Toxicity of Cyclohexadepsipeptides

The moulds Fusarium subglutinans and F. proliferatum can produce a number of toxic metabolites such as beauvericin (BEA) and enniatin (ENN), concomitantly detected in cereal probes. BEA and ENN are cyclohexadepsipeptide ionophores with cytotoxic, antibiotic and hypolipidaemic properties. Newly in vitro synthetized cyclohexadepsipeptides of the ENN type show potential antihelmintic properties. Therefore, it is of interest whether the therapeutically promising properties of the two cyclohexadepsipeptides might prevail the obviously unspecific cytotoxic effects, and thus to clarify the mechanism of cytotoxic action. In isolated ventricular myocytes (BEA, ENN) and unilamellar vesicles (BEA) both mycotoxins showed channel forming activity, but differing in ion selectivity, substates, conductivity and kinetics. The influence of ENN`s molecular structure on single channel current properties will be investigated in different mammalian cell membranes and unilamellar vesicles by testing ENN homologues, and studying their effect on the cell regulatory effects, channel stability and lifetimes. So far, experiments with the patch-clamp technique and the fluorescence- imaging method revealed a complex mechanism of the effect of BEA on ion flux and the regulation of intracellular calcium, sodium and potassium ion concentrations. It seems that compensatory mechanisms of the cell can prevent cytolysis until a certain critical point. Although some steps in this mechanism remain to be studied, they cannot be elucidated solely with the patch-clamp method and fluorescence imaging. Therefore, we intend to investigate the action of BEA and ENN in isolated mammalian cells with the confocal laser scanning microscopy in order to clarify the compensatory mechanisms on the subcellular level. Furthermore the incorporation of BEA and ENN into the cell membrane should be studied by means of the solid-state NMR in order to support the interpretation of patch clamp experiments concerning channel conformation.

The cyclohexadepsipeptide mycotoxins beauverin (BEA) and enniatin (ENN) interact with mammalian cells and artificial membranes, in which they incorporate forming cation selective channels. Through their ionophoric properties BEA and ENN greatly disturb the physiological ionic balance and pH in a variety of cells, and inhibit cellular metabolism by mitochondrial depolarization and cytolysis. A model for the BEA and ENN pore was proposed which assumes BEA or ENN molecules vertically stacked, their central cavities forming a long hydrophilic pore spanning the membrane. Although divalent cations are conducted in the absence of monovalent cations, in their presence they rather act as blocking agents. Differences in pore kinetics may be explained in terms of channel stability and lifetimes being directly related to membrane properties. The moieties in the cyclohexadepsipetide molecule (BEA, ENN A, ENN A1, ENN B, ENN B1) clearly affect single channel kinetics. As the physiological ionic balance is disturbed, cellular compensatory mechanisms are activated to retain homeostasis. Regarding the raised intracellular calcium concentration it should be noted that sustained calcium elevation usually results in dysfunction of the contractile mechanism, which is assumed to be the cause of the observed reduction in force of contraction in isolated papillary muscles. Compensatory efforts against the ionic imbalance caused by ionophoric challenge increase cellular ATP requirements. Shortening of the cardiac action potential duration and decline in contractility, observed in BEA-treated papillary muscle preparations, may be associated to an ATP deficit. Rigor cell shortening, which is usually accompanied by progressive elevation of cytosolic calcium and acidosis, was regularly observed after BEA. The consistent increase in the intracellular magnesium concentration observed following BEA application gives evidence for ATP breakdown and release of bound magnesium. Except its incorporation in the sarcolemma, BEA may also diffuse into the cell through the lipid membranes and incorporate into the mitochondrial membrane, and depolarizing it. In contrast to published data our experiments reveal that moniliformin (MON) is not a specific cardiotoxic secondary metabolite, but also affects smooth muscle preparations. MON exerts no electrophysiological effects, and it does not influence intracellular ion concentrations, ATP and pH. No synergistic cytotoxic effect of the concurrently in cereals identified Fusarium mycotoxins MON, BEA and ENN was found. BEA and ENN concentration-dependently inhibit bone resorption in osteoclasts and decrease the cell number, BEA showing a stronger effect than ENN. As opposed to the destruxins, BEA and ENN affect osteoclastogenesis. These findings obtained in the course of toxicological investigations of secondary metabolites reveal interesting new insights into a possible therapeutic impact of these mycotoxins.