Experimental Basis for the High Oral Toxicity of Dinophysistoxin 1: A Comparative Study of DSP

Toxins 2014, 6, 211-228; doi:10.3390/toxins6010211 toxins ISSN 2072-6651 www.mdpi.com/journal/toxins Article Experimental Basis for the High Oral Toxicity of Dinophysistoxin 1: A Comparative Study of DSP Diego A. Fernández 1, M. Carmen Louzao 1,*, María Fraga 1, Natalia Vilariño 1, Mercedes R. Vieytes 2 and Luis M. Botana 1,* 1 Department of Pharmacology, Faculty of Veterinary, University of Santiago de Compostela, University Campus, Lugo 27002, Spain; E-Mails: [email protected] (D.A.F.); [email protected] (M.F.); [email protected] (N.V.) 2 Department of Physiology, Faculty of Veterinary, University of Santiago de Compostela, University Campus, Lugo 27002, Spain; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mails: [email protected] (M.C.L.); [email protected] (L.M.B.); Tel./Fax: +34-982-822-233 (M.C.L. & L.M.B.). Received: 31 October 2013; in revised form: 23 December 2013 / Accepted: 28 December 2013 / Published: 3 January 2014 Abstract: Okadaic acid (OA) and its analogues, dinophysistoxin 1 (DTX1) and dinophysistoxin 2 (DTX2), are lipophilic and heat-stable marine toxins produced by dinoflagellates, which can accumulate in filter-feeding bivalves. These toxins cause diarrheic shellfish poisoning (DSP) in humans shortly after the ingestion of contaminated seafood. Studies carried out in mice indicated that DSP poisonous are toxic towards experimental animals with a lethal oral dose 2–10 times higher than the intraperitoneal (i.p.) lethal dose. The focus of this work was to study the absorption of OA, DTX1 and DTX2 through the human gut barrier using differentiated Caco-2 cells. Furthermore, we compared cytotoxicity parameters. Our data revealed that cellular viability was not compromised by toxin concentrations up to 1 μM for 72 h. Okadaic acid and DTX2 induced no significant damage; nevertheless, DTX1 was able to disrupt the integrity of Caco-2 monolayers at concentrations above 50 nM. In addition, confocal microscopy imaging confirmed that the tight-junction protein, occludin, was affected by DTX1. Permeability assays revealed that only DTX1 was able to significantly cross the intestinal epithelium at concentrations above 100 nM. These data suggest a higher oral toxicity of DTX1 compared to OA and DTX2. OPEN ACCESS Toxins 2014, 6 212 Keywords: okadaic acid; dinophysistoxin-1; dinophysistoxin-2; Caco-2; intestinal permeability; trans-epithelial electric resistance; occludin; annexin V; Luminex; immunoassay 1. Introduction During harmful algae blooms, bivalves accumulate marine toxins, being a serious threat for human consumers of edible shellfish. Toxins causing diarrhetic shellfish poisoning (DSP) are produced by several marine dinoflagellates of the genera, Prorocentrum and Dinophysis, which are of worldwide distribution [1]. DSP is characterized by diarrhea, gastrointestinal distress, nausea, vomiting and, frequently, abdominal pain. These symptoms usually appear from 30 min to 12 h after ingestion of contaminated seafood [2]. However, as of now, there have been no records of human fatalities related to acute DSP intoxication [3]. Toxins causing DSP were first reported by Yasumoto et al., in 1978 [2]. These toxins are lipophilic and heat-stable polyether compounds that can be accumulated in the hepatopancreas of filter-feeding bivalve species, such as oysters, clams, scallops or mussels [4,5]. The main compound is okadaic acid (OA); it was first isolated from the marine sponge, Halichondria okadai, and its structure (Figure 1) was studied in 1981 by Tachibana et al., [6]. Other DSP toxins are the isomeric compound, dinophysistoxin-2 (DTX2), the methylated derivative, dinophysistoxin-1 (DTX1), and the toxins acylated at the C-7 hydroxyl group with long-chain fatty acids, collectively known as dinophysistoxin-3 (DTX3) [3,7–10]. Figure 1. Chemical structure of (A) okadaic acid (OA); (B) dinophysistoxin-1 (DTX1); and (C) DTX2. Toxins 2014, 6 213 The mechanism of action of this group of toxins is mainly the potent inhibition of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A) [11–14]. The different inhibitory potencies of DSP toxins on protein phosphatases have been determined allowing the establishment of inhibition equivalency factors (IEFs). The IEFs were calculated as the ratio of the IC50 for OA to the IC50 for DTX1 or DTX2. The IEF of DTX1 for PP1 is in the 0.4–0.9 range, whereas for PP2A, it is between 0.9 and 2.4 [15–18], indicating more inhibition of this phosphatase than that exerted by OA. Regarding DTX2, the IEF for PP2A is lower, with reported values ranging from 0.4 to 0.6 [18,19]. According to the current studies, the rest of the derivatives have even less inhibition effect over both phosphatases [15,20–23]. In the present work, we have studied and compared some cytotoxicity parameters of OA, DTX1 and DTX2. Our final goal was to determine the toxin absorption through the human intestine by using human colon adenocarcinoma (Caco-2) cells. 2. Results 2.1. Cytotoxic Effect of OA, DTX1 and DTX2 in Differentiated Caco-2 Monolayers Metabolic activity assay: Cytotoxic effects of OA, DTX1 and DTX2 (1, 10, 20, 40, 100, 200, 500 and 1000 nM) in differentiated Caco-2 cells have been evaluated in a time-dependent manner by the AlamarBlue metabolic activity assay. Incubation with 0.01% Triton X-100 has been used as a positive control of death. Data are shown in Figure 2 as the percent of fluorescence vs. control. Differentiated Caco-2 cells treated with OA, DTX1 or DTX2 did not show any decrease in fluorescence intensity (Figure 2A–C). Therefore, none of the toxins affect the cell metabolism significantly, even at the highest concentration and incubation time tested (1000 nM for 72 h), indicating no change in cell viability. Cell membrane integrity assay: Some fluorescent methods allow a non-destructive evaluation of apoptosis by detecting changes in the structure of cell membranes. Annexin V has a high affinity for phosphatidylserine (PS), a phospholipid component located on the inner surface of the lipid bilayer of the cellular plasma membrane. During early apoptosis processes, this PS migrates to the outer portion, becomes exposed on the surface of the cell and can be detected by binding to annexin V labeled with a fluorescent dye. The annexin V assay was carried out in differentiated Caco-2 monolayers that were treated for 24 h with 100 nM of OA, DTX1, DTX2 or vehicle. Incubation with 0.005% Triton X-100 was used as a positive control of cell death and 1 μM staurosporine (STP) and 100 mM sodium butyrate (NaBT) as inductors of cell apoptosis. Control confocal microscopy images (Figure 3A) showed normal Caco-2 monolayers with no fluorescent signal, as expected. Lack of fluorescence is also found in cells incubated with OA, DTX1 and DTX2 (Figure 3B–D), revealing that no apoptotic processes were undergoing on the Caco-2 monolayers under these conditions. As for the apoptosis inductor treatments, cells incubated with 1 μM STP and 100 mM NaBT (Figure 3E,F) showed evident FITC green fluorescence zones, indicating structural changes in the plasma membrane integrity. The treatment with 0.005% Triton X-100 (Figure 3G) showed a heavily damaged cell monolayer with noticeable loss of substrate adhesion and fluorescent green dots corresponding to the FITC-marked annexin V. Toxins 2014, 6 214 Figure 2. Viability of Caco-2 cells treated with OA, DTX1 and DTX2. Differentiated Caco-2 cells were incubated with increasing concentrations of (A) OA, (B) DTX1 and (C) DTX2 for 6, 8, 10, 12, 24, 48 and 72 h. 0.01% Triton X-100 treatment is also represented (D). Results are expressed as the percent of viability vs. control. Reported values are the mean ± SEM of three independent experiments performed in duplicate. 2.2. Effect of OA, DTX1 and DTX2 on the Trans-Epithelial Electrical Resistance (TEER) of Differentiated Caco-2 Monolayers We assessed tight junction formation stability through measurements of trans-epithelial electrical resistance (TEER). Therefore, only inserts with TEER higher than 300 Ωcm2, which indicates that the Caco-2 monolayer is intact and suitable for drug permeability assays [24], were used for further testing. We studied the changes that OA, DTX1 and DTX2 induced in the TEER of the Caco-2 monolayer as a function of time. Differentiated Caco-2 monolayers were incubated with 10, 50 and 100 nM OA, DTX1 and DTX2. TEER was measured immediately before the toxins addition and after 3, 6, 12 and 24 h of incubation with them. OA and DTX2 did not induce any change in the TEER values of Caco-2 monolayers at any concentration or time tested (Figure 4). However, 50 nM DTX1 (Figure 4E) significantly decreased the TEER value when incubated for 24 h. This decrease was much more noticeable at 100 nM for 12 and 24 h (Figure 4H), indicating a clear disturbance on the Caco-2 monolayer. Toxins 2014, 6 215 Figure 3. Confocal imaging of differentiated Caco-2 monolayers treated with OA, DTX1 and DTX2 and labeled with FITC-conjugated annexin V (left columns). Transmission images of the same cells are presented in the right columns. (A) shows control cells; while (B–G) are photographs from Caco-2 monolayers incubated for 24 h with 100 nM OA, 100 nM DTX1, 100 nM DTX2, 1 μM staurosporine, 100 mM sodium butyrate and 0.005% Triton X-1000, respectively. The images are representative of three independent experiments. Image magnification is 40×. The scale bar is 25 μm. Toxins 2014, 6 216 Figure 4. Effects of OA, DTX1 and DTX2 on the trans-epithelial electric resistance (TEER) of differentiated Caco-2 monolayers. Figure shows the TEER ratio between toxin-treated monolayers and control monolayers. (A,D,G) Monolayers incubated with 10, 50 and 100 nM OA; (B,E,H) 10, 50 and 100 nM DTX1; and (C,F,I) 10, 50 and 100 nM DTX2. (*) Indicates statistically different value (p < 0.05) versus time 0 h. Reported values are the mean ± SEM of three independent experiments performed in duplicate. 2.3. Confocal Microscopy Imaging for Visualizing Occludin in Caco-2 Cells Treated with OA, DTX1 and DTX2 The observed decrease of TEER in the Caco-2 cells when incubated with 50 nM DTX1 for more than 12 hours suggests some damage to the monolayer that has been often linked to tight-junction disruption. Tight-junction strands are composed of membrane integral proteins, occludin being the best-studied member. We evaluate the integrity of such tight-junctions in Caco-2 cells treated with 100 nM OA, DTX1 and DTX2 for 24 h by labeling occludin. Immunofluorescent staining in control conditions (Figure 5A) showed occludin located mainly at the cell border. Neither 100 nM OA nor 100 nM DTX2 treatments (Figure 5B,D) had a significant effect on occludin location. However, Caco-2 cells incubated with 100 nM DTX1 presented fragmented and less uniform staining of occludin that even forms small vesicles at some points and showed marked indentations between adjacent cells. (Figure 5C). Toxins 2014, 6 217 Treatment with 0.005% Triton X-100, 100 mM NaBT and 1 μM STP also for 24 h, which disrupted the barrier function, resulted in a redistribution of occludin with adjacent diffuse intracellular staining and a granular appearance (Figure 5E–G). Figure 5. Effects of OA, DTX1 and DTX2 on occludin distribution in differentiated Caco-2 monolayers. Occludin from Caco-2 monolayers were labeled with anti-occludin, mouse monoclonal antibody (Alexa Fluor® 488). (A) Control monolayers and monolayers incubated with (B) 100 nM OA; (C) 100 nM DTX1; (D) 100 nM DTX2; (E) 1 μM staurosporine; (F) 100 mM sodium butyrate; and (G) 0.005% Triton X-1000 for 24 h. Representative images of three independent experiments. Image magnification is 40×. The scale bar is 25 μm. 2.4. Permeability of Caco-2 Monolayers to OA, DTX1 and DTX2 With the aim of evaluating the transport of OA, DTX1 and DTX2 through the cell monolayer, we performed the permeability assays as described in Section 4.7. Caco-2 cell monolayers were incubated with OA, DTX1 and DTX2 for 3, 6, 12 and 24 h. Figure 6 shows the amount of toxin quantified in Toxins 2014, 6 218 samples taken from the apical (insert) and basolateral (well) side of the monolayer. According to the results, almost all the OA and DTX2 remain in the apical side (Figure 6 A,D and C,F). Thus, the toxins were unable to cross the Caco-2 monolayer under the experimental conditions used. Despite this, Caco-2 monolayers incubated with 100 nM DTX1 showed an increase in the amount of toxin in the basolateral side after 24 h of incubation (Figure 6E). DTX1 showed the highest permeability concurrent with the highest decrease in transepithelial electrical resistance (TEER) (Figure 4H). Likewise, in the absence of a cell monolayer (shown as no cells in Figure 6), all the toxins easily flow over inserts. Figure 6. Permeability of differentiated Caco-2 monolayers to OA, DTX1 and DTX2. Toxin content in samples taken from wells and inserts with or without Caco-2 monolayers was quantified. The cells were incubated with 50 and 100 nM of (A,D) OA; (B,E) DTX1; and (C,F) DTX2. White bars show the amount of toxin in the well/receiver compartments, while black bars represent the toxin found in the inserts/donor compartments. The grey bars indicate the amount of toxin initially used in the treatments. Reported values are the mean ± SEM of three independent experiments performed in duplicate. 3. Discussion At present, the Caco-2 cell line is one of the best available models of human enterocytes in vitro, which may represent in vivo intestinal epithelium well [25]. Despite their tumoral origin, these cells are able to spontaneously differentiate to small intestine enterocytes after 21 days of culture, expressing, for instance, the brush border enzyme activities [26]. In the present study, we have evaluated the cytotoxic responses of differentiated Caco-2 cells to OA and compared to the cytotoxicity of DTX1 Toxins 2014, 6 219 and DTX2. OA cytotoxicity was not detected in the viability assays (Figure 2), in agreement with the data of Tripuraneni et al. [27] and Ehlers et al. [28]. In addition, our data are in accordance with Okada et al. [29], who detected no cell membrane damage nor LHD release with 1–2 μM OA. On the other hand, treatments with DTX1 and DTX2 up to 1 μM for 72 h did not show noticeable cytotoxic effects on differentiated Caco-2 cells. Moreover, we observed no significant apoptotic induction on cells treated with 100 nM OA, DTX1 and DTX2 for 24 h (Figure 3). When Caco-2 cells are cultured on inserts, they display the typical properties of an epithelial barrier, allowing for studies of permeability [30]. In these studies, TEER is a measure of the ionic movement across the paracellular pathway that can provide an indirect assessment of tight-junctions establishment and stability. Tight-junctions prevent the passage of molecules and ions through the spaces between cells. Tripuraneni et al. [27] found that high concentrations of OA (≥600 nM) decreased the TEER of Caco-2 monolayers, disrupting the barrier function of intestinal cells and increasing paracellular permeability in the absence of any noticeable cytotoxic response. However, Ehlers et al. [28] reported a lack of OA effect on the integrity of the monolayer based on the absence of TEER changes when differentiated Caco-2 monolayers were treated with up to 200 nM OA for 24h. Our results revealed that even though OA and DTX2 did not induce any change (Figure 4A,D,G and 4C,F,I), 50 nM DTX1 started to decrease TEER values, lowering them to 50% after 12 h treatment with 100 nM DTX1 and to 70% after 24 h (Figure 4H). This suggests a clear effect of DTX1 on the integrity of Caco-2 monolayers when compared to the same concentrations of OA and DTX2. Nevertheless, as we could observe, a decrease in TEER does not necessarily imply cellular toxicity. Okada et al. in 2000 [29] also found a significant TEER reduction in Caco-2 cells in just 30 to 90 min, but with concentrations of 1 to 2 μM of OA and without cell damage or death. Wang et al. observed notable damage to the intestinal ultrastructure after oral administration of OA to mice, which was repaired within 24 h, which also indicate no cell death [31]. Thus, one of the possible reasons for the decrease of TEER in DTX1-treated Caco-2 monolayers could be the damage of tight-junctions among cells. Tight-junctions form the physical barrier to the diffusion of substances through the paracellular space [32]. Their structure consists of assemblies of trans-membrane proteins, mainly claudins and occludin [33,34], which are associated with scaffolding proteins of the zonula occludens family [35] and signaling proteins, such as protein kinases and phosphatases [36,37]. It has also been reported in the involvement of peri-junctional actin and myosin light chains in the regulation of the barrier functions of tight-junctions [33,38]. Phosphorylation and dephosphorylation processes on components of the tight-junctions, especially on occludin, have a high impact on paracellular permeability, where PP2A and PP1 seem to be key enzymes [39–41]. Seth et al. [39] reported that protein phosphatase inhibitors and reduced expression of PP2A and PP1 accelerated the calcium-induced increase in transepithelial electrical resistance and the barrier to inulin permeability and also enhanced the junctional organization of occludin and ZO-1 during tight-junction assembly in Caco-2 cells. In this context and based on our results, apart from the previously reported lack of a TEER decrease, we observed no modifications on the distribution of occludin when cells were incubated with OA and DTX2 (Figure 5B,D). 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