Hemoglobin-catalyzed lipid peroxidation in the presence of mercuric chloride

In order to elucidate the possible mechanism of initiation of peroxidative processes in Hg2+-treated erythrocytes, the effect of HgCl2 on hemoglobin-catalyzed peroxidation of phospholipid liposomes was studied. It was demonstrated that HgCl2 significantly increases the rate of hemoglobin-catalyzed peroxidation. The addition of superoxide dismutase and catalase partially inhibits this effect. Furthermore, it was found that HgCl2 potentiates the hemoglobin oxidation. A suggestion was made that the acceleration of hemoglobin-catalyzed peroxidation by HgCl2 is associated at least in part with the increased production of superoxide anion radicals from hemoglobin.     

Differential effects of mercurial compounds on excitable tissues.

Sarcoplasmic reticulum (SR), Ca2+ plus Mg2+-ATPase, and Ca2+-ionophore were obtained from white rabbit skeletal muscles. Methylmercury inhibited the Ca2+ plus Mg2+-ATPase and Ca2+-transport but had no effect on the Ca2+-ionophore. Mercuric chloride inhibited all three functions (i.e., ATPase, transport and ionophoric activity). The mechanism of HgCl2 inhibition of the Ca2+-ionophore was by competition with Ca2+ for Ca2+-ionophoric site whereas its inhibition of the enzyme and Ca2+-transport was due to the blockage of essential sulfhydryl (--SH) groups. Ca2+ plus Mg2+-ATPase and Ca2+-transport were more sensitive to methylmercury than to HgCl2. Acetylcholine receptor (AChR) was obtained for the electric organ of T. californica. Methylmercury inhibited the ACh binding to AChR WITH Ki = 5.7 - 10(-6) M. This effect was not due to mercuric ion alone since mercuric chloride up to 10(-4) M did not affect ACh binding to AChR. It is concluded that: the Ca2+ plus Mg2+-ATPase and Ca2+-transport contain --SH groups essential for their activity, and that the two functions are tightly coupled; the Ca2+-ionophore contains no --SH groups essential for its activity; CH3HgCl inhibition of Ca2+ plus Mg2+-ATPase and Ca2+-transport is partly due to its reactivity with --SH groups in hydrophobic environment; the Ca2+-transport is inhibited by HgCl2 through two processes, one which is the blockage of --SH groups and another which is the inhibition of the Ca2+-ionophoric site; and the inhibition of ACh binding to AChR is due to the blockage of --SH groups in hydrophobic environment, which is inaccessible to Hg2+. Our data present for the first time a molecular basis for the myopathy associated with mercurial compounds toxicity.     

Fluidity and osmotic sensitivity changes of phospholipase A2-treated liposomes

Membrane fluidity and osmotic sensitivity were examined in DPPC liposomes treated with phospholipase A2 (PL.A2) in the presence of Ca2+ or Mg2+. The amount of liposome phospholipid hydrolyzed differed with the two ions. Embedded DPH, a rod-like fluorescent probe, was employed in the determination of membrane fluidity. Membrane fluidity decreased according to the degree of phospholipid hydrolization in liposomes by PL.A2. The reciprocal value of absorption at 450 nm was measured as the index of osmotic sensitivity of liposomes. Intact sonicated liposomes showed osmotic insensitivity. PL.A2-treated liposomes in which about 40% of total phospholipid was hydrolyzed showed osmotic sensitivity. No change in the membrane fluidity was obtained when PL.A2-treated liposomes were exposed to hypertonic or hypotonic solution. These results suggested that the motion of the acyl-chain of phospholipids and free fatty acids was resisted in PL.A2-treated liposomes. The resistance may be due to a phase separation between phospholipids and free fatty acids. The pore for water permeation might be induced in the border between phase-separated domains in PL.A2-treated liposomes.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

A study on the reaction of human erythrocytes with hydrogen peroxide

Membrane fluidity of human erythrocytes treated with H2O2 (1--20 mM) was studied using three kinds of fatty acid spin labels. A strongly immobilized signal appeared on exposure of erythrocytes to H2O2 but was not observed in either H2O2- or Fenton's reagent-treated ghosts or lipid vesicles prepared from H2O2-treated erythrocytes, indicating that the appearance of this signal necessitates the reaction of hemoglobin with H2O2 and is not due to lipid peroxidation. The ESR spectrum of maleimide-prelabeled erythrocytes showed an isotropic signal and the rotational correlation time (tau c) increased as the concentration of H2O2 was increased. Furthermore, maleimide labeling of H2O2-pretreated erythrocytes showed a strongly immobilized component, in addition to a weakly immobilized component. From the relative ratio of the signal intensity of hemoglobin and membrane proteins, it was found that label molecules bound predominantly to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of H2O2-treated erythrocytes demonstrated globin aggregation. Therefore, the changes in the ESR signal observed on H2O2 treatment may be due to some change in hemoglobin, such as globin aggregation or its binding to the membranes. The ESR spectrum of H2O2-treated erythrocytes at -196 degrees C is characterized by signals of nonheme ferric iron type (g equal to 4.3), low spin ferric iron, and free radical type at g equal to 2.00. At higher H2O2 concentrations, the ESR lines due to low spin ferric iron became broad and their peak heights decreased, compared with that at g equal to 2.00 or 4.3. These results indicate that oxidative stress such as decrease of membrane fluidity, lipid peroxidation, and globin aggregation in H2O2-treated erythrocytes is dependent on the reaction of hemoglobin with H2O2.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe³⁺-chelates, which initiated reactions that were dependent on membrane charge: Fe³⁺-EDTA and Fe³⁺-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe³⁺-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe³⁺-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Generation of toxic phospholipid(s) during oxyhemoglobin-induced peroxidation of phosphatidylcholines

When egg yolk diacylglycerophosphocholine (PC) liposomes were incubated with human oxyhemoglobin, peroxidation of liposomal lipid was induced, as monitored by an increase of thiobarbituric acid (TBA)-reactive substances, an increase of lipid hydroperoxides and the generation of chemiluminescence in the presence of luminol. During the reaction, cytotoxic substance(s), which induced shedding of acetylcholinesterase-enriched vesicles from human erythrocytes, were produced. Formation of TBA-reactive substances and lipid hydroperoxides preceded generation of chemiluminescence, conversion of oxyhemoglobin to methemoglobin and production of the toxic substances. Either superoxide dismutase or catalase could suppress generation of chemiluminescence, but not other events. Methemoglobin or ferrous ion plus ascorbate could induce peroxidation of the liposomes without production of the cytotoxic substance(s). Synthetic PCs containing both saturated and polyunsaturated fatty acyl chains caused the production of cytotoxic products which induced shedding of vesicles from erythrocytes, whereas those containing only polyunsaturated fatty acyl chains did not, suggesting that the molecular species which can produce cytotoxic products may be phospholipids containing both saturated and polyunsaturated fatty acids. The mechanism of oxyhemoglobin-induced peroxidation of lipids will be also discussed.     

The Mechanism of Iron (III) Stimulation of Lipid Peroxidation

A study conducted on Fe²⁺ autoxidation showed that its rate was extremely slow at acidic pH values and increased by increasing the pH; it was stimulated by Fe³⁺ addition but the stimulation did not present a maximum at a Fe²⁺/Fe³⁺ ratio approaching 1:1. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation was able to oxidize deoxyribose; the increased Fe²⁺ oxidation observed at higher pHs was paralleled by increased deoxyribose degradation. The species generated during Fe³⁺-catalyzed Fe²⁺ autoxidation could not initiate lipid peroxidation in phosphatidylcholine liposomes from which lipid hydroperoxides (LOOH) had been removed by treatment with triph-enylphosphine. Neither Fe²⁺ oxidation nor changes in the oxidation index of the liposomes due to lipid peroxidation were observed at pHs where the Fe³⁺ effect on Fe²⁺ autoxidation and on deoxyribose degradation was evident. In our experimental system, a Fe²⁺/Fe³⁺ ratio ranging from 1:3 to 2:1 was unable to initiate lipid peroxidation in LOOH-free phosphatidylcholine liposomes. By contrast Fe³⁺ stimulated the peroxidation of liposomes where increasing amounts of cumene hydroperoxide were incorporated. These results argue against the participation of Fe³⁺ in the initiation of LOOH-independent lipid peroxidation and suggest its possible involvement in LOOH-dependent lipid peroxidation.     

An investigation into thee mechanism of citrate---FE-dependent lipid peroxidation

Chelation by citrate was found to promote the autoxidation of Fe²⁺, measured as the disapperance of 1,10-phenanthroline-chelatable Fe²⁺. The autoxidation of citrate---²⁺ could in turn promote the peroxidation of microsomal phospholipid liposomes, as judged by malondialdehyde formation. At low citrate---Fe²⁺ ratios the autoxidation of Fe²⁺ was slow and the formation of malondialdehyde was preceded by a lag phase. The lag phase evidence of this, linear initial rates of lipid peroxidation were obtained via the combination of citrate---Fe²⁺ and citrate---Fe³⁺, optimum activity occurring at a Fe³⁺---Fe²⁺ ratio of 1:1. Evidence is also presented to suggest that the superoxide and the hydrogen peroxide that are formed during the autoxidation of citrate---Fe²⁺ can either stimulate or inhibit lipid peroxidation by affecting the yield of citrate---Fe³⁺ from citrate---Fe²⁺. No evidence was obtained for the participation of the hydroxyl radical in the initiation of lipid peroxidation by citrate---Fe²⁺.     

Possible contribution of oxyhemoglobin to the iron-induced hemolysis simultaneous effect of iron and hemoglobin on lipid peroxidation

The mechanism of iron toxicity in iron overloaded patients is not well established. A hypothesis was put forward that free radical processes are involved. Our earlier study indicates that iron-induced hemolysis is preceded by peroxidation of the membrane lipids. In the present work the simultaneous effect of iron and hemoglobin on lipid peroxidation was studied. It was found that in hemoglobin-containing liposome suspensions Fe2+ in concentrations above 10(-5) M inhibits the peroxidation, while Fe3+ drastically potentiates it, with concomitant transformation of oxyhemoglobin to methemoglobin. The experiments with scavengers of activated oxygen indicate superoxide anion radical (O-.2), hydroxyl radical (OH.) and singlet oxygen (1O2) participation. The possible mechanism of the phenomenon is discussed. A conclusion is drawn that the toxic effect of Fe3+ may be associated not only with iron--membrane interaction, but also with increased methemoglobin formation and O-.2 release.     

Reactivity of propylene oxides towards deoxycytidine and identification of reaction products

In order to elucidate the mechanism of the stimulative effect of molybdenum on mercury-mediated renal metallothionein induction, the levels of translatable metallothionein mRNA (MT mRNA) in the kidneys of rats treated with saline or Na2MoO4 or HgCl2 or Na2MoO4 and HgCl2 were measured by translation experiments in cell-free protein synthesizing systems. The time course of accumulation of mercury in renal nuclei of rats given HgCl2 with or without Na2MoO4-pretreatment was also investigated. Molybdenum, itself, did not elevate levels of MT mRNA compared to saline controls at all time points (0, 6 and 14 h after exposure to HgCl2) but rapidly elevated the levels of the mRNA more than Hg-dosed rats when HgCl2 was also administered. On the other hand, the time course study in renal nuclei showed that the mercury content of nuclei was consistently lower in Mo-Hg-dosed rats than in Hg-dosed rats at all time points (4, 8 and 24 h after exposure to HgCl2). These results suggest that the stimulative effect of molybdenum on mercury-mediated metallothionein induction is coupled with an increase of the mRNA coding for the low molecular weight protein and that such an increase in the levels of translatable MT mRNA is not due to the difference in uptake of mercury into renal nuclei.     

Exercise-induced oxidative stress affects erythrocytes in sedentary rats but not exercise-trained rats.

Oxidant stress is one of the factors proposed to be responsible for damaged erythrocytes observed during and after exercise. The impact of exertional oxidant stress after acute exhaustive treadmill running on erythrocyte damage was investigated in sedentary (Sed) and exercise-trained (ET) rats treated with or without antioxidant vitamins C and E. Exhaustive exercise led to statistically significant increments in the levels of thiobarbituric acid-reactive substance (TBARS) and H2O2-induced TBARS in Sed rats and resulted in functional and structural alterations in erythrocytes (plasma hemoglobin concentrations, methemoglobin levels, and rise in osmotic fragility of erythrocytes with decrease in erythrocyte deformability). Administration of antioxidant vitamin for 1 mo before exhaustive exercises prevented lipid peroxidation (TBARS, H2O2-induced TBARS) in Sed rats without any functional or structural alterations in erythrocytes. Parameters indicating erythrocyte lipid peroxidation and deterioration after exhaustive exercise in rats trained regularly with treadmill running for 1 mo were not different from those in Sed controls. Erythrocyte lipid peroxidation (TBARS) increased in exhausted-ET rats compared with ET controls; however, the plasma hemoglobin, methemoglobin levels, and erythrocyte osmotic fragility and deformability did not differ. Exhaustive exercise-induced lipid peroxidation in ET rats on antioxidant vitamin treatment was prevented, whereas functional and structural parameters of erythrocytes were not different from those of the ET controls. We conclude that exertional oxidant stress contributed to erythrocyte deterioration due to exercise in Sed but not in ET rats.     

Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions

Lead (Pb2+) ions accelerate the lipid peroxidation observed when Fe2+ ions are added to phospholipid liposomes at pH 5.5 or pH 7.4, although Pb2+ ions alone do not induce any peroxidation. Similarly, aluminium (Al3+) ions increase Fe2+-dependent liposomal peroxidation at pH 5.5. Both Pb2+ and Al3+ accelerate the peroxidation of erythrocytes induced by high concentrations of H2O2 in the presence of azide, and they also increase the peroxidation that occurs when Fe2+ or Fe2+-ADP is added to rat liver microsomes at pH 7.4. It is proposed that increased lipid peroxidation may contribute to the toxic actions of Pb2+ in humans.     

The antioxidant action of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a chemiluminescence probe to detect superoxide anions

The antioxidant effect of 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α]pyrazin-3-one (MCLA), a Cypridina luciferin analog that acts as a chemiluminescence probe to detect O^⋅−₂, was investigated. MCLA produced a lag in oxygen consumption induced by cumene hydroperoxide in microsomes or by 2,2′-azobis (2-amidinopropane) dihydrochloride in liposomes and disappeared during the duration of the lag. MCLA profoundly inhibited the propagation reaction in Fe²⁺-dependent lipid peroxidation in liposomes, and MCLA disappearance accompanied by suppression of oxygen consumption markedly occurred in liposomes susceptible to peroxidation. Thiobarbituric acid-reactive substances in all systems used were also suppressed by MCLA dose dependently. These results indicate that MCLA has an antioxidant property through scavenging free radicals.     

An investigation into the role of hydroxyl radical in xanthine oxidase-dependent lipid peroxidation

A model lipid peroxidation system dependent upon the hydroxyl radical, generated by Fenton's reagent, was compared to another model system dependent upon the enzymatic generation of superoxide by xanthine oxidase. Peroxidation was studied in detergent-dispersed linoleic acid and in phospholipid liposomes. Hydroxyl radical generation by Fenton's reagent (FeCl₂ + H₂O₂) in the presence of phospholipid liposomes resulted in lipid peroxidation as evidenced by malondialdehyde and lipid hydroperoxide formation. Catalase, mannitol, and Tris-Cl were capable of inhibiting activity. The addition of EDTA resulted in complete inhibition of activity when the concentration of EDTA exceeded the concentration of Fe²⁺. The addition of ADP resulted in slight inhibition of activity, however, the activity was less sensitive to inhibition by mannitol. At an ADP to Fe²⁺ molar ratio of 10 to 1, 10 mm mannitol caused 25% inhibition of activity. Lipid peroxidation dependent on the enzymatic generation of superoxide by xanthine oxidase was studied in liposomes and in detergent-dispersed linoleate. No activity was observed in the absence of added iron. Activity and the apparent mechanism of initiation was dependent upon iron chelation. The addition of EDTA-chelated iron to the detergent-dispersed linoleate system resulted in lipid peroxidation as evidenced by diene conjugation. This activity was inhibited by catalase and hydroxyl radical trapping agents. In contrast, no activity was observed with phospholipid liposomes when iron was chelated with EDTA. The peroxidation of liposomes required ADP-chelated iron and activity was stimulated upon the addition of EDTA-chelated iron. The peroxidation of detergent-dispersed linoleate was also enhanced by ADP-chelated iron. Again, this peroxidation in the presence of ADP-chelated iron was not sensitive to catalase or hydroxyl radical trapping agents. It is proposed that initiation of superoxide-dependent lipid peroxidation in the presence of EDTA-chelated iron occurs via the hydroxyl radical. However, in the presence of ADP-chelated iron, the participation of the free hydroxyl radical is minimal.     

Effect of methyl mercury on phosphorylation, transport, and oxidation in mammalian mitochondria.

the toxic effects of CH3HgCL on mitochondria of mammalian organs including human and rat liver were examined. [203Hg]CH3HGCl was bound mainly to mitochondrial proteins. The binding was not effected by the energy state of mitochondria. The state 3 respiration, oxidative phosphorylation and 32Pi-ATP exchange reaction were inhibited by 10 to 50 nmol of CH3HgCl per mg of mitochondrial protein, while NADH-and succinate-dehydrogenase and ATPase were more resistant to it The difference spectrum of the treated mitochondria indicated that the point of inhibition was located after flavin and before cytochrome b. Mitochondrial swelling was induced by CH3HgCl, in accordance with previous morphological observations in vivo. The swelling, stimulation of ATPase and energy-dependent H+ extrusion cauded by CH3HgCl were equally dependent on K+. Under these conditions, uptake of K+ by mitochondria was increased and the membrane potential was dissipated. Unlike the case with other organomercuric compounds, transport of phosphate was not inhibited by CH3HgCl. When tested on liposomes, CH3HgCl itself was not lipid-soluble, as some organomercuric compounds are, and was not an uncoupler or a K+-carrier. It was concluded that protein bound CH3HgS-induced K+ uptake into mitochondria and the resulting loss of membrane potential was the major cause of uncoupling, though at higher concentrations, the electron transport system was also inhibited.     

Ion permeation of AQP6 water channel protein. Single channel recordings after Hg2+ activation

Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 microm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 microm HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by 22Na+ influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.     

Hg2+ and Cu+ are ionophores, mediating Cl-/OH- exchange in liposomes and rabbit renal brush border membranes.

Organometals, including organomercurials, are capable of mediating Cl-/OH- exchange across lipid membranes by forming neutral ion pairs. In this study, the ability of inorganic metals to catalyze Cl-/OH- exchange was examined. In the presence of an inwardly directed chloride gradient, HgCl2 at concentrations as low as 30 nM resulted in quenching of acridine orange fluorescence in liposomes, indicating liposomal acidification. In the presence of the reducing agent, ascorbate, CuSO4 at concentrations as low as 0.6 microM also mediated chloride-dependent liposomal acidification. Copper in the absence of ascorbate, iron (with or without ascorbate), cobalt, cadmium, zinc, nickel, and lead were without an effect. 36Cl efflux from rabbit renal brush border membrane vesicles was also markedly stimulated by micromolar concentrations of mercury or copper plus ascorbate. Vesicle integrity was not altered by the concentrations of mercury or copper employed in these studies. In the absence of ascorbate, CuCl stimulated chloride efflux only under anaerobic conditions, confirming that it is the reduced form of copper (Cu+) that mediates chloride transport across the membrane. In the presence of mercury or reduced copper, an inside alkaline pH gradient stimulated the uphill accumulation of 36Cl and 82Br, respectively, confirming Cl-/OH- exchange. Studies in liposomes and brush border membranes demonstrate that this is an electroneutral process. These results show that Hg2+ and Cu+ are capable of acting as ionophores, mediating electroneutral Cl-/OH- exchange in liposomes and brush border membrane vesicles. This effect could contribute to the toxicity of these two metals.     

Inhibition of Membrane Lipid Peroxidation by a Radical Scavenging Mechanism: a Novel Function for Hydroxyl-Containing Ionophores

In the present study we show that K⁺/H⁺ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe²⁺-citrate and 2,2'-azobis(2-amidinopropane) di-hydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidyl-choline (PC) liposomes containing negatively charged lipids—dicetyl phosphate (DCP) or cardiolipin (CL)—and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na⁺ than for K⁺, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O₂ consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by buty-lated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, vali-nomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (S_eff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe²⁺-citrate-induced production of radicals derived from piperazine-based buffers, demonstrating their property as radical scavengers. Both Fe²⁺-citrate and ABAP promote a much more pronounced decrease of LAS fluorescence in PC/CL liposomes than in dimyristoyl phosphatidyl-choline (DMPC, saturated phospholipid)-DCP liposomes, indicating that the ionophore also scavenges lipid peroxyl radicals. A slow decrease of fluorescence is observed in the latter system, for all lipid compositions in sucrose medium, and in the absence of membranes, indicating that the primary radicals stemming from both inductors also attack the ionophore. Altogether, the data lead to the conclusion that the membrane-incorporated cation complexes of NIG, LAS and MON inhibit lipid peroxidation by blocking initiation and propagation reactions in the lipid phase via a free radical scavenging mechanism, very likely due to the presence of alcoholic hydroxyl groups in all three molecules and to the attack of the aromatic moiety of LAS.     

Peroxidation of liposomal palmitoyllinoleoylphosphatidylcholine (PLPC), effects of surface charge on the oxidizability and on the potency of antioxidants

Peroxidation of membrane phospholipids is an important determinant of membrane function. Previously we studied the kinetics of peroxidation of the polyunsaturated fatty acid (PUFA) residues in model membranes (liposomes) made by sonication of palmitoyllinoleoylphosphatidylcholine (PLPC). Since most biomembranes are negatively-charged, we have now studied the effect of negative surface charge on the kinetics of peroxidation of liposomes made of PLPC and 9% of one of the negatively-charged phospholipids phosphatidylserine (PS) or phosphatidic acid (PA). Peroxidation was initiated by either CuCl2 or AAPH and continuously monitored spectrophotometrically. The following results were obtained: (i) The negative charge had only a slight effect on AAPH-induced peroxidation, but accelerated markedly copper-induced peroxidation of the liposomes, probably by increasing the binding of copper to the membrane surface. (ii) Ascorbic acid (AA) inhibited AAPH-induced but promoted copper-induced peroxidation in all the studied liposomes, probably by enhancing the production of free radicals upon reduction of Cu(II) to Cu(I). (iii) alpha-tocopherol (Toc) inhibited AAPH-induced peroxidation in all the studied liposomes, whereas the effect of tocopherol on copper-induced peroxidation varied from being pro-oxidative in PA-containing liposomes, to being extremely anti-oxidative in PS-containing liposomes, even at very low tocopherol concentrations. The significance of the latter unusual protective effect, which we attribute to recycling of tocopherol by a PS-Cu complex, requires further investigation.