Preliminary study of the effects of Okadaic Acid in the intestinal tract of mouse Diego A. Fernández 1* , Andres C. Vieira 1 , M. Carmen Louzao 1* , José Manuel Cifuentes 2 , Natalia Vilarino 1 , Juan A. Rubiolo 3 , Albina Román 4 , Mercedes R. Vieytes 3 and Luis M. Botana 1* 1 Universidad de Santiago de Compostela, Facultad de Veterinaria, Departamento de Farmacología, Spain 2 Universidad de Santiago de Compostela, Facultad de Veterinaria, Departamento de Anatomía, Spain 3 Universidad de Santiago de Compostela, Facultad de Veterinaria, Departamento de Fisiología Animal, Spain 4 Universidad de Santiago de Compostela, Unidad de Microscopía Electrónica y Confocal, Spain Introduction Diarrheic Shellfish Poisoning (DSP) toxins that include Okadaic Acid (OA) and its analogues (Dinophysis toxins) are produced by several marine dinoflagellates of the genera Dinophysis and Prorocentrum (Reguera et al., 2012). These toxins are especially dangerous during harmful algae bloom because filter-feeders bivalves such as mussels, scallops, oysters, clams and other marine organisms can bio-accumulate them posing a serious threat for the human health regarding shellfish consumption worldwide (Yasumoto and Murata, 1990;FAO, 2004). The main symptoms of DSP usually appear early, between 30 minutes to 12 hours after ingestion and they include diarrhea, nausea, vomiting, abdominal pain and gastrointestinal distress (Yasumoto et al., 1978). They disappear in 2-3 days and so far there are no documented reports of human fatalities due to DSPs. OA is a polyether derivative of a C38 fatty acid that was first isolated from the marine black sponge Halichondria okadai, usually found across the Pacific coast of Japan. OA structure was studied in 1981 by Tachibana et al. (Tachibana et al., 1981). It has been widely accepted since 1990 that one of the main mechanisms of action of OA and its derivatives is the strong inhibition of serine/threonine protein phosphatases PP1 and PP2A (Takai et al., 1987;Honkanen et al., 1994;Dawson and Holmes, 1999;Louzao et al., 2005). However, recent papers suggested that it should be re-evaluated (Louzao et al., 2003; Louzao et al., 2005;Vilarino et al., 2008;Espina et al., 2010) especially when focusing on the exact triggering mechanism of the diarrheic effects (Munday, 2013). In this preliminary work, we have studied the possible effects that OA could cause in the intestinal tract when the toxin is ingested. For this purpose, samples of the small intestine of mice treated by gavage with this DSP toxin were examined and compared to control samples using images obtained with a transmission electron microscope (TEM) to discern any structural changes induced by OA in such tissue. Material and Methods Animals and Treatments: All animal procedures were conducted according to the principles approved by the Institutional Animal Care Committee of the Universidad de Santiago de Compostela. A total of 6 CD-1 female mice (Charles River Inc., Barcelona, Spain), weighing 18–23 g were used. Mice were divided in treated specimens (3 mice) and controls (3 mice). The animals were kept in a controlled environment with a stable temperature (23 ± 2 °C) and humidity (60%–70%) for one week before the experiment, as well as fed with a diet for rodents containing 18.5% of protein (Harlan®). Twelve hours before the treatment all mice were singly housed and fed with physiological solution supplemented with glucose, in order to have fasted animals. The Okadaic Acid (OA) for the treatments had a purity of 99% (Laboratorio Cifga S.A., Lugo, Spain) and was initially dissolved in ethanol. The toxin was then dissolved in physiological solution and administered by gavage to all the experimental mice at a concentration of 1000 µg/kg. Control animals were treated with the same physiological solution and vehicle (2.5 % ethanol). Then all the mice were granted with free access to food and sacrificed after 24 hours in a carbon dioxide chamber. Immediately, samples of the small intestine were collected and stored in a stabilization solution and stored until further use. Preparation of samples for Transmission electron microscopy (TEM): Intestine samples were treated with TEM fixative for 30 min at 4 °C in an orbital shaker at low speed. Fixative was then removed and the samples were rinsed three times with 0.2 M cacodylate trihydrate buffer. Postfixation was carried out in 1% OsO4. Then the samples were rinsed again in 0.2 M cacodylate trihydrate that was removed before the dehydration in graded ethanol solutions, including one bath of ethanol 70% with uranyl acetate, cleared in propylene oxide and finally embedded in Epon 812 (Momentive Specialty Chemicals Inc., Houston TX, USA). Ultrathin sections of the samples were obtained with a Leica Ultracut UCT ultramicrotome from Leica Microsystems GmbH (Wetzlar, Germany) and counterstained with uranyl acetate and lead citrate. Ultrastructural analysis was performed with a JEOL JEM-1011 Transmission Electron Microscope (Jeol Ltd, Tokyo, Japan). Results and discussion There is some controversy regarding the effects of OA and DSP toxins in the intestinal tract with highly variable results. Some authors found that when OA reaches the small intestine it could causes different degrees of damage to the epithelium including edema, Journals A-Z By Subjects Events Jobs People Videos News Blogs Images Books About Submit Register Login Search EVENT ABSTRACT Back to Event