Subacute Cardiotoxicity of Yessotoxin: In Vitro and in Vivo Studies Sara F. Ferreiro, † Natalia Vilariñ o,* ,† Cristina Carrera, †,§ M. Carmen Louzao, † Antonio G. Cantalapiedra, ‡,§ Germa ́ n Santamarina, ‡,§ J. Manuel Cifuentes, ∥ Andre ́ s C. Vieira, † and Luis M. Botana* ,† † Departamento de Farmacología, ‡ Departamento de Ciencias Clínicas Veterinarias, § Hospital Veterinario Universitario Rof Codina and ∥ Departamento de Anatomía y Producció n Animal, Facultad de Veterinaria, Universidade de Santiago de Compostela, 27002 Lugo, Spain * S Supporting Information ABSTRACT: Yessotoxin (YTX) is a marine phycotoxin produced by dinoflagellates and accumulated in filter feeding shellfish. Although no human intoxication episodes have been reported, YTX content in shellfish is regulated by many food safety authorities due to their worldwide distribution. YTXs have been related to ultrastructural heart damage in vivo, but the functional consequences in the long term have not been evaluated. In this study, we explored the accumulative cardiotoxic potential of YTX in vitro and in vivo. Preliminary in vitro evaluation of cardiotoxicity was based on the effect on hERG (human ether-a-go-go related gene) channel trafficking. In vivo experiments were performed in rats that received repeated administrations of YTX followed by recordings of electrocardiograms, arterial blood pressure, plasmatic cardiac biomarkers, and analysis of myocardium structure and ultrastructure. Our results showed that an exposure to 100 nM YTX for 12 or 24 h caused an increase of extracellular surface hERG channels. Furthermore, remarkable bradycardia and hypotension, structural heart alterations, and increased plasma levels of tissue inhibitor of metalloproteinases-1 were observed in rats after four intraperitoneal injections of YTX at doses of 50 or 70 μg/kg that were administered every 4 days along a period of 15 days. Therefore, and for the first time, YTX-induced subacute cardiotoxicity is supported by evidence of cardiovascular function alterations related to its repeated administration. Considering international criteria for marine toxin risk estimation and that the regulatory limit for YTX has been recently raised in many countries, YTX cardiotoxicity might pose a health risk to humans and especially to people with previous cardiovascular risk. ■ INTRODUCTION Yessotoxin (YTX) is a polycyclic ether first isolated from the scallop Patinopecten yessoensis. 1 YTXs are produced by the phytoplanktonic dinoflagellates Protoceratium reticulatum, Lin- gulodinium polyedrum, and Gonyaulux spinifera. 2−4 Although no human intoxications have been reported, many food safety authorities have established maximum levels of YTXs in shellfish destined to human consumption. 5 Mice in vivo toxicological data point to the heart as one of the main target organs of YTXs due to ultrastructural alterations described in cardiomyocytes after oral and intraperitoneal administrations in acute and short-term studies. 6−12 However, acute evaluation of YTX effects on heart function did not evidence cardiac dysfunction following intravenous administration of YTX to rats. 13 In vitro data demonstrate that YTX induces cell death in many cell types, including rat cardiomyocytes, and alterations of calcium movement, cyclic nucleotides, E-cadherin, and cytoskeleton. 14−18 These toxicological data suggest that YTX repeated exposures might affect heart function in the long term, mainly considering the mitochondrial damage in cardiomyo- cytes. Actually, the study of the toxicological significance of these ultrastructural changes in the heart is recommended in the last EFSA report on YTXs. 19 The European Medicines Agency (EMA) recommendations and several expert publications establish the guidelines for cardiotoxicity evaluation. 20,21 In vitro evaluation of human ether-a-go-go related gene (hERG) channel function by patch clamp has been the in vitro method of choice for the screening of potential acute cardiotoxicity; but hERG trafficking is also being evaluated nowadays for the detection of chronic effects. 20 In vivo heart functionality is par excellence monitored by electrocardiography, which can evidence structural alterations that affect the normal cardiac impulse generation or propagation. 22 Arterial blood pressure (ABP) measurement is also recommended in hemodynamic response evaluation. Structural alterations of the heart have been traditionally studied by histopathological analysis, but quantification of Received: January 12, 2016 Published: April 22, 2016 Article pubs.acs.org/crt © 2016 American Chemical Society 981 DOI: 10.1021/acs.chemrestox.6b00012 Chem. Res. Toxicol. 2016, 29, 981−990