Interaction of aflatoxin B1 with model lipid biomembranes.

THEUMER, MG; RUBINSTEIN HR; PERILLO, MA

M¨¦rida

Congreso; VI Latinamerican Congress of Mycotoxins, II International Symposium on Fungal and Algal Toxins in Industry.; 2010

Sociedad Latinoamericana de Micotoxicolog¨ªa.

Background: Most of the mycotoxicoses in human beings and in animals arise after consumption of diets contaminated with any or several fungal metabolites.  Aflatoxin B1 (AFB1) absorption from the gastrointestinal system results in its immediate transport to the liver, which may contribute to aflatoxin hepatotoxicity. The lipophilicity of this mycotoxin is crucial in the determination of its uptake into living cells, including hepathocytes, to then exert its toxic effects. Non-ionic diffusion (Muller & Petzinger, 1988) and membrane transport (Tachampa et al., 2008) appears to be the main entry pathway of AFB1 into cells. However the initial toxin-membrane interactions, and the changes that such process may induce in the latter, are scarcely known.   Aim: To characterize the interaction of AFB1 with model lipid biomembranes, in order to evaluate the probable modulation of the cell plasma membrane functionality by the toxin.   Materials and methods: Penetration of AFB1 in lipidic interfaces: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dpPC, Avanti Polar Lipids, USA) monomolecular layers (MML) at the air-water interface were prepared and monitored as described (Theumer et al., 2008). The lateral surface pressure (p) was measured by the Wilhelmy plate method. The penetration of AFB1 (Sigma-Aldrich, USA) dissolved in EtOH was evaluated varying: a) the toxin concentration (0-8.4 ¦ÌM) injected in the subphase of MMLs at similar initial molecular packing (pi ¡Ö 10 mN/m), and b) the MML molecular packing (5-35 mN/m) and injecting the AFB1 (3 ¦ÌM) in the subphase. Steady state fluorescence: Multilamellar vesicles (MLV) were prepared by hydration of a dpPC film with bidistilled water, intensive vortexing and heating at 50 ¡ãC. The fluorescent probes DPH (2 ¦ÌM) and TMA-DPH (6 ¦ÌM) were added to the dpPC MLV suspension and incubated for 1 h at room temperature. The effects of AFB1 (0-8.9 ¦ÌM) on the DPH and TMA¨CDPH steady-state fluorescence anisotropy were studied. Anisotropy values were calculated from the emission fluorescence intensities at ¦Ëem = 430 nm (¦Ëex = 356 nm) (Theumer et al., 2008).   Results and discussion: When injected in the subphase of dpPC Langmuir films at similar molecular packing (pi ¡Ö 10 mN/m), the mycotoxin (0-8.4 ¦ÌM, corresponding to 0-42 ¦ÌL of EtOH used as vehicle) induced a linear increase in the lateral surface pressure (p) of the MML. Although the addition of EtOH alone, in amounts equivalent to those applied in the AFB1 containing samples, also increased p, its effect was significantly lower than that observed with AFB1 (slope 0.0374 mN/m ¦ÌL−1 EtOH). Moreover, in dpPC Langmuir films the maximal p allowing the AFB1 penetration (pcut-off) was p ¡Ö 30 mN/m, which was determined by monitoring the increments in the p values seven minutes at the plateau in p value reached after the injection of AFB1 (3 ¦ÌM) in the subphase of the dpPC MML at different initial molecular packing (pi 5-35 mN/m). Surface pressure changes may be interpreted either as a toxin penetration in the monolayer and/or an interfacial deformation accompanying the toxin adsorption. The possible disturbances in the membrane environment due to the incorporation of AFB1 to dpPC MLVs were studied using DPH and TMA-DPH as probes. AFB1 (0-8.9 ¦ÌM) decreased in a dose-dependent manner, the fluorescence anisotropy of TMA-DPH (from 0.399¡À0.020 to 0.044¡À0.002) and of DPH (from 0.341¡À0.002 to 0.090¡À0.003). Steady-state fluorescence anisotropy provides information about the organization of the membrane environment around the fluorescent probe. DPH is known to be located within the hydrocarbon chain region of the membrane core and its parent compound, TMA-DPH, stabilizes its DPH moiety at the polar head group region of bilayers. The changes in the fluorescence anisotropy of both probes indicate that AFB1 localize deeply in the bilayers membrane structure with a consequent decrease in the molecular order and an increase in the molecular mobility at the surface, and also affecting the membrane dynamics at the hydrocarbon chain region.   Conclusion: AFB1 modified the molecular order and mobility of the model lipid biomembranes studied. Then a modulation of the cell membrane functionality should be expected by its sole interaction with the AFB1.   References: Muller, N. & Petzinger, E. 1988. Hepatocellular uptake of aflatoxin B1 by non-ionic diffusion. Inhibition of bile acid transport by interference with membrane lipids. Biochimica Biophysica Acta, 938:334-344.   Tachampa, K., Takeda, M., Khamdang, S., Noshiro-Kofuji, R., Tsuda, M., Jariyawat, S., Fukutomi, T., Sophasan, S., Anzai, N., & Endou, H. 2008. Interactions of organic anion transporters and organic cation transporters with mycotoxins. Journal of Pharmacological Sciences, 106:435-443.   Theumer, M. G., Clop, E. M., Rubinstein, H. R., & Perillo, M. A. 2008. The lipid-mediated hypothesis of fumonisin B1 toxicodynamics tested in model membranes. Colloids and Surfaces B Biointerfaces, 64:22-33.   Acknowledgements: SeCyT-UNC, Ministerio de Ciencia y Tecnolog¨ªa de C¨®rdoba, Foncyt and Conicet. MGT and MAP are career investigators from Conicet.