Intestinal transport of Cylindrospermopsin using the Caco-2 cell line

1 Intestinal transport of Cylindrospermopsin using the Caco-2 cell line Silvia Pichardo1, Vicenta Devesa2, María Puerto1, Dinoraz Vélez2, Ana M. Cameán1 1 Area of Toxicology, Faculty of Pharmacy, University of Seville, Profesor García González n°2, 41012 Seville. Spain. 2 Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Av. Agustín Escardino 7, 46980 Paterna (Valencia), Spain. *Corresponding author: Silvia Pichardo Sánchez Area of Toxicology, Faculty of Pharmacy, University of Seville, Profesor García González n°2, 41012 Seville. Spain. E-mail address: spichard[email protected] Tel: +34 954 556762 Fax: +34 954 556422 2 Abstract Cylindrospermopsin (CYN) is a cyanotoxin produced by various cyanobacterial species. It is a water soluble zwitterion, stable at extreme temperatures and pH. Despite the main route of exposure to CYN is through drinking water and food, there is a lack of data concerning its intestinal absorption and the mechanisms implicated. The aim of this study was to characterize the mechanisms involved in the intestinal absorption of CYN, using Caco-2 human cell line as a model of the intestinal epithelium. The results obtained in the present work increases the limited knowledge regarding CYN transport across the intestinal epithelium and identifies the paracellular route as an important pathway in CYN absorption. A minor carrier-mediated transcellular transport has been evidenced. This transport is not affected by low temperatures, suggesting that an active mechanism is not involved. Moreover, the transport through the intestinal monolayer is H+ and GSH dependent and Na+ independent. The transport characteristics elucidated in this study prepare the ground for future studies directed at identifying transporters involved in the intestinal absorption of this toxin. Keywords: Cylindrospermopsin; Caco-2; intestinal transport; glutathione, biliar salts. 3 1. Introduction Cylindrospermopsin (CYN) is a cyanotoxin produced by a number of cyanobacterial species including Cylindrospermopsis raciborskii (Ohtani et al., 1992), Umezakia natans (Terao et al., 1994), Aphanizomenon ovalisporum (Shaw et al., 1999), Raphidiopsis curvata (Li et al., 2001), Anabaena bergii (Stüken et al., 2006), Aphanizomenon flosaquae (Preussel et al., 2006), and Anabaena lapponica (Bazin et al., 2010). The toxin structure consists of a tricyclic guanidine linked to hydroxymethyluracil group (Ohtani et al., 1992). CYN is a zwitterion highly water soluble (Sivonen and Jones, 1999), being stable to extreme temperatures and pH (Chiswell et al., 1999). Exposure to CYN is likely to happen since the 90% of this cyanotoxin is released from cyanobacterial cells to the water (Chiswell et al., 1999; Rücker et al., 2007). Human can be intoxicated by this cyanotoxin mainly by the oral route through the ingestion of contaminated drinking water (Kuiper-Goodman et al., 1999; Gutiérrez- Praena et al., 2012) and also by food contaminated with CYN (Kittler et al., 2012; Gutiérrez-Praena et al., 2013). Two severe human poisoning events have been reported. In North Queensland (Australia), a bloom of Cylindrospermopsis raciborskii in a drinking water reservoir resulted in human poisoning undergoing damage in liver and kidney (Hawkins et al., 1985). Also, in the Brazilian dialysis clinic tragedy in 1996, the presence of CYN and microcystins in dialysis water resulted in more than 50 deaths (Carmichael et al., 2001). Despite the oral route is the main pathway of human exposure to CYN, there is a lack of data concerning the intestinal absorption of this cyanotoxin. Regarding the mechanisms of transport implicated in the absorption, due to the small size of the molecule, a limited passive diffusion has been suggested (Runnegar et al., 2002). In addition, some studies have indicated the participation of a facilitated transport mechanism (Froscio et al., 2009) and an active transport, the latter making use of bile acid transport systems (Chong et al. 2002). In order to better understand the mechanisms of action of this toxin and to 4 attempt to reduce these toxic effects, it is necessary to characterize its intestinal absorption. The study of the intestinal absorption can be addressed using in vivo or in vitro models. However, due to the ease and accuracy of in vitro models, they are extensively used, being the most employed the Caco-2 cell line (Rocha et al., 2013). These cells are derived from a colon adenocarcinoma which differentiate spontaneously after 14-15 days of culture (Calatayud et al., 2011). When Caco-2 cells are differentiated they exhibit morphological and functional features of mature enterocytes (Hidalgo et al., 1989), and they can even express some transporter also present in the human small intestine (Maubon et al., 2007). All these features have converted Caco-2 cells into a suitable model for studying the mechanisms of intestinal absorption. Indeed, this model is currently used for in vitro study of absorption of pharmaceuticals (Sevin et al., 2013; Nohr et al., 2014) and minerals (Ca, Fe, Zn) (Calatayud et al., 2011); however, it is not extensively used in the study of absorption and bioavailability of cyanotoxins. The use of Caco-2 monolayers has been practically restricted to microcystins (Zeller et al., 2011; Henri et al., 2014), and there is only an approach to the evaluation of the intestinal permeability of CYN (Fernandez et al., 2014). Considering the lack of data in this field, the present work aimed to investigate the mechanisms involved in the intestinal absorption of CYN using Caco-2 human cells as a model of intestinal epithelium. In order to address this objective, transport assays were carried out both in absorptive and secretory directions. It has been evaluated the participation of paracellular and transcellular (active or passive) pathways, analysing the influence of parameters such as concentration of H+, Na+ and GSH. Taking into account the information obtained in these assays and the data from literature, the participation of some transporters has also been investigated by means of assays of competitive inhibition using natural ligands of these transporters (taurocholate and L-carnitine). 5 2. Materials and methods 2.1. Cyanotoxin standard. The cyanotoxin cylindrospermopsin standard (purity > 95%) was supplied by Alexis Corporation (Lausen, Switzerland). Stock solution of CYN (100 µg/mL) was prepared in sterilized milliQ water and maintained at 4oC until analysis. 2.2. Culture of Caco-2 cells. Caco-2 cell line, derived from a human colon carcinoma, was acquired from the American Type Culture Collection (ATCC® HTB-37, Manassas, VA, USA). Cells were cultured in 75 cm2 flasks using Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (4.5 g/L) at pH 7.2, supplemented with 10% (v/v) of heat-inactivated fetal bovine serum, 1% (v/v) non-essential amino acids, 1 mM sodium pyruvate, 10 mM HEPES (N- 2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), 100 U/ml of penicillin, 0.1 mg/ml of streptomycin and 0.0025 mg/l of amphotericin B (DMEMc); and incubated at 37º C and 5% flow of CO2. The medium was changed every 2–3 days. When the cell monolayer reached 80% confluence, they were detached with a solution of trypsin (0.5 mg/mL) and EDTA (ethylene diamine tetraacetic acid, 0.2 mg/mL) and reseeded at a density of 5×104 cells/cm². The cells were used between passages 21 and 34. All the reagents used were acquired from Hyclone (Fisher, Spain). For CYN transport assays, Caco-2 cells were seeded on 6-well plates with inserts with a polyester membrane (diameter 24 mm, pore size 0.4 µm; Transwell, Corning, Cultex, Spain). The porous support of the insert in which Caco-2 cells were seeded separates the well into two compartments: apical (upper) and basolateral (lower). The apical (A) compartment represents the intestinal lumen and the basolateral (B) compartment the serous compartment (blood or lymphatic vessels). The cells were seeded at a density of 7.5×104 cells/cm² and they were supplemented with 1.5 mL of DMEMc in the apical chamber and 2 ml of DMEMc in the basolateral chamber. The medium was changed 6 every 2–3 days until cell differentiation was achieved (14-15 days post seeding). The formation of Caco-2 monolayers was evaluated during the differentiation by measuring transepithelial electrical resistance (TEER) using a Millicell-ERS voltohmmeter (Millipore Corp, Madrid, Spain). The cells were considered suitable for the assays when the TEER values were greater than 250  cm2. 2.3. Cell viability assays. The effect of various concentrations of CYN on the viability of Caco-2 cells was evaluated by using sodium resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt, Sigma). The cells were seeded at a density of 2.5×104 cells/cm2 in 24-well plates and supplemented with 1 mL of DMEMc. After differentiation took place, cells were exposed to various concentrations of CYN (0.8, 2, 5, 10, 20 µg/mL prepared in DMEMc without fetal bovine serum) for 24 and 48 h. After exposure, the medium was withdrawn and the culture was washed with phosphate buffered saline (PBS, Hyclone). Then 500 µL of resazurin solution (10 µg/mL in DMEMc without serum) were added and it was incubated for 2 h at 37°C, 5% CO2 and 95% relative humidity. A volume of 100 µL for each condition studied was transferred to a 96-well plate and resazurin reduction was measured colorimetrically (570 and 600 nm) using a PowerWave HT microplate scanning spectrophotometer (BioTek Instruments, USA). 2.4. Transport assay: Calculation of permeability coefficients (Papp). The transport assay was performed in Hanks’ balanced salt solution with NaCO3 (HBSS) (Hyclone) supplemented with 10 mM of HEPES (pH 7.2). The study was conducted in apical-basolateral direction (A-B), absorptive direction, and in the basolateral-apical direction (B-A), the secretory one. The standard solutions of CYN (0.8 µg/mL) prepared in HBSS-10 mM HEPES were added to the donor compartment (apical or basolateral, depending on the transport direction). At the established times (15, 30, 45, 60, and 120 7 min), the contents of the acceptor compartment were totally removed and replaced by an equal volume of fresh medium. CYN was determined in aliquots of the acceptor medium removed at each time and in the donor medium removed at the end of the experiment following the protocol described in section 2.12. The apparent permeability coefficients (Papp) were calculated by using equation 1. Papp = (dC/dt) (Vr/ACo) (Eq.1) where: dC/dt is the flow (µg/s) determined by the linear slope of the equation that governs the variation in the concentrations of CYN, corrected with dilution, against time. Vr is the volume of the acceptor compartment (2 mL in A-B direction or 1.5 mL in B-A direction). A is the surface occupied by the cell monolayer (4.67 cm2). Co is the initial concentration of CYN in the donor compartment (µg/mL). The efflux ratio (Er) was calculated using equation 2, Er = Papp(B-A)/Papp(A-B) (Eq. 2) where: Papp(B-A) is the apparent permeability coefficient in the basolateral-apical direction (cm/s). Papp(A-B) is the apparent permeability coefficient in the apical-basolateral direction (cm/s). 2.5. Study of paracellular transport of CYN. The participation of the paracellular pathway in transport of CYN was evaluated by modulating the cell junctions in both directions (A-B and B-A). For this purpose, the cell monolayer was incubated with 5 mM EDTA in PBS free of Ca2+ and Mg2+ (Hyclone) for 5 min. Then, the standard of CYN (0.8 mg/L), prepared in medium consisting of 50% of 8 HBSS free of Ca2+ and Mg2+ (Hyclone) and 50% of HBSS with Ca2+ and Mg2+, both supplemented with 10 mM HEPES, was added to the apical compartment. The acceptor medium was collected at various times (15, 30, 45, 60 and 120 min) and the concentration of CYN was determined in order to evaluate the Papp in both directions (equation 1). At the same time, the efficiency of the EDTA in modulating the cell junctions was monitored by determining the Papp of Lucifer Yellow (LY) before and after the EDTA treatment. LY is a fluorescent compound transported mainly across intercellular junctions which evidence the integrity of the monolayer. 2.6. Effect of temperature on transport of CYN. The study of CYN transport at 4°C and 37°C was conducted in the A-B and B-A directions. For this study, 0.8 µg/mL of CYN prepared in HBSS supplemented with 10 mM HEPES was added to the donor compartment. At various times (30, 45, 60, 90 and 120 min), the medium was removed from the acceptor compartment and replaced with the same quantity of fresh medium. The concentration of CYN was quantified in the aliquots that were removed and the Papp (equation 1) was determined. 2.7. Effect of pH on permeability of CYN. The effect of pH on transport of CYN (0.8 µg/mL) in the A-B direction was studied with a pH in the acceptor/donor compartments of 5.5/7.2 and then compared with the assays previously made at pH 7.2/7.2. The medium at pH 5.5 was prepared with HBSS supplemented with 20 mM o-2-(N-morpholine) ethanesulfonic acid (MES, Sigma), whereas the medium at pH 7.2 was prepared with HBSS supplemented with 10 mM HEPES. At various times (30, 45, 60, 90 and 120 min) the acceptor medium was collected and, after determining the concentration of CYN, the Papp was calculated (equation 1). 9 2.8. Influence of Na+ ions and GSH upon transport of CYN. The influence of Na+ ions was analyzed in A-B direction. A control medium was prepared [10 mM HEPES, 130 mM NaCl (Panreac), 10 mM KCl (Panreac), 1 mM MgSO4 (Sigma), 5 mM glucose (Panreac) and 1 mM CaCl2 (Panreac)], together with a medium without NaCl in which the latter salt was replaced by an equimolar concentration of choline chloride [(CH3)3N(Cl)CH2CH2OH, Sigma]. The CYN treatment (0.8 mg/L) was prepared in control medium or in medium without NaCl, and was added to the apical compartment (1.5 mL). Control medium (2 mL) was added to the basolateral compartment. The influence of GSH upon the transport of CYN in the A-B direction was evaluated by co-exposing Caco-2 cells to 0.8 mg/L CYN and GSH (Sigma) at various concentrations (0.1, 1, 10 mM) prepared in HBSS with 10 mM HEPES. After 120 min, apical and basolateral media were collected and the concentration of CYN was determined (section 2.12). 2.9. Inhibition studies of CYN transport with taurocholate and L-carnitine. The inhibition assays were carried out at pH 7.2 for L-carnitine and at pH 5.5 on the apical side for taurocholate. The cell monolayer was preincubated with the corresponding inhibitor added to the apical compartment: 0.2mM sodium taurocholate or 0.012 mM L- carnitine (Sigma, Spain). Then, without eliminating the inhibitor, we added the standard solutions of CYN (0.8 mg/L final concentration). At various times (30, 60 and 90 min), the medium was removed from the acceptor compartment and replaced with the same quantity of fresh medium. The concentration of CYN was quantified in the aliquots that were removed and the Papp (equation 1) was determined. 2.10. Monolayer integrity during the transport assays. Monolayer integrity was verified by measuring TEER at different times and determining the apparent permeability coefficient (Papp) of LY. This compound was added at a 16 for a better understanding of risks that these compounds can exert after intake. In order to explore the intestinal uptake of compounds, in vitro studies provide an interesting alternative to in vivo studies (Calatayud et al., 2011). There are several studies that correlate data of apparent permeability coefficients obtained in vitro using Caco-2 cell model with the human intestinal absorption rate (Artursson and Karlsson, 1991; Yee, 1997). Hence, the evaluation of this parameter can provide us preliminary information of what might be occurring in vivo, although in many cases, extrapolation is difficult (Tavelin et al., 2003). According to Yee (1997), Papp < 1×10-6 cm/s in Caco-2 cells indicates a low in vivo absorption (0-20%), Papp between 1-10×10-6 cm/s suggests a moderate absorption (20- 70%), and Papp > 10×10-6 cm/s reveals a high in vivo absorption (70-100%). Comparing the results obtained in the present work for CYN in the A-B direction using Caco-2 cells, the absorption in vivo would be generally low (<20%). This fact corroborates the results obtained previously by Fernández et al. (2014) using the same cellular model, reporting a limited passage of the toxin through the intact intestinal epithelium. Since no toxicokinetic data performed on laboratory animals is available, the results obtained in the present work cannot be compared with any in vivo data. Solute transport through the intestinal epithelium can occur by two pathways: transcellular or paracellular through intercellular junctions. Paracellular transport involves only passive diffusion mechanism, whereas transcellular transport can be mediated by passive, facilitated or active processes (Sugano et al., 2010). The studies performed in the present study show a significant increase in the permeability of CYN with the opening of intercellular junctions, especially in absorptive direction, indicating that this toxin is transported by paracellular pathway. The increases in the transport of CYN observed after opening the paracellular passage are similar or higher to those observed for LY, compound mainly transported through this route. This finding suggests that this pathway is important in the transport of CYN across the intestinal epithelium. 17 This would also explain why the values of Papp of CYN in absorptive direction deduced using Caco-2 cells are low. The monolayer formed by Caco-2 cells has tighter cell junctions than those in the human small intestine (Tavelin et al., 2003), and therefore it may not be the best model for studying the bioavailability of substances that move paracellularly, since underestimation of the intestinal transport could be observed. Besides the paracellular transport, a transcellular pathway could also participate in CYN transport. The evaluation of the possible participation of transcellular transport by passive diffusion using artificial lipid membranes shows that this type of transport does not occur or is negligible (section 3.7). This was an expected finding considering the hydrophilic nature of this cyanotoxin. In the case of the transcellular transport mediated by carrier (active or facilitated), initially the assays were focused on the assessment of the participation of active transport mechanisms. Low temperatures applied to cellular systems are considered general inhibitors of metabolism and ATP generation, and hence, of active transport (Calatayud et al., 2011). The study of CYN transport at low temperatures shows differences with respect to the data obtained at 37° C only in secretory direction (B-A) (Figure 3), suggesting the possible involvement of a transcellular transport energy-dependent in this direction, a fact that corroborates the deductions derived from the efflux ratio analysis (section 3.1). However, the data do not support the presence of an active transport in absorptive direction, which could be involved in CYN transport but in a limited extent. Chong et al (2002) suggested that CYN and bile acids share transport mechanisms since primary cells of murine liver showed lower cytotoxicity to CYN when co-exposed with bile acids, taurocholate and cholate. In the intestine, particularly in the apical membrane of the small intestine, there are several bile acid transporters: ASBT (SLC10A2) and different isoforms of OATP transporter family (Trauner and Boyer, 2002; Alrefai and Gill, 2007). The ASBT transporters participate in a sodium-dependent transport of bile salts (Dawson, 2011). The present study has revealed no influence of Na+ ions in the transport 18 of CYN (section 3.5, data not shown), which could discard the participation of ASBT in the uptake of CYN through the apical domain. Concerning OATPs, several isoforms have been described in the apical membrane of the intestinal epithelium: OATP1A2 or OATP-A and OATP2B1 or OATP-B (Roth et al., 2012). This family of carriers performs a transport ATP- and Na+-independent (Roth et al., 2012). Some studies has reported the influence of GSH in the transport mediated by these carries. Physiologic GSH efflux stimulates OATP1-mediated substrate uptake into hepatocytes as well as into choroid plexus epithelial cells (Gao et al., 1999). Similar to OAPT1, OATP2-mediated taurocholate transport was stimulated by high intracellular GSH concentrations (Li et al., 2000). Furthermore, the optimum pH for the activity of OATP-B is around 5-5.5 (Kobayashi et al., 2003), is a H+-dependent transporter. Our results show that this type of transport (independent of Na+ and ATP, and dependent of H+ and GSH) could participate in the transport of CYN from the apical side. However, the participation of this carrier was not evidenced in the assays performed with taurocholate, substrate with high affinity for OATPB (Hagenbuch and Meier, 2003), since the presence of this bile acid does not reduce the permeability of CYN at acid pHs. Furthermore, Chong et al. (2002) showed that the protective effect of bile salts from CYN exposures was in a lesser extent than those observed for other toxins such as microcystin and lophyrotomin. Based on these results, the latter authors suggested that in addition to bile salts transporters, other mechanisms of transport of CYN may exist. It should be highlighted that CYN is a zwitterion and could enter the cell through the specific transporters for such molecules. The organic cation/zwitterion transporters (OCTNs) operate as uniporters which mediate facilitated diffusion or as Na+/zwitterion cotransporters (Koepsell, 2013). Again, Na+-dependent transport is discarded and the possibility of a facilitated transport mediated by OCTN rises. In the apical membrane of the intestinal epithelium the presence of various isoforms of OCTNs (OCTN1 and OCTN2) have been described, which mediate the transport of molecules such as L- 19 carnitine. The inhibition studies using this substrate do not reduce the A-B transport of CYN; therefore these carriers cannot be pointed as CYN transporters from the apical to basolateral side. Summarising all the data obtained in the present work, CYN transport in absorptive (A- B) and secretory (B-A) directions using Caco-2 as a model of intestinal epithelium is reduced. Paracellular passive diffusion is predominant in apical-basolateral direction. In the secretory transport the involvement of active transport (P-glicoprotein, multidrug resistance proteins) seems to be likely, although an important role of the paracellular pathway have also been evidenced. The characteristics of the cell model, reduced paracellular space and high levels of expression of P-glycoprotein, may be in part the reason of the low transport observed from the apical to the basolateral side. Further studies could be desirable using models with greater pore diameter in the intercellular space and with an expression level of efflux proteins closer to that of the human intestine, such as co-cultures Caco-2/HT29-MTX (Hilgendorf et al., 2000) or monocultures of NCM-460 (Sahi et al., 1998). In vivo assays are also needed to confirm the results derived from cell lines and to select the most suitable in vitro models for further studies. Regarding the mechanisms of transport, in addition to the paracellular transport, a minor transcellular transport has been evidenced in the apical-basolateral direction which could be H+ and GSH-dependent, facilitated (not dependent on energy) and Na+-independent. The attempts made to target a specific transporter (bile salts or zwitterions carriers) have been unsuccessful, possibly due to the accentuated paracellular route. The identification of transporters responsible of the transcellular passage of CYN may require other approaches, such as the use of Xenopus laevis oocytes microinjected or overexpression of transporters in certain cell types. 5. Acknowledgements 20 This work was funded by the Spanish ‘Ministerio de Economía y Competitividad’ through projects AGL2015-64558R and AGL2012-33461, co-financed by FEDER Funds. The authors wish to thank the Cell Culture Service and the Mass Spectrometry Service of CITIUS from the Universidad de Sevilla for technical support. The author also wish to acknowledge the European Cooperation in Science and Technology, COST Action ES 1105 "CYANOCOST- Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and management" for adding value to this study through networking and knowledge sharing with European experts and researchers in the field. 21 6. References Alrefai, W.A., Gill, R.K., 2007. Bile acid transporters: structure, function, regulation and pathophysiological implications. Pharmaceut. Res. 24, 1803-1823. Artursson, P., Karlsson, J., 1991. Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Commun. 175, 880-885. Bazin, E., Mourot, A., Humpage, A.R., Fessard, V., 2010. Genotoxicity of a freshwater cyanotoxin, Cylindrospermopsin, in two human cell lines: Caco-2 and HepaRG. Environ. Mol. Mutagen. 51, 251-259. Calatayud, M., Devesa, V., Montoro, R., Vélez, D., 2011. In vitro study of intestinal transport of arsenite, monomethylarsonous acid, and dimethylarsinous acid by Caco-2 cell line. Toxicol. Lett. 204, 127-133. Carmichael, W.W., Azevedo, S.M.F.O., An, J.S., Molica, R.J.R., Jochimsen, E.M., Lau, S., Rinehart, K.I., Shaw, G.R., Eaglesham, G.K., 2001. Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ. Health Persp. 39, 341-344. Chiswell, R.K., Shaw, G.R., Eaglesham, G., Smith, M.J., Norris, R.L., Seawright, A.A., Moore, M.R., 1999. Stability of cylindrospermopsin, the toxin from the cyanobacterium, Cylindrospermopsis raciborskii: effect of pH, temperature, and sunlight on decomposition. Environ. Toxicol. 14, 155-161. Chong, M.W., Wong, B.S., Lam, P.K., Shaw, G.R., Seawright, A.A., 2002. Toxicity and uptake mechanism of cylindrospermopsin and lophyrotomin in primary rat hepatocytes. Toxicon 40, 205-211. Dawson, P.A., 2011. Role of the intestinal bile acid transporters in bile acid and drug disposition. Handb. Exp. Pharmacol. 201, 169-203. 22 Fernández, D.A., Louzao, M.C., Vilariño, N., Fraga, M., Espiña, B., Vieytes, M.R., Botana, L.M., 2014. Evaluation of the intestinal permeability and cytotoxic effects of cylindrospermopsin. Toxicon 91, 23-34. Froscio, S.M., Cannon, E., Lau, H.M., Humpage, A.R., 2009. Limited uptake of the cyanobacterial toxin cylindrospermopsin by Vero cells. Toxicon 54, 862-868. Gan, L.S., Hsyu, P.H., Pritchard, J.F., Thakker, D., 1993. Mechanism of intestinal absorption of ranitidine and ondansetron: transport across Caco-2 cell monolayers. Pharmaceut. Res. 10, 1722-1725. Gao, B., Stieger, B., Noe, B., Fritschy, J.M., Meier, P.J., 1999. Localization of the organic anion transporting polypeptide 2 (Oatp2) in capillary endothelium and choroid plexus epithelium of rat brain. J. Histochem. Cytochem., 47: 1255-1264. Gutiérrez-Praena, D., Pichardo, S., Jos, A., Moreno, F.J., Cameán, A.M., 2012. Biochemical and pathological toxic effects induced by the cyanotoxin cylindrospermopsin on the human cell line Caco-2. Water Res. 46, 1566-1575. Gutiérrez-Praena, D., Jos, A., Pichardo, S., Moreno, I.M., Cameán, A.M., 2013. Presence and bioaccumulation of microcystins and cylindrospermopsin in food and the effectiveness of some cooking techniques at decreasing their concentrations: A review. Food Chem. Toxicol. 53, 139-152. Guzmán-Guillén, R., Prieto-Ortega, A.I., González, A.G., Soria-Díaz, M.E., Cameán, A.M., 2012. Cylindrospermopsin determination in water by LC-MS/MS: optimization and validation of the method and application to real samples. Environ. Toxicol. Chem. 31, 2233-2238. Hagenbuch, B., Meier, P.J., 2003. The superfamily of organic anion transporting polypeptides. BBA–Biomembranes 1609, 1-18. Hawkins, P.R., Runnegar, M.T., Jackson, A., Falconer, I.R., 1985. Severe hepatotoxicity caused by the tropical cyanobacterium (blue-green algae) Cylindrospermopsis 23 raciborskii (Woloszynska) Seenaya and Subba Raju isolated from a domestic water supply reservoir. Appl. Environ. Microb. 50, 1292-1295. Henri J, Huguet A, Delmas JM, Besson A, Sanders P, Fessard V. 2014. Low in vitro permeability of the cyanotoxin microcystin-LR across a Caco-2 monolayer: with identification of the limiting factors using modelling. Toxicon 91, 5-14. Hidalgo, I.J., Borchardt, R.T., 1990. Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco-2. Biochim. Biophys. Acta 1028, 25-30. Hilgendorf, C., Spahn-Langguth, H., Regårdh, C.G, Lipka, E., Amidon, G.L., Langguth, P., 2000. Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport. J. Pharm. Sci. 89, 63-75. Hubatsch, I., Ragnarsson, E.G.E., Artursson, P., 2007. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protocols 2, 2011-2019. Kansy, M., Senner, F., Gubernator, K., 1998. Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J. Med. Chem. 41, 1007-1010. Kittle, K., Schreiner, M., Krumbein, A., Manzei, S., Koch, M., Rohn, S., Maul, R., 2012. Uptake of the cyanobacterial toxin cylindrospermopsin in Brassica vegetables. Food Chem. 133, 875–879. Kobayashi, D., Nozawa, T., Imai, K., Nezu, J., Tsuji, A., Tamai, I., 2003. Involvement of human organic anion transporting polypeptide OATP-B (SLC21A9) in pH- dependent transport across intestinal apical membrane. J. Pharmacol. Exp. Ther. 306, 703-708. Koepsell, H., 2013. The SLC22 family with transporters of organic cations, anions and zwitterions. Mol. Aspects Med. 34, 413-435. 24 Kristl, A., 2009. Membrane permeability in the gastrointestinal tract: The interplay between microclimate pH and transporters. Chem. Biodivers. 6, 1923-1932. Kuiper-Goodman, T., Falconer, I., Fitzgerald, J., 1999. Human health aspects. in: Chorus, I. and Bartram, J. (Eds.), Toxic cyanobacteria in water. A guide to their public health consequences, monitoring and management. London, pp. 113-153. Li, L., Meier, P.J., Ballatori, N., 2000. Oatp2 mediates bidirectional organic solute transport: a role for intracellular glutathione. Mol. Pharmacol. 58(2), 335-340. Li, R., Carmichael, W.W., Brittain, S., Eaglesham, G.K., Shaw, G.R., Watanabe, M.M., 2001. First report of the cyanotoxins cylindrospermopsin and deoxycylindrospermopsin from Raphidiopsis curvata (Cyanobacteria). J. Phycol. 37, 1121-1126. Maubon, N., Le Vee, M., Fossati, L., Audry, M., Le Ferrec, E., Bolze, S., Fardel, O., 2007. Analysis of drug transporter expression in human intestinal Caco-2 cells by realtime PCR. Fundam. Clin. Pharmacol. 21, 659-663. Nohr, M.K., Hansen, S.T., Brodin, B., Holm, R., Nielsen, C.U., 2014. The absorptive flux of the anti-epileptic drug substance vigabatrin is carrier-mediated across Caco-2 cell monolayers. Eur. J. Pharm. Sci. 51, 1-10. Nugent, S., Kumar, D., Rampton, D., Evans, D., 2001. Intestinal luminal pH in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylates and other drugs. Gut 48, 571-577. Ohtani, I., Moore, R.E., Runnegar, M.T., 1992. Cylindrospermopsin: a potent hepatotoxin from the blue-green algae Cylindrospermopsis raciborskii. J. Am. Chem. Soc. 114, 7941-7942. Preussel, K., Stüken, A., Wiedner, C., Chorus, I., Fastner, J., 2006. First report on cylindrospermopsin producing Aphanizomenon flos-aquae (Cyanobacteria) isolated from two German lakes. Toxicon 47, 156-162. 25 Rocha, R.A., Devesa, V., Vélez, D., 2013. In vitro study of intestinal transport of fluoride using the Caco-2 cell line. Food Chem. Toxicol. 55, 156-163. Roth, M., Obaidat, A., Hagenbuch, B., 2012. OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies. Br. J. Pharmacol. 165, 1260-1287. Rücker, J., Stüken, A., Nixdorf, B., Fastner, J., Chorus, I., Wiedner, C., 2007. Concentrations of particulate and dissolved cylindrospermopsin in 21 Aphanizomenon-dominated temperate lakes. Toxicon 50, 800-809. Runnegar, M.T., Xie, C., Snider, B.B., Wallace, G.A., Weinreb, S.M., Kuhlenkamp, J., 2002. In vitro hepatotoxicity of the cyanobacterial alkaloid cylindrospermopsin and related synthetic analogues. Toxicol. Sci. 67, 81-87. Sahi, J., Nataraja, S.G., Layden, T.J., Goldstein, J.L., Moyer, M.P., Rao, M.C., 1998. Cltransport in an immortalized human epithelial cell line (NCM460) derived from the normal transverse colon. Am. J. Physiol. 275, C1048-1057. Sevin, E., Dehouck L., Fabulas-da Costa, A., Cecchelli, R., Dehouck, M.P., Lundquist, S., Culot, M., 2013. Accelerated Caco-2 cell permeability model for drug discovery. J. Pharmacol. Toxicol. Methods 68, 334-339. Shaw, G.R., Sukenik, A., Livne, A., Chiswell, R.K., Smith, M.J., Seawright, A.A., Norris, R.L., Eaglesham, G.K., Moore, M.R., 1999. Blooms of cylindrospermopsin containing cyanobacterium Aphanizomenum ovalisporum (Forti), in newly constructed lakes, Queensland, Australia. Environ. Toxicol. 14, 167-177. Sivonen, K., Jones, G., 1999. Cyanobacterial toxins. In: Chorus, I., Bartram, J. (Eds.), Toxic Cyanobacteria in Water: A Guide to their Public Health Consequences, Monitoring and Management. WHO, pp. 41-111. Stüken, A., Rücker, J., Endrulat, T., Preussel, K., Hemm, M., Nixdorf, B., Karsten, U., Wiedner, C., 2006. Distribution of three alien cyanobacterial species (Nostocales) 0 10 20 30 40 50 60 70 80 CYN CYN + 0.1 mM GSH CYN + 10 mM GSH ng CYN/mg protein * * Figure 5