Different modes of ozone-induced lipid oxidation in Candida utilis yeast cells and isolated membrane preparations

Significant differences in the development of ozonolysis of lipids in membrane preparations and intact cells of the Candida utilis yeast were revealed. First, unlike isolated membranes, in which lipid modifications can be initiated by low ozone doses (< 0.5 micromol O3/mg protein) and develop proportionally to the treatment dose, in intact yeast cells, even the most ozone-sensitive sterols and nitrogen-containing phospholipids (phosphatidylcholine and phosphatidylethanolamine) did not undergo oxidative destruction at doses up to 6.0 micromol O3/mg protein. Second, the peculiarity of the ozone-initiated lipid modification in intact cells was that different classes of lipids exhibited different sensitivity to ozone. With an increase in the ozone dose, neutral lipids (sterols) and nitrogen-containing phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) were modified to a greater extent. Third, the accumulation of lipid peroxidation products upon ozone treatment of cells, in contrast to the isolated membranes, was absent at low ozone doses and was recorded only after the lethal damage. It is suggested that these differences are related to both the function of antioxidative enzymes (catalase, superoxide dismutase, peroxidase, etc.) and the difference between the structural states (i.e., stability and accessibility to oxidation) of lipids in the isolated membranes and the intact cells.     

Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Uric acid protects membranes and linolenic acid from ozone-induced oxidation

Aqueous preparations of linolenic acid, bovine serum albumin, and bovine erythrocyte membrane fragments were bubbled with ozone in the presence or absence of uric acid. Ozonation of the membrane fragments or the bovine serum albumin did not result in protein degradation. After 15 min of ozonation, the absorbance of the thiobarbituric acid-reactive material increased by 0.34 in the linolenic acid preparation and by 0.08 in the suspension of membrane fragments. In the presence of uric acid, these changes in absorbance were reduced to 0.14 for the fatty acid and to 0.01 for the membrane fragments. This result indicates that uric acid protects lipids from ozone-induced oxidation.     

Overexpression of SIPK in tobacco enhances ozone-induced ethylene formation and blocks ozone-induced SA accumulation

Ozone induces rapid activation of SIPK, a mitogen-activated protein kinase (MAPK) in tobacco. Through transgenic manipulation it has previously been shown that overexpression of SIPK leads to enhanced ozone-induced lesion formation with concomitant accumulation of ROS. In spite of this hypersensitive phenotype, the effect of this altered SIPK expression on the levels of various hormones that regulate ozone-induced cell death has remained unexplored. The response of both salicylate and ethylene, the major phytohormones that modulate ozone-induced cell death, have now been analysed in SIPK-OX tobacco plants. Ozone treatment strongly induced ethylene formation in the sensitive SIPK-OX plants at ozone concentrations that failed to elicit stress ethylene release in WT plants. By contrast, SIPK-overexpressing plants displayed no ozone-induced SA accumulation, whereas WT plants accumulated SA upon ozone exposure. Epistatic analysis of SIPK-OX function suggests that the ozone-induced cell death observed in SIPK-OX plants is either independent, or upstream, of SA accumulation.     

Antioxidant-mediated inhibition of the heat shock response leads to apoptosis

We examined the hypothesis that reactive oxygen species (ROS) contribute to the induction of heat shock proteins (hsps) during stress response. Exposure of HL-60 human myelocytic cells to 42 degrees C induced both hsp72 and hsp27. In the presence of the antioxidant molecules pyrrolidine dithiocarbamate or 1,10-phenanthroline induction of hsp72 and 27 was significantly decreased, while N-acetyl-L-cysteine caused a slight reduction. Prevention of hsp induction was associated with heat sensitization and increased caspase activity, indicating that the cells were undergoing apoptosis. These data suggest that ROS contribute to the induction of hsps and furthermore, that hsp induction and apoptosis are mutually exclusive events within the same cell.     

Human SP-A genetic variants and bleomycin-induced cytokine production by THP-1 cells: effect of ozone-induced SP-A oxidation

Surfactant protein A (SP-A) plays a role in innate host defense. Human SP-A is encoded by two functional genes (SP-A1 and SP-A2), and several alleles have been characterized for each gene. We assessed the effect of in vitro expressed human SP-A genetic variants, on TNF-alpha and IL-8 production by THP-1 cells in the presence of bleomycin, either before or after ozone-induced oxidation of the variants. The oligomerization of SP-A variants was also examined. We found 1) cytokine levels induced by SP-A2 (1A, 1A(0)) were significantly higher than those by SP-A1 (6A(2), 6A(4)) in the presence of bleomycin. 2) In the presence of bleomycin, ozone-induced oxidation significantly decreased the ability of 1A and 1A/6A(4), but not of 6A(4), to stimulate TNF-alpha production. 3) The synergistic effect of bleomycin/SP-A, either before or after oxidation, can be inhibited to the level of bleomycin alone by surfactant lipids. 4) Differences in oligomerization were also observed between SP-A1 and SP-A2. The results indicate that differences among SP-A variants may partly explain the individual variability of pulmonary complications observed during bleomycin chemotherapy and/or in an environment that may promote protein oxidation.     

Dissection of the ozone-induced calcium signature

The cellular responses of plants to numerous extracellular stimuli are mediated by transient elevations in the concentration of cytosolic free calcium ([Ca2+]cyt). We have addressed the question of how cells can use this apparently ubiquitous system to initiate so many specific and appropriate end responses. We show that the pollutant gas ozone elicits a biphasic Ca2+ response in intact Arabidopsis plants and a subsequent increase in expression of the gene encoding the antioxidant defence enzyme glutathione-S-transferase (GST). The second of the two [Ca2+]cyt peaks, but not the first, could be eliminated either by pre-treatment of plants with lanthanum chloride, or by reducing the duration of ozone fumigation. Under these conditions, ozone-induced GST expression was abolished. These data provide a functional dissection of the ozone Ca2+ signalling pathway and indicate that the second ozone-induced [Ca2+]cyt peak provides the necessary information to direct expression of GST.     

Vanadate-stimulated NADH oxidation in plasma membrane

The rate of NADH oxidation with oxygen as the acceptor is very low in mouse liver plasma membrane and erythrocyte membrane. When vanadate is added, this rate is stimulated 10- to 20-fold. The absorption spectrum of vanadate does not change with the disappearance of NADH. The reaction is inhibited by superoxide dismutase, and there is no activity under an argon atmosphere. This indicates that oxygen is the electron acceptor and the reaction is mediated by superoxide. The vanadate stimulation is not limited to plasma membrane. Golgi apparatus and endoplasmic reticulum show similar increase in NADH oxidase activity when vanadate is added. The endomembranes have significant vanadate-stimulated activity with both NADH and NADPH. The vanadate-stimulated NADH oxidase in plasma membrane is inhibited by compounds, which inhibit NADH dehydrogenase activity: catechols, anthracycline drugs and manganese. This activity is stimulated by high phosphate and sulfate anion concentrations.     

Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage

Potato peels are waste by-product of the potato processing industry. They are reportedly rich in polyphenols. Our earlier studies have shown that extracts derived from potato peel (PPE) possess strong antioxidant activity in chemical and biological model systems in vitro, attributable to its polyphenolic content. The main objective of this study was to investigate the ability of PPE to protect erythrocytes against oxidative damage, in vitro. The protection rendered by PPE in erythrocytes was studied in terms of resistance to oxidative damage, morphological alterations as well as membrane structural alterations. The total polyphenolic content in PPE was found to be 3.93 mg/g powder. The major phenolic acids present in PPE were predominantly: gallic acid, caffeic acid, chlorogenic acid and protocatechuic acid. We chose the experimental prooxidant system: FeSO₄ and ascorbic acid to induce lipid peroxidation in rat RBCs and human RBC membranes. PPE was found to inhibit lipid peroxidation with similar effectiveness in both the systems (about 80–85% inhibition by PPE at 2.5 mg/ml). While PPE per se did not cause any morphological alteration in the erythrocytes, under the experimental conditions, PPE significantly inhibited the H₂O₂-induced morphological alterations in rat RBCs as revealed by scanning electron microscopy. Further, PPE was found to offer significant protection to human erythrocyte membrane proteins from oxidative damage induced by ferrous–ascorbate. In conclusion, our results indicate that PPE is capable of protecting erythrocytes against oxidative damage probably by acting as a strong antioxidant.     

Analysis of unsaturated lipids by ozone-induced dissociation

Recent developments in analytical technologies have driven significant advances in lipid science. The sensitivity and selectivity of modern mass spectrometers can now provide for the detection and even quantification of many hundreds of lipids in a single analysis. In parallel, increasing evidence from structural biology suggests that a detailed knowledge of lipid molecular structure including carbon-carbon double bond position, stereochemistry and acyl chain regiochemistry is required to fully appreciate the biochemical role(s) of individual lipids. Here we review the capabilities and limitations of tandem mass spectrometry to provide this level of structural specificity in the analysis of lipids present in complex biological extracts. In particular, we focus on the capabilities of a novel technology termed ozone-induced dissociation to identify the position(s) of double bonds in unsaturated lipids and discuss its possible role in efforts to develop workflows that provide for complete structure elucidation of lipids by mass spectrometry alone: so-called top-down lipidomics.     

Modification of glycophorin A during oxidation of erythrocyte membrane

Human erythrocyte ghosts were oxidized with tert-butyl hydroperoxide and subsequently treated with tritiated borohydride to label the membrane proteins modified during the membrane oxidation. From the ghosts, oxidized-and-tritiated glycophorin A was isolated and characterized. No intermolecular cross-links were observed as analyzed by sodium dodecylsulfate gel electrophoresis. But, the number of lysine residues was significantly reduced and susceptibility to proteinases such as trypsin, chymotrypsin and pronase was lower than that of control glycophorin A. Trypsinization of the oxidized-and-tritiated glycophorin A gave insoluble and soluble trypsin fragments. After dansylation, N-terminal amino acids of the trypsin-fragments were determined. Dansyl amino acids from the insoluble trypsin fragments were not identical with those from control insoluble counterparts in the membrane-spanning region of glycophorin A molecule. Fractionation by gel filtration of dansyl-soluble trypsin fragments, and the N-terminal amino acid analysis of the fractionated peptides indicated that the peptides derived from the glycosylated region located in the outside of the membrane matrix were identical with those from control soluble counterparts. The results suggest that the glycosylated outside region of glycophorin A was modified only slightly but the hydrophobic membrane-spanning region was extensively modified during membrane oxidation, most likely by oxidized lipids.     

Effect of membrane composition on lipid oxidation in liposomes

To study the effect of membrane composition on the oxidation of liposomes, different systems were prepared by adding one component at time to phosphatidylcholine (Epikuron 200). In particular, the effect of cholesterol and its ester, cholesterol stearate, on membrane structure and oxidation was studied. A first screening of the structure and net charge of the different preparation was made by means of z-potential and size measurements. Then the liposomes were oxidized by using a hydrophilic radical initiator, the (2,2-azobis(2-amidinopropane) hydrochloride, AAPH, which thermally decomposes to give a constant radical flux in water. The oxidation of liposomes, monitored by following the absorbance of the primary products of oxidation at 234 nm, was shown to be dependent on the composition of the liposomal bilayer and so on its biophysical properties. In addition, size and z-potential measurements gathered in the time course of the peroxidation reaction, revealed that the oxidation induced a modification of the superficial characteristics of the membrane bilayer so as to change its charge at the shear plane (z-potential). This behaviour was shared by all liposomal preparations independent of the composition. The change in sizes of the different liposomal preparation, instead, followed different trends, being more stable both in control samples and in oxidized ones when cholesterol was present. From the analysis of the results, it can be concluded that cholesterol affects the oxidation induced by hydrophilic radical initiator of model membranes by changing the biophysical properties of the phospholipid bilayer. The rigidity induced by cholesterol at temperatures above the T_m makes the membrane more resistant to radical attack from an external aqueous phase and this in turn delays the start of the reaction. The decrease of z-potential of the liposomal particles induced by the oxidation process can be an important clue to understand the mechanisms involved in the etiology of important diseases.     

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.     

Membrane oxidation assay--a novel lipoxygenase-based evaluation of membrane oxidizability

A membrane oxidation assay is presented which uses isolated erythrocyte membranes ("ghosts") and lipoxygenase as a selective catalyst for the transfer of oxygen to cis - cis -1,4-pentadiene-moieties. The latter are, for instance, present in linoleic and arachidonic acids, both of which are integral parts of membranes. These non-conjugated double bonds represent energetically favorable sites for oxidative attack and therefore, may be rearranged and partially consumed during oxidative stress. Consequently, the measurement of oxygen consumption in the course of the lipoxygenase-mediated oxidation provides a tool for the quick and reliable determination of such double bonds. Significant inter-individual differences have been noted in 11 subjects, which also correlate to the total radical antioxidant parameter (TRAP) values obtained. This assay will be helpful in the assessment of oxidizable structures in erythrocyte membranes that may be diminished as a consequence of oxidative damage suffered by an individual. In conclusion, a simple and rapid assay for the assessment of the oxidizability of erythrocyte membranes is presented complementing the TRAP assay for plasma antioxidative status.     

Inhibition of chemokine receptor function by membrane cholesterol oxidation

Membrane cholesterol is required to maintain chemokine receptor conformation and function for CXCR4 and CCR5. We previously demonstrated that chemokines preferentially bind to receptors within lipid rafts, which are cholesterol- and sphingolipid-rich membrane microdomains. To further elucidate the role of cholesterol in chemokine receptor function, we examined the effects of membrane cholesterol oxidation by cholesterol oxidase (CO), which enzymatically converts cholesterol to 4-cholesten-3-one. Here, we demonstrate that CO treatment (0.25-2.0 U/ml) of human T cells inhibits CXCL12 (SDF-1alpha) and CCL4 (MIP-1beta) binding to cell surface CXCR4 and CCR5, respectively, resulting in the inhibition of chemokine-mediated intracellular calcium mobilization and chemotaxis. The effects were significantly enhanced by cotreatment with low-dose sphingomyelinase (SMase) (0.125 mU/ml), which produced little inhibitory effect by itself. CO and SMase treatment also inhibited HIV-1 infection through CXCR4, but not virus replication. Similar to the removal of membrane cholesterol, CO/SMase treatment induced conformation changes in the chemokine receptors as detected by differential loss in binding of epitope-specific monoclonal antibodies. We conclude that the native form of cholesterol with the hydroxyl group at C3 is critical to CXCR4 and CCR5 conformation and function.     

Nociceptive mechanisms modulate ozone-induced human lung function decrements

We have previously suggested that ozone(O₃)-induced pain-relatedsymptoms and inhibition of maximal inspiration are due to stimulationof airway C fibers (M. J. Hazucha, D. V. Bates, and P. A. Bromberg.J. Appl.Physiol. 67: 1535-1541, 1989). If this were so,pain suppression or inhibition by opioid-receptor agonists shouldpartially or fully reverseO₃-induced symptomatic and lung functional responses. The objectives of this study were to determine whether O₃-induced pain limitsmaximal inspiration and whether endogenous opioids contribute tomodulation of the effects of inhaledO₃ on lung function. Theparticipants in this double-blind crossover study were healthyvolunteers (18-59 yr) known to be "weak" (WR;n = 20) and "strong"O₃ responders (SR;n = 42). They underwent either two 2-hexposures to air or two 2-h exposures to 0.42 parts/millionO₃ with moderate intermittentexercise. Immediately afterpost-O₃ spirometry, the WR wererandomly given either naloxone (0.15 mg/kg iv) or saline, whereas SRrandomly received either sufentanil (0.2 µg/kg iv) or saline.O₃ exposure significantly(P < 0.001) impaired lung function.In SR, sufentanil rapidly, although not completely, reversed both thechest pain and spirometric effects (forced expiratory volume in 1 s;P < 0.0001) compared with saline.Immediate postexposure administration of saline or naloxone had nosignificant effect on WR. Plasma -endorphin levels were not relatedto an individual's O₃responsiveness. Cutaneous pain variables showed a nonsignificantweak association with O₃responsiveness. These observations demonstrate that nociceptive mechanisms play a key role in modulatingO₃-induced inhibition ofinspiration but not in causing lack of spirometric response toO₃ exposure in WR.

     

Differences in biochemical properties and in biological function between human SP-A1 and SP-A2 variants, and the impact of ozone-induced oxidation

The human surfactant protein A (SP-A) locus consists of two functional genes, SP-A1 and SP-A2, with several alleles characterized for each gene. Functional variations between SP-A1 and SP-A2 variants either before or after ozone exposure have been observed. To understand the basis of these differences, we studied SP-A1 and SP-A2 variants by comparing coding sequences, oligomerization patterns under various conditions, composition of oligomers with regard to amino terminal sequence isoforms, biological activity (regulation of phosphatidylcholine (PC) secretion by alveolar type II cells), and the impact of ozone-induced oxidation. We found that (i) the SP-A1 (6A(4)) allele is the most divergent from all SP-A2 alleles, particularly from the SP-A2 (1A(1)). (ii) Differences exist in oligomerization among SP-A1, SP-A2, and coexpressed SP-A1/SP-A2, with higher order multimers (i.e., consisting of more subunits) observed for SP-A1 than for SP-A2 variants. Differences among SP-A1 or SP-A2 gene products are minimal. (iii) Amino acid variants in the amino terminal sequences are observed after signal peptide removal, including variants with an extra cysteine. (iv) Oxidation is observed after ozone exposure, involving several SP-A residues that include cysteine, methionine, and tryptophan. (v) The SP-A2 variant (1A(0)) and the coexpressed protein 1A(0)/6A(2) inhibit ATP-stimulated PC secretion from alveolar type II cells to a greater extent than SP-A1 (6A(2)), a biologic activity that was susceptible to ozone treatment.     

Protection of membrane cholesterol by sphingomyelin against free radical-mediated oxidation

Although the free radical-mediated oxidation of free cholesterol (FC) is critical in the generation of regulatory sterols and in atherogenesis, the physiological regulation of this process is poorly understood. We tested the hypothesis that sphingomyelin (SM), a major phospholipid of cell membranes, which is closely associated with FC, protects FC against oxidation, because of its unique structure, and affinity to the sterol. We employed phosphatidylcholine (PC) liposomes containing varying amounts of SM, and either radioactive FC or a fluorescent analog, dehydroergosterol (DHE), and determined the oxidative decay of the sterol in presence of 2,2′-azo-bis(2-amidinopropane hydrochloride) (AAPH). Incorporation of 25 mol% of SM in the liposomes inhibited the oxidation of FC or DHE by up to 50%. This inhibition was specific for SM among phospholipids, and was abolished by sphingomyelinase treatment. SM was not degraded during the oxidation reaction, and its effect was not dependent on the nature of the oxidizing agent, because it also inhibited sterol oxidation by FeSO₄/ascorbate, and by cholesterol oxidase. These studies show that SM plays a physiological role in the regulation of cholesterol oxidation by free radicals.