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Las ficotoxinas son compuestos tóxicos producidos por determinadas especies de microalgas y acumuladas predominantemente en moluscos filtradores llegan al hombre a través de la red trófica. Las intoxicaciones por consumo de alimentos contaminados con estas toxinas entrañan un importante riesgo para la seguridad alimentaria. La regulación del contenido de ficotoxinas en los alimentos destinados al consumo humano debe basarse en datos toxicológicos que garanticen la protección de la salud humana. La toxicidad aguda de muchas de las toxinas emergentes y la toxicidad crónica de todas ellas en general son en gran medida desconocidas. En relación a los efectos cardiotóxicos de las ficotoxinas se sabe que algunas de ellas modifican la función cardíaca in vivo. Sin embargo, se desconocen los efectos cardíacos de la mayoría de estos compuestos; a pesar de que en algunos casos la información existente sugiere cardiotoxicidad. El objetivo propuesto para esta Tesis Doctoral ha sido el estudio del potencial cardiotóxico agudo y subagudo de las toxinas marinas pertenecientes a los grupos ácido ocadaico (AO) y dinofisistoxinas (DTXs), yesotoxinas (YTXs) y azaspirácidos (AZAs) mediante experimentos in vitro e in vivo recomendados por expertos y guías especializadas. La cardiotoxicidad in vitro se determinó mediante la evaluación de la actividad de estas toxinas en el canal cardíaco hERG (human ether-a-go-go related gen), concretamente su capacidad para alterar las corrientes medidas mediante técnicas electrofisiológicas o el tráfico celular a largo plazo. Los estudios in vivo analizaron la función y estructura cardíacas en ratas después de la administración aguda o subaguda de las toxinas. Los resultados in vitro muestran que el AO, la YTX y el AZA-2 no poseen actividad de relevancia fisiológica en el canal hERG. Los estudios in vivo realizados evidencian un efecto cardiotóxico agudo para el AZA-2 que se manifiesta como alteraciones funcionales en el ritmo y la conducción del corazón. Además, la exposición repetida a YTX y AZA-1 provoca alteraciones funcionales y ultraestructurales a nivel cardiovascular. El daño cardíaco se manifestó también por la activación de la autofagia o la apoptosis en los tejidos cardíacos de las ratas tratadas con YTX o AZA-1 respectivamente. En conclusión, los resultados de esta Tesis Doctoral proporcionan por primera vez una significación fisiopatológica funcional a los efectos cardíacos histopatológicos descritos con anterioridad para la YTX y confirman un potencial cardiotóxico in vivo de los AZAs que hasta el momento no se había descrito. Esta información toxicológica debe de ser considerada por el riesgo que ambas toxinas pueden suponer para la seguridad alimentaria y por ende para la salud pública.

INTRODUCTION Marine algal blooms caused by the overgrowth of marine phytoplankton have increased in frequency and severity in the last years; supposing a worldwide public health problem. Yessotoxin (YTX) is a marine biotoxin produced by dinoflagellates and accumulated in filter feeding shellfish through the tropic chain. YTXs are worldwide distributed and regulated by safety authorities although no human toxicity outburst has been unequivocally related to them (Tubaro et al., 2010). The toxicological studies available report that YTX causes ultrastructural alterations in cardiac muscle in vivo (Terao et al., 1990) and that induces apoptosis and several alterations in many cell lines in vitro; however, its mechanism of action still remains unknown (Tubaro et al., 2010). HERG is the recommended target when cardiotoxicity is evaluated in vitro. The hERG (human ether-a-go-go related gene) encodes the cardiac potassium channel responsible of the rapid delayed rectifier K+ current (Ikr) (Sanguinetti et al., 1995). HERG channel dysfunction is related to repolarization disorders and fatal heart arrhythmias (Sanguinetti et al., 1995). Drugs can alter hERG by different mechanisms such as direct block or by disruption of trafficking. HERG trafficking is a chronic effect that usually takes hours or even days to occur. Presently, there are drugs that can cause direct channel block (Redfern et al., 2003), disrupt hERG trafficking (Guo et al., 2007) or both (Han et al., 2011). YTX is not an acute hERG channel blocker (manuscript under revision), however its effects on hERG channel trafficking have not been studied yet. The aim of this study was to evaluate YTX effects on extracellular hERG channel protein levels and their functional implications. MATERIAL AND METHODS Reagents: Yessotoxin (purity ≥ 97.9%) (Figure 1A) is certified reference material supplied by Laboratorio CIFGA S.A. (Lugo, Spain). Annexin V-FITC apoptosis detection kit was from Immunostep (Salamanca, Spain). Cy3® goat anti-rabbit IgG was from Invitrogen® (Madrid, Spain). Monoclonal anti--tubulin was from Sigma-Aldrich Química S.A. (Madrid, Spain). Anti-Kv 11.1 (HERG) (extracellular) was from Alomone Labs (Jerusalem, Israel). The anti-hERG antibody HERG (H-175) was from Santacruz Biotechnology, Inc. (Santa Cruz, CA). Goat anti-rabbit IgG was from Millipore® Iberica S.A. (Madrid, Spain). Ionflux extracellular solution, Ionflux intracellular solution and Ionflux plates were obtained from Fluxion Bioscences Inc. (South San Francisco, CA). Cell line: A PrecisionTM hERG CHO (chinese hamster ovary) Recombinant cell line (Millipore® Iberica S.A., Madrid, Spain) was used. Cell culture conditions and passage routine were as previously published (Ferreiro et al., 2014). Imaging flow citometry: Toxin and/or carrier (DMSO) at desired concentrations were added to hERG-CHO cultures for 6, 12 or 24 h at 37 ºC. All samples were incubated with FITC-annexin-V. The cells were then incubated with the anti-Kv 11.1 (1:100) antibody and with Cy3® goat anti-rabbit IgG (1:200). Flow cytometry data were acquired using an ImageStream multispectral imaging flow cytometer and quantitative measurements were done using the data analysis software IDEAS (Amnis Corporation, Seattle, WA). Automated patch clamp: The effect of YTX on hERG channel activity was tested using an IonFlux 16 automated patch clamp system (Fluxion Bioscences Inc, South San Francisco, CA). Toxin and/or carrier at desired concentrations were added to hERG-CHO cultures for 6 and 12 at 37 ºC. The cells were prepared at 20x106 cells/ml in EC solution. The voltage protocol applied to record hERG currents was as follows: cells were clamped at -80 mV for 100 ms, pulsed to -100 mV for 90 ms and to -50 mV for 50 ms, then depolarized to +20 mV for 5 s and repolarized to -50 mV for 5 s, and finally returned to -80mV. Western blot analysis: HERG channel protein levels in hERG-CHO cells were evaluated by western blot analysis. Cell cultures were incubated with toxin or carrier at the desired concentrations for 6 or 12 h at 37 ºC. HERG channel proteins were detected with an anti-hERG antibody (1:20000) and with an HRP-labeled goat anti-rabbit IgG antibody (1:15000). The membranes were reblotted with -Tubulin for normalization. Chemiluminescence was detected using a Dyversity Imaging System and quantification was done with the Genetools software (Syngene, Cambridge, UK). Data analysis: Data were plotted as mean ± SEM. Statistical significance was determined by using t test for unpaired data and ANOVA was used for multiple comparisons. P < 0.05 was considered for significance. RESULTS YTX effects on plasma membrane hERG levels The chronic effect of YTX on plasma membrane hERG levels was studied in hERG-CHO cells. Cells were incubated with 100 nM YTX or carrier for 6, 12 and 24 hours. Cells treated with YTX for 12 and 24 hours underwent a statistically significant increase of hERG channel levels on the cell surface versus carrier controls. No effects on annexin-V-related fluorescence, which was used as a positive marker of apoptosis, were shown in the same conditions (Figure 1B). Staurosporine, a compound that causes apoptosis in CHO cells (Godard et al., 1999), did not cause hERG or annexin-V-related fluorescence increases at a concentration of 500 nM for 12 and 24 hours. However, 1 µM staurosporin induced a stadistically significant increase of annnexin-V-related fluorescence without increasing hERG-related fluorescence levels (data not shown). YTX effects on hERG trafficking The YTX chronic effects on hERG channel trafficking were evaluated by western blot. HERG channels usually display two protein bands, the immature core-glycosylated channel (135 kDa) and the mature fully-glycosylated channel (155 kDa) (Zhou et al., 1998). The intensity of these bands was compared for toxin-treated and carrier-control cells. -tubulin was used for normalization. HERG-CHO cells were treated with 100 nM YTX or carrier for 6 and 12 hours. The intensity of the core-glycosylated form in YTX-treated cells for 6 and 12 hours was decreased versus controls, being statistically significant for the treatment of 12 hours. In these conditions, the intensity of the fully-glycosylated hERG was slightly increased, but it was not statistically different (Figure 1C). YTX chronic effects on hERG currents The functional relevance of the increase of plasma membrane hERG channel levels was explored using automated patch clamp techniques. HERG-CHO cells were incubated with 100 nM YTX or carrier for 6 and 12 hours. Then hERG current was activated using an adequate voltage protocol. The amplitude of Ikr was slightly increased in YTX-treated cells for the treatment of 12 hours but these results were not statistically different versus control levels (Figure 1D). DISCUSSION Currently, published evidence indicates that hERG channel dysfunction can be due to more than one mechanism for many drugs (Guth, 2007). Alterations of hERG channel trafficking are considered an important factor in hERG-related cardiotoxicity. Actually, a screening study revealed that almost 40% of the drugs that block Ikr have also trafficking effects (Wible et al., 2005). Although YTX does not block hERG channels, it has been historically described as cardiotoxic due to in vivo damage to cardiomyocytes. Our results show that YTX induces a significant increase of hERG channel levels on the extracellular side of the plasma membrane in vitro. YTX causes cell death in many cell lines (Korsnes and Espenes, 2011) and the alterations of surface hERG levels might be related to the apoptotic process. However, annexin-V, a relatively early marker of apoptosis (Vermes et al., 1995), occurs later than the increase of surface hERG. Additionally, staurosporine triggered apoptosis without a simultaneous increase of surface hERG, so events are not necessarily related. Therefore YTX-induced elevated hERG in the plasma membrane seem to be independent of apoptosis. Functional implications of hERG currents have been described after alterations of cell surface hERG density (Guth, 2007). YTX did not cause significant alterations of hERG currents. Furthermore the hERG levels after YTX treatment were duplicated, so the effect on currents should be clearly evidenced if these channels were functional. The hERG channels on the cell surface are regulated by its production, translocation to the plasma membrane and degradation. The increase of extracellular channel could be a consequence of a higher production and externalization or a slower degradation. Higher synthesis in our cell model would not be physiologically relevant but our results demonstrated that the amount of immature hERG is reduced instead of increased. Fully glycosylated hERG seems slightly increased in these conditions but it is far from the almost three-fold surface hERG increase. HERG channel is fully glycosylated in the Golgi and externalized to the plasma membrane in vesicles (Smyth and Shaw, 2010) so the amount of fully glycosylated hERG detected by western blot is bigger than the amount detected by flow cytometry. If the externalized fraction of fully glycosylated hERG is small, the elevation of extracellular hERG levels will probably not be detected by Western blot. These data suggest that an impairment of channel internalization could be responsible for the increase of extracellular hERG. Many studies are being done to describe hERG channel degradation pathways, but the exact mechanism by which mature hERG channels are internalized is still unknown (Guo et al., 2012). In summary in vitro chronic treatment with YTX causes an increase of hERG channels on the plasma membrane probably due to a reduction of channel internalization. No functional effects have been observed on hERG currents but more studies are needed to better characterize the effects that YTX can exert on this channel and to know the consequences that can suppose for human health.