Cyclic polyether phycotoxins in vitro studies: effects of yessotoxin on a primary culture of rat cardiomyocytes-comparison of ciguatoxins and brevetoxins potency on human VGSC of brain and peripheral sensory neurons expressed in HEK293 cells

Yessotoxins (YTXs) are ladder-shaped polycyclic ether toxins, structurally related to brevetoxins and ciguatoxins (Ciminiello and Fattorusso, 2008). The parent compound of this class, yessotoxin, has been initially isolated from the scallop Patinopecten yessoensis (Murata et al., 1987). Only later their natural source has been identified in the phytoplanktonic dinoflagellates Protoceratium reticulatum (= Gonyaulax grindley) (Satake et al., 1997), Lingulodinium polyedrum (= Gonyaulax polyedra) (Tubaro et al., 1998; Paz et al., 2004) and Gonyaulax spinifera (Rhodes et al., 2006). When environmental conditions promote the growth of these species, their toxins accumulate in edible tissues of filter feeding shellfish exposed to these dinoflagellates, thus entering in the food chain. No human toxicity has been reported for YTXs, although YTXs contaminated-shellfish were worldwide recorded, thus, yessotoxin toxicological potential is still unknown. Toxicological in vivo studies revealed high toxicity in mice after intraperitoneal administration (LD50~?g/Kg), whilst very low toxicity (no lethality) was found after acute or repeated oral administration. Both routes are associated with clear evidence of ultrastructural cardiac alteration in rodent cardiac muscle, soon after toxin administration (Aune et al., 2002; Tubaro et al., 2003). Notwithstanding many in vitro studies highlighted numerous intracellular targets, YTX mechanism of action is unclear and the effects on the cardiac functional properties remain unknown. This study was performed on neonatal rat cardiomyocytes to study toxin effects on various fundamental aspects of cardiac muscle cells activity: cell beating, intracellular Ca2+ and cAMP levels, cell vitality, mitochondrial membrane potential and type of cell death occurrence. Results showed a time- and concentration-dependent reduction in the beating frequency (0.3 ?M YTX, 1 h; p<0.05), neither associated to the uncoupling between the membrane electrical activity and Ca2+ release from intracellular stores nor to the impairment of the mechanisms controlling the Ca2+ homeostasis, nor to altered intracellular cyclic AMP levels. However, a decrease in the firing frequency (about 50%) occurred together with a 50% reduction of the number of beating cardiomyocytes. A time- and concentration-dependent decrease in cell viability (0.1 µM YTX; 24 h) was observed, that evolved in two phases: at 24 h a significant (p<0.001) increase in mitochondrial activity (0.0001-1 ?M YTX) together with membrane hyperpolarization (0.01-1 ?M YTX; p<0.001) occurred, with subsequent reduced cell viability and mitochondrial depolarization (0.01-1 ?M YTX; p<0.001) starting from 48 h. YTX effect on mitochondrial potential wasn't affected by peripheral benzodiazepine receptor ligands PK-11195 and/or 4-chloro-diazepam (100 nM) after 24-48 h. Increasing concentrations of YTX induced the appearance of nuclear apoptotic bodies in a time-dependent way (0.001-0.1 ?M YTX; 5-24 h), but no caspase activation (0.001-0.1 ?M YTX; 5-72 h). Further viability experiments showed an irreversible cell damage, since no recovery occurred after up to 71 h in YTX-free medium. Moreover, 1 h exposure to 1 ?M YTX was sufficient to inhibit beating activity and to cause irreversible reduction of cardiac cells viability. Propidium iodide uptake experiments showed a significant (p<0.01) increase of necrotic cells after 24 h (0.01 µM), but not after 5 h YTX exposure. These results show a very cell-specific response to YTX if compared to previous studies, and a severe damage to in vitro cardiomyocytes. Thus, although no human intoxication due to YTX contamination has been reported so far, the toxicological potential of this compound should be better investigated. Studying this toxin is limited by its non-commercial supply: YTX needed for this experiments was a kind gift of Professor T. Yasumoto.