Frontiers in Marine Science (Jun 2014)
Preliminary study of the effects of Okadaic Acid in the intestinal tract of mouse
Abstract
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, ulceration, separation and destruction of villi of epithelial cells to cite a few (Ito et al., 2002;Le Hegarat et al., 2006). Nevertheless, other authors were unable to discern any significant damage or ultrastructure changes that could be related to the presence of OA (Tubaro et al., 2003;Vieira et al., 2013). In this preliminary work we found no clear effects of OA on small intestine tissue. TEM images of control mice samples presented the normal morphology of intestinal enterocytes, as expected. This is, cells with big and irregular nuclei with well-defined nucleoli, rough endoplasmic reticulum cisternae surrounding the numerous mitochondria, some inclusions in the cytoplasm, noticeable indentations between adjacent cells with some desmosomes and tight-junctions visible and the most remarkable attribute, the presence of microvilli in the apical side of the plasma membrane that forms the brush border. However, there were some control samples where all these attributes were not so clear and even some vacuoles appear in certain fields. On the other hand, most samples of OA-treated mice showed a similar aspect to control cells, specially the microvilli in the apical side. Some vacuoles were also found in the cytoplasm of the cells. In Figure 1, representative images of the small intestine of control (A) and 1000 μg/kg OA-treated mice (B). The following structures are indicated: Microvilli/brush border (MV), desmosomes (D), amorphous cytoplasmic inclusions (I), vacuoles (V), mitochondria (M), and nucleus (N). In a recent paper Wang et al. (Wang et al., 2012) reported that a dose of 750 μg/kg OA produced microvilli damage on small intestine cells of ICR mice as early as 3 hours after treatment. However, the cells were able to gradually recover and after 24 hours they presented an apparently normal microvilli again. They also reported that the levels of serine/threonine protein phosphatases were reduced at 3 hours but normal after 24 hours. Nevertheless the expression of other proteins such as Villin 1 and hnRNP F were significant altered by OA suggesting that the diarrheic effects of this toxin were not only related to protein phosphatases as it has been extensively reported. Besides the microvilli alteration they noticed dilation of the rough endoplasmic reticulum after 3 hours of oral administration, and swelling, ridge disappearance and destroyed structure of the mitochondria after 6 hours. The small OA effect on intestinal cells could be related to the intestinal absorption of the toxin. It was recently reported that this toxin is not well absorbed by the gastro-intestinal tract (Ehlers et al., 2011;Fernandez et al., 2014). In accordance with that it was registered a rapid excretion and elimination of OA in other in vivo studies (Matias et al., 1999). Our results support the little morphological effect of OA on intestinal cells. However, more interdisciplinary research is needed to obtain precise and reliable data to clarify the effects of OA in the intestinal epithelium and its relation with the diarrhea.
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