Mechanism of the membrane interaction of polynuclear platinum anticancer agents. Implications for cellular uptake

The interaction between phospholipids and polynuclear platinum drugs was studied as a mechanism model for cellular uptake of anticancer drugs. The interaction was studied by differential scanning calorimetry (DSC), 31P nuclear magnetic resonance spectroscopy (NMR), inductively coupled plasma optical emission spectroscopy (ICP-OES), and electrospray ionization mass spectrometry (ESI-MS). The transition temperature, enthalpy, and entropy of negatively charged phospholipids DPPS, DPPA, and DPPG were changed upon reaction with the trinuclear platinum complex [{trans-PtCl(NH3)2}2mu-Pt(NH3)2{H2N(CH2)6NH2}2](NO3)4 (I, BBR3464) and the dinuclear analogue [{trans-PtCl(NH3)2}mu-{(NH2)(CH2)3NH2(CH2)4(NH2)}Cl3 (II, BBR3571). This suggests that these platinum complexes interacted not only with the phosphate headgroup but also with the region of the fatty acid tail of liposomes and finally changed the fluidity of the membrane. Both noncovalent (presumably electrostatic and hydrogen bonding) and covalent interactions were involved in the reactions of the negatively charged phospholipids DPPA, DPPS, and DPPG with the highly positively charged platinum complexes. In contrast, few differences were seen for the zwitterionic phospholipids DPPC and DPPE. The binding ratio of BBR3464 to DPPA liposomes was higher than the ratio of BBR3464 to DPPS liposomes, and similar differences were seen for BBR3571. The binding ratios of the platinum complexes to negatively charged phospholipids DPPA, DPPS, and DPPG were slightly lower in a 100 mM chloride solution than in a chloride-free solution. The binding of BBR3464 and BBR3571 with the liposomes was significantly stronger than that with cis-[PtCl2(NH3)2], cisplatin. ESI-MS confirmed that the products of the incubation of BBR3464 with DPPA and DPPS correspond to chloride displacement and formation of [Pt3(NH3)6{NH2(CH2)6NH2}2(DPPA)2]2+ (1) and [Pt3(NH3)6{NH2(CH2)6NH2}2(DPPS)2]2+ (2), respectively. Similar observations were made for BBR3571. 31P NMR spectra confirmed that the site of binding for DPPA was the phosphate oxygen, whereas for DPPS, a binding site of the nitrogen of the serine side chain is indicated. Noncovalent interactions were also confirmed by use of the analogue [{Pt(NH3)3}2mu-Pt(NH3)2{H2N(CH2)6NH2}2](NO3)6 (III, 0,0,0/t,t,t). The implications of these results for the mechanism of cellular uptake of polynuclear platinum complexes are discussed.     

Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies

In biological membranes, the anionic characteristics of the polar headgroup of phosphatidic acids are responsible for structural changes induced by Ca2+ in many cellular processes. The very simple headgroup structure of dipalmitoylphosphatidic acid (DPPA) offers particular advantages as a model to study the interactions between Ca2+ and natural phosphatidic acids such as cardiolipin and phosphatidylserine. The effects of calcium ions on DPPA membranes have been studied as a function of temperature by potentiometry and by Raman, ESR and 31P-NMR spectroscopies. The protons in monosodic DPPA liposomes have been considered as a probe to detect pH variations resulting from introduction of Ca2+ inside the membrane. This method has also allowed us to determine the stoichiometry of this reaction: 2 DPPA(H) + Ca2+----Ca(DPPA)2 + 2H+. 31P-NMR spectroscopy has been used to detect reorganization-condensation phenomena in multilamellar vesicles of DPPA under the influence of calcium and temperature. Furthermore, the temperature profiles obtained from Raman spectra for Ca(DPPA)2 membranes provide conclusive evidence that Ca2+ induces major reorganization of the phosphatidic acid component into a highly ordered phase. Quantitative estimates of the degree of motional restriction of spin-labeled soaps embedded inside membranes composed of DPPA with or without Ca2+ have been made using ESR technique. These results are discussed and compared to those found previously for a natural phosphatidic acids such as phosphatidylserine.     

Mn~(2+)和Cu~(2+)与脂质体和脂酶体相互作用的ESR研究

用ESR实验研究了Mn~(2 )、Cu~(2 )与DOPC,DPPC,SPL,DOPA,DPPA脂质体及其与H~ -ATP酶复合体重组的脂酶体的相互作用.通过Mn~(2 )—ESR谱线强度以及Cu~(2 )—ESR谱g因子的测量得出,磷脂分子头部不同的化学组成及其脂酰链的不同状态决定了Mn~(2 )、Cu~(2 )与膜脂结合的强弱程度,通过脂质体和脂酶体中自旋标记物5NS—ESR谱的测量进一步得出Mn~(2 )的结合增大了膜脂排列的序参数,而酶复合体的嵌入都导致与膜脂结合的Mn~(2 )比例减小.因而,当Mn~(2 )与脂酶体相互作用时,膜脂的排列最终达到一个平衡状态.在中性磷脂脂酶体的膜与Mn~(2 )之间,这种相互作用不明显.     

Tannic acid inhibits in vitro iron-dependent free radical formation

The antioxidant activity of tannic acid (TA), a plant polyphenol claimed to possess antimutagenic and anticarcinogenic activities, was studied by monitoring (i) 2-deoxyribose degradation (a technique for OH detection), (ii) ascorbate oxidation, (iii) ascorbate radical formation (determined by EPR analysis) and (iv) oxygen uptake induced by the system, which comprised Fe(III) complexes (EDTA, nitrilotriacetic acid (NTA) or citrate as co-chelators), ascorbate and oxygen. TA removes Fe(III) from the co-chelators (in the case of EDTA, this removal is slower than with NTA or citrate), forming an iron-TA complex less capable of oxidizing ascorbate into ascorbate radical or mediating 2-deoxyribose degradation. The effectiveness of TA against 2-deoxyribose degradation, ascorbate oxidation and ascorbate radical formation was substantially higher in the presence of iron-NTA (or iron-citrate) than with iron-EDTA, which is consistent with the known formation constants of the iron complexes with the co-chelators. Oxygen uptake and 2-deoxyribose degradation induced by Fe(II) autoxidation were also inhibited by TA. These results indicate that TA inhibits OH formation induced by Fe(III)/ascorbate/O(2) mainly by arresting Fe(III)-induced ascorbate oxidation and Fe(II) autoxidation (which generates Fe(II) and H(2)O(2), respectively), thus limiting the production of Fenton reagents and OH formation. We also hypothesize that the Fe(II) complex with TA exhibits an OH trapping activity, which explains the effect of TA on the Fenton reaction.     

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.     

Enzyme behaviour and molecular environment. The effects of ionic strength, detergents, linear polyanions and phospholipids on the pH profile of soluble cytochrome oxidase.

The activity vs. pH profile for the oxidation of ferrocytochrome c by purified cytochrome oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) was investigated as a function of ionic strength (from 10 to 200 mM) in the absence and in the presence of various perturbants: Tween 20, linear polyanions (RNA, heparin, polyglutamic acid) and phospholipids (asolectin, phosphatidylcholine, phosphatidic acid and cardiolipin). The activation induced by Tween 20 and "zero net charge" phospholipid liposomes was not pH dependent. On the other hand, linear polyanions and polyanionic liposomes strongly perturbed the pH profile, mostly at low ionic strength, by shifting the pH optimum about 1.7 pH units towards alkaline pH values. This effect was reversed by increasing ionic strength. These observations are interpreted in the light of polyelectrolyte theory. Since these results show striking with membrane-bound enzyme, it is concluded that in vivo cytochrome oxidase is located within polyanionic sites of the micochondrial membrane. The activation broght about by phospholipids may result from two posible processes: creation of a hydrophobic environment by the non-polar tails, preventing autoaggregation; and creation of a suitable polyelectrolytic environment by the polar heads (of non zero net charge), increasing the intrinsic reaction rate.     

Interaction of pulmonary surfactant protein A with phospholipid liposomes: a kinetic study on head group and fatty acid specificity.

Recent work on surfactant protein A (SP-A) has shown that Ca(2+) induces an active conformation, SP-A, which binds rapidly to liposomes and mediates their aggregation. Employing sensitive real time assays, we have now studied the lipid binding characteristics of the SP-A liposome interaction. From the final equilibrium level of the resonant mirror binding signal, an apparent dissociation constant of ca. K(d)=5 microM is obtained for the complex between SP-A and dipalmitoylphosphatidylcholine (DPPC) liposomes. At nanomolar SP-A concentrations, this complex is formed with a subsecond (0.3 s) reaction time, as measured by light-scattering signals evoked by photolysis of caged Ca(2+). With palmitoyloleoylphosphatidylcholine (POPC), the complex formation proceeds at half the rate, compared to DPPC, leading to a lower final equilibrium level of SP-A lipid interaction. Distearoylphosphatidylcholine (DSPC) shows a stronger interaction than DPPC. Regarding the phospholipid headgroups, phosphatidylinositol (PI) and sphingomyelin (SM) interact comparable to DPPC, while less interaction is seen with phosphatidylethanolamine (PE) or with phosphatidylglycerol (PG). Thus both headgroup and fatty acid composition determine SP-A phospholipid interaction. However, the protein does not exhibit high specificity for either the polar or the apolar moiety of phospholipids.     

Polyamine inhibition of lipoperoxidation. The influence of polyamines on iron oxidation in the presence of compounds mimicking phospholipid polar heads.

Polyamines appear to inhibit peroxidation of vesicles containing acidic phospholipids. A correlation exists between polyamine binding to phospholipid vesicles and its protective effect. However, phosphatidylinositol-containing vesicles which bind spermine are not protected by the polyamine [Tadolini, Cabrini, Landi, Varani & Pasquali (1985) Biogenic Amines 3, 97-106]. In the present paper I tested the hypothesis that polyamines, in particular spermine, by forming a ternary complex with iron and the phospholipid polar head may change the susceptibility of Fe2+ to autoxidation and thus its ability to generate free oxygen radicals. Different compounds mimicking phospholipid polar heads were studied, namely AMP, mimicking phosphatidic acid, CDP-choline, mimicking phosphatidylcholine, and glycerophosphoinositol, mimicking phosphatidylinositol. The results support the proposed hypothesis. In the presence of CDP-choline or of glycerophosphoinositol, spermine poorly affects Fe2+ autoxidation, whereas a considerable inhibition is observed in the presence of AMP. The ability of other phosphorus-containing compounds (ATP, ADP, cyclic AMP, sodium phosphate) to affect Fe2+ autoxidation in the presence of polyamines was also evaluated to understand the molecular mechanism of this phenomenon. It is proposed that polyamines may be part of the passive cellular defence mechanism against the oxidative damage caused by Fe2+.     

Antioxidant properties of S-adenosyl-L-methionine in Fe(2+)-initiated oxidations

S-Adenosylmethionine (SAM) is protective against a variety of toxic agents that promote oxidative stress. One mechanism for this protective effect of SAM is increased synthesis of glutathione. We evaluated whether SAM is protective via possible antioxidant-like activities. Aerobic Hepes-buffered solutions of Fe2+ spontaneously oxidize and consume O2 with concomitant production of reactive oxygen species and oxidation of substrates to radical products, e.g., ethanol to hydroxyethyl radical. SAM inhibited this oxidation of ethanol and inhibited aerobic Fe2+ oxidation and consumption of O2. SAM did not regenerate Fe2+ from Fe3+ and was not consumed after incubation with Fe2+. SAM less effectively inhibited aerobic Fe2+ oxidation in the presence of competing chelating agents such as EDTA, citrate, and ADP. The effects of SAM were mimicked by S-adenosylhomocysteine, but not by methionine or methylthioadenosine. SAM did not inhibit Fe2+ oxidation by H2O2 and was a relatively poor inhibitor of the Fenton reaction. Lipid peroxidation initiated by Fe2+ in liposomes was associated with Fe2+ oxidation; these two processes were inhibited by SAM. However, SAM did not show significant peroxyl radical scavenging activity. SAM also inhibited the nonenzymatic lipid peroxidation initiated by Fe2+ + ascorbate in rat liver microsomes. These results suggest that SAM inhibits alcohol and lipid oxidation mainly by Fe2+ chelation and inhibition of Fe2+ autoxidation. This could represent an important mechanism by which SAM exerts cellular protective actions and reduces oxidative stress in biological systems.     

The involvement of superoxide anions in the nitro blue tetrazolium chloride reduction mediated by NADH and phenazine methosulfate

Oxygen inhibits competitively the reduction of nitro blue tetrazolium chloride (nitro BT) by NADH and phenazine methosulfate (PMS). The oxygen-dependent inhibition is stronger in the presence of superoxide dismutase, whereas cyanide counteracts the oxygen interference. On the other hand, the oxidation of NADH mediated by PMS and dioxygen is affected only marginally by superoxide dismutase and cyanide. Therefore, it is concluded that the involvement of superoxide anions occurs at the level of nitro BT reduction via a nitro blue tetrazolinyl radical, as has been suggested by Picker and Fridovich [1984) Arch. Biochem. Biophys. 228, 155-158) and not at the level of PMS oxidation. The inhibition of the oxygen interference in the nitro BT reduction by cyanide is dependent on the cyanide concentration, whereas in nitrogen cyanide has no effect on the reduction. It is caused by competition between cyanide and oxygen to reduce or oxidize the nitro BT radical to either formazan with concomitant cyanogen production or nitro BT, respectively. For the histochemical localization and analysis of electropherograms of NAD(P)+-dependent dehydrogenase activities, the interference of oxygen can be avoided by anaerobic incubations or by the use of 5 mM nitro BT when incubating aerobically.     

Diphtheria toxin induces leakage of acidic liposomes

Diphtheria toxin (DT) induces the leakage of dipalmitoylphosphatidic acid (DPPA) membranes but not neutral dipalmitoylphosphatidylcholine (DPPC) membranes. Cholesterol incorporated into liposomes enhances the membrane leakage induced by DT in acidic DPPA membranes but not in neutral DPPC membranes. Membrane leakage was determined by assaying the release of TEMPOcholine, a cationic spin probe from the multilamellar vesicles by using electron spin resonance methods. The effect of DT on membrane leakage is noticeable at 3 micrograms/ml concentrations, and reaches a plateau of about 20% leakage at 20 micrograms/ml. This saturation phenomenon led to the postulation that DT binds to the first shell of DPPA membranes and induces the leakage of TEMPOcholine limited to this layer of DPPA multimellar vesicles.     

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.     

Protecting or promoting effects of spermine on DNA strand breakage induced by iron or copper ions as a function of metal concentration

Polyamines are ubiquitous polycations that participate in cellular processes such as growth, differentiation and cell death. Among the different functions ascribed to these organic cations, the polyamine spermine is known to protect DNA from the damage produced by reactive oxygen species (ROS) generated by different agents including copper ions. We have found that spermine exerts opposite effects on DNA strand breakage induced by Fenton reaction depending on metal concentration. Whereas at low concentration of the transition metals, 10 microM copper or 50 microM Fe(II), 1 mM spermine exerted a protective role, at metal concentrations higher than 25 microM copper or 100 microM Fe(II), spermine stimulated DNA strand breakage. The promotion of the damage induced by spermine was independent of DNA sequence but decreased by increasing the ionic concentration of the media or by the presence of metal-chelating agents. Moreover, spermine did not increase the oxidation of 2-deoxyribose by metal/H2O2 when DNA was substituted by 2-deoxyribose as a target for damage. Our results corroborate that spermine may protect DNA and 2-deoxyribose from the damage induced by ROS but also demonstrate that under certain conditions spermine may promote DNA strand breakage. The fact that this promoting effect of spermine on ROS-induced damage was observed only in the presence of DNA suggests that this polyamine under certain conditions may facilitate the interaction of copper and iron ions with DNA leading to the formation of ROS in close proximity to DNA.     

Calcium and spermine interaction with phospholipid bilayers: a 15N NMR study

The binding of Ca2+ to membrane models composed of diplamitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) 15N-labeled in the polar head group was investigated at pH 8.5 and pH 9.4 by 15N-NMR spectroscopy. Both phospholipids exhibit a decrease in the chemical shift anisotropy, indicating changes of the order parameter of the C-N bond and decrease in half-height width. Binding of Ca2+ induces a chemical shift change for the DPPE signal which indicates a decrease in the pKa of the amino group. The binding of spermine was also investigated for mixed phase (DPPC/DPPE) at pH 8 and pH 9.4; a decrease in the DPPE pKa was also noted. The signals of both phospholipids are broadened and the line shapes are more complex because of the lower mobility and the higher steric bulk of this molecule. The results show the value of 15N-NMR in the study of mixed liposomes and indicate that the deprotonation of membrane surface could constitute a necessary step for fusion processes.     

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on *OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as beta-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against *OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for *OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of *OH from H2O2. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H2O2. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on *OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.     

Nuclear magnetic resonance and thermal studies on the interaction between salicylic acid and model membranes

DSC and (1H and 31P) NMR measurements are used to investigate the perturbation caused by the keratolytic drug, salicylic acid (SA) on the physicochemical properties of the model membranes. Model membranes (in unilamellar vesicular (ULV) form) in the present studies are prepared with the phospholipids, dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidylethanolamine (DPPE), dipalmitoyl phosphatidic acid (DPPA) and mixed lipid DPPC-DPPE (with weight ratio, 2.5:2.2). These lipids have the same acyl (dipalmitoyl) chains but differed in the headgroup. The molar ratio of the drug to lipid (lipid mixture), is in the range 0 to 0.4. The DSC and NMR results suggest that the lipid head groups have a pivotal role in controlling (i) the behavior of the membranes and (ii) their interactions with SA. In the presence of SA, the main phase transition temperature of (a) DPPE membrane decreases, (b) DPPA membrane increases and (c) DPPC and DPPC-DPPE membranes are not significantly changed. The drug increases the transition enthalpy (i.e., acyl chain order) in DPPC, DPPA and DPPC-DPPE membranes. However, the presence of the drug in DPPC membrane formed using water (instead of buffer), shows a decrease in the transition temperature and enthalpy. In all the systems studied, the drug molecules seem to be located in the interfacial region neighboring the glycerol backbone or polar headgroup. However, in DPPC-water system, the drug seems to penetrate the acyl chain region also.     

Phospholipid dependence of calcium ion effects on electrophoretic mobilities of liposomes

Electrophoretic light scattering (ELS) and depolarization of fluorescence have been used to determine the effect of membrane fluidity on the binding of Ca2+ to liposomes. ELS was used to measure the electrophoretic mobilities of the liposomes. Fluorescence depolarization was used to determine membrane fluidity. Zero to 30 mol% phosphatidylserine (PS) was incorporated into liposomes containing, as bulk phospholipids, one of the following: dimyristoyl-phosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), or hydrogenated egg phosphatidylcholine (H egg PC). The binding of Ca2+ to the liposomes appears to be influenced by membrane fluidity. Liposomes containing bulk phospholipids whose phase transition temperature is higher than the experimental temperature exhibit enhanced binding of CA2+.     

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.     

EPR spin trapping and 2-deoxyribose degradation studies of the effect of pyridoxal isonicotinoyl hydrazone (PIH) on ⋅OH formation by the Fenton reaction

The search for effective iron chelating agents was primarily driven by the need to treat iron-loading refractory anemias such as β-thalassemia major. However, there is a potential for therapeutic use of iron chelators in non-iron overload conditions. Iron can, under appropriate conditions, catalyze the production of toxic oxygen radicals which have been implicated in numerous pathologies and, hence, iron chelators may be useful as inhibitors of free radical-mediated tissue damage. We have developed the orally effective iron chelator pyridoxal isonicotinoyl hydrazone (PIH) and demonstrated that it inhibits iron-mediated oxyradical formation and their effects (e.g. 2-deoxyribose oxidative degradation, lipid peroxidation and plasmid DNA breaks). In this study we further characterized the mechanism of the antioxidant action of PIH and some of its analogs against ^⋅OH formation from the Fenton reaction. Using electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap for ^⋅OH we showed that PIH and salicylaldehyde isonicotinoyl hydrazone (SIH) inhibited Fe(II)-dependent production of ^⋅OH from H₂O₂. Moreover, PIH protected 2-deoxyribose against oxidative degradation induced by Fe(II) and H₂O₂. The protective effect of PIH against both DMPO hydroxylation and 2-deoxyribose degradation was inversely proportional to Fe(II) concentration. However, PIH did not change the primary products of the Fenton reaction as indicated by EPR experiments on ^⋅OH-mediated ethanol radical formation. Furthermore, PIH dramatically enhanced the rate of Fe(II) oxidation to Fe(III) in the presence of oxygen, suggesting that PIH decreases the concentration of Fe(II) available for the Fenton reaction. These results suggest that PIH and SIH deserve further investigation as inhibitors of free-radical mediated tissue damage.