Vítor M.C. Madeira

Reactivity of dietary phenolic acids with peroxyl radicals: Antioxidant activity upon low density lipoprotein peroxidation

The interaction of four phenolic acids, representative of three chemical groups present in human diet, with peroxyl radicals was studied in vitro in a low density lipoprotein (LDL) oxidation model. The controlled oxidation of LDL was initiated by free radicals generated from a hydrophilic azo initiator and followed by monitoring the oxygen consumption and the fluorescence quenching of cis-parinaric acid previously incorporated into LDL. The hydroxycinnamic acid derivatives, chlorogenic and caffeic acids, have high stoichiometric numbers and reactivity with peroxyl radicals as compared with trolox, the water-soluble analogue of vitamin E, whereas ellagic acid (a tannic compound) compares with trolox effects. Protocatechuic acid (a hydroxybenzoic acid derivative) exhibits a complex reaction with peroxyl radicals, as indicated by UV spectroscopy, resulting in undefined inhibition periods of LDL oxidation and low reactivity with peroxyl radicals. Presumably, secondary radicals of these compounds are unable to initiate LDL oxidation. The antioxidant activity of the various phenolic compounds is discussed in terms of structure-activity relationships.

Interaction of insecticides with lipid membranes.

The permeability of liposome membranes is increased by organophosphorus and organochlorinated insecticides at concentrations of 10(-5)--10(-4) M. The order of effectiveness is similar to the toxicity of the compounds to mammals, and is the following for permeation of non-electrolytes and for valinomycin-induced permeation of K+: parathion greater than 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) approximately aldrin greater than malathion greater than lindane. The degree of effectiveness for X-537A-induced permeation of Ca2+ was the following: aldrin greater than or equal to DDT greater than parathion greater than malathion greater than lindane. The organophosphorus compound, ethyl azinphos (10(-4) M), dramatically increases the permeability of liposome membranes to all the tested substances, probably as a consequence of surfactant effects. Some organochlorinated insecticides appear to react with cation ionophores and modulate their motion across lipid membranes. It is suggested that the insecticides may exert some of their toxic actions by modifying certain mechanisms in the cell membrane.

Membrane fluidity as affected by the insecticide lindane

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to study the interaction of lindane with model and native membranes. Lindane disorders the gel phase of liposomes reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC), since it broadens and shifts the main phase transition, but no apparent effect is detected in the fluid phase. These effects of lindane are more pronounced in bilayers of short-chain lipids, e.g., DMPC. In equimolar mixtures containing DMPC and DSPC, lindane preferentially interacts with the more fluid lipid species inducing lateral phase separations. However, in mixtures of DMPC and DPPC, the insecticide only broadens and shifts the main phase transition, i.e., an effect similar to that observed in bilayers of pure lipids. Lindane has no apparent effect in DMPC bilayers enriched with high cholesterol content (greater than or equal to 30 mol%), whereas disordering effects can still be detected in bilayers with low cholesterol (less than 30 mol%). Apparently, lindane does not perturb the fluid phase of representative native membranes, namely, mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes in agreement with the results obtained in fluid phospholipid bilayers, despite the reasonable incorporation of the insecticide in these membranes, as previously reported (Antunes-Madeira, M.C. and Madeira, V.M.C. (1985) Biochim. Biophys. Acta 820, 165-172).

Activation energies of the ATPase activity of sarcoplasmic reticulum

Summary Arrhenius plots of Ca ++ -stimulated ATP hydrolysis by sarcoplasmic reticulum (SR) show breaks (T t ) at 16.7°C and 11.5°C for rabbit and lobster preparations, respectively. The energies of activation (Eact) are about 10 and 19.5 Kcal/mole above and below T t , respectively, and are similar for both lobster and rabbit SR. The antibiotic filipin increases T t by about 7°C for both preparations, but the Eact above and below the new T t values remain similar to those of the controls. Desintegrated membranes do not show breaks in Arrhenius plots and the Eact assume relatively high values. The Ca ++ ionophore X-537A does not affect either the T t values nor the Eact.

The anticancer drug tamoxifen induces changes in the physical properties of model and native membranes

The interactions of tamoxifen with lipid bilayers of model and native membranes were investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py(3)Py). The effects of TAM of liposomes of DMPC, DPPC and DSPC are temperature dependent. In the fluid phase, TAM reduces dynamics of the upper bilayer region as observed by Py(3)Py and has no effect on the hydrophobic region as detected by DPH. In the gel phase, the effects of TAM evaluated by Py(3)Py are not discernible for DMPC and DPPC bilayers, whereas DSPC bilayers become more fluid. However, DPH detects a strong fluidizing effect of TAM in the hydrophobic region of the above membrane systems, where DPH distributes, as compared with the small effects detected by Py(3)Py. TAM decreases the main phase transition temperature but does not extensively broaden the transition thermotropic profile of pure lipids, except for bilayers of DMPC where TAM induces a significant broadening detected with the two probes. In fluid liposomes of sarcoplasmic reticulum lipids and native membranes, TAM induces an ordering effect, as evidenced by Py(3)Py, failing DPH to detect any apparent effect as observed for the fluid phase of liposomes of pure lipid bilayers. These findings confirm the hydrophobic nature of tamoxifen and suggest that the localization and effects of TAM are modulated by the order and fluidity of the bilayer. These changes in the dynamic properties of lipids and the non-specific interactions with membrane lipids, depending on the order or fluidity of the biomembrane, may be important for the multiple cellular effects and action mechanisms of tamoxifen.

Partition of lindane in synthetic and native membranes

Partition coefficients of the insecticide gamma-1,2,3,4,5,6-hexachlorocyclohexane (trivially, lindane) were determined in model and native membranes. Partition in egg phosphatidylcholine bilayers decreases linearly with temperature, over a range (10-40 degrees C) at which the lipid is in the liquid-crystalline state. Addition of 50 mol% cholesterol dramatically decreases partition (2100 falls to 100, at 10 degrees C) and abolishes the temperature dependence. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) are accompanied by a sharp increase in lindane partition. Apparently, the insecticide is easily accommodated in bilayers of short-aliphatic-chain lipids, since the partitions were 2450, 600 and 50 in DMPC, DPPC and DSPC, respectively, at temperatures 10 Cdeg below the midpoint of their transitions. The lindane partition sequence in native membranes is as follows: mitochondria, sarcoplasmic reticulum, myelin, brain microsomes and erythrocytes. This sequence correlates reasonably well with the relative content of cholesterol and is similar in liposomes of total extracted lipids, although the absolute partitions showed decreased values. Therefore, the presence of proteins in native membranes contributes to the insecticide partition, probably by favouring its interaction with lipids.

Changes induced by malathion, methylparathion and parathion on membrane lipid physicochemical properties correlate with their toxicity.

Perturbations induced by malathion, methylparathion and parathion on the physicochemical properties of dipalmitoylphosphatidylcholine (DPPC) were studied by fluorescence anisotropy of DPH and DPH-PA and by differential scanning calorimetry (DSC). Methylparathion and parathion (50 microM) increased the fluorescence anisotropy evaluated by DPH-PA and DPH, either in gel or in the fluid phase of DPPC bilayers, but mainly in the fluid phase. Parathion is more effective than methylparathion. On the other hand, malathion had almost no effect. All the three xenobiotics displaced the phase transition midpoint to lower temperature values and broadened the phase transition profile of DPPC, the effectiveness following the sequence: parathion>methylparathion>>malathion. A shifting and broadening of the phase transition was also observed by DSC. Furthermore, at methylparathion/lipid molar ratio of 1/2 and at parathion/lipid molar ratio of 1/7, the DSC thermograms displayed a shoulder in the main peak, in the low temperature side, suggesting coexistence of phases. For higher ratios, the phase transition profile becomes sharp as the control transition, but the midpoint is shifted to the previous shoulder position. Conversely to methylparathion and parathion, malathion did not promote phase separation. The overall data from fluorescence anisotropy and calorimetry indicate that the degree of effect of the insecticides on the physicochemical membrane properties correlates with toxicity to mammals. Therefore, the in vivo effects of organophosphorus compounds may be in part related with their ability to perturb the phospholipid bilayer structure, whose integrity is essential for normal cell function.

A rapid and ultrasensitive method to measure Ca++ movements across biological membranes

An electrometric system was used to measure Ca++ uptake by sarcoplasmic reticulum vesicles (SR). The method permits continuous recording of Ca++ uptake and thus the valuation of kinetic parameters. Furthermore, the ultrasensitivity of the method permits to follow changes in Ca++ concentration below 10−6 M.

Effects of parathion on membrane organization and its implications for the mechanisms of toxicity.

The effects of the organophosphorus insecticide parathion (O,O-diethyl O-(p-nitrophenyl)phosphorothioate) on the physical state of synthetic and native membranes was investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and by its anionic propionic acid derivative (DPH-PA), probing the outer regions of the bilayer. Parathion disorders the gel phase of liposomes reconstituted with dimyristoylphosphatidylcholine (DMPC), broadening the transition profile and shifting the temperature midpoint of the phase transition, as detected by both probes. The insecticide strongly orders the fluid phase either in the hydrophobic core or in the outer regions of the membrane, as evaluated by DPH and DPH-PA, respectively. These ordering effects of parathion were further confirmed in fluid models of egg-yolk phosphatidylcholine. Parathion increases to some extent the ordering promoted by cholesterol in DMPC bilayers, but high cholesterol concentrations (> or = 30 mol%) prevent parathion interaction. The results in native membranes correlate reasonably with those obtained in models of synthetic lipids. Thus, parathion does not exert detectable effects in cholesterol-rich membranes, namely, erythrocytes, but moderate ordering effects of parathion are detected by both probes in brain microsomes, i.e., membranes with a lower content of cholesterol. Again, in agreement with the models of synthetic lipids, pronounced ordering effects of parathion are detected in cholesterol-poor membranes, e.g., sarcoplasmic reticulum and mitochondria.

Interaction of Ca2+ and Mg2+ with synaptic plasma membranes

Abstract 1. 1. Synaptosomal fractions were isolated from sheep brain by sucrose density gradient centrifugation and were characterized by their succinate dehydrogenase and acetylholinesterase activities. Five different fractions were collected. Succinate dehydrogenase activity increases from the top to the bottom of the gradients,while the acetylcholinesterase activity is maximal for the top and minimal for the bottom fractions. 2. 2. Synaptic membranes bind Ca2+,Mg2+ and other cations. We found two Ca2+ and Mg2+ binding components. The maximum Ca2+ binding capacities for each component are 115 and 90 nmoles/mg of protein, while for Mg2+ the corresponding values are 80 and 90 nmoles/mg protein. The affinity constants are 0.87 and 74 μM for Ca2+, and 0.61 and 49 μM for Mg2+. 3. 3. Chlorpromazine at concentrations of 0.075 and 0.5 mM decreases the binding of Ca2+ by about 50 and 80%, respectively. Tetracaine has a much smaller effect; thus, at a concentration of 1 mM it displaces only 30% of the total Ca2+ bound. Procaine and the alkaloid caffeine were without effect on the Ca2+ bound. Acetylcholine displaces only 20% of the total Ca2+ bound when present at relatively high concentrations (5 mM). 4. 4. Divalent cations increase the fluorescence of 8-anilinonaphthalene-1-sulfonic acid in aqueous suspensions of synaptic membranes. The order of efficiency of the cations is Zn2+ ≈Cd2+ > Ca2+ ≈Sr2+ >Mg2+.