Fumonisin B1 In Vitro Partitioning Into Natural Membranes.

MARTIN G. THEUMER, ; HéCTOR R. RUBINSTEIN; MARíA A. PERILLO

Mendoza

Congreso; Strategies to reduce the impact of mycotoxins in Latin America in a global context; 2011

International Society for Mycotoxicology (ISM) and The Latin American Society for Mycotoxicology (SLAM).

Previous studies carried out in our laboratory showed that fumonisin B1 (FB1) can interact with artificial model membranes inducing molecular organizational changes which could be somehow involved in the toxicity of this mycotoxin. Erythrocytes have been used as appropriate cellular models to study the drug?biomembrane interaction. It has already been well established that mature red blood cells undergo one of two types of membrane transformation (exvagination or invagination), and a resultant shape change of the cells under in vitro action of some drugs according to their polarity properties. The purpose of the present work was to investigate whether the ability of FB1 to affect the curvature of model membranes could be exerted in natural membranes and transduced into changes in cellular morphology. Fresh blood samples were obtained from male Wistar rats by cardiac puncture using heparin as anticoagulant. After centrifugation, the upper layer was removed, the erythrocytes were washed and resuspended in PBS (pH=7.4) in order to get a hematocrit of 3-4%. Then the erythrocytes were incubated at 37 °C up to 1 hour with FB1 (1-50 ìM), and then fixed for two hours at 4 °C with a p-formaldehyde solution. Morphological examination of the erythrocytes was carried out with a microscope equipped with differential interference contrast. A biconcave disc shape was given a score of 0. Echinocytes and stomatocytes were given scores of +1 to +4 (exvagination) or -1 to -4 (invagination) according to Fujii et al. (1979). A temporal dependence in the morphological changes in erythrocytes incubated with all the FB1 concentrations assessed was observed. The toxin induced stomatocytes in a dose-dependent manner, and the degree of the morphological alteration of cell shape was directly correlated with the FB1 concentration tested. Packing properties of molecules in bilayers and non-bilayers structures depend on thermodynamic factors coupled to molecular geometry. Steric restrictions are imposed on the interfacial spontaneous curvature derived from the complementary shape of the molecules in a self-assembled structure. This molecular dynamics may be relevant in a wide spectrum of biological phenomena involving curvature changes that participate in many cellular processes mediated by membrane interactions, recombination and signaling (Garcia et al., 2000). These experiments suggest a preferential partitioning of FB1 into the negatively charged internal leaflet of the bilayer which, indirectly, involves the previous permeation of the toxin (predicted as zwitterions at physiological pH) through the membranes. Since the internal leaflet of the natural membranes are negatively charged, mainly due to the contribution of negative aminophospholipids, the probable protonation of the toxin in the cytoplasm could precede its preferential distribution into the cytoplasmic half of the bilayer. Bibliography: Fujii, T., Sato, T., Tamura, A., Wakatsuki, M., and Kanaho, Y. (1979). Shape changes of human erythrocytes induced by various amphipathic drugs acting on the membrane of the intact cells. Biochem Pharmacol 28, 613-620. Garcia, D. A., Quiroga, S., and Perillo, M. A. (2000). Flunitrazepam partitioning into natural membranes increases surface curvature and alters cellular morphology. Chem Biol Interact 129, 263-277.