Dimyristoylphosphatidylcholine Large Unilamellar Vesicles Research Articles

Antioxidant capacity of Ugni molinae fruit extract on human erythrocytes: an in vitro study.

Ugni molinae is an important source of molecules with strong antioxidant activity widely used as a medicinal plant in Southern Chile-Argentina. Total phenol concentration from its fruit extract was 10.64 ± 0.04 mM gallic acid equivalents. Analysis by means of HPLC/MS indicated the presence of the anthocyanins cyanidin and peonidin, and the flavonol quercitin, all in glycosylated forms. Its antioxidant properties were assessed in human erythrocytes in vitro exposed to HClO oxidative stress. Scanning electron microscopy showed that HClO induced an alteration in erythrocytes from a normal shape to echinocytes; however, this change was highly attenuated in samples containing U. molinae extracts. It also had a tendency in order to reduce the hemolytic effect of HClO. In addition, X-ray diffraction experiments were performed in dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine bilayers, classes of lipids preferentially located in the outer and inner monolayers, respectively, of the human erythrocyte membrane. It was observed that U. molinae only interacted with DMPC. Results by fluorescence spectroscopy on DMPC large unilamellar vesicles and isolated unsealed human erythrocyte membranes also showed that it interacted with the erythrocyte membrane and DMPC. It is possible that the location of U. molinae components into the membrane outer monolayer might hinder the diffusion of HClO and of free radicals into cell membranes and the consequent decrease of the kinetics of free radical reactions.

Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 μM; (c) X-ray diffraction studies showed that PPA in the 0.1–0.5 mM range induced increasing structural perturbation to DMPC, but no effects on DMPE multibilayers were detected.

Phospholipid Bilayers as Molecular Models for Drug‐Cell Membrana Interactions.

Since cell membranes are highly complex entities, there has been extensive interest in employing simple models to mimic biological cell membranes. Phospholipids are amphipatic molecules with long hydrophobic acyl chains and zwitterionic polar heads which are assembled into different types of molecular aggregates. The most relevant is the bilayer because of its relation with cell membranes. Among a large variety of relevant biological functions, the cell membrane acts as a diffusion barrier and protects the cell interior. Both structure and functions of the cell membrane are susceptible to alterations as a consequence of interactions with extrinsic chemical species such as therapeutic drugs. On the other hand, pharmacologic activity of a drug frequently involves an intial interaction with cell membranes even if they are not their final targets. With the aim to better understand the molecular mechanisms of the interaction of therapeutic drugs with cell membranes we utilize human erythrocyte membranes and molecular models of red blood cell membranes. Human erythrocytes are an ideal cell system for studying basic drug-membrane interactions, because they are composed of only one membrane and have no internal organelles. On the other hand, although less specialized than many other cell membranes, they carry on enough functions in common with them such as active and passive transport, and the production of ionic and electric gradients, to be considered representative of the plasma membrane in general. Molecular models of cell membranes consist of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, representative of phospholipid classes located in the outer and inner monolayers of cell membranes, particularly of the human erythrocyte, respectively. The capacity of drugs to interact with membrane bilayers is currently evaluated in our labs by X-ray diffraction on DMPC and DMPE multilayers; DMPC large unilamellar vesicles (LUV) and isolated unsealed human erythrocyte membranes (IUM) are studied by fluorescence spectroscopy, and intact human erythrocytes are observed by scanning electron microscopy (SEM). We have been using these systems and techniques in order to determine the interaction with and the membraneperturbing effects of different compounds of therapeutic interest. As an example of our research, results obtained with the nonsteroidal anti-inflammatory drug diclofenac, the calcium channel blocker verapamil and the neuroleptic chlorpromazine will be presented. Acknowledgements FONDECYT (grant 1090041). L4.2

Structural effects of Zn2+ on cell membranes and molecular models

Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, intact human erythrocytes were observed with scanning electron microscopy (SEM), and neuroblastoma cell morphology was observed under inverted microscope. This study presents evidence that 0.1mM Zn and higher concentrations affect cell membrane and molecular models.

Human erythrocytes are affected in vitro by flavonoids of Aristotelia chilensis (Maqui) leaves

Aristotelia chilensis (Mol.) Stuntz ( A. chilensis), also known as maqui, is a plant of the Elaeocarpaceae family that grows in central and southern Chile as well as southwestern Argentina. Infusions of its leaves have long been used in the traditional native herbal medicine to treat different ailments. Phytochemical studies of the plant's chemical composition of the plant indicate the presence of indolic alkaloids, flavonoids, cianidine glucosides, delfidine, malvidine, petunidine, cumarines and triterpenes . These compounds, particularly the flavonoids, have antioxidant properties. In order to evaluate the mechanisms of its toxicity and their antioxidant properties, the leaves’ aqueous extracts were induced to interact with human red cells, their isolated unsealed membranes (IUM), and molecular models of the human erythrocyte membrane. These consisted of multibilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, and large unilamellar vesicles (LUV) of DMPC. The capacity of A. chilensis aqueous extracts to perturb the bilayer structure of DMPC and DMPE was evaluated by X-ray diffraction, DMPC LUV and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). Results of the present study indicate that aqueous extracts of A. chilensis induced an alteration of human erythrocyte morphology from the normal discoid shape to an echinocytic form, changes that are explained in terms of the extract interaction with the membrane's outer phospholipid monolayer.

Cr(III) exerts stronger structural effects than Cr(VI) on the human erythrocyte membrane and molecular models

Chromium exists in many oxidation states, of which only the hexavalent Cr(VI) and the trivalent Cr(III) ions are stable under environmental conditions. It is generally reported that Cr(VI) is highly toxic while Cr(III) is relatively innocuous, although others have reported just the opposite. On the other hand, despite the many studies on chromium toxicity, and particularly after the knowledge that Cr(VI) anions readily enter the erythrocytes where they are reduced to Cr(III), there are practically no reports on the structural effects induced by chromium compounds on the erythrocyte membrane. With the aim to better understand the molecular mechanisms of the interaction of Cr(III) and Cr(VI) with cell membranes, CrCl3, and K2CrO4 were incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of the erythrocyte membrane. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylcholine (DMPE), phospholipid classes present in the outer and inner monolayers of the erythrocyte membrane, respectively. The capacity of Cr(III) and Cr(VI) to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed with scanning electron microscopy (SEM). In all these systems, it was found that Cr(III) induced considerably higher structural perturbations than Cr(VI).

Monomethylarsonate (MMA v) exerts stronger effects than arsenate on the structure and thermotropic properties of phospholipids bilayers

Methylation of inorganic arsenic has been regarded as a detoxification mechanism because its metabolites monomethylarsonic acid (MMA v) and dimethylarsinic acid (DMA v) are supposed to be less toxic than inorganic arsenite and arsenate. In recent years, however, this interpretation has been questioned. Additionally, there are insufficient reports concerning the effects of arsenic compounds on cell membrane structure and functions. With the aim to better understand the molecular mechanisms of the interaction of MMA v and arsenate with cell membranes, we have utilized molecular models consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of many cell membranes including that of the human erythrocyte. The capacity of MMA v and arsenate to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction; the modifications of their thermotropic behavior were followed by differential scanning calorimetry (DSC), while DMPC large unilamellar vesicles (LUV) were studied by fluorescence spectroscopy. It was found that MMA v and arsenate did not structurally perturb DMPC bilayers; however, DMPE bilayers did suffer structural perturbations by MMA v. DSC measurements also revealed that DMPE's thermotropic properties were significantly affected by arsenicals, where MMA v was more effective than arsenate, whilst only slight modifications were observed in the case of DMPC-MMA v system.

Effects of lithium on the human erythrocyte membrane and molecular models

The mechanism whereby lithium carbonate controls manic episodes and possibly influences affective disorders is not yet known. There is evidence, however, that lithium alters sodium transport and may interfere with ion exchange mechanisms and nerve conduction. For these reasons it was thought of interest to study its perturbing effects upon membrane structures. The effects of lithium carbonate (Li +) on the human erythrocyte membrane and molecular models have been investigated. The molecular models consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. This report presents the following evidence that Li + interacts with cell membranes: a) X-ray diffraction indicated that Li + induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC and DMPE; b) experiments performed on DMPC large unilamellar vesicles (LUV) by fluorescence spectroscopy also showed that Li + interacted with the lipid polar groups and hydrophobic acyl chains, and c) in scanning electron microscopy (SEM) studies on intact human erythrocytes the formation of echinocytes was observed, effect that might be due to the insertion of Li + in the outer monolayer of the red cell membrane.

Large Disk Intermediate Precedes Formation of Apolipoprotein A-I−Dimyristoylphosphatidylcholine Small Disks

Small approximately 8.5 nm disks formed spontaneously when dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUVs) were incubated with apolipoprotein A-I (apoA-I) (100:1 molar ratio). However, in a time course study, the transient production of approximately 11 nm large disks was detected and isolated by gel filtration. The intermediate large disks contained three apoA-I molecules and were stable over time; however, when additional apoA-I was added, they formed small disks containing two molecules of apoA-I. The reaction kinetics of apoA-I with DMPC LUVs was monitored by fluorescence resonance energy transfer, and two phases were observed, supporting the presence of the intermediate in the formation of small disks. The lipid dynamics of LUVs and disks were assayed, revealing the presence of sequestered lipid-protein domains upon apoA-I binding to DMPC LUVs. In addition, the lipids in the intermediate large disks were more constrained than those in the small disks. We propose that apoA-I binds with DMPC LUVs to form small lipid-protein domains on the LUV; then the domains are released to form large disks, which can mature in the presence of additional apoA-I to form small disks. Thus, the formation of small apoA-I lipid disks proceeds through the formation of a large disk intermediate.

Human erythrocytes are affected by the organochloride insecticide chlordane

Chlordane is a widely used organochlorine insecticide. In order to evaluate its perturbing effect upon the morphology of human erythrocytes it was caused to interact with human red cells and molecular models of cell membranes. These consisted in bilayers of dimyristoylphosphatidylethanolamine (DMPE) and of dimyristoylphosphatidylcholine (DMPC), representative of phospholipid classes located in the inner and outer monolayers of the erythrocyte membrane, respectively. Scanning electron microscopy (SEM) observations indicated that this pesticide induced a significant alteration in the shape of the erythrocytes as they changed their discoid shape to spherocytes. According to the bilayer couple hypothesis, the shape changes induced in erythrocytes by foreign molecules are due to differential expansion of their two monolayers. The fact that chlordane produced spherocytes would indicate that the pesticide was equally located in the outer and the inner moieties of the red cell membrane. This conclusion was supported by the results obtained from X-ray diffraction studies. These showed that the hydrophobic and polar head regions of DMPC bilayers were perturbed when the insecticide was in a 1:10 molar ratio with respect to the lipid. These results were confirmed by the fluorescence experiments performed in DMPC large unilamellar vesicles (LUV). Chlordane produced a sharp decrease in the anisotropy and general polarization parameters in the 0–0.1 mM range, implying an increase in the fluidity at the acyl chain and polar region of DMPC. On the other hand, the bilayer structure of DMPE was perturbed in a fashion similar to that observed by X-ray diffraction in DMPC, a fact that explains the morphological change induced by chlordane to the human erythrocytes.

Structural effects of titanium citrate on the human erythrocyte membrane

The structural effects of titanium citrate on the human erythrocyte membrane were studied through its interaction with intact erythrocytes and isolated unsealed human erythrocyte membranes (IUM). The studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Titanium citrate induced shape changes in erythrocytes, which were damaged and ruptured leaving empty and retracted membranes. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar head group and the acyl chain packing arrangements of the membrane phospholipid bilayer. Titanium citrate also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that titanium citrate induced structural perturbation of the polar head group and of the hydrophobic acyl regions of DMPC, while the effects on DMPE bilayers were negligible. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles. All these findings indicate that the structural perturbations induced by titanium to human erythrocytes can be extended to other cells, thereby affecting their functions.

Cadmium-induced changes in the membrane of human erythrocytes and molecular models

The structural effects of cadmium on cell membranes were studied through the interaction of Cd 2+ ions with human erythrocytes and their isolated unsealed membranes (IUM). Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Cd 2+ induced shape changes in erythrocytes, which took the form of echinocytes. According to the bilayer couple hypothesis, this result meant that Cd 2+ ions located in the outer monolayer of the erythrocyte membrane. Fluorescence spectroscopy measurements in IUM indicated a disordering effect at both the polar headgroup and the acyl chain packing arrangements of the membrane phospholipid bilayer. Cd 2+ ions also interacted with molecular models of the erythrocyte membrane consisting in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing classes of phospholipids located in the outer and inner monolayers the erythrocyte membrane, respectively. X-ray diffraction indicated that Cd 2+ ions induced structural perturbation of the polar headgroup and of the hydrophobic acyl regions of DMPC, while the effects of cadmium on DMPE bilayers were much milder. This conclusion is supported by fluorescence spectroscopy measurements on DMPC large unilamellar vesicles (LUV). All these findings point to the important role of phospholipid bilayers in the interaction of cadmium on cell membranes.

Aluminum fluoride affects the structure and functions of cell membranes

No useful biological function for aluminum has been found. To the contrary, it might play an important role in several pathologies, which could be related to its interactions with cell membranes. On the other hand, fluoride is a normal component of body fluids, soft tissues, bones and teeth. Its sodium salt is frequently added to drinking water to prevent dental caries. However, large doses cause severe pathological alterations. In view of the toxicity of Al 3+ and F − ions, it was thought of interest to explore the damaging effects that AlF 3 might induce in cell membranes. With this aim, it was incubated with human erythrocytes, which were examined by phase contrast and scanning electron microscopy, and molecular models of biomembranes. The latter consisted of large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and bilayers of DMPC and dimyristoylphosphatidylethanolamine (DMPE) which were studied by fluorescence spectroscopy and X-ray diffraction, respectively. In order to understand the effects of AlF 3 on ion transport (principally sodium and chloride) we used the isolated toad skin to which electrophysiological measurements were applied. It was found that AlF 3 altered the shape of erythrocytes inducing the formation of echinocytes. This effect was explained by X-ray diffraction which revealed that AlF 3 perturbed the structure of DMPC, class of lipids located in the outer monolayer of the erythrocyte membrane. This result was confirmed by fluorescence spectroscopy on DMPC LUV. The biphasic (stimulatory followed by inhibitory) effects on the isolated skin suggested changes in apical Cl − secretion and moderate ATPase inactivation.

Effects of lead on the human erythrocyte membrane and molecular models

Lead has no biological function; however, low, and particularly, high levels of exposure have a number of negative consequences for human health. Despite the number of reports about lead toxicity, very little information has been obtained regarding its effects on cell membranes. For this reason, the structural effects of lead on the human erythrocyte membranes were investigated. This aim was attained by making lead ions interact with intact erythrocytes, isolated unsealed erythrocyte membranes (IUM) and molecular models. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representing phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane. The results, obtained by electron microscopy, fluorescence spectroscopy and X-ray diffraction, indicated that (a) lead particles adhered to the external and internal surfaces of the human erythrocyte membrane; (b) lead ions disturbed the lamellar organization of IUM and DMPC large unilamellar vesicles (LUV) and (c) induced considerable molecular disorder in both lipid multilayers, the effects being much more pronounced in DMPC.

The Local Anesthetic Proparacaine Modifies Sodium Transport in Toad Skin and Perturbs the Structures of Model and Cell Membranes

Experimental results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of proparacaine to isolated toad skin, which may reflect an inhibition of the active transport of ions. This finding was explained on the basis of the results obtained from membrane models incubated with proparacaine. These consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), phospholipid multilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively, and in large unilamellar vesicles (LUV) of DMPC X-ray diffraction showed that proparacaine interaction with DMPC and DMPE bilayers perturbed both structures, especially DMPC. This result, confirmed by fluorescence spectroscopy of DMPC LUV at 18 degrees C, demonstrated that the local anesthetic (LA) could interact with the lipid moiety of cell membranes. However, effects observed by scanning electron microscopy (SEM) of human erythrocytes and by fluorescence spectroscopy of IUM might also imply proparacaine-protein interactions. Thus, the LA may alter epitheial sodium channels through interaction with the lipid matrix and with channel protein residues.

Structural Effects of the Local Anesthetic Bupivacaine Hydrochloride on the Human Erythrocyte Membrane and Molecular Models

ABSTRACT The interaction of the local anesthetic bupivacaine with the human erythrocyte membrane and molecular models is described. The latter consisted of isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC), and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representatives of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Optical and scanning electron microscopy revealed that bupivacaine induced erythrocyte spheroechinocytosis. According to the bilayer couple hypothesis, this result implied that bupivacaine inserted in the outer monolayer of the erythrocyte membrane. Experiments performed on IUM and DMPC LUV by fluorescence spectroscopy and X-ray diffraction on DMPC and DMPE multilayers confirmed this result. Changes in the molecular organization of membranes alter lipid–protein interactions and induce functional perturbation of membrane proteins such as Na + channels. Since local anesthetics may control the influx of Na + into the human erythrocyte, in order to relate the structural perturbations induced by bupivacaine in these systems to Na + transport, the interaction of this anesthetic with isolated toad skin was also studied. Electrophysiological measurements indicated a significant decrease in the potential difference and in the short-circuit current of the skin after the application of the anesthetic, reflecting inhibition of the active transport of ions. These results suggest that bupivacaine-induced conformational changes of the lipid molecules alter the lipid–protein boundaries of the outer moiety of the erythrocyte membrane, thus interfering with the function of neighboring sodium channels.

Dibucaine-induced modification of sodium transport in toad skin and of model membrane structures.

The interaction of the local anesthetic dibucaine with the isolated toad skin and membrane models is described. The latter consisted of human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), large unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results indicate a significant decrease in the potential difference (PD) and in the short-circuit current (Isc) after the application of dibucaine in toad skin, which may be interpreted as reflecting inhibition of the active transport of ions. This finding might be explained on the basis of the results obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, dibucaine induced structural perturbations in IUM, DMPC LUV and phospholipid multilayers. Scanning electron microscopy revealed that dibucaine induced erythrocyte stomatocytosis. According to the bilayer couple hypothesis an echinocytic type of shape change would have been expected given the preferential interaction of dibucaine with DMPC. Although it is still premature to define the molecular mechanism of action of dibucaine, the experimental results confirm the important role played by the phospholipid bilayers in the association of the anesthetic with cell membranes.

The Organophosphorus Insecticide Parathion Changes Properties of Natural and Model Membranes

Abstract The interaction of parathion, a widely used organophosphorus insecticide, with cell membranes of the isolated toad skin, human erythrocytes, and membrane molecular models is described. The latter consisted of multilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and large unilamellar vesicles (LUV) of DMPC. They represent phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. Results obtained from electrophysiological measurements of the toad skin supported the deduction that the main site with which parathion interacted to reduce ion transport is situated on the outward facing barrier of the epithelium. Fluorescence spectroscopy showed that the insecticide interacted with and altered the structure of isolated unsealed human erythrocyte membranes. The increase of the fluorophor DPH anisotropy and of laurdan general polarization indicated an ordering effect on the acyl chains and a decrease in the molecular dynamics and/or water penetration at the lipid polar head group region. However, in DMPC LUV parathion at increasing concentrations enhanced the polar head group ordering but decreased that of their acyl chains. X-ray analysis of DMPC and DMPE multilayers showed that both structures were deeply perturbed by a parathion:lipid ratio as low as 1:200. Nonetheless, increasing parathion concentrations induced different results in both lipid bilayers.

HgCl 2 disrupts the structure of the human erythrocyte membrane and model phospholipid bilayers

The structural effects of Hg(II) ions on the erythrocyte membrane were studied through the interactions of HgCl 2 with human erythrocytes and their isolated resealed membranes. Studies were carried out by scanning electron microscopy and fluorescence spectroscopy, respectively. Hg(II) induced shape changes in erythrocytes, which took the form of echinocytes and stomatocytes. This finding means that Hg(II) locates in both the outer and inner monolayers of the erythrocyte membrane. Fluorescence spectroscopy results indicate strong interactions of Hg(II) ions with phospholipid amino groups, which also affected the packing of the lipid acyl chains at the deep hydrophobic core of the membrane. HgCl 2 also interacted with bilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction indicated that Hg(II) ions induced molecular disorder to both phospholipid bilayers, while fluorescence spectroscopy of dimyristoylphosphatidylcholine large unilamellar vesicles confirmed the interaction of Hg(II) ions with the lipid polar head groups. All these findings point to the important role of the phospholipid bilayers in the interaction of Hg(II) on cell membranes.

Toxic effects of the fungicide benomyl on cell membranes

This paper examines the toxicity of the fungicide benomyl towards cell membranes. Approaches to this aim were the study of its acute effects on the stimulatory response of a frog neuroepithelial synapse and on membrane models. The latter consisted of large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE). Results showed that benomyl at concentrations as low as 10 μM decreased the stimulatory response of the potential difference (PD) and the short-circuit current (SCC) of the frog sympathetic junction. It is concluded that benomyl caused a dose-dependent reduction in the response of a sympathetic junction of the frog to stimulation leading to Cl − channel perturbation. This finding might be explained from those obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, similar (0.01–1.0 mM) concentrations induced structural perturbations in DMPC large unilamellar vesicles and multilayers, respectively. Although it is still premature to define the precise molecular mechanism of benomyl toxicity, the experimental results confirm the important role played by the phospholipid bilayers in the interaction of the pesticide with cell membranes.