Characterization of the dinophysistoxin-2 acute oral toxicity in mice to define the Toxicity Equivalency Factor Paula Abal a , M. Carmen Louzao a, * , Jos  e Manuel Cifuentes b , Natalia Vilari ~ no a , Ines Rodriguez a , Amparo Alfonso a , Mercedes R. Vieytes c , Luis M. Botana a, ** a Departamento de Farmacología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27002, Spain b Departamento de Anatomía, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27002, Spain c Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo 27002, Spain article info Article history: Received 22 November 2016 Received in revised form 14 February 2017 Accepted 15 February 2017 Available online 20 February 2017 Keywords: Dinophysistoxin-2 Intestinal tight junctions Oral toxicity Toxicity Equivalency Factor Ultrastructural effects abstract Ingestion of shellfish with dinophysistoxin-2 (DTX2) can lead to diarrheic shellfish poisoning (DSP). The official control method of DSP toxins in seafood is the liquid chromatography-mass spectrometry analysis (LC-MS). However in order to calculate the total toxicity of shellfish, the concentration of each compound must be multiplied by individual Toxicity Equivalency Factor (TEF). Considering that TEFs caused some controversy and the scarce information about DTX2 toxicity, the aim of this study was to characterize the oral toxicity of DTX2 in mice. A 4-Level Up and Down Procedure allowed the characterization of DTX2 effects and the estimation of DTX2 oral TEF based on determination of the lethal dose 50 (LD50). DTX2 passed the gastrointestinal barrier and was detected in urine and feces. Acute toxicity symptoms include diarrhea and motionless, however anatomopathology study and ultrastructural images restricted the toxin effects to the gastro- intestinal tract. Nevertheless enterocytes microvilli and tight junctions were not altered, disconnecting DTX2 diarrheic effects from paracellular epithelial permeability. This is the first report of DTX2 oral LD 50 (2262 mg/kg BW) indicating that its TEF is about 0.4. This result suggests reevaluation of the present TEFs for the DSP toxins to better determine the actual risk to seafood consumers. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction Phycotoxins produced by harmful microalgae can be ingested by filter-feeding shellfish, mainly bivalve mollusks, and accumulate in the tissues reaching humans through food web. Consumption of mollusks that contain the phycotoxins okadaic acid (OA), dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2) or dinophysistoxin-3 (DTX3) can lead to diarrheic shellfish poisoning (DSP) with symptoms such as diarrhea, nausea, vomiting and abdominal pain (Yasumoto et al., 1978). Several coastal areas worldwide have been affected by the presence of DSP toxins including European, Latin American, North American, Western Af- rican and Asian coasts (Reguera et al., 2014). Therefore although DSP is not considered fatal, it represents an increasing global concern both regarding health and economic impacts related to the ban on selling contaminated bivalve mollusks (Reguera et al., 2014; Van Dolah, 2000). Toxicological information related to DSP toxins has been ob- tained mainly with OA, the representative compound. OA was the first toxin of this group described and isolated from the black sponge Halichondria okadai (Tachibana et al.,1981). OA is also one of the most frequent marine toxins; therefore its potential toxic ef- fects have been investigated in several in vivo and in vitro studies (Valdiglesias et al., 2013). Data on OA toxicokinetics in humans is scarce. Most studies have been performed in rodents where OA is absorbed by the gastrointestinal tract (Ito et al., 2002; Matias et al., 1999). However, the amount of OA that reaches the blood stream depends on several factors, for instance toxin concentration or bioavailability. Recently, a limited passage of OA, DTX1 and DTX2 from the “luminal” to the “blood side” was detected using the human intestinal Caco-2 cell model to simulate the intestinal barrier (Ehlers et al., 2011; Fern andez et al., 2014). * Corresponding author. ** Corresponding author. E-mail addresses: mcarmen.louzao@usc.es (M.C. Louzao), luis.botana@usc.es (L.M. Botana). Contents lists available at ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox http://dx.doi.org/10.1016/j.fct.2017.02.023 0278-6915/© 2017 Elsevier Ltd. All rights reserved. Food and Chemical Toxicology 102 (2017) 166e175