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.

Effects of DDE on the fluidity of model and native membranes: implications for the mechanisms of toxicity.

2,2-Bis(p-chlorophenyl)-1,1-dichloroethylene (DDE) interaction with model and native membranes was studied by means of fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH), probing the bilayer core, and by intramolecular excimerization of 1,3-di(1-pyrenyl) propane (Py(3)Py), probing the outer regions of the bilayer. In the gel phase of DMPC bilayers, DDE induces concentration-dependent fluidizing effects into the hydrophobic core, but no effects are detected in the outer regions of the membrane, as evaluated by DPH and Py(3)Py, respectively. Regarding the fluid phase, DDE has no apparent effect on the bilayer center, but it induces a limited ordering effect on the outer regions. Similar effects are described for bilayers of DPPC and DSPC. Unlike DPH, Py(3)Py is very sensitive to DPPC and DSPC pretransitions, not abolished by DDE (50 microM), as opposite to the effects observed with lindane (Antunes-Madeira, M.C., Almeida, L.M. and Madeira, V.M.C. (1990) Biochim. Biophys. Acta 1022, 110-114), but similar to those observed with DDT (Antunes-Madeira, M.C., Almeida, L.M. and Madeira, V.M.C. (1991) Pestic. Sci. 33, 347-357). DDE inhibits to some extent the cholesterol-induced ordering in DMPC bilayers and high cholesterol concentrations (> or = 30 mol%) do not prevent DDE interaction, as evaluated by DPH. On the other hand, the effects of DDE reported by Py(3)Py depend on temperature and cholesterol contents of DMPC bilayers. For cholesterol levels ranging from 10 to 50 mol% and temperatures below the phase transition of DMPC, Py(3)Py fails to detect any significant effect. Nevertheless, above the phase transition, Py(3)Py detects either ordering effects of DDE at low cholesterol contents (< 30 mol%) or fluidizing effects at high cholesterol levels (> or = 30 mol%). The results in native membranes correlate reasonably with those obtained in models of synthetic lipids. Thus, DPH does not detect any apparent effect of DDE in relatively fluid native membranes of sarcoplasmic reticulum, but detects moderate disordering effects in membranes of brain microsomes and erythrocytes, i.e., membranes with high cholesterol. On the other hand, Py(3)Py reports ordering effects of DDE in fluid membranes of sarcoplasmic reticulum, an effect similar to that observed in fluid systems of synthetic lipids without or with low cholesterol. Additionally, as described for models, Py(3)Py detects disordering effects of DDE in cholesterol rich membranes, namely, brain microsomes.