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). ROS can induce lipid peroxidation (LPO) resulting in the destruction of biomembranes. Thus, murine W256 tumour progressing and tumour regressing animal models were used to study the involvement of plasma inflammatory mediators and oxidative burst of circulating granulocytes in malignant destruction and detrimental tumour growth. The involvement of LPO-derived aldehydes (i.e. acrolein, 4-hydroxy-2-nonenal and malondialdehyde) and myeloperoxidase (MPO) appearance in the granulocyte anti-cancer response were further evaluated. The results obtained revealed a significant increase in neutrophil elastase in animals with regressing tumour. Furthermore, the presence of MPO in tumour microenvironment was accompanied by the formation of acrolein only 5 h after tumour transplantation and its presence increased during tumour regression. Later, at an early stage of tumour regression, the presence of other LPO-derived aldehydes were also observed. The results obtained suggest that elevated neutrophil elastase and initiation of LPO may play an important role in the tumour development leading to tumour regression.

Distribution and time-course of 4-hydroxynonenal, heat shock protein 110/105 family members and cyclooxygenase-2 expression in the hippocampus of rat during trimethyltin-induced neurodegeneration.

Trimethyltin (TMT), an organotin compound considered a useful tool to obtain an experimental model of neurodegeneration, exhibits neurotoxicant effects selectively localised in the limbic system and especially in the hippocampus, which are different in the rat and in mice. In the rat hippocampus, we investigated the expression of aldehyde 4-hydroxynonenal, a major bioactive marker of membrane lipid peroxidation, heat shock protein (HSP) 110/105 family members, markers of oxidative stress, and the neuroinflammatory marker cyclooxygenase-2 after TMT-intoxication at various time points after treatment. Our data show that TMT-induced neurodegeneration in the rat hippocampus is associated specifically with oxidative stress and lipid peroxidation, but not with HSP expression, indicating species-specific differences in the neurotoxicity of TMT between rats and mice.

Down-modulation of nuclear localisation and pro-fibrogenic effect of 4-hydroxy-2,3-nonenal by thiol- and carbonyl-reagents.

Among the oxidative breakdown products of omega-6 unsaturated fatty acids, the aldehyde 4-hydroxy-2,3-nonenal (HNE) is receiving increasing attention for its potential pathophysiological implication, which at least partly lies on the demonstrated ability to modulate gene expression of a number of genes. Here we show that a marked down-modulation of HNE nuclear localisation in cells of a macrophage line (J774-A1) can be afforded by treatment with sulfydryl and carbonyl reagents without significantly interfering with cell viability. As regards the addition of thiol-group reagents to the cell suspension, N-ethylmaleimide (NEM) led to a sustained decrease of HNE nuclear localisation, while 4-(chloromercuri)-benzene-sulfonic acid (PCMBS) gave a similar but more transient effect. Hydroxylamine (HYD), a carbonyl-group reagent, was also able to inhibit HNE nuclear localisation. The actual efficacy of the inhibitors used was then tested on the HNE-induced stimulation of transforming growth factor beta1 (TGFbeta1) production by J774-A1 cells. Indeed, the thiol reagents NEM and PCMBS, both markedly down-modulating HNE nuclear localisation, were able to inhibit HNE-induced increase of TGFbeta1 protein synthesis. The carbonyl reagent HYD was less effective on this respect, producing strong but incomplete protection against HNE-induced TGFbeta1 increase. Taken together, the results indicate that sulfydryl groups are involved in the process of HNE cellular internalisation, while both sulfydryl and carbonyl groups are involved in the process of HNE nuclear translocation, and consequently in the modulation of gene expression by the aldehyde. Further, an actual demonstration is provided that HNE-induced effect on gene regulation can be efficiently counteracted by suitable interference with HNE biochemistry.

Involvement of lipid peroxidation, oncogene expression and induction of apoptosis in the antitumorous activity of ferric-sorbitol-citrate.

We described before that iron-containing, anti-anaemic drug, ferric-sorbitol-citrate complex (FSC) inhibited proliferation of various murine cancer cells in vitro and caused tumour regression in vivo, but did not affect proliferation of the non-malignant cells. The aim of this study was to evaluate further the anticancer activity mechanism of FSC using human colon cancer cell line CaCo2. After treatment with FSC for 72 hours impaired proliferative ability and viability of CaCo2 cells as observed. Growth modification caused by FSC involved diminished expression of Bcl-2, and over-expression of mp53 proto-oncogenes, accompanied by increased incidence of apoptosis. Immunostaining the cells applying monoclonal antibodies for lipid peroxidation product 4-hydroxynonenal (HNE) showed that FSC-iron increased intracellular HNE, but did not induce severe HNE-mediated oxidative stress. Thus, antitumorous mechanism of FSC involves modulation of oncogene expression and induction of apoptosis apparently not triggered by lipid peroxidation-mediated oxidative stress, although FSC might restore endogenous HNE production in the CaCo2 cells to level resembling physiological for various non-malignant cells and tissues. Higher dose of FSC increased also number of intracellular ferritin positive CaCo2 cells.

4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor.

Immunohistochemical analysis of the distribution of the lipid peroxidation product 4-hydroxynonenal (HNE) in the brain of baboons exposed to experimental hemorrhagic traumatic shock or sepsis showed that systemic oxidative stress and the thereby generated HNE affect the blood:brain barrier and the regulation of cerebral blood flow determining secondary brain damage. Similarly, HNE was determined during ischemia in the brain blood vessels of rats exposed to ischemia/reperfusion injury of the brain. After reperfusion, HNE disappeared from the blood vessels but remained in neurones and in glial cells. Since HNE modulates cell proliferation and differentiation (including proto-oncogene expression), it is postulated that HNE might have prominent local and systemic effects that are not only harmful but beneficial, too, determining the outcome of various pathophysiological conditions based on oxidative stress.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

The presence of 4-hydroxynonenal/protein complex as an indicator of oxidative stress after experimental spinal cord contusion in a rat model.

OBJECT: The authors tested the hypothesis that breach of the blood-spinal cord barrier (BSCB) will produce evidence of oxidative stress and that a similar staining pattern will be seen between 4-hydroxynonenal (HNE)/protein complexes and extravasated immunoglobulin G (IgG). METHODS: Adult female Fischer 344 rats, each weighing 200 to 225 g, were subjected to a spinal cord contusion at T-10 by means of a weight-drop device. Spinal cord tissue was assessed for oxidative stress by localizing extravasated plasma contents with a monoclonal antibody for rat IgG and protein conjugation with HNE, which is an aldehyde byproduct of lipid peroxidation. The animals were killed at 1 and 6 hours, and 1, 2, and 7 days after surgery. Maximum HNE/protein staining was observed at 2 days postinjury, and HNE/protein and IgG manifested similar staining patterns. Analysis revealed a graduated but asymmetrical rostral-caudal response relative to the T-10 injury site. Both HNE/protein complex and IgG staining revealed that the caudal levels T-11 and T-12 stained significantly more intensely than the rostral levels T-9 and T-8, respectively. A higher percentage of neurons positive for HNE/protein immunostaining was observed in spinal cord levels caudal to the injury site compared with equidistant rostral regions. Protein dot-blot assays also revealed a similar asymmetrical rostral-caudal HNE/protein content. To analyze the timing of the BSCB breach, another group of animals received identical contusions, and horseradish peroxidase (HRP) was injected 10 minutes before or at various times after injury (1, 3, and 6 hours, and 1, 2, and 7 days). Maximum HRP permeability was seen immediately after injury, with a significant decrease occurring by 1 hour and a return to control levels by 2 days posttrauma. CONCLUSIONS: Data from this study indicate possible compromise of neuronal, axonal, glial, and synaptic function after trauma, which may be a factor in motor deficits seen in animals after spinal cord contusion. The colocalization of the IgG stain with the HNE/protein stain is consistent with the hypothesis of a mutual cause-effect relationship between BSCB and oxidative stress in central nervous system trauma.

Evaluation of a platelet apheresis technique for the preparation of leukocyte-reduced platelet concentrates.

OBJECTIVES: Reduction of the white blood cell (WBC) contamination in platelet concentrates (PC) protects patients from the immunological and infectious side effects of platelet transfusion caused by WBC. This can be done either by filtration of the PC or by improved apheresis techniques that yield WBC-poor preparations. METHODS: To evaluate an improved technique for platelet collection, we carried out 201 separations in 89 healthy cytapheresis donors using the new COBE Spectra leukoreduction system (LRS) and compared the results with those of standard dual-needle separations obtained with the same cell separator. RESULTS: A small but statistically significant difference was found in platelet collection efficiency in comparison with the standard non-LRS software procedures (LRS: 52.6 vs. 56.3% for the reference). However, median WBC contamination was only 0.01 x 10(6) WBC per LRS product. This significant (p < 0.0005) improvement corresponds to a 10-fold reduction of WBC as compared with the standard dual-needle technique. CONCLUSIONS: The COBE Spectra LRS system produced PCs with a platelet collection efficiency nearly equal to previous techniques and with a residual WBC content satisfying even the most stringent criteria for WBC-depleted blood components. As this purity is achieved without important platelet loss, conventional fiber filtration no longer seems necessary in this kind of PC.

4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4-hydroxynonenal potentiates oxidative cascades. Collectively, these findings suggest that 4-hydroxynonenal plays important roles in oxidative impairment of synaptic functions that would be expected to promote excitotoxic cascades.

Evidence that 4-hydroxynonenal mediates oxidative stress-induced neuronal apoptosis.

Oxidative stress is believed to play important roles in neuronal cell death associated with many different neurodegenerative conditions (e.g., Alzheimer's disease, Parkinson's disease, and cerebral ischemia), and it is believed also that apoptosis is an important mode of cell death in these disorders. Membrane lipid peroxidation has been documented in the brain regions affected in these disorders as well as in cell culture and in vivo models. We now provide evidence that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, is a key mediator of neuronal apoptosis induced by oxidative stress. HNE induced apoptosis in PC12 cells and primary rat hippocampal neurons. Oxidative insults (FeSO4 and amyloid beta-peptide) induced lipid peroxidation, cellular accumulation of HNE, and apoptosis. Bcl-2 prevented apoptosis of PC12 cells induced by oxidative stress and HNE. Antioxidants that suppress lipid peroxidation protected against apoptosis induced by oxidative insults, but not that induced by HNE. Glutathione, which binds HNE, protected neurons against apoptosis induced by oxidative stress and HNE. PC12 cells expressing Bcl-2 exhibited higher levels of glutathione and lower levels of HNE after oxidative stress. Collectively, the data identify that HNE is a novel nonprotein mediator of oxidative stress-induced neuronal apoptosis and suggest that the antiapoptotic action of glutathione may involve detoxification of HNE.

4-Hydroxynonenal, a product of lipid peroxidation, inhibits dephosphorylation of the microtubule-associated protein tau.

In Alzheimer's disease (AD) the microtubule-associated protein tau is excessively phosphorylated in degenerating neurons, but the mechanisms underlying the increased phosphorylation are unknown. Recent findings suggest that oxidative stress, and membrane lipid peroxidation in particular, contributes to the neurodegenerative process in AD. We now report that following exposure of cultured rat hippocampal neurons to 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, tau is resistant to dephosphorylation. Immunocytochemical and Western blot analyses using phosphorylation-sensitive tau antibodies showed that HNE treatment causes a moderate increase in basal levels of tau phosphorylation, and prevents tau dephosphorylation by alkaline phosphatase in neurons pretreated with the phosphatase inhibitor okadaic acid. Studies with anti-HNE antibodies showed that HNE binds directly to tau, and that HNE immunoreactivity localizes to cell bodies and axons, cell compartments that contain tau. These data suggest a role for HNE in altered tau phosphorylation and neurofibrillary degeneration in AD.

A prospective randomized study of three types of platelet concentrates in patients with haematological malignancy: corrected platelet count increments and frequency of nonhaemolytic febrile transfusion reactions.

We prospectively randomized 51 patients with haematological malignancy requiring platelet concentrates (PCs) to receive either single donor platelet-pheresis products (SD-PC), PCs made from pooled buffy coats (BC-PC) or pooled units of platelets made by the platelet-rich plasma method (PRP-PC). The leucocyte content of each type of PC was 0.33 (0.03-13.5), 5.68 (0.19-99.0) and 365 (65-910) x 10(6); median (range), respectively; P < 0.0001. All red cell transfusions were leucodepleted by filtration. Statistical comparison of the probability of the occurrence of a nonhaemolytic febrile transfusion reaction (NHFTR) following transfusion of PCs in patients in each group showed a significant decrease for the SD-PC and BC-PC groups (0.031 and 0.038, respectively) when compared with PRP-PC (0.171); P = 0.0001. The actual corrected platelet count increments (CCI) at 1-6 and 18-24 h post-transfusion for all three types of PC did not differ significantly. We conclude that transfusion of PRP-PC is associated with a significant increase in NHFTR.

Monoclonal antibodies for detection of 4-hydroxynonenal modified proteins.

A promising approach to study lipid peroxidation pathology is antibodies recognizing aldehydes which react with and became bound to amino acid side chains of proteins. We present in this study the characterization of several monoclonal antibodies which recognize 4-hydroxynonenal (HNE) modified proteins. Six out of 20 antibodies recognizing HNE modified BSA were able to detect HNE-protein adducts in peroxidized liver microsomes. Two of these antibodies were selected and characterized. Both antibodies could also detect HNE-protein adducts in oxidized low density lipoprotein. They exhibit no detectable cross reaction with proteins modified by malonaldehyde, nonanal, nonanal and 4-hydroxyhexenal. Protein bound 4-hydroxyoctenal and 4-hydroxydecenal were recognized to some extent. Further characterization revealed that the two antibodies are highly selective for HNE bound to histidine with only some cross reaction to HNE bound to lysine and cysteine. Preliminary quantitative ELISA-analysis showed that oxidized microsomes and oxidized LDL contain 12 nmol and 3 nmol HNE-histidine per mg protein respectively.

Cytotoxic and chemotactic potencies of several aldehydic components of oxidised low density lipoprotein for human monocyte-macrophages.

We have investigated the cytotoxic and chemotactic potencies of malondialdehyde (MDA), hexanal, 4-hydroxyhexenal (HHE), 4-hydroxynonenal (HNE) and 4-hydroxyoctenal (HOE), which are aldehydes found in oxidised low density lipoprotein (LDL), for human monocyte-macrophages. They were toxic in the following order: hexanal

Inhibition of melanoma B16-F10 growth by lipid peroxidation product 4-hydroxynonenal.

Since a gradual benign-to-malignant progression of murine melanoma B16 after exposure in vitro to hypoxia was described recently, the aim of this study was to test if exposing melanoma B16-F10 cells to aldehyde 4-hydroxynonenal (HNE), which is considered not only as one of the major "second toxic messengers" of oxygen free radicals (or oxidative stress), but as a normal constituent of many cells and tissues, might have opposite effects. Treatment of the tumor cells with 50 microM HNE in vitro or in vivo did not prevent development of the tumors, but inhibited their growth. Tumor growth inhibition was equal for in vitro and in vivo treatment, but appeared after a delay of almost one week, since there was no difference of the tumor volume to the control observed during the initial period of the tumor growth. Similarly, both HNE treatment of the tumor cells before transplantation and HNE treatment of the melanoma bearing mice resulted in equally prolonged survival time. Thus, the results obtained suggest that while hypoxia could increase the malignancy of the murine melanoma cells, exposing these cells to one of the major "second toxic messengers" of oxygen free radicals, HNE, has almost opposite effects and further indicate the possible use of the aldehyde in vivo.

Elevated serum neopterin levels in atherosclerosis.

Plasma levels of neopterin were determined in patients with different clinical stages of atherosclerosis. Non-hospitalized patients with atherosclerosis had serum and plasma neopterin levels within the normal range of the assay (6 +/- 2 nM). These values were not significantly different from those reported for healthy blood donors (5 +/- 2 nM). In contrast, about 50% (29 out of 61) of hospitalized patients undergoing conservative or surgical therapy had neopterin plasma levels, which exceeded the normal range (greater than 10 nM) up to 10-fold. The two groups differ on a significance level of P less than 0.01. For further evaluation hospitalized patients were subgrouped according to neopterin levels. In the subgroup with elevated neopterin levels patients with higher Frederickson types of atherosclerosis were overrepresented compared to patients with normal neopterin levels. Type 4 differed significantly from patients without pathological changes of lipoprotein (P less than 0.05). Only 3 patients suffered from minimal skin necrosis, two of them had elevated neopterin levels. Significantly more patients with peripheral artery occlusions had elevated neopterin levels than patients with occlusions of central arteries (P less than 0.05). All other criteria used for comparison (sex, age, smoking, antioxidant status, diabetes, hypertension, adipositas, hyperuricemia) did not vary significantly in both subgroups. These data indicate that neopterin plasma levels might be a valuable parameter in activity staging and therapeutic follow up of atherosclerotic patients. Additionally, an involvement of the nonspecific immune system in atherogenesis is suggested by the increased plasma neopterin concentrations.

Purine catabolism in polymorphonuclear neutrophils. Phorbol myristate acetate-induced accumulation of adenosine owing to inactivation of extracellularly released adenosine deaminase.

Since physiological concentrations (0.1-1 microM) of adenosine influence the functions of human polymorphonuclear neutrophils (PMNs), we investigated the metabolism of adenosine in suspensions of stimulated and unstimulated PMNs. Stimulation with phorbol myristate acetate (PMA, 1 microM), but not by zymosan (0.5 mg/ml) or N-formyl-methionyl-leucyl-phenylalanine (fMLP, 1 microM), provoked an accumulation of endogenous adenosine at a rate of 2.3 +/- 1.0 amol/cell per minute. A similar accumulation was observed with both unstimulated and stimulated PMNs after the addition of deoxycoformycin (dCF, 1-100 microM), an inhibitor of adenosine deaminase. Exogenous adenosine (10 microM) was deaminated at a rate of 9.8 +/- 3.7 amol/cell per minute in control or zymosan or fMLP-stimulated PMN suspensions. This deamination was nearly completely suppressed when the PMNs had been stimulated with PMA. In contrast, the activity of adenosine deaminase in PMN lysates (231 +/- 72 amol/cell per minute) was not modified by PMA stimulation. alpha, beta-Methyleneadenosine 5'-diphosphate (AMPCP, 2.5 mM), an inhibitor of membranous ecto-5'-nucleotidase, profoundly inhibited endogenous adenosine accumulation under all conditions. PMA stimulation also provoked an inactivation of extracellular adenosine deaminase, purine nucleoside phosphorylase, and lactate dehydrogenase in PMN suspensions. We concluded that PMNs, even when not stimulated, continuously produce adenosine by dephosphorylation of extracellularly released adenylates; and that stimulation of PMNs by PMA causes adenosine accumulation owing to the inactivation of adenosine deaminase released by broken cells.

Allopurinol kinetics in humans as a means to assess liver function: design of a loading test.

A six-compartment model of allopurinol and oxipurinol kinetics, after intravenous allopurinol injection in the human, is studied further to improve the blood and urine specimen collection schedule for clinical use. The effects of various error sources are also investigated by simple techniques like real data set truncation and adding normally distributed random errors to data obtained from simulation of allopurinol and oxipurinol plasma curves with preset parameters. All parameters estimation is performed with the NONLIN parameter estimation program. Main interest was focused on estimation of the fractional rate constant of transport from the central "extracellular" compartment to the metabolically active compartment. This parameter is regarded as a lumped measure of liver perfusion and liver cell membrane transport. The blood sampling schedule can be reduced to six specimens collected over 60 min, without affecting the accuracy and precision of estimated clinical parameters. The maximum allowable coefficient of variation for preanalytical errors and the analytical within-run and between-run errors are around 5, 4, and 5%, respectively. Analytical between-run bias up to 20% does not affect the estimate of the principal parameter, when both allopurinol and oxipurinol are biased in the same direction. Collection and analysis of urine samples was shown to be unnecessary.