RAL was raised to 20:1. tNE significantly increased the Km of the reaction and kinetic analyses indicated a mixed competitive/noncompetitive inhibition. By contrast, analogous reactions catalyzed by adult rat hepatic cytosol (ARHC) were highly resistant to inhibition by the same aldehydes. Significant inhibition (> 40% reduction of t-RA generation) by 4HNE, NA, and tNE were achieved at high molar ratios of aldehyde/t-RAL (> 175:1). Hexanal did not inhibit the reaction significantly even at very high ratios of aldehyde/t-RAL (> 2,000:1). Interestingly, when reduced glutathione (GSH, 10 mM) alone or GSH plus glutathione S-transferase (GST) were added to RCC-catalyzed reactions, additions of tNE or 4HNE showed either no significant inhibition or a partial lack of inhibition. Results suggested that GSH-dependent conjugation with 4HNE proceeded slowly compared to conjugation with tNE. To test the hypothesis that GST-catalyzed GSH conjugation can effectively prevent inhibition of t-RA synthesis by aldehydic products of lipid peroxidation, triethyltin bromide (TEB, a potent inhibitor of GST, 20 microM) was added to ARHC-catalyzed reactions when hexanal or tNE were present in the incubations. Eighty and 60% of hexanal and tNE inhibition, respectively, were observed. This was apparently due to TEB blockage of GST-catalyzed GSH conjugation reactions and thus strongly supported the stated hypothesis.

Topics: Aldehydes; Animals; Embryo, Mammalian; Female; Glutathione; Glutathione Transferase; Kinetics; Lipid Peroxidation; Pregnancy; Rats; Rats, Sprague-Dawley; Tretinoin

The effects of nonanal, trans-2-nonenal and 4-hydroxy-2,3-trans-nonenal on the formation of thromboxane B2 (TXB2), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid in washed rabbit platelets were examined. Nonanal and trans-2-nonenal at concentrations ranging from 0.25 to 2 microM inhibited TXB2, HHT and 12-HETE formation, reducing the amounts of these three arachidonic acid metabolites by 50% at nonanal and trans-2-nonenal concentrations of approximately 0.25 microM. The inhibition of TXB2, HHT and 12-HETE formation induced by 4-hydroxy-2,3-trans-nonenal (50% inhibition by 4-hydroxy-2,3-trans-nonenal at a concentration of approximately 100 microM) was 400 times weaker than that induced by nonanal and trans-2-nonenal. These results suggest that nonanal and trans-2-nonenal can be modulators of platelet arachidonic acid metabolism by affecting the activity of cyclooxygenase and 12-lipoxygenase.

Topics: Aldehydes; Animals; Arachidonic Acid; Blood Platelets; Cyclooxygenase Inhibitors; Lipoxygenase Inhibitors; Male; Rabbits

Our work has evaluated the effects of certain lipid peroxidation products, i.e. 4-hydroxy-2,3-trans-nonenal (HNE), 4-hydroxy-2,3-trans-octenal (HOE), 2-trans-nonenal and nonanal, on phosphoinositide-specific phospholipase C (PL-C). The enzymatic activity has been determined in vitro by measuring the hydrolysis of labelled phosphatidylinositol-4,5-bisphosphate added to plasma membranes isolated from rat neutrophils. Concentrations of HNE between 10(-8) and 10(-6) M, and concentrations of HOE ranging from 10(-11) to 10(-8) M, were able to activate PL-C. Neither 2-trans-nonenal nor nonanal induced any change of PL-C activity. HNE, HOE and 2-nonenal, but not nonanal, have been shown to possess chemotactic properties towards rat neutrophils. The good correlation between the concentrations of HNE and HOE active on PL-C and those able to stimulate cell migration suggests that their chemotactic activity might be mediated by the activation of PL-C, as in the case of most chemoattractants. On the contrary, this mechanism of action cannot be applied to 2-nonenal, an aldehyde much more hydrophobic than HNE or HOE; its chemotactic activity might be the consequence of some perturbation of the lipidic environment of the cell membrane where it dissolves easily.

Topics: Aldehydes; Animals; Cell Membrane; Dose-Response Relationship, Drug; Enzyme Activation; Male; Neutrophils; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoric Diester Hydrolases; Rats; Rats, Wistar; Type C Phospholipases

The molecular structure of aldehydes is closely related to their antimicrotubular effect. Morphological modifications of the microtubular system in living cells after incubation with certain aldehydes are consistent with biochemical alterations detected in previous research. The microtubular arrangement was visualized by an immunofluorescence technique with antitubulin antibodies, while the content of tubulin in the cells was evaluated by a colchicine binding assay. 2-Nonenal behaved similarly to 4-hydroxynonenal, a lipid peroxidation product, disorganizing microtubular network in 3T3 fibroblasts and decreasing the amounts of tubulin able to bind labelled colchicine. Nonanal did not significantly impair the tubulin characteristics in the cells, despite the fact that it has been shown to be active on the purified microtubular system; benzaldehyde was ineffective. This would appear to explain the mechanisms of interaction of aliphatic aldehydes which might be suitable for use as antimicrotubular drugs.

Topics: 3T3 Cells; Aldehydes; Animals; Benzaldehydes; Cell Survival; Colchicine; Fluorescent Antibody Technique; Immunohistochemistry; Mice; Microtubules; Tubulin

Some aldehydes have previously been shown to alter the microtubular system in a concentration-dependent manner. This paper examines the effects of taxol on the impairment of in vitro functionality of microtubular protein and of cytoplasmic microtubules by C-9 aliphatic aldehydes. Taxol-induced polymerization was inhibited by aldehydes in a similar way to GTP-induced polymerization, even though to a different degree. The addition of taxol restored the ability of aldehyde-impaired tubulin to polymerize, but only in the presence of nonanal. Colchicine binding activity in the presence of taxol was slightly modified by aldehydes at 20 degrees C, while it increased at 37 degrees C, compared to controls. Immunofluorescence showed a low disruption of microtubules in fibroblasts incubated with aldehydes and pretreated with taxol, probably as a consequence of its stabilizing activity. Anyway, aldehydes studied behaved differently, indicating a structure-activity relationship.

Topics: 3T3 Cells; Aldehydes; Alkaloids; Animals; Binding Sites; Colchicine; Guanosine Triphosphate; Mice; Microtubule Proteins; Microtubules; Paclitaxel; Polymers; Structure-Activity Relationship; Temperature; Tubulin

4-Hydroxynonenal is one of the main breakdown products of lipid peroxidation. It has an antiproliferative effect, which may partly be the consequence of an interaction with cytoskeletal structures. Its effects on microtubular protein are compared with those of homologous aldehydes with the same number of carbon atoms, and with that of benzaldehyde. Unlike the other aliphatic aldehydes, this latter aldehyde does not impair microtubular functions at every concentration in the range. Nonanal has the greatest effect on tubulin polymerization, whereas it only slightly impairs colchicine binding activity. 2-Nonenal and 4-hydroxynonenal have less inhibiting effect on tubulin polymerization; their effect on colchicine binding activity is dose-dependent. The targets of 4-hydroxynonenal on tubulin are -SH groups; the action mechanism of other aldehydes has not yet been identified.

Topics: Aldehydes; Animals; Benzaldehydes; Cattle; Colchicine; Cysteine; In Vitro Techniques; Mercaptoethanol; Microtubule Proteins; Oxidation-Reduction; Protein Binding

Toxic aldehydes, such as 4-hydroxy-2-nonenal (4HNE) and 2-nonenal (2NE), formed during lipid peroxidation have been isolated and implicated in the cytotoxic effects of oxidative stress. We have investigated the cytotoxicity and metabolism of 4HNE and 2NE in control (HA-1) cells and in two H2O2-resistant Chinese hamster fibroblast cell lines. The H2O2-resistant cells were found to be significantly more resistant than HA-1 cells to the cytotoxicity of 4HNE, as determined by clonogenic cell survival (dose-modifying factors at 10% isosurvival of 2.0-3.0). The H2O2-resistant cells demonstrated a significant 2-3-fold increase in the amount of 4HNE removed (mol/cell) from culture media containing 72 microM-4HNE when compared with HA-1 cells. The enhanced ability of H2O2-resistant cells to metabolize 4HNE was abolished by heating the cells at 100 degrees C for 45 min. Similar results were obtained with 2NE. Total glutathione and glutathione transferase activity, believed to be involved in cellular detoxification of 4HNE, were found to be significantly increased (2-3-fold) in the resistant cells when compared with the HA-1 cells. These results show that cell lines adapted and/or selected in a highly peroxidative environment are also resistant to the cytotoxicity of aldehydes formed during lipid peroxidation. This resistance appears to be related to increased cellular metabolism of these aldehydes, possibly through the glutathione transferase system. These findings suggest that the formation of aldehydes due to lipid peroxidation may contribute significantly to the mechanisms of oxidant-induced injury and the selective pressure exerted by H2O2-mediated cytotoxicity in culture.

Topics: Aldehydes; Animals; Cell Line; Cell Survival; Drug Resistance; Hydrogen Peroxide; Kinetics; Lipid Peroxidation