Modification of human low-density lipoprotein by the lipid peroxidation product 4-hydroxynonenal

The effects of the lipid peroxidation product 4-hydroxynonenal on freshly prepared human low-density lipoprotein (LDL) were studied. At a fixed LDL concentration (5.7 mg/ml) the amount of 4-hydroxynonenal incorporated into the LDL increased with increasing aldehyde concentration from 28-30 (0.2 mM) to 140 (1 mM) mol per mol LDL, whereas at a fixed aldehyde concentration (0.2 mM) its incorporation into LDL decreased with increasing LDL concentration from 48 (1 mg LDL/ml) to 26 (12 mg LDL/ml) mol 4-hydroxynonenal bound per mol LDL. Of the total hydroxynonenal taken up 78% was bound to the protein and 21% to the lipid moiety; the remaining 1% was dissolved as free aldehyde in the lipid fraction. Amino acid analysis of the apolipoprotein B revealed that 4-hydroxynonenal attacks mainly the lysine and tyrosine residues and to a lesser extent also serine, histidine and cysteine. Treatment of LDL with 4-hydroxynonenal results in a concentration-dependent increase of the negative charge of the LDL particle as evidenced by its increased electrophoretic mobility. Moreover, 4-hydroxynonenal treatment leads to a partial conversion of the apolipoprotein B-100 into higher molecular weight forms most probably apolipoproteins B-126 and B-151. Compared to malonaldehyde, 4-hydroxynonenal exhibits a much higher capacity to modify LDL and it is therefore believed that this aldehyde is a more likely candidate for being responsible for LDL modification under in vivo lipid peroxidation conditions.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

Binding of the lipid peroxidation product 4-hydroxynonenal to human polymorphonuclear leukocytes

4-Hydroxynonenal (HNE) is produced during peroxidation of polyunsaturated fatty acids. It exerts a chemokinetic effect on human polymorphonuclear leukocytes (PMN). Investigations of this mechanism were performed. The results indicate that [³H]-HNE binding to PMN results both in non-specific bonds to the numerous SH groups of the cells and in binding to a saturable, reversible and specific HNE site. Scatchard analysis revealed that this is a single site with an apparent affinity constant of 319 nM and a density of 1·57 pmol (10⁶)^?1 cells. This specific binding site may be involved in the chemokinetic effect of HNE.     

Chemotactic activity of the lipid peroxidation product 4-hydroxynonenal and homologous hydroxyalkenals

The effect of the lipid peroxidation product 4-hydroxynonenal and homologous aldehydes (4-hydroxyoctenal, 4-hydroxyundecenal, 4-hydroxytetradecenal and 4-hydroxypentadecenal) on migration and polarization of rat neutrophils was examined. The most effective aldehydes were 4-hydroxyoctenal and 4-hydroxypentadecenal, which stimulated oriented migration at ED50 = 1.4 X 10(-12) M and 1.3 X 10(-12) M, resp., whereas the other aldehydes had ED50 between 1 X 10(-7) and 6 X 10(-11) M. The peptides fMet-Phe and fMet-Leu-Phe used as positive controls had ED50 values of 4.2 X 10(-7) M and 4.5 X 10(-10) M resp. The 4-hydroxyalkenals induced only a small increase of the percentage of polarized cell and did not enhance the random migration. The effects of 4-hydroxyalkenals were only observed when the incubation buffer contained bovine serum albumin (BSA), in the absence of BSA neither the aldehydes nor the peptides exhibited chemotactic properties. Since the aldehydes easily react with the sulfhydryl groups of the BSA to form the S-alkylated BSA in an equilibrium reaction, the chemotactic substance could either be the free aldehyde or the BSA-aldehyde adduct. The adduct prepared from BSA and 4-hydroxynonenal was chemotactic at doses of 0.65 to 0.0065 mg/ml, when tested in the presence of unmodified BSA. Since the adduct released free 4-hydroxyalkenal during the assay in the reverse reaction, it can not be decided whether the active principle is the aldehyde itself or the aldehyde attached to the BSA. From the effective doses of the aldehydes (10(-7) to 10(-12)M) and the BSA-aldehyde adduct it appears very unlikely that the BSA itself gained chemotactic properties through the alkylation of its sulfhydryl groups by the aldehyde.     

Metabolism of the lipid peroxidation product 4-hydroxynonenal by isolated hepatocytes and by liver cytosolic fractions.

The metabolism of the lipid peroxidation product 4-hydroxynonenal and of several other related aldehydes by isolated hepatocytes and rat liver subcellular fractions has been investigated. Hepatocytes rapidly metabolize 4-hydroxynonenal in an oxygen-independent process with a maximum rate (depending on cell preparation) ranging from 130 to 230 nmol/min per 10(6) cells (average 193 +/- 50). The aldehyde is also rapidly utilized by whole rat liver homogenate and the cytosolic fraction (140 000 g supernatant) supplemented with NADH, whereas purified nuclei, mitochondria and microsomes supplemented with NADH show no noteworthy consumption of the aldehyde. In cytosol, the NADH-mediated metabolism of the aldehyde exhibits a 1:1 stoichiometry, i.e. 1 mol of NADH oxidized/mol of hydroxynonenal consumed, and the apparent Km value for the aldehyde is 0.1 mM. Addition of pyrazole (10 mM) or heat inactivation of the cytosol completely abolishes aldehyde metabolism. The various findings strongly suggest that hepatocytes and rat liver cytosol respectively convert 4-hydroxynonenal enzymically is the corresponding alcohol, non-2-ene-1,4-diol, according to the equation: CH3-[CH2]4-CH(OH)-CH = CH-CHO + NADH + H+----CH3-[CH2]4-CH(OH)-CH = CH-CH2OH + NAD+. The alcohol non-2-ene-1,4-diol has not yet been isolated from incubations with hepatocytes and liver cytosolic fractions, but was isolated in pure form from an incubation mixture containing 4-hydroxynonenal, isolated liver alcohol dehydrogenase and NADH and its chemical structure was confirmed by mass spectroscopy. Compared with liver, all other tissues possess only little ability to metabolize 4-hydroxynonenal, ranging from 0% (fat pads) to a maximal 10% (kidney) of the activity present in liver. The structure of the aldehyde has a strong influence on the rate and extent of its enzymic NADH-dependent reduction to the alcohol. The saturated analogue nonanal is a poor substrate and only a small proportion of it is converted to the alcohol. Similarly, nonenal is much less readily utilized as compared with 4-hydroxynonenal. The effective conversion of the cytotoxic 4-hydroxynonenal and other reactive aldehydes to alcohols, which are probably less toxic, could play a role in the general defence system of the liver against toxic products arising from radical-induced lipid peroxidation.     

The lipid peroxidation product 4-hydroxynonenal is a potent inducer of the SOS response.

An important aspect of bacterial mutagenesis by several difunctional carbonyl compounds appears to be the induction of the SOS system. We tested the ability of a series of carbonyl compounds to induce expression of the SOS-regulated umu operon in Salmonella typhimurium TA1535/pSK1002. SOS-inducing potencies varied widely among the carbonyl compounds tested. 4-Hydroxynonenal, a product of lipid peroxidation, was the most potent SOS-inducer, with maximal induction observed at concentrations of 0.1-1 microM. Acrolein, crotonaldehyde and methacrolein induced little increase over background umu expression. Malondialdehyde, another product of lipid peroxidation, was a very weak SOS-inducer with a maximal response induced at a concentration of 28 mM. Substitution at the alpha-position of malondialdehyde, which abolishes frameshift mutagenicity, did not abolish SOS-inducing activity. Substitution of the hydroxyl group of malondialdehyde and alpha-methyl-malondialdehyde by a better leaving group (benzoyloxy) resulted in an approximately 250-fold higher SOS-inducing potency. Comparison of the present results to literature reports on bacterial mutagenicity indicates a poor correlation of the two properties between different classes of difunctional carbonyl compounds and even within the same class of difunctional carbonyl compounds.     

Effects of the lipid peroxidation product 4-hydroxynonenal on the aggregation of human platelets

The stimulation by ADP or arachidonic acid of the aggregation of human platelets in plasma was inhibited by 4-hydroxynonenal (HNE). This reduction of aggregation was time related, and was increased by prolonged preincubation of the platelets with the aldehyde. HNE was more potent than its homologue 4-hydroxypentenal (HPE). HNE was less active in decreasing the aggregation induced by calcium ionophore A23187 or collagen in comparison with ADP. HNE was inactive against aggregation of platelet-rich plasma (PRP) stimulated by thrombin whereas it potently inhibited the aggregation of washed platelets in response to both thrombin and collagen. Platelets were found to degrade HNE, and mechanisms additional to covalent binding to glutathione are indicated by the results obtained. The aldehydes, including HNE, generated by platelets originated principally from arachidonic acid metabolism.     

Micellar electrokinetic chromatography separation and laser-induced fluorescence detection of the lipid peroxidation product 4-hydroxynonenal

4-Hydroxnonenal (HNE) is a product of lipid peroxidation in biological systems that causes a variety of harmful biological effects. A method for identifying HNE based on derivatization with the fluorescent reagent dansylhydrazine (5-(dimethylamino)naphthalene-1-sulphonehydrazine (DNSH) followed by micellar electrokinetic chromatography separation laser-induced fluorescence detection has been developed. The derivatization reaction has also been investigated for significant experimental parameters and rat brain homogenates with induced lipid peroxidation have been analysed for HNE contents. The limit of detection (3 S/N) was 30 nM or 0.3 fmol in the injected sample.     

Metabolism of lipid peroxidation product, 4-hydroxynonenal (HNE) in rat erythrocytes: role of aldose reductase

Lipid peroxidation represents a significant source of erythrocyte dysfunction and aging. Because the toxicity of lipid peroxidation appears to be in part due to aldehydic end products, we examined, in rat erythrocytes, the metabolism of 4-hydroxy-trans-2-nonenal (HNE), one of the most abundant and toxic lipid-derived aldehydes. Packed erythrocytes, 0.1 ml, completely metabolized 20 nmoles of HNE in 20 min. The glutathione conjugate of HNE and 4-hydroxynonanoic acid (HNA) represented 70 and 25% of the total metabolism, respectively. Approximately 70% of the metabolites were extruded to the medium. Upon electrospray ionization mass spectrometry, the glutathione conjugate resolved into two distinct species corresponding to glutathionyl HNE (GS-HNE) and glutathionyl 1,4-dihydroxynonene (GS-DHN). The concentration of GS-DHN formed was twice that of GS-HNE. Inhibition of aldose reductase by sorbinil and tolrestat led to a selective decrease in the formation of GS-DHN, although the extent of HNE glutathiolation was unaffected. Inhibitors of aldehyde or alcohol dehydrogenase, i.e., cyanamide and 4-methyl pyrazole, had no effect on the formation of HNA and GS-DHN, indicating that these enzymes are not significant participants in the erythrocyte HNE metabolism. Thus, oxidation to HNA, conjugation with glutathione, and further reduction of the conjugate by aldose reductase appear to be the major pathways of HNE metabolism in erythrocytes. These pathways may be critical determinants of erythrocyte toxicity due to lipid peroxidation-derived aldehydes.     

Induction of differentiation in human HL-60 cells by 4-hydroxynonenal, a product of lipid peroxidation

4-Hydroxynonenal (HNE) is the major diffusible toxic product generated by lipid peroxidation of cellular membranes. The level of lipid peroxidation and, consequently, the concentration of its products are inversely related to the rate of cell proliferation and directly related to the level of cell differentiation. In the present paper the effects of HNE on the proliferation and differentiation of the HL-60 human promyelocytic cell line have been investigated. Repeated treatment at 45-min intervals with HNE (1 microM) was performed to maintain the cells in the presence of the aldehyde for 7 1/2 or 9 h. The effect of HNE on cell proliferation and differentiation was compared with dimethyl sulfoxide (DMSO)-treated cells. HNE causes a strong inhibition of cell growth without affecting cell viability. Moreover, HL-60 cells acquire the capability to produce chemiluminescence after soluble (phorbol myristate acetate) or corpuscolate (zymosan) stimulation. The phagocytic ability has also been calculated by counting the number of cells that phagocytize opsonized zymosan. Values were 43 and 55% after 10 or 12 HNE treatments, respectively, and 88% in DMSO-treated cells. Myeloperoxidase activity, 5 days after treatment, decreased by 85% in either HNE- or DMSO-treated cells while acid phosphatase activity increased with respect to untreated cells. Results obtained indicate that HNE at concentrations close to those found in the normal tissues can induce inhibition of proliferation and induction of differentiation in the HL-60 cell line.     

A mechanism for the neuroprotective effect of apolipoprotein E: isoform-specific modification by the lipid peroxidation product 4-hydroxynonenal

Inheritance of the apolipoprotein E (apoE) epsilon4 allele increases the risk for Alzheimer's disease and may also influence the pathogenesis of other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). The influence of apoE genotype on disease susceptibility must ultimately be explained by the fact that apoE proteins differ in only two amino acids: apoE2 has two cysteine residues, apoE3 has one cysteine residue, and apoE4 has none. We previously reported increased protein modification by the lipid peroxidation product 4-hydroxynonenal (HNE), which covalently binds to proteins on cysteine residues, in human ALS lumbar spinal cord. We now report increased levels of HNE-modified apoE in lumbar spinal cord samples from mice expressing an ALS-linked mutation in Cu/Zn-superoxide dismutase relative to controls. Studies of interactions of pure apoE proteins with HNE showed that the isoforms differ in the amount of HNE they can bind, with the order E2 > E3 > E4. This correlated with the differential ability of apoE isoforms to protect against apoptosis induced by HNE in cultures of mouse spinal cord motor neurons and by the amyloid beta-peptide in cultures of rat hippocampal neurons. These data suggest that apoE plays a major role in detoxifying HNE, and the differential neuroprotective effect of its isoforms may help explain the relationship between apoE genotype and the susceptibility to neurodegenerative diseases.     

Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.     

Effect of 4-hydroxynonenal,a lipid peroxidation product,on exocytosis in HL-60 cells

Our work analysed the effect of 4-hydroxynonenal (HNE), a chemotactic aldehydic end-product of lipid peroxidation, on exocytosis in HL-60 cells. We measured the release of beta-glucuronidase, an enzyme of azurophil granules, from the cells incubated at 37 degrees C for 10 min in the presence of HNE concentrations ranging between 10(-8) and 10(-5) M. The release of lactate dehydrogenase was assayed to test cell viability. HNE (1 microM) was able to induce a significant and strong stimulation of beta-glucuronidase secretion without leading to cytotoxic effects. The finding that HNE could increase the exocytotic secretion from HL-60 cells together with its known chemotactic property supports the hypothesis that this lipid peroxidation product may play an important role as a chemical mediator of inflammation; moreover it is noteworthy that micromolar concentrations of HNE have actually been found in exudates from acute and chronic inflammations.     

Detection of 4-hydroxynonenal as a product of lipid peroxidation in native Ehrlich ascites tumor cells

4-Hydroxynonenal, which is a major product of lipid peroxidation in rat liver microsomes, was detected in native Ehrlich ascites tumor cells. Its formation was stimulated either by ferrous ions or by Fe(II)-histidinate. The identification was based on chromatographic (TLC/HPLC) and ultraviolet-spectroscopic evidence using synthetic 4-hydroxynonenal as reference. Highest values of 4-hydroxynonenal concentration (about 0.1 microM in the cell suspension) after 30 min of incubation were observed with Fe(II)-histidinate as stimulant. Saturation was already reached after an incubation period of 10 min. The results confirm the expectation by Schauenstein and Esterbauer (in Submolecular Biology and Cancer, Ciba Foundation Series 67 (1979) pp. 225-244, Excerpta Medica, Amsterdam) that endogenous lipid peroxidation gives rise to a distinct intracellular level of alpha, beta-unsaturated aldehydes. A simple hypothetical mechanism for the formation of 4-hydroxynonenal from n-6-polyunsaturated fatty acids is presented.     

Permeability properties of isolated enterocytes from rat small intestine

Metabolic and permeability properties of enterocytes isolated by treatment of rat small intestine with hyaluronidase or EDTA were compared. No significant difference was observed in the ability of the two types of cell to produce lactate from glucose. However, while cells obtained with hyaluronidase accumulate alpha-methylglucoside, cells obtained with EDTA were unable to accumulate the sugar above the medium concentrations. When resuspended in a medium designed to resemble the intracellular medium, potentiometric measurements showed that cells obtained with hyaluronidase released Ca2+ to the medium while cells obtained with EDTA accumulated it. Using 45Ca transport assays, this was shown to be an ATP-dependent process, the accumulated 45Ca being totally released by the addition of the ionophore A23187. When cells obtained with EDTA were resuspended in a medium containing concentrations of free Ca2+ higher that 10 microM, the uptake was partially inhibited by sodium orthovanadate and also by oligomycin and antimycin. At free Ca2+ concentrations lower than 1 microM, the accumulation was inhibited up to 87% by sodium orthovanadate while mitochondrial inhibitors inhibited only 5%. Thus, it appears that during their preparation cells obtained with hyaluronidase retain their integrity while cells obtained with EDTA become permeable to Ca2+ and other ions. The usefulness of both types of preparation in metabolic and transport studies is discussed.     

Structural-functional analysis of diffusion in glucose absorption by rat small intestine enterocytes

To elucidate mechanisms providing transport of sugars across intestinal epithelium, on taking into account the current hypotheses (active transport, participation of paracellular transport and passive component of transcellular transport), it was important to reveal structural changes of tight junctions and distribution of the carriers of facilitated diffusion of GLUT2 and protein kinase C during absorption of glucose. On using confocal and electron microscopy, ultrastructural and immunocytochemical studies of enterocytes after perfusion of isolated rat small intestine fragment with 75 mM glucose (chronic experiment) have shown: 1) fluorescent labels of transporter GLUT2 and PKCbetaII are located in the apical area of enterocytes situated at the upper half of the villus. Antibodies against GLUT2, conjugated with gold, are revealed at the microvilli or apical membrane and in the area of terminal network; 2) no ultrastructural changes of the tight junction are detected on ultrathin sections and freeze--fracture replics. At the same time, fluorescent and gold labels against actin are concentrated in the vicinity of the lateral membrane in the tight junction area. The results obtained can serve a confirmation of a hypothesis that at high glucose concentrations GLUT2 participates in its transfer across the apical membrane.     

MicroRNA expression changes during human leukemic HL-60 cell differentiation induced by 4-hydroxynonenal,a product of lipid peroxidation

4-Hydroxynonenal (HNE) is one of several lipid oxidation products that may have an impact on human pathophysiology. It is an important second messenger involved in the regulation of various cellular processes and exhibits antiproliferative and differentiative properties in various tumor cell lines. The mechanisms by which HNE affects cell growth and differentiation are only partially clarified. Because microRNAs (miRNAs) have the ability to regulate several cellular processes, we hypothesized that HNE, in addition to other mechanisms, could affect miRNA expression. Here, we present the results of a genome-wide miRNA expression profiling of HNE-treated HL-60 leukemic cells. Among 470 human miRNAs, 10 were found to be differentially expressed between control and HNE-treated cells (at p < 0.05). Six miRNAs were down-regulated (miR-181a*, miR-199b, miR-202, miR-378, miR-454-3p, miR-575) and 4 were up-regulated (miR-125a, miR-339, miR-663, miR-660). Three of these regulated miRNAs (miR-202, miR-339, miR-378) were further assayed and validated by quantitative real-time RT-PCR. Moreover, consistent with the down-regulation of miR-378, HNE also induced the expression of the SUFU protein, a tumor suppressor recently identified as a target of miR-378. The finding that HNE could regulate the expression of miRNAs and their targets opens new perspectives on the understanding of HNE-controlled pathways. A functional analysis of 191 putative gene targets of miRNAs modulated by HNE is discussed.