Mechanism of beauvericin- and moniliformintoxicity

Research project P 14507 Mechanism of beauvericin- and moniliformin-toxicity Rosa LEMMENS-GRUBER 26.6.2000 The moulds F. subglutinans and F. proliferatum are the most prevalent Fusarium-species on cereals in Austria. From these species the mycotoxins beauvericin and moniliformin are produced. Concerning mycotoxicosis by beauvericin only scarce reports are available, while moniliformin is well known as a cardiotoxic acting mycotoxin, however, the reports about the action and mechanism are contradictory. Beauvericin is a cyclodepsipeptide with cytotoxic, antibiotic and insecticide properties, and a potent and specific inhibitor of cholesterol acyltransferase. Several papers report about a beauvericin-induced increase of ion permeability in biological membrane systems for potassium, sodium and calcium ions, while the only available study in mammalian tissue rather suggests a calcium- antagonistic effect. Whether beauvericin is an ionophoric or a calcium-antagonistic acting compound is still contradictory. Therefore, we will study the effect of beauvericin in isolated smooth an d heart muscle preparations and cells with electrophysiological methods (intracellular microelectrode technique, patch-clamp technique) in order to identify the mechanism of action. If action potential parameters will be changed, the distinct current(s) will be studied in detail with the patch clamp technique. In the case of ionophoric activity we will continue to study the channel forming activity in liposomes, clarifying ion selectivity, kinetics, blockade of the pore, etc. An interaction of beauvericin with calcium ions is known, however, the mechanism is not identified, which should be clarified by using the fluorescence imaging method, eventually combined with the patch-clamp technique. Moniliformin is a well known cardiotoxic acting mycotoxin. However, the mechanism of toxicity is not clear right now. Cytotoxicity is discussed in terms of hypoxia and altered cell metabolism causing ultrastructural damage with degeneration and necrosis in the heart muscle, since moniliformin affects a number of enzyms like pyruvate dehydrogenase, transketolase, glutathione peroxidase and reductase as well as alanine aminotransferase and alkaline phosphatase. Some authors found also effects on rate and force of contraction, an effect which could not be proved by others. Whether alterations in cardiac electrical conductance with subsequent arrhythmia are solely due to the changed functions of cardiac cell enzyms or are also due to a direct effect on ionic membrane currents is not clear, contradictory results are published. Therefore, we will perform experiments with moniliformin using electrophysiological methods (intracellular microelectrode technique, patch clamp technique, eventually combined with fluorescence imaging) in order to study whether moniliformin directly affects ionic membrane currents or whether cardiotoxicity by moniliformin only occurs secondary by hypoxia and enzyme inhibition.

The exact mechanism of action for the cytotoxic ionophore and antibiotic secondary metabolite Beauvericin, produced by various Fusarium species, was unclear. Data for the effect of beauvericin on mammalian tissue are scarce, and they hint towards an calcium-antagonistic activity. On the other hand the fact that moniliformin is cardiotoxic, was published in a number of papers, however, the data are contradictory. As both mycotoxins are detected concomitantly in cereal probes we also studied the effects of the two secondary metabolites in combination. We studied the electromechanical and -physiological effects on different isolated mammalian tissue and cells as well as in liposomes. Ionophores can change the ion permeability of a membrane via a carrier mechanism and/or by inducing pores. Single channel current analysis (patch-clamp method in the inside-out configuration) revealed that beauvericin incorporates into cell membranes and liposomes, and acts as an pore-inducing agent. Mono- und divalent cations are conducted through this beauvericin-induced pore with the selectivity K+ > Na+ > Li + >>> Ca2+ > Mg 2+. Trivalent cations block the beauvericin-induced pore, and anions are not conducted. The currents carried by cations differ in conductivity and kinetics. Comparison of these parameters in ventricular myocytes and liposomes revealed a dependency on the properties of the membrane. The beauvericin-induced pore in its open state has substates, indicating different configurations of the open state. From the electrophysiological data we can deduce a model for a narrow pore consisting of three vertically stacked beauvericin molecules. Cations can flow through the pore along the electrochemical gradient, and they induce an initial imbalance of intracellular physiological ions. This effect is compensated by cellular mechanisms, but gradually depletion of ATP, mitochondrial depoarisation and cytolysis due to calcium overload appear. For moniliformin up to a concentration of 100 M weak, but significant electromechanical effects and non- significant electrophysiological effects were observed. The results are interesting in view of food safety, because beauvericin and moniliformin are concomitantly detected mycotoxins in cereals, contaminating feed and food. Therefore, the question arises whether these two mycotoxins are relevant toxicologic agents. In experiments on isolated cells the combined application of both secondary metabolites enhanced the cytotoxic effect. However, the cells are able to compensate beauvericin-induced intracellular physiological ion imbalance over a wide range of concentration. As some papers report about an antibiotic and antilipidaemic activity of beauvericin the knowledge about the mechanism of cytotoxicity is also important in this context.