Pathophysiology of neutrophil-mediated extracellular redox reactions.

Neutrophil granulocyte leukocytes (neutrophils) play fundamental role in the innate immune response. In the presence of adequate stimuli, neutrophils release excessive amount of reactive oxygen species (ROS) that may induce cell and tissue injury. Oxidative burst of neutrophils acts as a double-edged sword.

4-Hydroxy-nonenal-A Bioactive Lipid Peroxidation Product.

This review on recent research advances of the lipid peroxidation product 4-hydroxy-nonenal (HNE) has four major topics: I. the formation of HNE in various organs and tissues, II. the diverse biochemical reactions with Michael adduct formation as the most prominent one, III.

Elevated neutrophil elastase and acrolein-protein adducts are associated with W256 regression.

The involvement of granulocytes in immune response against cancer is not well understood. Depending on the cytokine milieu in which they act and on their oxidative burst, granulocytes may play either an inhibitory or stimulatory role in tumour growth. Unsaturated fatty acids, essential components of cellular membranes and storage lipids, are susceptible to granulocyte-derived reactive oxygen species (ROS).

The involvement of granulocytes in spontaneous regression of Walker 256 carcinoma.

We described before that oxidative burst of granulocytes is cytotoxic for melanoma B16F10 and for Walker 256 carcinoma (W256). Therefore, we assumed that granulocytes could also be important mechanism of the host defence against tumour. In current study we report massive granulocyte infiltration at the site of W256 transplanted in the hind limb of Sprague-Dawley associated with spontaneous tumour regression observed for 22/25 rats (87%).

4-hydroxynonenal: a membrane lipid oxidation product of medicinal interest.

A comprehensive focus on 4-hydroxynonenal (HNE) as candidate molecule in a variety of pathophysiological conditions occurring in humans is here provided. Despite an active, now well characterized, metabolism in most cells and tissues, HNE can be easily detected and quantified by means of several methods, although with different sensitivity. Measurements of HNE and/or stable metabolites in biological fluids are already applied as lipid peroxidation/oxidative stress markers in a huge number of human disease processes, often sustained by inflammatory reactions.

Oxidative burst of neutrophils against melanoma B16-F10.

Intensive oxidative burst was determined by chemiluminescence of peripheral blood neutrophils of mice that were intramuscularly injected with melanoma B16-F10 and/or subcutaneously with Sephadex G-200. The neutrophils from papula developed at the site of Sephadex injection were cytotoxic for the B16-F10 cells in vitro. However, survival of Sephadex injected tumour-bearing mice was lower than of control animals bearing B16-F10, while their tumours grew faster and were less necrotic.

Oxidative burst and anticancer activities of rat neutrophils.

It is assumed that oxidative damage caused by reactive oxygen species (ROS) from activated neutrophil granulocytes may contribute to pathology of tumors. ROS are crucial in neutrophil-mediated tumor cell lysis. The present study is focused on the oxidative burst and antitumorous activities of neutrophils when challenged with Walker carcinoma W256.

Expression and synthesis of TGFbeta1 is induced in macrophages by 9-oxononanoyl cholesterol, a major cholesteryl ester oxidation product.

Within the broad variety of compounds generated via oxidative reactions in low density lipoproteins (LDL) and subsequently found in the atherosclerotic plaque, are aldehydes still esterified to the parent lipid and termed core-aldehydes. The most represented cholesterol core-aldehyde in LDL is 9-oxononanoyl cholesterol (9-ONC), an oxidation product of cholesteryl linoleate. Here we report that 9-ONC, at concentration actually detectable in biological material, significantly up-regulates the expression and the synthesis of the pro-fibrogenic cytokine transforming growth factor beta1 (TGFbeta1) by cultured macrophages.

Basic aspects of the biochemical reactivity of 4-hydroxynonenal.

4-hydroxynonenal (HNE), a major lipid peroxidation product of n-6 polyunsaturated fatty acids, which was discovered by the late Hermann Esterbauer, is a remarkable trifunctional molecule. Both the hydroxy group and the conjugated system consisting of a C=C double bond and a carbonyl group contribute to the high reactivity of HNE. Most of the biochemical effects of HNE can be explained by its rapid reactions with thiol and amino groups.

The formation of phosphatidylcholine oxidation products by stimulated phagocytes.

Phagocytic cells produce a variety of oxidants as part of the immune defence, which react readily both with proteins and lipids, and could contribute to the oxidation of low density lipoprotein in atherosclerosis. We have investigated the oxidation of phospholipid vesicles by neutrophils and mononuclear cells, to provide a model of lipid oxidation in the absence of competing protein. Phorbol 12-myristate 13-acetate-stimulated neutrophils were incubated with phospholipid vesicles containing dipalmitoyl phosphatidylcholine, palmitoyl-arachidonoyl phosphatidylcholine (PAPC) and stearoyl-oleoyl phosphatidylcholine, before extraction of the lipids for analysis by HPLC coupled to electrospray mass spectrometry.

Mechanisms of iron-induced oxidative modifications of creatine kinase in rat brain in vitro. possible involvement of HNE.

The model of oxidative stress induced by Fe/ascorbate in rat brain in vitro was used to compare the antioxidant capacity of known antioxidants. Creatine kinase (CK) was selected as a marker of protein injury in such studies. Of the antioxidant enzymes (catalase, superoxide dismutase), oxygen radical scavengers (mannitol, glutathione), and the chelator (EDTA) tested in this work and this system, only catalase and glutathione prevented the injury induced by oxidative stress, indicating that H2O2 and the glutathione peroxidase reaction were involved in the preventive effect.

Differential influence of the lipid peroxidation product 4-hydroxynonenal on the growth of human lymphatic leukaemia cells and human periopherial blood lymphocytes.

The purpose of this study was to compare the influence of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids, on the proliferation of malignant CEM-NKR lymphatic leukaemia cells and normal human lymphocytes (HPBM). Furthermore the growth modulating effect of phytohemagglutinin (PHA) on both cell lines was examined. The effects of HNE were monitored 18 hours and 3 days after incubation, using two different DNA-synthesis assays ([3H]-thymidine- and BrdU- incorporation) and a mitochondrial dehydrogenases activity assay (MTT).

The reactions of hypochlorous acid, the reactive oxygen species produced by myeloperoxidase, with lipids.

Myeloperoxidase (MPO), an abundant enzyme in phagocytes, has been implicated in the pathogenesis of various inflammatory diseases including atherosclerosis. The major oxidant produced by MPO, hypochlorous acid (HOCl), is able to modify a great variety of biomolecules by chlorination and/or oxidation. In this paper the reactions of lipids (preferentially unsaturated fatty acids and cholesterol) with either reagent HOCl or HOCl generated by the MPO-hydrogen peroxide-chloride system are reviewed.

An improved method for the sensitive monitoring of low density lipoprotein modification by myeloperoxidase.

The aim of this investigation was to compare an improved fluorometric method with an UV absorbance assay for their ability to monitor low density lipoprotein (LDL) modification by myeloperoxidase (MPO) and to evaluate determining factors influencing the modification of LDL. Using absorbance at 234 nm to study the kinetics of LDL aggregation, and a native fluorescence assay for protein oxidation, we found that all components of the MPO/H2O2/Cl- system may have rate determining effects on LDL modification. While the lipoprotein modification rate correlated positively with enzyme concentration, variation of the concentration of H2O2 had a biphasic effect on the maximal rate of LDL modification with both methods.

Hypochlorite induces the formation of LDL(-), a potentially atherogenic low density lipoprotein subspecies.

Oxidation of low density lipoprotein (LDL) induced by hypochlorous acid (HOCl) leading to LDL(-), a minimally oxidized subspecies of LDL, was investigated. LDL(-) is characterized by its greater electronegativity and oxidative status, and is found in plasma in vivo. Its concentration was found to be elevated under conditions that predispose humans to atherosclerosis.

Postischemic reperfusion of the spinal cord: prolonged reperfusion alleviates the metabolic alterations induced by 25 min ischemia in the cervical and thoracolumbal segments.

In recent years, increasing amount of information has indicated that in some tissues the main damage due to oxidative stress does not occur during reperfusion but during the ischemic episode of the ischemia/reperfusion event. In this respect, serious doubts were also expressed about the origin of the increased amounts of free radicals which were believed to form and reported to appear in the perfusate during the first minutes of reperfusion. Moreover, speculative explanations were only available for a second increase in lipid peroxidation which was reported to occur after postischemic reperfusions exceeding 60 min.

4-Hydroxynonenal in the pathomechanisms of oxidative stress.

Here we review the current knowledge on the biochemistry and molecular pathology of oxidative stress with specific regard to a major aldehydic end-product stemming from peroxidation of biomembranes, that is 4-hydroxynonenal (HNE). This multifunctional molecule, which derives from the most represented class of polyunsaturated fatty acids in the membranes, is potentially able to undergo a number of reactions with proteins, phospholipids, and nucleic acids. Despite an active metabolism in most of the cell types, HNE can be detected in several biological tissues by means of sufficiently precise methods, although with different sensitivity.

Detection of phospholipid oxidation in oxidatively stressed cells by reversed-phase HPLC coupled with positive-ionization electrospray [correction of electroscopy] MS.

Measurement of lipid peroxidation is a commonly used method of detecting oxidative damage to biological tissues, but the most frequently used methods, including MS, measure breakdown products and are therefore indirect. We have coupled reversed-phase HPLC with positive-ionization electrospray MS (LC-MS) to provide a method for separating and detecting intact oxidized phospholipids in oxidatively stressed mammalian cells without extensive sample preparation. The elution profile of phospholipid hydroperoxides and chlorohydrins was first characterized using individual phospholipids or a defined phospholipid mixture as a model system.

Differential regulation of endothelin secretion and endothelin receptor mRNA levels in JAR, JEG-3, and BeWo choriocarcinoma cell lines and in human trophoblasts, their nonmalignant counterpart.

Endothelin (ET) secretion and expression of both ET-A and ET-B receptor subtypes have been found in a number of primary cancers. The present study tested (1) whether choriocarcinoma cells and their nonmalignant counterpart, the trophoblast, secrete ET-1 and express ET-A and ET-B receptors; (2) whether ET-1 secretion and receptor mRNA levels are regulated by the same factors in nonvascular tissues as in vascular tissues; and (3) whether such regulation is similar in malignant and nonmalignant cells. All cells secreted ET-1 in similar amounts (approximately 0.

Comparison of HOCl traps with myeloperoxidase inhibitors in prevention of low density lipoprotein oxidation.

In this study, the production of the highly toxic oxidant hypochlorous acid (HOCl) by the phagocytic enzyme myeloperoxidase (MPO) was quantitated and the concomitant alterations of low density lipoprotein (LDL) were analyzed in view of the potential role of LDL in atherosclerosis. Using the monochlorodimedone assay, it was found that HOCl is produced in micromolar concentrations. The kinetics of the decrease of tryptophan fluorescence appeared to be a sensitive method to monitor LDL alterations under near in vivo conditions.

Kinetics of chlorination of monochlorodimedone by myeloperoxidase.

The phagocyte-derived enzyme myeloperoxidase has been recently implicated in the pathogenesis of atherosclerosis, because it catalyzes the reaction of hydrogen peroxide with chloride ions to give the highly toxic oxidant hypochlorous acid. The aim of this study was to determine the dependence of this reaction on the concentration of hydrogen peroxide and of the enzyme by means of the photometric monochlorodimedone assay. The initial rate of hypochlorous acid formation increased less than proportionally with increasing myeloperoxidase concentrations.

Kinetics of tryptophan oxidation in plasma lipoproteins by myeloperoxidase-generated HOCl.

The relative susceptibility of the apoprotein components of human lipoproteins [high-density lipoprotein (HDL) and low-density lipoprotein (LDL)] and their subclasses to oxidation by the myeloperoxidase/H2O2/Cl- system in vitro was studied by measuring the decrease in rate of tryptophan fluorescence. Whereas the lipoprotein-modification rate showed a saturation type of dependence on the concentration of myeloperoxidase, a biphasic dependence on the concentration of the lipoproteins was found. High concentrations of H2O2 were also found to inhibit tryptophan oxidation in LDL but to a lesser extent in HDL.

Impaired proliferation and DNA synthesis of a human tumor cell line (HeLa) caused by short treatment with the anti-anemic drug jectofer (ferric-sorbitol-citrate) and the lipid peroxidation product 4-hydroxynonenal.

Anti-anaemic drug, ferric-sorbitol-citrate complex (FSC), inhibit tumour cell growth through the mechanisms which are complex and not entirely understood. The probable mechanisms of described effects of iron is iron-induced oxidative stress of the treated cells. Hence, the effects of FSC on HeLa cell growth in vitro were compared with the biological activity of one of the major mediators of the oxygen free radicals--aldehyde 4-hydroxinonenal (HNE), to see if the effects of FSC and of HNE resemble each other.

Correlation of low-density lipoprotein modification by myeloperoxidase with hypochlorous acid formation.

Myeloperoxidase is an enzyme in phagocytes which catalyzes several redox reactions. A major product is hypochlorous acid which appears to be important in inflammatory processes such as atherosclerosis. The aim of this study was to investigate whether the kinetics of low-density lipoprotein modification by the myeloperoxidase/hydrogen peroxide/chloride system in vitro conform to the established kinetics of hypochlorous acid formation and to compare the results with known in vivo data.