15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid and 4-hydroxy-2-nonenal

In this study, we investigated the possible link between lipid peroxidation (LPO) and the formation of protein carbonyls (PCOs) during depletion of brain glutathione (GSH). To this end, rat brain slices were incubated with the GSH depletor diethyl maleate (DEM) in the absence or presence of classical LPO scavengers: trolox, caffeic acid phenethyl ester (CAPE), and butylated hydroxytoluene (BHT). All three scavengers reduced DEM-induced lipid oxidation and protein carbonylation, suggesting that intermediates/products of the LPO pathway such as lipid hydroperoxides, 4-hydroxynonenal and/or malondialdehyde are involved in the process. Additional in vitro experiments revealed that, among these products, lipid hydroperoxides are most likely responsible for protein oxidation. Interestingly, BHT prevented the carbonylation of cytoskeletal proteins but not that of soluble proteins, suggesting the existence of different mechanisms of PCO formation during GSH depletion. In pull-down experiments, beta-actin and alpha/beta-tubulin were identified as major carbonylation targets during GSH depletion, although other cytoskeletal proteins such as neurofilament proteins and glial fibrillary acidic protein were also carbonylated. These findings may be important in the context of neurological disorders that exhibit decreased GSH levels and increased protein carbonylation such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.

Topics: Aldehydes; Animals; Blotting, Western; Brain Chemistry; Cytoskeletal Proteins; Free Radical Scavengers; Glutathione; In Vitro Techniques; Leukotrienes; Lipid Peroxidation; Lipid Peroxides; Male; Malondialdehyde; Nerve Tissue Proteins; Oxidation-Reduction; Protein Carbonylation; Rats; Sulfhydryl Compounds

The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFkappaB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC(50) = 13 +/- 1.5 microm and 1 +/- 0.2 microm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G(1), impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on (1)inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase.

Topics: Aldehydes; Apoptosis; Arachidonate 15-Lipoxygenase; bcl-2-Associated X Protein; Blotting, Western; Catalysis; Cell Adhesion; Cell Cycle; Cell Division; Cell Line; Dose-Response Relationship, Drug; G1 Phase; Humans; Inflammation; Inhibitory Concentration 50; Kinetics; Leukotrienes; Lipid Peroxides; Lipoxygenase; Promoter Regions, Genetic; Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Ribonucleotide Reductases; Selenoproteins; Thioredoxin-Disulfide Reductase; Time Factors

Ni(2+), a toxic, carcinogenic and allergenic agent, affected both the kinetic and chemical courses of the Fe(2+)-induced oxidation of arachidonic acid (AA) in 0.05 M phosphate buffer (pH 7.4) and at 37 degrees C. At 10 microM concentration, Ni(2+) decreased the rate of oxidation of peroxide-free AA (200 microM) promoted by 50 microM Fe(2+), as determined by measurement of thiobarbituric acid reactive species (TBARS) and 1H NMR analysis. However, in the presence of low levels of peroxides (e.g. 2%), Ni(2+) exerted a significant stimulatory effect on Fe(2+)-induced AA oxidation and TBARS formation. 1H NMR analysis showed that Ni(2+) (10 microM) enhanced formation of genotoxic alkenals including 4-hydroxy-2-nonenal (4-HNE, GC/MS evidence) by Fe(2+)-promoted degradation of both AA and 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) methyl esters. The observed stimulatory effects of Ni(2+) on peroxide breakdown and cytotoxic aldehyde formation provide an attractive explanation to the enhanced sensitization capacity of nickel in inflammatory states compared to normal states.

Topics: Aldehydes; Arachidonic Acid; Cations, Divalent; Gas Chromatography-Mass Spectrometry; Iron; Leukotrienes; Lipid Peroxidation; Lipid Peroxides; Magnetic Resonance Spectroscopy; Models, Chemical; Nickel; Oxidation-Reduction; Thiobarbituric Acid Reactive Substances

The mechanism of arachidonic acid (AA)-induced apoptosis in vascular smooth muscle cells (VSMCs) was studied in the A-10 rat aortic smooth muscle cell line. Treatment of serum-deprived VSMCs with 50 microM AA for 24 h resulted in a loss of cell viability. The apoptotic effect of AA was characterized by annexin V binding, sub-G1 population of cells, cell shrinkage and chromatin condensation. AA-induced VSMC death was attenuated by antioxidants alpha-tocopherol and glutathione, the hydrogen peroxide (H2O2) scavenger catalase and by serum proteins, albumin and gamma globulins. Moreover, the AA peroxidation products, 12(S)-hydroperoxyeicosatetraenoic acid (HPETE), 15(S)-HPETE, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) caused VSMC apoptosis. These data suggest an oxidative mechanism of AA-induced VSMC death. The apoptotic effect of AA was pH-dependent, being inhibited by extracellular alkalinization to pH 8.0. AA inhibited serum-stimulated cell cycle progression in quiescent cells, but not in proliferating cells. In conclusion, AA, through its oxidation products causes VSMC apoptosis. Antioxidants, by inhibiting VSMC apoptosis, may prevent consequent pathological events such as atherosclerotic plaque rupture.

Topics: Aldehydes; Animals; Antioxidants; Aorta; Apoptosis; Arachidonic Acid; Cell Line; Cell Survival; Culture Media, Serum-Free; DNA; Leukotrienes; Lipid Peroxides; Malondialdehyde; Myocytes, Smooth Muscle; Rats

Unsaturated fatty acids constitutive of cardiac membranal lipid matrix are one of the primary targets for reactive oxygen species generated during ischemia-reperfusion cycle. Lipid peroxidation is a cascade of intricate reactions involving the successive formations of fatty acids hydroperoxides and aldehydic compounds such as alkenals derived from the oxidative fragmentation of these hydroperoxides. The potential deleterious effects of different classes of lipid peroxidation products on cardiac cells were compared using three in vitro approaches: (i) cardiomyocyte integrity, (ii) electromechanical activity of papillary muscle, and (iii) atrial contractility. The following products of lipid peroxidation were tested: (i) photoperoxidized arachidonic acid pooling hydroperoxidized derivatives and aldehydic compounds, (ii) fatty acids hydroperoxides, and (iii) 4-hydroxynonenal, a characteristic alkenal derived from the oxidative fragmentation of hydroperoxidized n-6 fatty acids. Only fatty acids hydroperoxides induced drastic loss of cellular integrity and severe disturbances in electromechanical activity of cardiomyocytes. 4-hydroxynonenal induced only a slight leak of lactate dehydrogenase at high concentrations and did not modify the electromechanical behavior of cardiac preparations. Under our conditions, monohydroperoxidized fatty acids but not 4-hydroxynonenal induced acute cardiac cell damages. In conclusion, lipid hydroperoxides can be considered both as markers of oxidative injury and relay sources of oxidative stress.

Topics: Aldehydes; alpha-Linolenic Acid; Animals; Arachidonic Acid; Atrial Function; Biomechanical Phenomena; Cells, Cultured; Electrophysiology; Fatty Acids; Heart; Hydrogen Peroxide; Leukotrienes; Lipid Peroxidation; Lipid Peroxides; Myocardial Contraction; Papillary Muscles; Photochemistry; Rats; Rats, Wistar

The mechanism of the formation of 4-hydroxynonenal through the NADPH-linked microsomal lipid peroxidation was investigated. The results were as follows: 4-hydroxynonenal arises exclusively from arachidonic acid contained in the polar phospholipids, neither arachidonic acid of the neutral lipids nor linoleic acid of the polar or neutral lipids are substrates for 4-hydroxynonenal generation. This finding results from the estimation of the specific radioactivity of 4-hydroxynonenal produced by microsomes prelabelled in vivo with [U-14C]arachidonic acid. Phospholipid-bound 15-hydroperoxyarachidonic acid would have the structural requirements needed for 4-hydroxynonenal (CH3-(CH2)4-CH(OH)-CH=CH-CHO). Microsomes supplemented with 15-hydroperoxyarachidonic acid and NADPH, ADP/iron converted only minimal amounts (0.6 mol%) of 15-hydroperoxyarachidonic acid into 4-hydroxynonenal; similarly, 15-hydroperoxyarachidonic acid incubated at pH 7.4 in the presence of ascorbate/iron yielded only small amounts of 4-hydroxynonenal with a rate orders of magnitude below that observed with microsomes. Phospholipid-bound 15-hydroperoxyarachidonic acid is therefore not a likely intermediate in the reaction pathway leading to 4-hydroxynonenal. The rate of 4-hydroxynonenal formation is highest during the very initial phase of its formation and the onset does not show a lag phase, suggesting a transient intermediate predominantly formed during the early phase of microsomal lipid peroxidation. After 60 min of incubation, 204 nmol polyunsaturated fatty acids (20 nmol 18:2, 143 nmol 20:4, 41 nmol 22:6) were lost per mg microsomal protein and the incubation mixture contained 206 nmol lipid peroxides, 71.6 nmol malonic dialdehyde and 4.6 nmol 4-hydroxynonenal per mg protein. Under artificial conditions (pH 1.0, ascorbate/iron, 20 h of incubation) not comparable to the microsomal peroxidation system, 15-hydroperoxyarachidonic acid can be decomposed in good yields (15 mol%) into 4-hydroxynonenal. Autoxidation of arachidonic acid in the presence of ascorbate/iron gave after 25 h of incubation 2.8 mol% (pH 7.4) and 1.5 mol% (pH 1.0) 4-hydroxynonenal. The most remarkable difference between the non-enzymic system and the enzymic microsomal system is that the latter forms 4-hydroxynonenal at a much higher rate.

Topics: Aldehydes; Animals; Arachidonic Acid; Arachidonic Acids; Ferrous Compounds; Hydrogen-Ion Concentration; Kinetics; Leukotrienes; Lipid Peroxides; Male; Malondialdehyde; Microsomes, Liver; NADP; Rats; Rats, Inbred Strains; Time Factors