Aluminum ions stimulate the oxidizability of low density lipoprotein by Fe2+: implication in hemodialysis mediated atherogenic LDL modification

Objective: Al³⁺ stimulates Fe²⁺ induced lipid oxidation in liposomal and cellular systems. Low-density lipoprotein (LDL) oxidation may render the particle atherogenic. As elevated levels of Al³⁺ and increased lipid oxidation of LDL are found in sera of hemodialysis patients, we investigated the influence of Al³⁺ on LDL oxidation.

Materials and methods: Using different LDL modifying systems (Fe²⁺, Cu²⁺, free radical generating compounds, human endothelial cells, hemin/H₂O₂ and HOCl), the influence of Al³⁺ on LDL lipid and apoprotein alteration was investigated by altered electrophoretic mobility, lipid hydroperoxide-, conjugated diene- and TBARS formation.

Results: Al³⁺ could stimulate the oxidizability of LDL by Fe²⁺, but not in the other systems tested. Al³⁺ and Fe²⁺ were found to bind to LDL and Al³⁺could compete with Fe²⁺ binding to the lipoprotein. Fluorescence polarization data indicated that Al³⁺ does not affect the phospholipid compartment of LDL.

Conclusions:The results indicate that increased LDL oxidation by Fe²⁺ in presence of Al³⁺ might be due to blockage of Fe²⁺ binding sites on LDL making more free Fe²⁺ available for lipid oxidation.     

DNA Single Strand Breakage by H2O2 and Ferric or Cupric Ions: Its Modulation by Histidine

The role of histidine on DNA breakage induced by hydrogen peroxide (H₂O₂) and ferric ions or by H₂O₂ and cupric ions was studied on purified DNA. L-histidine slightly reduced DNA breakage by H₂O₂ and Fe³⁺ but greatly inhibited DNA breakage by H₂O₂ and Cu²⁺. However, only when histidine was present, the addition of EDTA to H₂O₂ and Fe³⁺ exhibited a bimodal dose response curve depending on the chelator metal ratio. The enhancing effect of histidine on the rate of DNA degradation by H₂O₂ was maximal at a chelator metal ratio between 0.2 and 0.5, and was specific for iron. When D-histidine replaced L-histidine, the same pattern of EDTA dose response curve was observed. Superoxide dismutase greatly inhibited the rate of DNA degradation induced by H₂O₂, Fe³⁺, EDTA and L-histidine involving the superoxide radical.

These studies suggest that the enhancing effect of histidine on the rate of DNA degradation by H₂O₂ and Fe³⁺ is mediated by an oxidant which could be a ferrous-dioxygen-ferric chelate complex or a chelate-ferryl ion.     

Ascorbic Acid Oxidation and DNA Scission Catalyzed by Iron and Copper Chelates

The asorbic acid (AH^-) auto-oxidation rates catalyzed by copper chelates of 1,10-phenanthroline (OP) or by iron chelates of bleomycin (BLM) are only slightly higher than the oxidation rates catalyzed by the metal ions. AH^- oxidation in the presence of DNA is accompanied by degradation of the DNA. The rates of DNA scission by the metal chelates are markedly higher than the rates induced by the free metal ions. AH^- oxidation is slowed down in the presence of DNA which forms ternary complexes with the chelates. The ternary complexes react slowly with AH^- but induce DNA double strand breaks more efficiently than the free metal chelates. With OP, DNA is degraded by the reaction of the ternary complex, DNA-(OP)₂Cu(I), withH₂O₂

AH^- oxidation in the presence of DNA was biphasic, showing a marked rate increase after DNA was cleaved. We suggest that this sigmoidal pattern of the oxidation curves reflects the low initial oxidative activity of the ternary complexes, accelerating as DNA is degraded.

Using O₂^-produced by pulse radiolysis as a reductant, we found that AH^- oxidation with (OP)₂Cu(II) induced more DNA double strand breaks per single strand break than bipyridine-copper.

The site specific DNA damaging reactions indicated by these results are relevant to the mechanism of cytotoxic activities of bleomycin and similar antibiotics or cytotoxic agents.     

Carvedilol inhibition of lipid peroxidation. A new antioxidative mechanism

To define the molecular mechanism(s) of carvedilol inhibition of lipid peroxidation we have utilized model systems that allow us to study the different reactions involved in this complex process.

Carvedilol inhibits the peroxidation of sonicated phosphatidylcholine liposomes triggered by FeCl₂ addition whereas atenolol, pindolol and labetalol are ineffective. The inhibition proved not to be ascribable (a) to an effect on Fe²⁺ autoxidation and thus on the generation of oxygen derived radical initiators; (b) to the scavenging of the inorganic initiators O^·-₂ and ^·OH; (c) to an effect on the reductive cleavage of organic hydroperoxides by FeCl₂; (d) to the scavenging of organic initiators. The observations that (a) carvedilol effectiveness is inversely proportional to the concentration of FeCl₂ and lipid hydroperoxides in the assay; (b) the drug prevents the onset of lipid peroxidation stimulated by FeCl₃ addition and; (c) it can form a complex with Fe³⁺, suggest a molecular mechanism for carvedilol action. It may inhibit lipid peroxidation by binding the Fe³⁺ generated during the oxidation of Fe²⁺ by lipid hydroperoxides in the substrate. The lag time that carvedilol introduces in the peroxidative process would correspond to the time taken for carvedilol to be titrated by Fe³⁺; when the drug is consumed the Fe³⁺ accumulates to reach the critical parameter that stimulates peroxidation. According to this molecular mechanism the antioxidant potency of carvedilol can be ascribed to its ability to bind a species, Fe³⁺, that is a catalyst of the process and to its lipophilic nature that concentrates it in the membranes where Fe³⁺ is generated by a site specific mechanism.     

2-Hydroxy-Succinaldehyde, a Lipid Peroxidation Product Proving that Polyunsaturated Fatty Acids are Able to React with Three Molecules of Oxygen

2-Hydroxy-succinaldehyde was detected by a GC/MS analysis of trapped aldehydic compounds obtained after Fe²⁺/ascorbate lipid peroxidation of arachidonic acid. Precursor molecules of aldehydes are hydroperoxy compounds. Thus the generation of the two aldehydic groups in 2-hydroxysuccinaldehyde requires a precursor molecule with two hydroperoxy groups. The hydroxy group in 2-position is generated by a third hydroperoxidation reaction. The detection of 2-hydroxysuccinaldehyde—although found only in traces—is the first example for triple dioxigenation of unsaturated fatty acid. Linolenic acid produces 2-hydroxysuccinaldehyde in much lower amounts than arachidonic acid. A similar oxidation of linoleic acid was not observed.     

Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.     

Lipid Peroxidation. Definition of Experimental Conditions for Selective Study of the Propagation and Termination Phases

To find experimental conditions to selectively study the propagation phase of lipoperoxidation we studied the lipoperoxidation, catalyzed by FeCl₂, of liposomes in a buffering condition where Fe²⁺ autoxidation and oxygen active species generation does not occur. Liposomes from egg yolk phosphatidylcholine. prepared by vortex mixing, do not oxidize Fe²⁺: on the contrary they oxidize Fe²⁺ when prepared by ultrasonic irradiation. Dimyristoyl phosphatidylcholine liposomes prepared by ultrasonic irradiation do not oxidize Fe²⁺. During sonication polyunsaturated fatty acid residues autoxidize and lipid hydroperoxides (LOOH) are generated. Only when LOOH are present in the liposimes Fe²⁺ oxidizes and its rate of oxidation depends on the amount of LOOH in the assay. The reaction results in the generation of both LOOH and thiobarbituric acid reactive material (TBAR): it is inhibited by butylated hydroxytoluene and has a acidic pH optimum; it is not inhibited by catalase and OH' scavengers. The reaction studied. thus, appears to be the chain branching and propagation phase of lipoperoxidation. When we studied the dependence of Fe²⁺ oxidation, LOOH and TBAR generation on FeCl₂ concentration, we observed that at high FeCl₂ concentrations the termination phase of lipoperoxidation was prevalent. Thus. by selecting the appropriate FeCl₂ concentration the proposed experimental system allows study of either the propagation or the termination phase of lipoperoxidation.     

DNA strand scission by the nephrotoxin [2,2'-bipyridine]-3,3',4,4'-tetrol-1,1'-dioxide and related compounds in the presence of iron

The capacity of non-illuminated nephrotoxin orellanine ([2,2'-bipyridine]-3,3',4,4'-tetrol-1,1'-dioxide) to induce DNA damage in the presence of ferrous iron and dioxygen has been evaluated. Maximal single-strand breaks in plasmid DNA were obtained with a metal to ligand ratio 1:3. Instantaneous oxidation of Fe²⁺ in presence of orellanine under air was responsible for oxy-radical production concomitant to a stable ferric complex Fe(III)Or₃ formation, leading to oxidative DNA breakage at physiological pH. DNA damage was lowered in the presence of SOD and catalase or DMSO, indicating a set of reactions that leads to oxyradical generation. Iron chelators such as DTPA and EDTA had no protecting effect, Desferal slightly protected. GSH acted as an oxy-radical scavenger, whereas cysteine induced stronger damage.

Closely related bipyridine compounds were also studied in presence of Fe²⁺ and O₂ using a combination of spin-trapping and DNA-nicking experiments, none of which were able to chelate iron and induce damage at pH 7. Both catecholic moieties and aminoxide groups are required for observing breakage at physiological pH.     

Haemoglobin and Myoglobin as Inhibitors of Hydroxyl Radical Generation in a Model System of “Iron Redox” Cycle

Methionine was oxidized to ethylene by an “Iron Redox” system containing H₂O₂, Fe-EDTA and ascorbate. generating hydroxyl radicals or another species of similar reactivity. Oxy or met forms of haemoglobin and myoglobin were found to inhibit methionine oxidation. Methionine oxidation was elevated in the “Iron Redox” system by increasing ascorbic acid concentration. However, in the presence of metmyoglobin or methaemoglobin, the increases in ascorbic acid did not lower the haemproteins' inhibitory effects but rather increased them.

The pro-oxidative or anti-oxidative activities of haemproteins in biological oxidative reactions seem to be dependent on compartmentalization and on the presence and concentrations of reducing compounds and H₂O₂.     

Iron (III) Stimulation of Lipid Hydroperoxide-Dependent Lipid Peroxidation

In an experimental system where both Fe²⁺ autoxidation and generation of reactive oxygen species is negligible, the effect of FeCl₂ and FeCl₃ on the peroxidation of phosphatidylcholine (PC) liposomes containing different amounts of lipid hydroperoxides (LOOH) was studied; Fe²⁺ oxidation, oxygen consumption and oxidation index of the liposomes were measured. No peroxidation was observed at variable FeCl₂/FeCl₃ ratio when PC liposomes deprived of LOOH by triphenyl-phosphine treatment were utilized. By contrast, LOOH containing liposomes were peroxidized by FeCl₂. The FeCl₂ concentration at which Fe²⁺ oxidation was maximal, defined as critical Fe²⁺ concentration [Fe²⁺]*, depended on the LOOH concentration and not on the amount of PC liposomes in the assay. The LOOH-dependent lipid peroxidation was stimulated by FeCl₃, addition; the oxidized form of the metal increased the average length of radical chains, shifted to higher values the [Fe²⁺]* and shortened the latent period. The iron chelator KSCN exerted effects opposite to those exerted by FeCl₃ addition. The experimental data obtained indicate that the kinetics of LOOH-dependent lipid peroxidation depends on the Fe²⁺/Fe³⁺ ratio at each moment during the time course of lipid peroxidation. The results confirm that exogenously added FeCl₃ does not affect the LOOH-independent but the LOOH-deendent lipid peroxidation; and suggest that the Feg, endogenously generated exerts a major role in the control of the LOOH-dependent lipid peroxidation.     

Anthracycline-Induced Oxygen Consumption and Oxidative Damage in Rat Liver Microsomes are not Necessarily Coupled: A study with 8 structurally related anthracyclines

The stimulative effect of 8 anthracyclines (the parent compounds daunorubicin and doxorubicin and 6 structurally closely related anthracyclines) on the production of thiobarbituric acid (TBA)-reactive material was investigated in liver microsomes. Except for daunorubicinone and doxorubicinone, all derivatives stimulated NADPH-dependent production of TBA-reactive material. Doxorubicinone had no effect, daunorubicinone inhibited TBA-reactivity at concentrations up to 50 μM. However, the latter two compounds stimulated oxygen consumption in the presence of EDTA to a degree comparable to that induced by the parent compounds. Since the oxygen uptake under these circumstances represents redox cycling of the drugs, apparently redox cycling and production of TBA-reactive material were not coupled for these compounds.

Spectral measurements showed no decisive role for interaction with free iron (Fe³⁺) ions in the non-coupling of redox cycling and production of TBA reactive material. Evidence for a role of bound iron ions was not obtained.

It is discussed that for the aglycones oxygen consumption and production of TBA reactive material might be non-coupled through their different interaction with microsomal RNA.     

The lactate-dependent enhancement of hydroxyl radical generation by the Fenton reaction

The effect of lactic acid (lactate) on Fenton based hydroxyl radical (^·OH) production was studied by spin trapping, ESR, and fluorescence methods using DMPO and coumarin-3-carboxylic acid (3-CCA) as the ^·OH traps respectively. The ^·OH adduct formation was inhibited by lactate up to 0.4mM (lactate/iron stoichiometry = 2) in both experiments, but markedly enhanced with increasing concentrations of lactate above this critical concentration. When the H₂O₂ dependence was examined, the DMPO-OH signal was increased linearly with H₂O₂ concentration up to 1 mM and then saturated in the absence of lactate. In the presence of lactate, however, the DMPO-OH signal was increased further with higher H₂O₂ concentration than 1 mM, and the saturation level was also increased dependent on lactate concentration. Spectroscopic studies revealed that lactate forms a stable colored complex with Fe³⁺ at lactate/Fe³⁺ stoichiometry of 2, and the complex formation was strictly related to the DMPO-OH formation. The complex formation did not promote the H₂O₂ mediated Fe³⁺ reduction. When the Fe³⁺-lactate (1:2) complex was reacted with H₂O₂, the initial rate of hydroxylated 3-CCA formation was linearly increased with H₂O₂ concentrations. All the data obtained in the present experiments suggested that the Fe³⁺-lactate (1:2) complex formed in the Fenton reaction system reacts directly with H₂O₂ to produce additional ^·OH in the Fenton reaction by other mechanisms than lactate or lactate/Fe³⁺ mediated promotion of Fe³⁺/Fe²⁺ redox cycling.     

Mechanisms of Saccharomyces Cerevisiae PMA1 H+-ATPase inactivation by Fe2+, H2O2 and Fenton reagents

Although considerably more oxidation-resistant than other P-type ATPases, the yeast PMA1 H⁺-ATPase of Saccharomyces cerevisiae SY4 secretory vesicles was inactivated by H₂O₂, Fe²⁺, Fe- and Cu-Fenton reagents. Inactivation by Fe²⁺ required the presence of oxygen and hence involved auto-oxidation of Fe²⁺ to Fe³⁺. The highest Fe^2- (100 μM) and H₂O₂ (100 mM) concentrations used produced about the same effect. Inactivation by the Fenton reagent depended more on Fe²⁺ content than on H₂O₂ concentration, occurred only when Fe²⁺ was added to the vesicles first and was only slightly reduced by scavengers (mannitol, Tris, NaN₃, DMSO) and by chelators (EDTA, EGTA, DTPA, BPDs, bipyridine, 1, 10-phenanthroline). Inactivation by Fe- and Cu- Fenton reagent was the same; the identical inactivation pattern found for both reagents under anaerobic conditions showed that both reagents act via OH^·. The lipid peroxidation blocker BHT prevented Fenton-induced rise in lipid peroxidation in both whole cells and in isolated membrane lipids but did not protect the H⁺-ATPase in secretory vesicles against inactivation. ATP partially protected the enzyme against peroxide and the Fenton reagent in a way resembling the protection it afforded against SH-specific agents. The results indicate that Fe²⁺ and the Fenton reagent act via metal-catalyzed oxidation at specific metal-binding sites, very probably SH-containing amino acid residues. Deferrioxamine, which prevents the redox cycling of Fe²⁺, blocked H⁺-ATPase inactivation by Fe²⁺ and the Fenton reagent but not that caused by H₂O₂, which therefore seems to involve a direct non-radical attack. Fe-Fenton reagent caused fragmentation of the H⁺-ATPase molecule, which, in Western blots, did not give rise to defined fragments bands but merely to smears.     

Direct NAD(P)H hydrolysis into ADP-ribose(P) and nicotinamide induced by reactive oxygen species: a new mechanism of oxygen radical toxicity

The effect of different oxygen radical-generating systems on NAD(P)H was determined by incubating the reduced forms of the pyridine coenzymes with either Fe²⁺-H₂O₂ or Fe³⁺-ascorbate and by analyzing the reaction mixtures using a HPLC separation of adenine nucleotide derivatives. The effect of the azo-initiator 2,2'-azobis(2-methylpropionamidine)dihydrochloride was also tested. Results showed that, whilst all the three free radical-producing systems induced, with different extent, the oxidation of NAD(P)H to NAD(P)⁺, only Fe²⁺-H₂O₂ also caused the formation of equimolar amounts of ADP-ribose(P) and nicotinamide. Dose-dependent experiments, with increasing Fe²⁺ iron (concentration range 3-180 μM) or H₂O₂ (concentration range 50-1000 μM), were carried out at pH 6.5 in 50 mM ammonium acetate. NAD(P)⁺, ADP-ribose(P) and nicotinamide formation increased by increasing the amount of hydroxyl radicals produced in the medium. Under such incubation conditions NAD(P)⁺/ADP-ribose(P) ratio was about 4 at any Fe²⁺ or H₂O₂ concentration. By varying pH to 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0 and 7.4, NAD(P)⁺/ADP-ribose(P) ratio changed to 5.5, 3.2, 1.8, 1.6, 2.0, 2.5, 3.0, 5.4 and 6.5, respectively. Kinetic experiments indicated that 90-95% of all compounds were generated within 5s from the beginning of the Fenton reaction. Inhibition of ADP-ribose(P), nicotinamide and NAD(P)⁺ production of Fe²⁺-H₂O₂-treated NAD(P)H samples, was achieved by adding mannitol (10-50 mM) to the reaction mixture. Differently, selective and total inhibition of ADP-ribose(P) and nicotinamide formation was obtained by performing the Fenton reaction in an almost completely anhydrous medium, i.e. in HPLC-grade methanol. Experiments carried out in isolated postischemic rat hearts perfused with 50 mM mannitol, showed that, with respect to values of control hearts, this hydroxyl radical scavenger prevented reperfusion-associated pyridine coenzyme depletion and ADP-ribose formation. On the basis of these results, a possible mechanism of action of ADP-ribose(P) and nicotinamide generation through the interaction between NAD(P)H and hydroxyl radical (which does not involve the C-center where “conventional” oxidation occurs) is presented. The implication of this phenomenon in the pyridine coenzyme depletion observed in postischemic tissues is also discussed.     

Oxygen Toxicity. The Influence of Adenine-Nucleotides and Phosphate on Fe2+ Autoxidation

Fecl₂, in Na phosphate buffer autoxidizes forming active oxygen species which damage deoxyribose. Di-and triphosphate adenine-nucleotides inhibit both Fe²⁺ autoxidation and deoxyribose damage in Na phosphate buffer pH 7.4. The inhibition is related to the number of charges of the adenine-nucleotide molecule: ATP at pH 7.4 is a better inhibitor than ADP; at a pH (6.5) close to the pK's of the third and fourth charge of ADP and ATP, ADP inhibition is greatly decreased whereas ATP inhibition is slightly affected. The extent of ATP inhibition of Fe²⁺ autoxidation depends both on ATP/Mg²⁺ and ATP/Fe²⁺ ratios in the reaction mixture. Formation of a Fe²⁺ -nucleotide complex appears to be the mechanism through which ATP and ADP inhibit autoxidation and thus the generation of active oxygen species. These findings are discussed in relation to physiological and pathological fluctuations of nucleotide concentrations.     

The oxidation of chlorophyll a in alcohols

Oxidation potentials for the chlorophyll (Chl/Chl⁺) couple, relative to that of the Fe²⁺/Fe³⁺ couple, were determined in methanol, ethanol, and isopropanol from the initial extent of reaction of chlorophyll with FeCl₃. The presence of 4,7-dimethylphenanthroline was necessary to get sufficient oxidation of chlorophyll in isopropanol. The values were +0.75 V, +0.865 V, and approx. +1.0 V, respectively, based on an assumed value of +0.77 V for the Fe²⁺/Fe³⁺ potential. It is suggested that alcohols, especially methanol, may stabilize Chl⁺ by adding to the carbonyl group or the conjugated double bond system.     

Chemical synthesis,iron redox interactions and charge transfer complex formation of alkylitaconic acids from Ceriporiopsis subvermispora

In 1999, we first reported that a white rot fungus, Ceriporiopsis subvermispora produced a series of novel alkylitaconic acids (ceriporic acids). In the present paper we synthesized the metabolite, 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B) by Grignard reaction to analyze chemical properties of the alkylitaconates. Mass spectrometer (MS) and nuclear magnetic resonance (NMR) spectra of the synthetic compound was identical to those of the fungal metabolite isolated. The dicarboxylic acid inhibited autoxidation of Fe²⁺ to Fe³⁺ as well as reduction of Fe³⁺ to Fe²⁺ by the strong natural reductants, cysteine, glutathione, and ascorbic acid. The formation of charge transfer complexes (CTCs) between 1-heptadecene-2,3-dicarboxylic acid and oxidized intermediates from phenolic substrates were also observed. Thus, we herein report that the new class of lipid-related metabolites produced by C. subvermispora are potential metabolites participating in the control of iron redox reactions and CTCs formation from oxidized lignin fragments.     

Photo-Fenton氧化法处理废水的原理及影响因素

Photo-Fenton高级氧化技术是处理难降解有毒有机废水的一种有效的方法。本文阐述了该氧化法的原理及其影响因素,photo-Fenton氧化法在反应中会产生大量羟自由基(·OH),它是一种非常活泼及非选择性物种,其氧化电位为2.8V,氧化能力很强,能够引发水溶液中大部分有机物的氧化还原反应。其优点是操作简便及无二次污染等,反应产物Fe³⁺可与OH反应形成Fe(OH)₃沉淀而对环境无害。缺点是反应必须在pH≤3条件下进行,且H₂O₂消耗量大而导致价格昂贵,处理成本较高等。     

Ginkgo Biloba Extract EGB 761 or Trolox C Prevent the Ascorbic Acid/FE2+ Induced Decrease in Synaptosomal Membrane Fluidity

The ability of synaptosomes, prepared from striata, to take up ³H-dopamine declined rapidly during incubation at 37°C, in an oxygenated Krebs-Ringer medium with 0.1 mM ascorbic acid. Ascorbic acid was responsible for this decrease. Its effectiveness after a 60 min incubation was concentration dependent from 1 μM and virtually complete for 0.1 mM. Furthermore, a decrease of synaptosomal membrane fluidity was revealed by measurements of fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene. This decrease was potentiated by Fe²⁺ ions (1 μM). In contrast, it was prevented by the Fe²⁺ ion chelator, desferrioxamine (0.1 mM), by the Ginkgo biloba extract EGb 761 [2-16 μg/ml], as well as by the flavonoid quercetin (0.1 μM). This preventive effect was shared by trolox C (from 0.1 mM). It is concluded that peroxidation of neuronal membrane lipids induced by ascorbic acid/Fe²⁺ is associated with a decrease in membrane fluidity which, in turn, reduces the ability of the dopamine transporter to take up dopamine.     

Effects of hypochlorite on cultured respiratory epithelial cells

Neutrophils and eosinophils are involved in the pathogenesis of many respiratory diseases. The enzymes myeloperoxidase and eosinophil peroxidase catalyze the reaction of H₂O₂ with Cl to produce the reactive oxygen species HOCl.

Normal human bronchial epithelial (NHBE) cells were exposed to 0.18-0.90 mM HOCl for 48 h, and studied with immunohistochemical, metabolic and morphological studies.

The ability of the cells to attach to each other and/or to the matrix was altered. Immunohistochemical studies showed a decreased amount of desmosomes and focal adhesion sites, although the morphology of the cells was not affected. The ability of the mitochondria to oxidize glucose was reduced. HOCl-exposed cells had an increased production of NO, probably by an increased activity of cNOS, due to increased intracellular Ca²⁺. The antioxidant N-acetylcysteine inhibited both the NO production and the effects of HOCl on glucose oxidation. The cNOS-inhibitor N-propyl-L-arginine inhibited HOCl-induced NO production. X-ray microanalysis showed an increase in the intracellular Na⁺/K⁺ ratio, which indicates cell damage.

In conclusion, exposure to HOCl results in cell detachment and metabolic alterations in normal human bronchial epithelial cells. Oxygen radicals could in part mediate the effects. Oxygen radicals could hence contribute to the observed epithelial damage in respiratory diseases.