Ethylazinphos interaction with membrane lipid organization induces increase of proton permeability and impairment of mitochondrial bioenergetic functions.

Ethylazinphos increases the passive proton permeability of lipid bilayers reconstituted with dipalmitoylphosphatidylcholine (DPPC) and mitochondrial lipids. A sharp increase of proton permeability is detected at insecticide/lipid molar ratios identical to those inducing phase separation in the plane of DPPC bilayers, as revealed by differential scanning calorimetry (DSC). Ethylazinphos progressively depresses the transmembrane potential (DeltaPsi) of mitochondria supported by piruvate/malate, succinate, or ascorbate/TMPD. Additionally, a decreased depolarization induced by ADP depends on ethylazinphos concentration, reflecting a phosphorylation depression. This loss of phosphorylation is a consequence of a decreased DeltaPsi. A decreased respiratory control ratio is also observed, since ethylazinphos stimulates state 4 respiration and inhibits ADP-stimulated respiration (state 3). Ethylazinphos concentrations up to 100 nmol/mg mitochondrial protein increase the rate of state 4 together with a decrease in DeltaPsi, without significant perturbation of state 3 and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP)-uncoupled respiration. For increased insecticide concentrations, the state 3 and FCCP-uncoupled respiration are inhibited to approximately the same extent. The perturbations are more pronounced when the energization is supported by pyruvate/malate and less effective when succinate is used as substrate. The present data, in association with previous DSC studies, indicate that ethylazinphos, at concentrations up to 100 nmol/mg mitochondrial protein, interacts with the lipid bilayer of mitochondrial membrane, changing the lipid organization and increasing the proton permeability of the inner membrane. The increased proton permeability explains the decreased oxidative phosphorylation coupling. Resulting disturbed ATP synthesis may significantly underlie the mechanisms of ethylazinphos toxicity, since most of cell energy in eukaryotes is provided by mitochondria.

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.

Perturbations induced by alpha- and beta-endosulfan in lipid membranes: a DSC and fluorescence polarization study.

The interaction of alpha- and beta-endosulfan isomers with lipid bilayers was searched by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6- and 12-(9-anthroyloxy) stearic acids (2-AS, 6-AS and 12-AS) and 16-(9-anthroyloxy) palmitic acid (16-AP). Both endosulfan isomers, at insecticide/lipid molar ratios ranging from 1/40 to 1/1, shift the phase transition midpoint to lower temperature values and broaden the transition profile of dipalmitoylphosphatidylcholine (DPPC) bilayers. At insecticide/lipid molar ratios of 1/40, the isomers fully abolish the bilayer pretransition. Conversely to beta-endosulfan, alpha-endosulfan promotes a new phase transition, centered at 35.4 degrees C, in addition to the main phase transition of DPPC. Therefore, the alpha-isomer may undergo a heterogeneous distribution in separate domains in the plane of the membrane, whereas the beta-isomer may undergo a homogeneous distribution. Fluorescence polarization data indicate that alpha-endosulfan increases the lipid structural order in the regions probed by 2-AS and decreases it in the regions probed by 6-AS, 12-AS and 16-AP. On the other hand, the beta-isomer produces disordering effects in the upper regions of the bilayers, probed by 2-AS, and ordering in deeper regions, probed by 6-AS, 12-AS and 16-AP, mainly in the gel phase. The incorporation of cholesterol into DPPC bilayers progressively decreases the effects of beta-isomer which are vanished at 20 mol% cholesterol. However, this and higher cholesterol concentrations did not prevent alpha-endosulfan membrane interaction, as revealed by DSC and fluorescence polarization. The distinct effects promoted by alpha- and beta-endosulfan are discussed in terms of molecular orientation and positioning within the bilayer. Apparently, the alpha-isomer preferentially locates closer to the phospholipid headgroups whereas the beta-isomer distributes in deeper domains of the bilayer.

Biophysical perturbations induced by ethylazinphos in lipid membranes.

Perturbations induced by ethylazinphos on the physical organization of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol membranes were studied by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6-, 12-(9-anthroyloxy) stearic acids and 16-(9-anthroyloxy) palmitic acid. Ethylazinphos (50 and 100 microM) increases the fluorescence polarization of the probes, either in the gel or in the fluid phase of DPPC bilayers, and this concentration dependent effect decreases from the surface to the bilayer core. Additionally, the insecticide displaces the phase transition to a lower temperature range and broadens the transition profile of DPPC. A shifting and broadening of the phase transition is also observed by DSC. Furthermore at insecticide/lipid molar ratios higher than 1/7, DSC thermograms, in addition to the normal transition centered at 41 degrees C, also display a new phase transition centered at 45.5 degrees C. The enthalpy of this new transition increases with insecticide concentration, with a corresponding decrease of the main transition enthalpy. Ethylazinphos in DPPC bilayers with low cholesterol (< or = 20 mol%) perturbs the membrane organization as described above for pure DPPC. However, cholesterol concentrations higher than 20 mol% prevent insecticide interaction, as revealed by fluorescence polarization and DSC data. Apparently, cholesterol significantly modulates insecticide interaction by competition for similar distribution domains in the membrane. The present results strongly support our previous hypothesis that ethylazinphos locates in the cooperativity region, i.e. the region of C1-C9 atoms of the acyl chains, and extends to the lipid-water interface, where it increases lipid packing order sensed across all the thickness of the bilayer. Additionally, and, on the basis of DSC data, a lateral regionalization of ethylazinphos is here tentatively suggested.

Interaction of ethylazinphos with the physical organization of model and native membranes.

The interaction of ethylazinphos with the physical organization of model and native membranes was investigated by means of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its propionic acid derivative (DPH-PA). Ethylazinphos shifts the phase transition midpoint to lower temperature values and broadens the phase transition profile of bilayers reconstituted with dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC, DSPC), as detected by DPH and DPH-PA. Additionally, both probes detect significant effects of ethylazinphos in the fluid phase of the above lipid bilayers. The insecticide perturbations are more pronounced in bilayers of short-chain lipids, e.g., DMPC, in correlation with the higher partition in these membranes. On the other hand, the insecticide increases to some extent the ordering promoted by cholesterol in the fluid phase of DMPC, but high cholesterol concentrations (> or = 30 mol%) almost prevent insecticide interaction, as revealed by DPH and DPH-PA. In agreement with the results in models of synthetic lipids, the increase of intrinsic cholesterol in fluid native membranes depresses the partition values of ethylazinphos and consequently its effects.

Partition of DDE in synthetic and native membranes determined by ultraviolet derivative spectroscopy.

Partition coefficients of DDE (2,2-bis(p-chlorophenyl)-1,1-dichloroethylene) were determined, in model and native membranes, as a function of temperature, lipid chain length, cholesterol content and DDE concentration, by means of second derivative ultraviolet spectrophotometry. DDE incorporation increases with the temperature, since the partition values in dimyristoylphosphatidylcholine (DMPC), at 24, 30 and 37 degrees C, are 5722 +/- 138, 10356 +/- 763 and 14006 +/- 740, respectively. The insecticide incorporates better into bilayers of DMPC as compared with DPPC (dipalmitoylphosphatidylcholine). The partition decreases from 10355 +/- 763 in DMPC to 6432 +/- 613 in DPPC, at temperatures 5-7 degrees C above the midpoint of their transitions. The addition of cholesterol to fluid membranes of DMPC depresses the partition of DDE. In agreement with the results in models of synthetic lipids, the partition of DDE into native membranes increases with the temperature and decreases with the intrinsic cholesterol. It is concluded that a fluid membrane favors the accumulation of DDE.

Amiodarone effects on membrane organization evaluated by fluorescence polarization.

The effects of amiodarone (0-100 microM) on the physical state of synthetic and native membranes were investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and of its anionic propionic acid derivative (DPH-PA), probing the outer regions of the bilayer. In the gel phase of dimyristoylphosphatidylcholine (DMPC) bilayers, amiodarone broadens the transition profile and shifts the phase transition midpoint to lower temperature values, as evaluated by both probes. On the other hand, the drug orders the fluid phase of the lipid either in hydrophobic core or in the outer regions of the bilayer, as detected by DPH and DPH-PA, respectively. The effects of amiodarone on the thermotropic behaviour of DPPC confirm and extend data in DMPC. Cholesterol concentration modulates to a great extent the effects of amiodarone in the fluid phase of DMPC. Thus, both probes, DPH and DPH-PA, detect either ordering effects of amiodarone for low cholesterol concentrations (< or = 20 mol%) or disordering amiodarone effects at higher cholesterol levels (> 20 mol%). In agreement with the results in models of synthetic lipids, the ordering effects of amiodarone in fluid native membranes of mitochondria and brain microsomes are depressed with the increase in intrinsic cholesterol. The ordering effects in mitochondria may induce bioenergetic dysfunctions and consequently disturbances in the electromechanic functioning of myocardium.

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.