1. (-ΔF/Fo) BEAControl 10 μM ENN ENNControl 10 μM BEA CYCLODEPSIPEPTIDE TOXICITY: ionophoric properties and effects on the metabolic state and ionic homeostasisK. Kouri, M. Kamyar, R. Lemmens-GruberDepartment of Pharmacology and Toxicology, University of Vienna, Althanstr. 14, 1090 Vienna, Austria [Ca2+]i 542  50 (nM) 2656  55* (nM) 2.80  0.24*** 1.68  0.09 [Na+]i 0.98  0.07 1.47  0.01 1.31  0.08*** 1.75  0.03* [K+]i 1.49  0.06 2.2  0.10* 2.6  0.05 1.21  0.08** 6 0 3 12 8 10 13 14 15 [Ca2+]i (Fig.5) Increase in fluorescence of FURA-loaded cardiomyocytes corresponds to an increase of [Ca2+]i [K+]i and [Na+]i (Fig.3) Reduction of fluorescence in PBFI-loaded cells corresponds to a reduction of [K+]i Increase of SBFI fluorescence intensity corresponds to an increase of [Na+]i 0 6 Fig.5:Effects of 10 μM ENN on the [Ca2+]i of ventricular myocytes. F340/F380 fluorescence ratio from 5 different cells are depicted against time. Fig.2:Original traces of ENN-induced currents in inside-out patches (Eh=-40 mV). The conducting ions are A) K+, B) Na+, C) Ca2+ and D) Mg2+. Downward deflections represent channel openings. Note that the divalent cations (in C & D) are conducted with faster kinetics. Effects on the cellular metabolic state (Figs.6, 7): Fig.3:Effects of 10 μM BEA on [K+]i and [Na+]i of ventricular myocytes. Relative F340/F380 fluorescence from averaged responses (n=5) is depicted against time. 2 pA 20 ms Alterations in intracellular ionic concentrations for Na+ & K+ (Fig.3): Fig.6:BEA-induced changes in ΔΨm and NADH autofluorescence imaged in cardiomyocytes loaded with TMRM.Upper panels = control.Lower panels = BEA effects. MeanS.E. *=p<0.05, **=p<0.005, ***=p<0.001 (n=5) 30 μM BEA (arrows) results in a reversible mitochondrial depolarization (TMRM) Fig.7:BEA-induced changes in ΔΨm and NADH in cardiomyocytes depicted against time. NADH: biphasic response; initial decrease followed by sustained increase. Irreversible. Alterations in pHi (Fig.8): Alterations in [Ca2+]i and in cellular morphology in relation to [Ca2+]i (Figs.4, 5): Fig.8:BEA-induced intracellular acidification in 2 different cardiomyocytes. A B A B Fig.4: BEA induced Ca2+ increase in FURA-loaded cardiomyocytes Left panel: Ca2+ increase at the cross sections of two adjacent cells. Right panel: The Ca2+-overload is accompanied by a decrease in cell-size. Time after BEA application is indicated in min. C D C D • Calcium increase in 5 to 10 min (10 μM BEA) • Rigor • Irreversible Ca2+ overload after 15 min • Irreversible hypercontracture • Cytolysis INTRODUCTION The cyclohexadepsipeptide antibiotics beauvericin (BEA) and enniatin (ENN), secondary metabolites of pathogenic fungi including the genus Fusarium, are aworldwide occurring phyto-pathogen of food and livestock feed. Consumption of contaminated food can cause mycotoxicosis of various symptoms. BEA & ENN effects on mammalian organisms are not known, although the above fungal species have been implicated in various diseases. We have previously reported BEA & ENN ionophoric properties and channel formation. METHODS Techniques: -Patch Clamp (inside-out patches) -Fluorescence Imaging, CLSM Preparations: -guinea pig ventricular myocytes Dyes: FURA2,PBFI, SBFI, BCECF; TMRM RESULTS Cationic Conduction(Figs.1, 2): BEA(Fig.1) & ENN (Fig.2) form cation-selective channels in mammalian cell membranes & conduct mono- and divalent cations. CONCLUSION • BEA- and ENN-ionophoric activity has a definite toxic effect on the cellular ion concentration and metabolic state. • The disturbed ionic homeostasis in order to be preserved requires metabolic upregulation and ATP consumption. • Cyclohexadepsipeptide-induced toxicity resembles ischaemia: Ca2+ & Na+ overload, cellular acidification, rigor, metabolic inhibition. Fig.1:Original traces of BEA-induced currents in inside-out patches (Eh=-40 mV). The conducting ions are A) K+, B) Na+, C) Ca2+ and D) Mg2+. Downward deflections represent channel openings. Note faster kinetics of the divalent cations.