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|Title: ||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|
|Authors: ||Dell'ovo, Valeria|
|Supervisor/Tutor: ||Tubaro, Aurelia|
|Co-supervisor: ||Florio, Chiara|
Ramsdell, John S.
Dechraoui Bottein, Marie-yasmine
|Issue Date: ||26-Mar-2010|
|Publisher: ||Università degli studi di Trieste|
|Abstract: ||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~μg/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 μM 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 μM YTX) together with membrane hyperpolarization (0.01-1 μM YTX; p<0.001) occurred, with subsequent reduced cell viability and mitochondrial depolarization (0.01-1 μM 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 μM YTX; 5-24 h), but no caspase activation (0.001-0.1 μM 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 μM 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.|
Brevetoxins (BTXs) and ciguatoxins (CTXs) are two classes of algal neurotoxins produced by the dinoflagellates Karenia brevis and Gambierdiscus toxicus, and in humans are responsible of Neurotoxic Shellfish Poisoning and Ciguatera Fish Poisoning, respectively. Both intoxications are mainly characterized by neurological and gastro-intestinal symptoms and, in more severe cases, cardiovascular symptoms. Pharmacological studies have shown that molecular target for the CTXs and BTXs is the site-5 on the voltage-gated sodium channel (NaV). Toxin binding modifies channel activation and inactivation mechanisms to a terminal excitotoxicity and cell swelling via continuous sodium influx, membrane depolarization and spontaneous action potentials.
NaV are responsible for action potential generation and propagation in excitable cells, playing a fundamental role in many higher processes such as cognition, cardiac conduction and sensitive perception. Mammalian NaVα-subunits have been identified in excitable tissues and named NaV1.1 through NaV1.9 as products of different genes and with different primary tissue distributions, cDNA sequences, protein structures, gating kinetics and pharmacological properties. This study focused on the NaV1.8 isoform, highly expressed in sensory dorsal root ganglion neurons and the NaV1.2 isoform, broadly expressed in neurons in the central nervous system, with the aim to better understand central and peripheral nervous system effects of these toxins in human poisoning episodes. The activity of BTXs (PbTx-1, PbTx-7, PbTx-3), and CTXs (P-CTX-1, C-CTX-1 and P-CTX-3C) was screened on the human embryonic kidney cell line (HEK293) stably expressing either NaV1.2 or NaV1.8 human isoforms.
Cells were transfected by a chemical approach with NaVα subunits cDNA gene inserted in a TrueClone pCMV6-Neo plasmid and complexed in a liposome transfection reagent. The mRNA level analysis using Real Time PCR technique showed the specific presence of both isoforms only in transfected cells. Product specificity was confirmed by bio-sizing and sequencing techniques. The cytotoxic effects of neurotoxin BTX and CTX were assessed by exposing cells to increasing toxin concentrations in presence of the sodium channel activator veratridine and the sodium-potassium ATPase inhibitor ouabain. Results showed for BTX A and BTX B types, as well as for P- and C-CTX, a dose-dependent toxic effect on both HEK-NaV1.2 and HEK-NaV1.8 clones while no observable effects were measured on the non-transfected cells. For both clones, EC50 values of either P-CTX-1 and PbTx-7 were one order of magnitude lower than those of other CTXs tested (10-13 vs 10-12 M) and other BTXs tested (10-10 vs 10-9 M).
Overall the toxic response of the peripheral NaV1.8 to the polyether toxins was similar to that of the NaV1.2 suggesting the absence of a tissue selectivity of the polyether toxins for the peripheral channel. A better understanding of the particular sensory abnormalities associated to CFP and NSP will require further isoform studies.
|PhD cycle: ||XXII Ciclo|
|PhD programme: ||SCUOLA DI DOTTORATO DI RICERCA IN SCIENZE E TECNOLOGIE CHIMICHE E FARMACEUTICHE|
voltage gated sodium channels
|Main language of document: ||en|
|Type: ||Tesi di dottorato|
|Scientific-educational field: ||BIO/15 BIOLOGIA FARMACEUTICA|
|Appears in Collections:||Scienze biologiche|
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