Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species

Electrophilic lipids, such as 4-hydroxynonenal (HNE), and the cyclopentenones 15-deoxy-Delta12,14 -prostaglandin J2 (15d-PGJ2) and 15-J2-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ2. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle.     

Protein oxidation: examination of potential lipid-independent mechanisms for protein carbonyl formation

Previous data indicated that diquat-mediated protein oxidation (protein carbonyl formation) occurs through multiple pathways, one of which is lipid dependent, and the other, lipid independent. Studies reported here investigated potential mechanisms of the lipid-independent pathway in greater detail, using bovine serum albumin as the target protein. One hypothesized mechanism of protein carbonyl formation involved diquat-dependent production of H₂O₂, which would then react with site-specifically bound ferrous iron as proposed by Stadtman and colleagues. This hypothesis was supported by the inhibitory effect of catalase on diquat-mediated protein carbonyl formation. However, exogenous H₂O₂ alone did not induce protein carbonyl formation. Hydroxyl radical-generating reactions may result from the H₂O₂-catalyzed oxidation of ferrous iron, which normally is bound to protein in the ferric state. Therefore, the possible reduction of site-specifically bound Fe³⁺ to Fe²⁺ by the diquat cation radical (which could then react with H₂O₂) was also investigated. The combination of H₂O₂ and an iron reductant, ascorbate, however, also failed to induce significant protein carbonyl formation. In a phospholipid-containing system, an ADP:Fe²⁺ complex induced both lipid peroxidation and protein carbonyl formation; both indices were largely inhibitable by antioxidants. There was no substantial ADP:Fe²⁺-dependent protein carbonyl formation in the absence of phospholipid under otherwise identical conditions. Based on the lipid requirement and antioxidant sensitivity, these data suggest that ADP:Fe²⁺-dependent protein carbonyl formation occurs through reaction of BSA with aldehydic lipid peroxidation products. The precise mechanism of diquat-mediated protein carbonyl formation remains unclear, but it appears not to be a function of H₂O₂ generation or diquat cation radical-dependent reduction of bound Fe³⁺. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 185–190, 1998     

Involvement of lipid oxidation products in the formation of fluorescent and cross-linked proteins

Age-related fluorescent and cross-linked proteins increase with lipid oxidation of tissues. The fluorophores and cross-links have been considered to be conjugated Schiff bases between amino groups of proteins and malonaldehyde. Our recent studies showed that the fluorophores produced in the in vitro reaction of proteins with malonaldehyde are 1,4-dihydropyridine-3,5-dicarbaldehydes, whose fluorescence characteristics are similar to but not always the same as those of the age-related fluorescent substances, and that the cross-linking is due to less fluorescent conjugated Schiff bases. The in vitro reaction of proteins with oxidized lipids produces fluorescent and cross-linked proteins similar to those in the aging cells or tissues. Monofunctional aldehydes such as alkanals, alk-2-enals and alka-2,4-dienals can also participate in the formation of the fluorophores and cross-links. The fluorescent substances produced from the reaction of primary amines or proteins with these aldehydes showed spectra close to those of the age-related fluorescent substances.     

Fluorescence formation from the interaction of DNA with lipid oxidation degradation products

To clarify the mechanism of fluorescence formation between DNA and lipid degradation products in the presence of ferric chloride and ascorbic acid, a number of carbonyl compounds and decomposition products of pure methyl linolenate hydroperoxides were examined. Keto derivatives of methyl ricinoleate, linoleate, and oleate, alkanals and 2-alkenals produced little or no fluorescence with DNA in the presence of ferric chloride-ascorbic acid. 2,4-Alkadienals were more active and 2,4,7-decatrienal was the most active. Mixtures of volatile aldehydes prepared from linolenate hydroperoxide decomposed either thermally or with iron and ascorbate had the same activity as 2,4,7-decatrienal. Higher molecular-weight products from the decomposition of methyl linolenate hydroperoxides showed relatively low activity. beta-Carotene, alpha-tocopherol and other antioxidants effectively reduced the amount of fluorescence formed by linolenate hydroperoxides. The results suggest that, in addition to hydroperoxide decomposition products, singlet oxygen and/or free radical species contribute significantly to the fluorescence formed from the interaction of methyl linolenate hydroperoxides with DNA in the presence of ferric chloride and ascorbic acid.     

Routes of formation and toxic consequences of lipid oxidation products in foods.

Lipid oxidation in foods is initiated by free radical and/or singlet oxygen mechanisms which generate a series of autocatalytic free radical reactions. These autoxidation reactions lead to the breakdown of lipid and to the formation of a wide array of oxidation products. The nature and proportion of these products can vary widely between foods and depend on the composition of the food as well as numerous environmental factors. The toxicological significance of lipid oxidation in foods is complicated by interactions of secondary lipid oxidation products with other food components. These interactions could either form complexes that limit the bioavailability of lipid breakdown products or can lead to the formation of toxic products derived from non-lipid sources. A lack of gross pathological consequences has generally been observed in animals fed oxidized fats. On the other hand, secondary products of lipid autoxidation can be absorbed and may cause an increase in oxidative stress and deleterious changes in lipoprotein and platelet metabolism. The presence of reactive lipid oxidation products in foods needs more systematic research in terms of complexities of food component interactions and the metabolic processing of these compounds.     

Stability of lipid oxidation products in the rat liver and pathways of their utilization

The ability of liver homogenates to utilize various lipid peroxidation products was studied. Conjugated dienes and TBA-reactive products of unsaturated fatty acid phospholipids and triglycerides were found to be more stable that the corresponding lipid hydroperoxides. It was shown that decomposition of lipid hydroperoxides in liver homogenates is due to their reduction to corresponding oxycompounds without activation of free radical reactions. The ability of lipid hydroperoxides to be reduced in liver homogenates is determined by their chemical structure and decreases in the following order: polyunsaturated fatty acids--phospholipids--triglycerides--cholesterol esters.     

Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway

Cyclopentenone prostaglandins A2 and J2 are reactive compounds that possess unique biological activities. However, the extent to which they are formed in vivo remains unclear. In this study, we explored whether D2/E2-isoprostanes undergo dehydration in vivo to form A2/J2-isoprostanes. Oxidation of arachidonic acid in vitro generated a series of compounds that were confirmed to be A2/J2-isoprostanes by mass spectrometric analyses. A2/J2-isoprostanes were detected in vivo esterified to lipids in livers from normal rats at a level of 5. 1 +/- 2.3 ng/g, and levels increased dramatically by a mean of 24-fold following administration of CCl4. An A2-isoprostane, 15-A2t-isoprostane, was obtained and found to readily undergo Michael addition with glutathione and to adduct covalently to protein. A2/J2-isoprostanes could not be detected in the circulation, even following CCl4 administration, which we hypothesized might be explained by rapid formation of adducts. This was supported by finding that essentially all the radioactivity excreted into the urine following infusion of radiolabeled 15-A2t-isoprostane into a human volunteer was in the form of a polar conjugate(s). These data identify a new class of reactive compounds that are produced in vivo as products of the isoprostane pathway that can exert biological effects relevant to the pathobiology of oxidant injury.     

Halogenated compounds as inducers of lipid peroxidation in tissue slices

Twenty-seven halogenated compounds were screened as potential inducers of lipid peroxidation in rat liver, kidney, spleen, and testes slices. In addition to the known lipid peroxidation inducers--carbon tetrachloride and bromotrichloromethane--the novel compounds carbon tetrabromide, p-bromobenzyl bromide, and benzyl bromide increased lipid peroxidation in each of the tissues studied. Lipid peroxidation was measured by release of thiobarbituric acid-reactive substances (TBARS) from the tissue slices. The amount of TBARS released from liver slices incubated with bromotrichloromethane, carbon tetrabromide, dichloromethane, bromobenzene, chloroform, bromoform, benzyl chloride, bromochloromethane, and carbon tetrabromide correlated with the lethality of these compounds as evaluated by their oral LD50 in rats. The lethality of a number of the compounds tested did not correlate with their capacity to induce lipid peroxidation.     

Novel carbonyl and nitrile products from reactive chlorinating species attack of lysosphingolipid

Lysosphingolipids are important lipid signaling molecules that are associated predominantly with high density lipoproteins (HDL) in human plasma. Further, HDL has been shown to be a target for the reactive chlorinating species (RCS) produced by myeloperoxidase (MPO). Accordingly, RCS attack of lysosphingolipids was characterized in these studies. It was shown that RCS attack of sphingosylphosphorylcholine results in the formation of 2-hexadecenal and 1-cyano methano phosphocholine. The structures were identified and confirmed predominantly using mass spectrometric analyses. Further, it was demonstrated that RCS attack of another bioactive lysosphingolipid sphingosine 1-phosphate also results in the formation of 2-hexadecenal from its sphingosine base. Using a synthetically prepared, deuterated 2-hexadecenal internal standard, it was determined that 2-hexadecenal quickly accumulated in HDL treated with MPO/RCS generating system. Thus, the present studies characterize the formation of a novel group of lipid products generated following RCS attack of lysosphingolipids.     

Systematic isolation and identification of membrane lipid oxidation products

This report discusses the analytical procedure by which it is possible to isolate and identify the oxidation products of cellular and subcellular membrane lipids. The key point of this procedure is the method used for the transmethylation of the lipid material isolated from the tissues. In effect, both the conversion of the glycerides into methyl esters and the reduction of the hydroperoxyl groups into the corresponding hydroxyl groups is performed in one step, without breaking any oxirane rings that may be present. The methyl esters containing functional groups introduced by oxidative processes are separated from the non-modified ones by preparative TLC and are identified by GLC and GC-MS.     

Peroxynitrite-induced oxidation and nitration products of guanine and 8-oxoguanine: structures and mechanisms of product formation.

Peroxynitrite induces DNA base damage predominantly at guanine (G) and 8-oxoguanine (8-oxoG) nucleobases via oxidation reactions. Nitration products are also observed, consistent with the generation of radical intermediates that can recombine with the (.)NO(2) formed during peroxynitrite degradation. The neutral G radical, G(.), reacts with (.)NO(2) to yield 8-nitroguanine (8-nitroG) and 5-nitro-4-guanidinohydantoin (NI), while for 8-oxoG we have proposed a reactive guanidinylidene radical intermediate. The products generated during peroxynitrite-mediated 8-oxoG oxidation depend on oxidant flux, with dehydroguanidinohydantoin (DGh), 2,4,6-trioxo-[1,3,5]triazinane-1-carboxamidine (CAC) and NO(2)-DGh predominating at high fluxes and spiroiminodihydantoin (Sp), guanidinohydantoin (Gh) and 4-hydroxy-2,5-dioxo-imidazolidine-4-carboxylic acid (HICA) predominating at low fluxes. Both product sets are observed at intermediate fluxes. It is therefore important in model systems to ensure that the relative concentrations are well controlled to minimize competing reactions that may not be relevant in vivo. Increasingly sophisticated systems for modeling peroxynitrite production in vivo are being developed and these should help with predicting the products most likely to be formed in vivo. Together with the emerging information on the genotoxic and mutational characteristics of the individual oxidation products, it may be found that the extent of tissue damage, mutational spectra and, hence, cancer risk may change as a function of peroxynitrite fluxes as different product combinations predominate.     

Antimutagenic compounds and their possible mechanisms of action

Mutagenicity refers to the induction of permanent changes in the DNA sequence of an organism, which may result in a heritable change in the characteristics of living systems. Antimutagenic agents are able to counteract the effects of mutagens. This group of agents includes both natural and synthetic compounds. Based on their mechanism of action among antimutagens, several classes of compounds may be distinguished. These are compounds with antioxidant activity; compounds that inhibit the activation of mutagens; blocking agents; as well as compounds characterized with several modes of action. It was reported previously that several antitumor compounds act through the antimutagenic mechanism. Hence, searching for antimutagenic compounds represents a rapidly expanding field of cancer research. It may be observed that, in recent years, many publications were focused on the screening of both natural and synthetic compounds for their beneficial muta/antimutagenicity profile. Thus, the present review attempts to give a brief outline on substances presenting antimutagenic potency and their possible mechanism of action. Additionally, in the present paper, a screening strategy for mutagenicity testing was presented and the characteristics of the most widely used antimutagenicity assays were described.     

Photochemical oxidation of chlorpromazine in the dark induced by enzymically generated triplet carbonyl compounds

Enzymically generated triplet acetone and ethanal transfer energy to chlorpromazine as indicated by (i) suppression of the acetone chemiphosphorescence (ii) concomitant formation of chlo$\text{r}$ promazine photoproducts, that is the radical cation and the sulfoxide (iii) inhibition of photoproduct formation by a very efficient competition for triplet carbonyl energy using the sodium salt of 9,10-dibromoanthracene-2-sulfonic acid.This is the first report of a photooxidation in the dark.     

Aspartic acid functions as carbonyl trapper to inhibit the formation of advanced glycation end products by chemical chaperone activity

Advanced glycation end products (AGEs) were implicated in pathology of numerous diseases. In this study, we present the bioactivity of aspartic acid (Asp) to inhibit the AGEs. Hemoglobin and bovine serum albumin (BSA) were glycated with glucose, fructose, and ribose in the presence and absence of Asp (100–200 μM). HbA₁c inhibition was investigated using human blood and characterized by micro-column ion exchange chromatography. The effect of methyl glyoxal (MG) on hemoglobin and BSA was evaluated by fluorescence spectroscopy and gel electrophoresis. The effect of MG on red blood cells morphology was characterized by scanning electron micrographs. Molecular docking was performed on BSA with Asp. Asp is capable of inhibiting the formation of fluorescent AGEs by reacting with the reducing sugars. The presence of Asp as supplement in whole blood reduced the HbA₁c% from 8.8 to 6.1. The presence of MG showed an increase in fluorescence and the presence of Asp inhibited the glycation thereby the fluorescence was quenched. MG also affected the electrophoretic mobility of hemoglobin and BSA by forming high molecular weight aggregates. Normal RBCs showed typical biconcave shape. MG modified RBCs showed twisted and elongated shape whereas the presence of ASP tends to protect RBC from twisting. Asp interacted with arginine residues of bovine serum albumin particularly ARG 194, ARG 198, and ARG 217 thereby stabilized the protein complex. We conclude that Asp has dual functions as a chemical chaperone to stabilize protein and as a dicarbonyl trapper, and thereby it can prevent the complications caused by glycation.     

Vanadium compounds as antiparasitic agents: An approach to their mechanisms of action

BackgroundParasitic infections are a public health problem since they have high morbidity and mortality worldwide. In parasitosis such as malaria, leishmaniasis and trypanosomiasis it is necessary to develop new compounds for their treatment since an increase in drug resistance and toxic effects have been observed. Therefore, the use of different compounds that couple vanadium in their structure and that have a broad spectrum against different parasites have been proposed experimentally.ObjectiveReport the mechanisms of action exerted by vanadium in different parasites.ConclusionIn this review, some of the targets that vanadium compounds have were identified and it was observed that they have a broad spectrum against different parasites, which represents an advance to continue investigating therapeutic options.     

Sulfur-centered reactive intermediates derived from the oxidation of sulfur compounds of biological interest

Sulphur compounds play a central role in the structure and activity of many vital systems. In the living cell, sulfur constitutes an essential part of the defense against oxidative damage and is transformed into a variety of sulfur free radical species. Many studies of the chemistry of sulfur-centered radicals using pulse radiolysis and photolysis techniques to detect and measure the kinetics of these radicals have been published and reviewed. This paper discusses the present state of research on the formation and reactivity of certain sulfur-centered radicals [RS*, RSS*, RS*+, (RSSR)*+] and their implications for biological systems.     

Singlet oxygen-mediated protein oxidation: evidence for the formation of reactive peroxides

Abstract

Singlet oxygen (molecular oxygen in its ¹Δ_g state, ¹O₂) can be readily generated in a number of in vitro systems, and is suspected to be a major causative agent in some diseases. ¹O₂ has been shown to be produced in myeloperoxidase- and eosinophil peroxidase-catalysed reactions^1–3 and has been reported to be generated by some cell types including neutrophils,⁴ eosinophils,^3,5 and macrophages.⁶ UV light has been shown to generate ¹O₂, and it has been postulated that ¹O₂ plays a role in the development of cataract,^7–9 sunburn and some skin cancers.¹⁰     

Molecular mechanisms of compounds affecting bacterial biofilm formation and dispersal

Bacteria can switch between planktonic forms (single cells) and biofilms, i.e., bacterial communities growing on solid surfaces and embedded in a matrix of extracellular polymeric substance. Biofilm formation by pathogenic bacteria often results in lower susceptibility to antibiotic treatments and in the development of chronic infections; thus, biofilm formation can be considered an important virulence factor. In recent years, much attention has been directed towards understanding the biology of biofilms and towards searching for inhibitors of biofilm development and of biofilm-related cellular processes. In this report, we review selected examples of target-based screening for anti-biofilm agents: We focus on inhibitors of quorum sensing, possibly the most characterized target for molecules with anti-biofilm activity, and on compounds interfering with the metabolism of the signal molecule cyclic di-GMP metabolism and on inhibitors of DNA and nucleotide biosynthesis, which represent a novel and promising class of biofilm inhibitors. Finally, we discuss the activation of biofilm dispersal as a novel mode of action for anti-biofilm compounds.