Neurotoxic assessment of Microcystin-LR, cylindrospermopsin andtheir combination on the human neuroblastoma SH-SY5Y cell line

Accepted Manuscript Neurotoxic assessment of Microcystin-LR, Cylindrospermopsin and their combination on the human neuroblastoma SH-SY5Y cell line M.G. Hinojosa, A.I. Prieto, D. Gutiérrez-Praena, F.J. Moreno, A.M. Cameán, A. Jos PII: S0045-6535(19)30397-2 DOI: 10.1016/j.chemosphere.2019.02.173 Reference: CHEM 23285 To appear in: Chemosphere Received Date: 29 October 2018 Accepted Date: 24 February 2019 Please cite this article as: M.G. Hinojosa, A.I. Prieto, D. Gutiérrez-Praena, F.J. Moreno, A.M. Cameán, A. Jos, Neurotoxic assessment of Microcystin-LR, Cylindrospermopsin and their combination on the human neuroblastoma SH-SY5Y cell line, (2019), doi: 10.1016/j. Chemosphere chemosphere.2019.02.173 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 1Neurotoxic assessment of Microcystin-LR, Cylindrospermopsin and their 2combination on the human neuroblastoma SH-SY5Y cell line 3Hinojosa MG1, Prieto AI1, Gutiérrez-Praena D1*, Moreno FJ2, Cameán AM1, Jos A1 41 Área de Toxicología, Facultad de Farmacia, Universidad de Sevilla, C/Profesor García 5González 2, 41012 Sevilla, Spain. 62 Área de Biología Celular, Facultad de Biología, Universidad de Sevilla, Avda. Reina 7Mercedes s/n, 41012 Sevilla, Spain 8 9 10 11 12 13 14 15 16 17 18 19 20 * Corresponding author: Daniel Gutiérrez Praena. Área de Toxicología, Facultad de 21 Farmacia, Universidad de Sevilla, C/Profesor García González 2, 41012 Sevilla, Spain. 22 Mail: [email protected] ACCEPTED MANUSCRIPT 2 23 Abstract 24 Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN) are produced by 25 cyanobacteria. Although being considered as a hepatotoxin and a cytotoxin, 26 respectively, different studies have revealed neurotoxic properties for both of them. The 27 aim of the present work was to study their cytotoxic effects, alone and in combination, 28 in the SH-SY5Y cell line. In addition, toxicity mechanisms such as oxidative stress and 29 acetylcholinesterase (AChE) activity, and morphological studies were carried out. 30 Results showed a cytotoxic response of the cells after their exposure to 0-100 µg/mL of 31 MC-LR or 0-10 µg/mL CYN in both differentiated and undifferentiated cells. Thus, 32 CYN resulted to be more toxic than MC-LR. Respect to their combination, a higher 33 cytotoxic effect than the toxins alone in the case of undifferentiated cells, and almost a 34 similar response to the presented by MC-LR in differentiated cells were observed. 35 However, after analyzing this data with the isobolograms method, an antagonistic effect 36 was mainly obtained. The oxidative stress study only showed an affectation of 37 glutathione levels at the highest concentrations assayed of MC-LR and the combination 38 in the undifferentiated cells. A significant increase in the AChE activity was observed 39 after exposure to MC-LR in undifferentiated cells, and after exposure to the 40 combination of both cyanotoxins on differentiated cells. However, CYN decreased the 41 AChE activity only on differentiated cultures. Finally, the morphological study revealed 42 different signs of cellular affectation, with apoptotic processes at all the concentrations 43 assayed. Therefore, both cyanotoxins isolated and in combination, have demonstrated to 44 cause neurotoxic effects in the SH-SY5Y cell line. 45 46 47 Keywords: MC-LR, CYN, MC-LR+CYN combination, SH-SY5Y cells, neurotoxicity ACCEPTED MANUSCRIPT 3 48 1. Introduction 49 Cyanobacteria are present in a variety of aquatic and terrestrial ecosystems due 50 to their adaptive ability, even in extreme conditions (Svircev et al., 2014). Under 51 favorable conditions of light, pH, nutrients (nitrogen and phosphorus) and interaction 52 with other organisms, they present the capability of forming blooms and producing 53 secondary metabolites called cyanotoxins, whose occurrence is increasing due to long 54 term climate change (Buratti et al., 2017). According to their target organ, these toxins 55 can be classified as hepatotoxins (e.g. microcystins, nodularins), dermatotoxins (e.g. 56 lungbyatoxin), neurotoxins (e.g. anatoxin-a, homoanatoxin, saxitoxins), irritant toxins 57 (e.g. lipopolysaccharides) and cytotoxins (e.g. cylindrospermopsin) (Testai et al., 2016). 58 The exposure to these metabolites can occur by different paths such as the oral route, 59 dermal contact or inhalation, although the oral route is the most significant one, since 60 intoxication may take place by the intake of contaminated water, food or dietary 61 supplements based on algae (Buratti et al., 2017). Among all cyanotoxins, microcystins 62 (MCs) and cylindrospermopsin (CYN) have focused great interest, since they have been 63 involved in the death of different animal species and humans (Azevedo et al., 2002; 64 Bourke et al., 1983; Carmichael et al., 2001; Malbrouck and Kestemont, 2006). 65 Microcystins are cyclic heptapeptides synthetized by several cyanobacterial 66 species such as Mycrocistis aeruginosa, Oscillatoria agardhii, Plankthotrix agardii, and 67 Planktothris rubescens, etc. (Sivonen and Jones, 1999). Up to date, more than 246 68 congeners of MCs are known, being MC-LR the most potent congener and frequently 69 identified (Spoof and Catherine, 2017). Mainly considered as a hepatotoxin, MC-LR 70 can also affect other organs such as kidneys, heart or brain (Li et al., 2011; Qiu et al., 71 2009; Zeng et al., 2014, 2018). One of the main MC-LR-mechanisms of action is the 72 inhibition of protein serine/threonine phosphatases, causing a cascade of effects such as ACCEPTED MANUSCRIPT 4 73 the deregulation of phosphoproteins, which lead to tumor promotion and apoptosis 74 (MacKintosh et al., 1990). In addition, many studies have also demonstrated its in vitro 75 cytotoxic potential in different cell lines from fish, mammals and humans (Ding et al., 76 2017; Feurstein et al., 2009; Gutiérrez-Praena et al., 2012; Meng et al., 2011, 2013; 77 Pichardo et al., 2007; Rozman et al., 2017). Moreover, several authors have also 78 described that this toxin induces oxidative stress by increasing reactive oxygen species 79 (ROS) and reducing glutathione (GSH) levels, leading to cell apoptosis (Li et al., 2015; 80 Liu et al., 2016; Puerto et al., 2011; Qian et al., 2018), although these effects have not 81 been studied using human neuronal cell lines yet. 82 Cylindrospermopsin is an alkaloid consisting in a tricyclic guanidine combined 83 to a hydroxylmethyl uracil group. This toxin presents a highly water-soluble structure, 84 being commonly found out of the cells (Falconer and Humpage, 2006). Several 85 cyanobaterial species are able to produce this toxin, such as Cylindrospermopsis 86 raciborskii, Umezakia natans, Chrysosporum ovalisporum, Anabaena bergii, etc. 87 (Banker et al., 1997; Harada et al., 1994; Schembri et al., 2001; Shaw et al., 1999). The 88 main target of this cytotoxin is the liver, although kidneys, lungs, thymus, marrow bone, 89 adrenal gland, gastrointestinal tract, immune, heart and nervous system have been also 90 described as potential targets (Falconer et al., 1999; Guzmán-Guillén et al., 2015; 91 Hawkins et al., 1985; Humpage et al., 2000; Terao et al., 1994). The most well-known 92 mechanism of action of CYN is the inhibition of protein and GSH synthesis (Froscio et 93 al., 2003; Runnegar et al., 1995; Terao et al., 1994). This cyanotoxin also enhances ROS 94 production, which could lead to apoptosis or DNA damage (Gutiérrez-Praena et al., 95 2011, 2012; Guzmán-Guillén et al., 2013; Puerto et al., 2011). In addition, some studies 96 have indicated the pro-genotoxic properties of CYN, being essential its previous ACCEPTED MANUSCRIPT 5 97 metabolic activation by the enzymatic cytochrome P-450 complex (CYP450) (Humpage 98 et al., 2005; Puerto et al., 2018; Žegura et al., 2011). 99 Both cyanotoxins have evidenced to induce neurotoxic effects in different 100 experimental models (Florzyck et al., 2014). Thus, MCs have shown to cause neuronal 101 damage in vitro in different rodent cell lines such as primary murine cerebellar granule 102 neurons (CGNs) and primary rat astrocytes (Feurstein et al., 2011; Rozman et al., 2017). 103 Furthermore, many in vivo studies have manifested a clear neurotoxic potential, mostly 104 of MC-LR, in different animal species such as rodents, fish and nematodes, affecting to 105 their behavior, enhancing ROS levels, and modifying proteins related to 106 neurodegenerative diseases (Baganz et al., 2004; Wang et al., 2013; Wu et al., 2017). In 107 fact, MC-LR has produced pathological damage in hippocampus, neuronal degenerative 108 changes, inflammation in memory-related brain regions and apoptosis in rats, 109 suggesting that this toxin can be related to Alzheimer´s disease in humans (Li et al., 110 2012a; 2012b; 2014). Actually, some studies confirm its transport through the blood- 111 brain-barrier using OATP1A2, a variant of the organic anion transport system (OATP), 112 because of its relatively large hydrophilic structure (Feurstein et al., 2009; Fischer et al., 113 2005; Menezes et al., 2013). On the contrary, neurotoxic effects of CYN are still not 114 well elucidated. In this sense, there is only one report studying its effects in different in 115 vitro murine cell lines (Takser et al., 2016). Meanwhile, some in vivo reports have 116 showed in vivo neurotoxic effects in snails, tadpoles and fish, such as behavioral 117 alterations or histopathological changes (da Silva et al., 2018; Guzmán-Guillén et al., 118 2015; Kinnear et al., 2007; Kiss et al., 2002; White et al., 2007). 119 Moreover, it is worthy to point out that the majority of studies concerning 120 cyanotoxins toxicity are focused on single purified toxins, setting apart the fact that 121 organisms are exposed simultaneously to a wide variety of cyanotoxins when they are ACCEPTED MANUSCRIPT 6 122 present in aquatic systems. In fact, several studies have described the concomitant 123 occurrence of MCs- and CYN-producing cyanobacteria, as well as the presence of both 124 cyanotoxins at the same time (Bittencourt-Oliveira et al., 2014; Bogialli et al., 2006; 125 Oehrle et al., 2010; Vasas et al., 2004). In the case of neurotoxicity, only a study 126 conducted by Takser et al. (2016) showed the effects of a combination of MCs and 127 CYN, although they also included the neurotoxin anatoxin-a in the combination (1:1:1). 128 The SH-SY5Y cell line is a commonly used neuronal model due to its 129 biochemical and functional properties, being very appropriate for neurotoxicity studies. 130 Moreover, the differentiation of this cell line provides functional, biochemical and 131 morphologically mature neurons, which are more similar to those present in the human 132 brain (Xie et al., 2010). For this reason, both types of SH-SY5Y cells are a very 133 interesting experimental model to assess the possible damage induced by MC-LR and 134 CYN in human neural cells. 135 Thus, considering all this, the aim of the present study was to assess the 136 neurotoxic potential of MC-LR, CYN and their combination in vitro using the human 137 neuroblastoma SH-SY5Y cell line, by exploring the cell viability, oxidative stress (ROS 138 and GSH levels), acetylcholinesterase (AChE) activity and morphological changes after 139 the exposure to these cyanotoxins. 140 2. Materials & Methods 141 2.1. Supplies and chemicals 142 MC-LR and CYN (both purity > 95% by HPLC) were purchased from Enzo Life 143 Sciences. Minimum essential medium (MEM), cell culture reagents, and fetal bovine 144 serum (FBS) were obtained from Gibco (Biomol, Sevilla, Spain). Nutrient Mixture F-12 ACCEPTED MANUSCRIPT 7 145 Ham, retinoic acid (RA), and brain-derived neurotrophic factor human (BDNF) were 146 purchased in Sigma-Aldrich (Madrid, Spain). 147 The MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- 148 sulphophenyl)-2H-tetrazolium salt) Cell Titer 96® AQueous One Solution Cell 149 Proliferation Assay was purchased in Promega (Biotech Iberica, Madrid, Spain). The 150 Bradford Reagent and the neutral red (NR) were purchased from Sigma-Aldrich 151 (Madrid, Spain). 152 2.2. Model system 153 SH-SY5Y cells derived from a human neuroblastoma were obtained from ATCC 154 (CRL-2266). They were maintained at 37˚C in an atmosphere containing 5% CO2 at 155 95% relative humidity (CO2 incubator, NuAire®, Spain) in a medium consisting in 156 MEM and F-12 (1:1) supplemented with 10% FBS, 1% non-essential amino acids, 1% 157 sodium piruvate, 1% L-glutamine 200 mM, and 1% penicillin/streptomycin solution. 158 Cells were grown near confluence in 75-cm2 plastic flasks and harvested weekly with 159 0.25% trypsin-EDTA (1X). Cells were quantified in a Neubauer chamber. SH-SY5Y 160 cells were plated at density of 2·105 cells/mL to perform all the experiments. 161 2.3. Cell differentiation 162 SH-SY5Y cells were differentiated using the protocol provided by Encinas et al. 163 (2000) with some modifications. Cells were plated at density of 5·102 cells/mL in plates 164 of 48 wells, changing the medium every 48 hours with 1% of FBS, 10 µM RA and 50 165 ng/mL BDNF, for a week. The differentiation process was evaluated by morphological 166 analysis. 167 2.4. Toxin test solutions ACCEPTED MANUSCRIPT 8 168 Stock solutions of 4 mg/mL MC-LR and 1 mg/mL CYN were prepared in 169 absolute ethanol and sterilized milliQ water, respectively. Both solutions were 170 maintained at -20˚C until their use. 171 2.5 Cytotoxicity assays 172 Undifferentiated SH-SY5Y cells were seeded in 96-well tissue-culture plates for 173 basal cytotoxicity tests and incubated at 37˚C for 24 h prior to exposure. Differentiated 174 cells were exposed after a week from the start of the differentiation process in the same 175 48-well plates where the differentiation process took place. From the stock solution of 176 MC-LR, serial dilutions in medium without serum were prepared (20, 40, 60, 80, 100 177 µg/mL MC-LR). From the stock solution of CYN, serial dilutions in medium without 178 serum were prepared (0.1, 0.2, 0.3, 0.4, 0.5, 0.75, 1, 2.5, 5, 10 µg/mL CYN). Vehicle 179 control (ethanol) for MC-LR and a negative control (non-treated cells) were also 180 included. After replacing the medium, exposure solutions were added to the plates, and 181 incubated at 37°C for 24 and 48 h. The basal cytotoxicity endpoints assayed were 182 protein content (PC), supravital dye neutral red cellular uptake (NR), and tetrazolium 183 salt reduction (MTS). All the assays in the present paper were performed by triplicate. 184 Total protein content (PC) was quantified in situ, according to the procedure 185 given by Bradford (1976), with modifications (Pichardo et al., 2007), in the same plates 186 where exposure originally took place. The culture medium was replaced by 200 μL 187 NaOH to dissolve the proteins and after 2 h of incubation at 37°C, 180 μL were 188 replaced by the same volume of Bradford reagent. After 30-min incubation at room 189 temperature, absorbance was read at 620 nm. 190 Neutral red uptake was performed according to Borenfreund and Puerner (1984) 191 in the undifferentiated cells. The culture medium was replaced by 100 μL modified ACCEPTED MANUSCRIPT 15 333 A significant increase of AChE activity on undifferentiated SH-SY5Y cells was 334 observed only after the highest concentration of MC-LR assayed (37 µg/mL). However, 335 neither CYN nor its combination with MC-LR induced significant changes in this 336 enzymatic activity (Fig. 8A). 337 In the case of the differentiated cells, a significant decrease of AChE activity 338 was shown after exposure to all CYN-concentrations assayed (0.075-0.3 µg/mL). 339 Nevertheless, although MC-LR did not produce significant changes, exposure to the 340 mixture at the highest concentrations (45 and 0.3 µg/mL of MC-LR and CYN, 341 respectively) led to an increase in this enzymatic activity (Fig. 8B). 342 3.5. Morphology study 343 Unexposed undifferentiated SH-SY5Y cells observed under phase-contrast 344 microscope showed scarce cytoplasmic projections, frequently two of them placed in 345 opposite directions. These projections usually present connections with nearby cells 346 (Fig. 9A). The light microscope revealed mitotic cells with big nuclei and nucleoli (Fig. 347 9B). Under electronic microscope, cells present irregular nuclei with heterochromatin 348 condensation and big nucleoli. In the cytoplasm stand out a big number of free 349 ribosomes, mitochondria were scarce and presented dense matrix (Fig. 9C). In the non- 350 treated differentiated cells, a high number of cytoplasmic projections was observed 351 under the phase-contrast microscope (Fig. 10A). Under light microscopy, cells 352 presented a fusiform shape with endoplasmic reticulum dilatations and lipidic vacuoles 353 (Fig. 10B). The electron microscope revealed a cell cytoplasm with a higher number of 354 microtubules and intermediate filaments than the undifferentiated cells (Fig. 10C). 355 3.5.1. Microscope observations of cells exposed to pure MC-LR ACCEPTED MANUSCRIPT 16 356 Undifferentiated cells exposed to 37 µg/mL MC-LR presented, under phase- 357 contrast, light and electronic microcopies, cell death signs, with a reduction of their size, 358 chromatin condensation, and numerous vacuoles in the cytoplasm in fragmentation 359 process. Moreover, apoptotic nuclei with chromatin and nucleolar segregation were 360 observed (Fig. 11A-D). At the lowest concentrations (18.5 and 9.25 µg/mL MC-LR), 361 the most characteristic observations under phase-contrast microscope were the 362 cytoplasmic projections by way of lamellipodium (Fig. 11E). The light microscope 363 revealed cellular cycle arrest in the mitosis phase and nuclei with irregular shape and 364 big nucleoli (Fig. 11F). Under the TEM, cells presented chromatin condensation in the 365 nuclear membrane, segregated nucleoli and formation of autophagosomes (Figs. 11G 366 and H). 367 Differentiated SH-SY5Y cells exposed to 45 µg/mL MC-LR showed an elevated 368 refringence under the phase-contrast microscope caused by numerous dead cells and 369 apoptotic bodies, as it could also be observed by light microscopy (Fig. 12A-B). Under 370 electron microscopy, the presence of a large quantity of confluent heterophagosomes, 371 nucleolar segregation, and numerous lipidic vacuoles were observed (Fig. 12C-D). At 372 the lowest concentrations assayed (22.5 and 11.25 µg/mL MC-LR), cells observed 373 under phase-contrast, light and electronic microcopies presented protein granules, 374 possibly caused by alterations in the protein folding, lipidic vacuoles and chromatin 375 condensation. Mitotic processes were also observed, although this phenomenon could 376 be stopped due to the adverse situation induced by MC-LR (Fig. 12E-J). 377 3.5.2. Microscope observations of cells exposed to pure CYN 378 At the highest concentration assayed (1 µg/mL), undifferentiated cell cultures 379 presented clear morphological alterations leading to cell death such as apoptotic bodies, ACCEPTED MANUSCRIPT 17 380 and heterochromatin condensation (Fig. 13A and B). Ultrastructurally, nuclei presented 381 irregular shape and, frequently, the presence of apoptotic nuclei is high, with 382 cytoplasmic fragmentation. In addition, segregated nucleolus was observed, being only 383 visible their fibrillar component (Fig. 13C). At 0.5 and 0.25 µg/mL CYN, no 384 remarkable morphological alterations were observed under light microscopy, where it is 385 possible to observe the cellular cycle in mitosis phase. The TEM showed euchromatic 386 nuclei and numerous mitochondria in the cytoplasm, a characteristic of undifferentiated 387 cells (Fig. 13D and E). 388 After differentiation, SH-SY5Y cells exposed to the highest CYN concentration 389 (0.3 µg/mL CYN) experienced a massive cell death, easily observable by phase-contrast 390 (high refraction) and light (many cellular debris) microscopies. No cell division was 391 observed (Fig. 14A-B). Ultrastructurally, apoptotic nuclei with heterochromatin 392 condensation were appreciated, together with the presence of heterophagosomes (Fig. 393 14C). After exposure to 0.15 and 0.075 µg/mL CYN, cells presented an increased 394 number of ribosomes compared to control cells. Moreover, an increment in lipidic 395 vacuoles and dilated endoplasmic reticuli was observed. Nuclei presented the typical 396 apoptotic morphology (Fig. 14D-I). 397 3.5.3. Microscope observations of cells exposed to the combination MC-LR/ CYN 398 Concerning the combinations, the combination with the highest CYN and MC- 399 LR concentrations (1 + 37 µg/mL) induced and intense cell death in undifferentiated 400 cells, mainly by apoptosis, appearing numerous cellular debris and apoptotic bodies. 401 Moreover, apoptotic nuclei with chromatin condensation were observed (Fig. 15A and 402 B). The combination composed by 0.5 + 18.5 µg/mL induced a moderate cell death by ACCEPTED MANUSCRIPT 18 403 apoptosis, were cells presented blisters in the surface, a typical observation in cells 404 dying by apoptosis (Fig. 15C and D). 405 Differentiated cells presented two extremes. At the highest concentration 406 assayed (0.3 + 45 µg/mL CYN + MC-LR), a remarkable cell death was observed, with 407 the presence of pre-apoptotic bodies all over the culture. On the other hand, when cells 408 were exposed to the other two concentrations, zones without any visible damage and 409 zones with intense cellular death were perceived (Fig. 16A-C). The TEM showed 410 numerous indicators of cellular damage in cells exposed to the highest exposure 411 concentration. Thus, lipidic degeneration, apoptotic nuclei, endoplasmic reticulum with 412 protein concentration, pre-apoptotic bodies, heterophagosomes, nuclear bodies, and 413 nucleolar segregation were observed (Fig. 16D-G). 414 4. Discussion 415 Microcystin-LR and CYN have been extensively studied in vitro in hepatic and 416 renal cell lines (Chen and Xie, 2016; McLellan and Manderville, 2017; Pichardo et al., 417 2017). However, the studies concerning neuronal cell lines are still scarce, although 418 some in vivo studies point out that these cyanotoxins could induce neurotoxic effects 419 (Guzmán-Guillén et al., 2015; Kist et al., 2012; Qian et al., 2018; Wu et al., 2016; 420 2017). Neurotoxicity of cyanotoxins has been reviewed, including the main mechanisms 421 and effects (Florczyk et al., 2014; Hu et al., 2016) although the molecular mechanisms 422 underlying these effects have not been still elucidated yet. In this sense, the present 423 work focused on the potential effects induced by MC-LR, CYN, and their combination 424 in the human neuronal SH-SY5Y cell line. For MC-LR, only Zhang et al. (2018) used 425 this experimental model but for different purposes, such as transport, bioaccumulation, 426 hyperphosphorylation of PP2A-dependent Tau sites, and cell death. In the case of CYN ACCEPTED MANUSCRIPT 19 427 and its combination with MC-LR, the present study shows their effects in vitro, 428 contributing to mend the lack of information about this matter. 429 Regarding to MC-LR, our results showed a decrease of the cell viability after 24 430 and 48 h of exposure to MC-LR in both undifferentiated and differentiated cells, being 431 the undifferentiated the most sensitive cells. According to our results, several authors 432 have described a reduction of cell viability in different neuronal cell lines exposed to 433 MC-LR (primary murine WBC, primary murine CGNs cells, primary hippocampal 434 neurons, RAW246.7 murine macrophage-like cells, BV-2 cells, N2a cells, GT1-7 cells 435 and SH-SY5Y cells) (Cai et al., 2015; Ding et al., 2017; Feurstein et al., 2009; 2011; Li 436 et al., 2015; Takser et al., 2016; Zhang et al., 2018). However, most of these authors 437 used MC-LR concentrations up to 10 µM for 24, 48 or 72 h of exposure, and although 438 they described decreases in cell viability, only Cai et al. (2015) referred an EC50 value 439 for pure MC-LR. These authors exposed cells up to 30 µM MC-LR, establishing an 440 EC50 of 10 µM MC-LR in primary hippocampal neurons after 24 h of exposure using 441 the MTT cytotoxicity assay. In this regard, the presence of organic anion transporting 442 polypeptide transporters (OATPs) has been described as an important requirement to 443 MC-LR toxicity. It is well known that the OATP1B subfamily members are MCs 444 transporters (Fischer et al., 2005). Fischer et al. (2005) suggested that OATP1A2 445 transporters expressed in brain capillary endothelial cells and in the cell membrane of 446 human neurons could be involved in MC-LR transport through the blood-brain barrier. 447 In addition, Ding et al. (2017) demonstrated the role of the Oatp1a5 transporting MC- 448 LR into neuronal cells. These facts support that MC-LR damage could only evolve if it 449 is transported into the nervous system through OATPs or other different transporters 450 (Feurstein et al., 2009; 2011). However, the presence of these transporters in the 451 nervous system is scarce and, in some cases, their number can vary with the ACCEPTED MANUSCRIPT 20 452 differentiation process (Rozman et al., 2017; Yagdiran et al., 2016). Thus, the low MC- 453 LR-toxicity in those cell lines (primary murine WBC, primary murine CGNs cells, 454 primary hippocampal neurons, RAW246.7 murine macrophage-like cells, BV-2 cells, 455 N2a cells, GT1-7 cells and SH-SY5Y cells) and the increment in the EC50 value in the 456 differentiated SH-SY5Y cells from our study, compared with the undifferentiated ones, 457 could be due to this fact. 458 The potential mechanisms by which MC-LR induces its neurotoxic effects 459 include effects on neurotransmitters, neurochannels, signal transduction, oxidative stress 460 and cytoskeleton disruption (Hu et al., 2016). The neurotoxicity of MCs seems a multi- 461 pathway process, although the molecular mechanisms remain evasive. In this sense, Cai 462 et al. (2015) described that MC-LR disrupt calcium homeostasis in neurons, inducing a 463 concentration-dependent increment of intracellular free Ca2+ levels from stores together 464 with a decrease in cell viability. In agreement with these findings, Li et al. (2015) 465 reported that increased intracellular Ca2+ levels led to an activation of the phosphatase 466 calcineurin, which result in apoptosis via dephosphorylation of the proapoptotic Bcl-2 467 family member Bad. This calcium release, together with the associated cytochrome C 468 release, also activates the caspases protein family, well-known apoptotic proteins. Thus, 469 Feurstein et al. (2011) and Rozman et al. (2017) demonstrated that MC-LR induced cell 470 death by apoptosis through the activation of caspase proteins in primary murine CGN 471 cells and primary rat astrocytes, respectively. These findings support our results, since 472 our most sensitive cytotoxicity biomarker was the MTS assay, which assesses the 473 mitochondrial health and its activity, which is related with cell death (Tait and Green, 474 2013). Zhang et al. (2018) demonstrated that 10 µM MC-LR with endoporter caused 475 cell death in SH-SY5Y cells by using the lactate dehydrogenase (LDH) release assay. In 476 this experimental model, the authors indicated that MC-LR induced phosphorylation of ACCEPTED MANUSCRIPT 21 477 protein Tau, promoting dissociation of Tau from microtubules and aggregation of 478 phospho-paired helical filaments-Tau, and consequently, neuronal degeneration and cell 479 death. Cytoskeleton disruption is considered to be one of the cytotoxicity triggering 480 caused by MC-LR (Hu et al., 2016) based on the alterations induced in vivo by the toxin 481 in diverse cytosqueletal proteins in brains of rats (Zhao et al., 2015), and in in vitro 482 experiments (Meng et al., 2011). 483 Cellular death can be also corroborated through morphological studies. In this 484 regard, the present work shows how MC-LR induced the most common characteristics 485 of cellular death such as cytoplasm fragmentation, chromatin condensation, and 486 nucleolar segregation, endoplasmic reticulum dilatation, lipidic vacuoles, and presence 487 of heterophagosomes, both in the undifferentiated and differentiated SH-SY5Y cells. In 488 this sense, Feurstein et al. (2011) found that MC-LR induced a slight impairment of the 489 neurite network in primary murine CGN cells. In addition, Meng et al. (2011) described 490 some apoptotic effects such as the reorganization of cytoskeletal architectures in 491 differentiated PC12 cells exposed to 10 µM MC-LR, which was also observable in the 492 differentiated SH-SY5Y cells from our study. Moreover, Zhang et al. (2018) described 493 neurites degeneration and cell death in SH-SY5Y cells exposed to 10 µM MC-LR. 494 Two other toxic mechanisms studied in the present work are the oxidative stress 495 generation and the AChE disruption. For the oxidative stress evaluation two related 496 parameters were studied, ROS generation and GSH depletion, since it has been stated 497 that free-radical damage is one of the toxic mechanisms of MC-LR (Meng et al., 2013). 498 In the present work, ROS levels did not suffer any alteration in undifferentiated cells 499 exposed to pure MC-LR. However, Meng et al. (2013) found that concentrations up to 500 10 µM MC-LR induced significant enhancement of ROS levels at early times, reaching 501 the highest levels at 3 h after the exposure. Nonetheless, these authors also found that ACCEPTED MANUSCRIPT 22 502 after this time, ROS levels started to decline to basal levels due to a rapid response of 503 cells (differentiated PC12 cells) against MC-LR. Moreover, in the present study, GSH 504 levels decreased after 24 h of exposure at all the concentrations assayed. This could 505 confirm the fact that ROS levels appeared unaffected since GSH could act directly 506 against ROS (Circu and Aw, 2010). These oxidative stress parameters were not 507 evaluated in differentiated SH-SY5Y cells since several authors have highlighted that 508 these cells are more resistant to oxidative stressors due to changes in mitochondrial 509 metabolism and antioxidant defenses (Cecchi et al., 2008; Cheung et al., 2009; 510 Schneider et al., 2011). In contrast, diverse in vivo studies have reported the implication 511 of oxidative stress in the neurotoxicity induced by MC-LR (Li et al., 2014 Mello et al., 512 2018, Wang et al., 2010; Zhao et al., 2015). 513 Concerning the AChE activity, this enzyme is a well-known biomarker of 514 neuronal damage, as the affectation of the cholinergic system could lead to a 515 malfunctioning of the locomotor system, behavior and cognitive processes (Kist et al., 516 2012). MCs may influence brain AChE indirectly via the inhibition of serine/threonine 517 phosphatases (Hu et al., 2016). In our study, a significant increase of AChE activity was 518 observed when undifferentiated SH-SY5Y cells were exposed to the highest 519 concentration of MC-LR (37 µg/mL). The increased AChE activity could lead to a 520 reduction of the cholinergic neurotransmission efficiency because of the lack of 521 acetylcholine in the synaptic space, possibly contributing to a progressive cognitive 522 impairment (Teodorak et al., 2015). Despite this, some other authors have demonstrated 523 that AChE plays a role by promoting or suppressing cell death. An enhanced AChE 524 activity take part in apoptosis, participating in the formation of apoptosomes or 525 influencing the expression of apoptotic genes (Park et al., 2004; Ben-Ari et al., 2006; 526 Zhu et al., 2007). Zhu et al. (2007) also stated that AChE expression during apoptosis is ACCEPTED MANUSCRIPT 23 527 associated with calcium mobilization. These facts are in agreement with our findings, 528 since it has been shown that MC-LR induced cell death by apoptosis at the highest 529 concentration assayed, correlating this effect with the AChE activity enhancement. On 530 the contrary, when differentiated SH-SY5Y cells were exposed to 45 µg/mL MC-LR, 531 no significant changes in the AChE activity were detected compared to the control 532 group. This could be due to structural and functional modifications of the cells after the 533 differentiation process, which could impede the effects of MC-LR over the AChE 534 activity. However, cell death by apoptosis was also observed in these cells, which 535 implies that not only the rise of the AChE could be involved in the cellular death, but 536 also other potential factors such as intracellular calcium levels etc., that should be 537 further investigated. Other authors have also evaluated the AChE activity disruption 538 induced by MC-LR, but their studies have been carried out in in vivo systems (Kist et 539 al., 2012; Qian et al., 2018; Wu et al., 2016; 2017), obtaining contradictory and not 540 conclusive results, depending on the administration route, or the experimental model 541 assayed, etc. Consequently, further research should be carried out in order to clarify the 542 effects of MCs on this key enzyme in the nervous system. 543 Concerning CYN, a very scarce number of studies have been carried out in order 544 to elucidate its neurotoxicity. However, due to its zwitterionic behavior and its small 545 size, CYN is likely to be taken by the cells through diffusion, being able the crossing 546 through the blood brain barrier (Florczyk et al., 2014; Valério et al. 2010). In the present 547 study, CYN showed EC50 values even lower than MC-LR both in undifferentiated and 548 differentiated cells. In agreement, Takser et al. (2016) found that pure CYN reached an 549 EC50 value between 0.1 and 10 µM CYN in N2a cells after 24, 48 and 72 h of exposure. 550 These authors also found that CYN induced almost a total cellular death at a 551 concentration of 10 µM in RAW264.7 and BV-2 cells. These findings are also in ACCEPTED MANUSCRIPT 24 552 agreement with the morphological results, which showed clear signs of cellular death by 553 apoptosis in both types of SH-SY5Y cells exposed to 1 µg/mL (2.4 µM) and 0.3 µg/mL 554 (0.7 µM) CYN. Relative to oxidative stress, although numerous works demonstrate in 555 vitro the oxidative stress induction by CYN in different cell lines (reviewed by Pichardo 556 et al., 2017), this work investigated the potential effects on ROS and GSH levels in the 557 undifferentiated SH-SY5Y cell line. However, no effects were found, which is in 558 disagreement with some in vivo studies, where oxidative stress was a main component 559 of the cellular damaged observed (Guzmán-Guillén et al., 2015; da Silva et al., 2018). 560 These discrepancies could be due to the different experimental model or the 561 concentrations used. 562 In relation to the AChE activity disruption, to our knowledge, no other papers 563 have been published concerning the effects of CYN on AChE activity in vitro. In the 564 present work, our results did not indicate any alteration at all CYN concentrations 565 assayed (up to 1 µg/mL) in undifferentiated cells. This is in agreement with da Silva et 566 al. (2018), who described no significant differences in the AChE activity in brain of fish 567 (Hoplias malabaricus) exposed to pure CYN after 7- and 14-days post treatment. In 568 addition, these authors also reported increased AChE activity by 44% in brain at 7 days 569 of exposure to aqueous CYN-producing cyanobacteria extracts, returning to control 570 levels after 14 days. However, when differentiated SH-SY5Y cells were exposed to 0- 571 0.3 µg/mL CYN, cultures showed a significant decrease of AChE activity at all CYN 572 concentrations. These observations are in agreement with the findings reported by 573 Guzmán-Guillén et al. (2015), who found a significant inhibition of 35% of AChE 574 activity in the brain of tilapia subchronically exposed to CYN (10 µg/L CYN) by 575 immersion in an A. ovalisporum culture for 14 days has been reported. In this study, 576 after a depuration process (7 days) a recovery of the enzyme was found. These contrary ACCEPTED MANUSCRIPT 31 718 Carmichael WW, Azevedo SMFO, An JS, Molica RJR, Jochimsen EM, Lau S, Rinehart 719 KL, Shaw GR, Eaglesham GK. Human fatalities form cyanobacteria: Chemical 720 and biological evidence for cyanotoxins. Environ Health Perspect. 2001; 721 109:663–8. Doi: 10.1289/ehp.01109663 722 Cecchi C, Pensalfini A, Liguri G, Baglioni S, Fiorillo C, Guadagna S, Zampagni M, 723 Formigli L, Nosi D, Stefani M. Differentiation increases the resistance of 724 neuronal cells to amyloid toxicity. Neurochem Res. 2008; 33:2516–31. Doi: 725 10.1007/s11064-008-9627-7 726 Chen L, Xie P. Mechanisms of Microcystin-induced cytotoxicity and apoptosis. Mini- 727 Reviews Med Chem. 2016;16:1018–31. Doi: 10.1016/j.aquatox.2014.01.021 728 Cheung YT, Lau WKW, Yu MS, Lai CSW, Yeung SC, So KF, Chang RCC. Effects of 729 all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in 730 neurotoxicity research. Neurotoxicol. 2009; 30:127-135. Doi: 731 10.1016/j.neuro.2008.11.001 732 Chou T. Theoretical basis, experimental design, and computerized simulation of 733 synergism and antagonism in drug combination studies. Pharmacol Rev. 2006; 734 58:621–81. Doi: 10.1124/pr.58.3.10. 735 Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined 736 effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul. 1984; 737 22:27–55. Doi: 10.1016/0065-2571(84)90007-4 738 Circu ML, Aw TY. Reactive oxygen species, cellular redox systems, and apoptosis. 739 Free Radic Biol Med. 2010; 48:749–62. Doi: 740 10.1016/j.freeradbiomed.2009.12.022 ACCEPTED MANUSCRIPT 32 741 Da Silva R de C, Grötzner SR, Moura Costa DD, Esquivel JR, Muelbert J, Freitas de 742 Magalhães V, Filipak F, de Oliveira CA. Comparative bioaccumulation and 743 effects of purified and cellular extract of cylindrospermopsin to freshwater fish 744 Hoplias malabaricus. J Toxicol Environ Heal - Part A Curr Issues. 2018; 745 81:620–32. Doi: 10.1080/15287394.2018.1469101 746 Ding J, Wang J, Xiang Z, Diao W, Su M, Shi W, Wan T, Han X. The organic anion 747 transporting polypeptide 1a5 is a pivotal transporter for the uptake of 748 microcystin-LR by gonadotropin-releasing hormone neurons. Aquat Toxicol. 749 2017; 182:1-10. Doi: 10.1016/j.aquatox.2016.11.005 750 Ellman, G. L., Courtney, K. D., Andres, V., & Featherstone, R. M. A new and rapid 751 colorimetric determination of acetylcholinesterase activity. Biochem. 752 Pharmacol. 1961; 7:88–95. Doi: 10.1016/0006-2952(61)90145-9 753 Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Ceña V, Gallego C, Comella JX. 754 Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived 755 neurotrophic factor gives rise to fully differentiated, neurotrophic factor- 756 dependent human neuron-like cells. J Neurochem. 2000; 75:991-1003. Doi: 757 10.1046/j.1471-4159.2000.0750991.x 758 Falconer IR. An overview of problems caused by toxic blue – green algae 759 (cyanobacteria) in drinking and recreational water. Environ Toxicol. 1999; 14:5– 760 12. Doi: 10.1002/(SICI)1522-7278(199902)14:1<5::AID-TOX3>3.0.CO;2-0 761 Falconer IR, Humpage R. Cyanobacterial (blue-green algal) toxins in water supplies: 762 cylindrospermopsin. Environ Toxicol. 2006; 21:299-304. Doi: 763 10.1002/tox.20194 ACCEPTED MANUSCRIPT 33 764 Feurstein D, Holst K, Fischer A, Dietrich DR. Oatp-associated uptake and toxicity of 765 microcystins in primary murine whole brain cells. Toxicol Appl Pharmacol. 766 2009; 234:247–55. Doi: 10.1016/j.taap.2008.10.011 767 Feurstein D, Stemmer K, Kleinteich J, Speicher T, Dietrich DR. Microcystin congener- 768 and concentration-dependent induction of murine neuron apoptosis and neurite 769 degeneration. Toxicol Sci. 2011; 124:424–31. Doi: 10.1093/toxsci/kfr243 770 Fischer WJ, Altheimer S, Cattori V, Meier PJ, Dietrich DR, Hagenbuch B. Organic 771 anion transporting polypeptides expressed in liver and brain mediate uptake of 772 microcystin. Toxicol Appl Pharmacol. 2005; 203:257–63. Doi: 773 10.1016/j.taap.2004.08.012 774 Florczyk M, Łakomiak A, Woźny M, Brzuzan P. Neurotoxicity of cyanobacterial 775 toxins. Environ Biotechnol. 2014; 10:26–43. Doi: 10.14799/ebms246 776 Froscio SM, Humpage AR, Burcham PC, Falconer IR. Cylindrospermopsin-induced 777 protein synthesis inhibition and its dissociation from acute toxicity in mouse 778 hepatocytes. Environ Toxicol. 2003; 18:243–51. Doi: 10.1002/tox.10121 779 Gutiérrez-Praena D, Pichardo S, Jos Á, María Cameán A. Toxicity and glutathione 780 implication in the effects observed by exposure of the liver fish cell line PLHC-1 781 to pure cylindrospermopsin. Ecotoxicol Environ Saf. 2011; 74:1567–72. Doi: 782 10.1016/j.ecoenv.2011.04.030 783 Gutiérrez-Praena D, Pichardo S, Jos Á, Moreno FJ, Cameán AM. Biochemical and 784 pathological toxic effects induced by the cyanotoxin Cylindrospermopsin on the 785 human cell line Caco-2. Water Res. 2012; 46:1566–75. Doi: 786 10.1016/j.watres.2011.12.044 ACCEPTED MANUSCRIPT 34 787 Gutiérrez-Praena D, Guzmán-Guillén R, Pichardo S, Moreno FJ, Vasconcelos V, Jos A, 788 Cameán AM. Cytotoxic and morphological effects of microcystin-LR, 789 cylindrospermopsin, and their combinations on the human hepatic cell line 790 HepG2. Environ Toxicol. 2018; 34:1-12. Doi: 10.1002/tox.22679 791 Guzmán-Guillén R, Prieto AI, Vasconcelos VM, Cameán Fernandez AM. 792 Cyanobacterium producing cylindrospermopsin cause histopathological changes 793 at environmentally relevant concentrations in subchronically exposed tilapia 794 (Oreochromis niloticus). Environ Toxicol. 2013; 90:1184–94. Doi: 795 10.1002/tox.21904 796 Guzmán-Guillén R, Manzano IL, Moreno IM, Ortega AIP, Moyano R, Blanco A, 797 Cameán AM. Cylindrospermopsin induces neurotoxicity in tilapia fish 798 (Oreochromis niloticus) exposed to Aphanizomenon ovalisporum. Aquat 799 Toxicol. 2015; 161:17–24. Doi: 10.1016/j.aquatox.2015.01.024 800 Harada K ichi, Ohtani I, Iwamoto K, Suzuki M, Watanabe MF, Watanabe M, Terao K. 801 Isolation of cylindrospermopsin from a cyanobacterium Umezakia natans and its 802 screening method. Toxicon. 1994; 32:73–84. Doi: 10.1016/0041- 803 0101(94)90023-X 804 Hawkins PR, Runnegar MTC, Jackson ARB, Falconer IR. Severe hepatotoxicity caused 805 by the tropical cyanobacterium supply reservoir. Appl Environ Microbiol. 1985; 806 50:1292–5. Doi: 10.1016/S1383-5718(00)00144-3 807 Hu Y, Chen J, Fan H, Xie P, He J. A review of neurotoxicity of microcystins. Environ 808 Sci Pollut Res. 2016; 23:7211–9. Doi: 10.1007/s11356-016-6073-y ACCEPTED MANUSCRIPT 35 809 Humpage AR, Fontaine F, Froscio S, Burcham P, Falconer IR. Cylindrospermopsin 810 genotoxicity and cytotoxicity: Role of cytochrome P-450 and oxidative stress. J 811 Toxicol Environ Heal - Part A. 2005; 68:739–53. 812 Jos A, Cameán AM, Pflugmacher S, Segner H. The antioxidant glutathione in the fish 813 cell lines EPC and BCF-2: response to model pro-oxidants as measured by three 814 different fluorescent dyes. Toxicol In Vitro. 2009; 23:546-53. 815 Kinnear SH, Fabbro LD, Duivenvoorden LJ, Hibberd EM. Multiple-organ toxicity 816 resulting from cylindrospermopsin exposure in tadpoles of the cane toad (Bufo 817 marinus). Environ Toxicol. 2007; 22:550-8. Doi: 10.1002/tox.20299 818 Kiss T, Vehovszky Á, Hiripi L, Kovács A, Vörös L. Membrane effects of toxins 819 isolated from a cyanobacterium, Cylindrospermopsis raciborskii, on identified 820 molluscan neurones. Comp Biochem Physiol - C Toxicol Pharmacol. 2002; 821 131:167–76. Doi: 10.1016/S1532-0456(01)00290-3 822 Kist LW, Rosemberg DB, Pereira TCB, De Azevedo MB, Richetti SK, De Castro Leão 823 J, Sarkis Yunes J, Bonan CD, Bogo MR. Microcystin-LR acute exposure 824 increases AChE activity via transcriptional ache activation in zebrafish (Danio 825 rerio) brain. Comp Biochem Physiol - C Toxicol Pharmacol; 2012; 155:247–52. 826 Doi: 10.1016/j.cbpc.2011.09.002 827 Li G, Cai F, Yan W, Li C, Wang J. A proteomic analysis of MCLR-induced 828 neurotoxicity: Implications for Alzheimer’s disease. Toxicol Sci. 2012; 829 127:485–95. Doi: 10.1093/toxsci/kfs114 830 Li XB, Zhang X, Ju J, Li Y, Yin L, Pu Y. Alterations in neurobehaviors and 831 inflammation in hippocampus of rats induced by oral administration of ACCEPTED MANUSCRIPT 36 832 microcystin-LR. Environ Sci Pollut Res. 2014; 21:12419–25. Doi: 833 10.1007/s11356-014-3151-x 834 Li X, Zhang X, Ju J, Li Y, Yin L, Pu Y. Maternal repeated oral exposure to microcystin- 835 LR affects neurobehaviors in developing rats. Environ Toxicol Chem. 2015; 836 34:64–9. doi:10.1002/etc.2765 837 Liu W, Chen C, Chen L, Wang L, Li J, Chen Y, Jin J, Kawan A, Zhang X. Sex- 838 dependent effects of microcystin-LR on hypothalamic-pituitary-gonad axis and 839 gametogenesis of adult zebrafish. Sci Rep. 2016; 6:1–12. Doi : 840 10.1038/srep22819 841 Lu H, Fernández-Franzón M, Font G, Ruiz MJ. Toxicity evaluation of individual and 842 mixed enniatins using an in vitro method with CHO-K1 cells. Toxicol In Vitro. 843 2013; 27:672–80. Doi: 10.1016/j.tiv.2012.11.009 844 MacKintosh C, Beattie KA, Klumpp S, Cohen P, Codd GA. Cyanobacterial 845 microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 846 2A from both mammals and higher plants. Febs. 1990; 264:187–92. Doi: 847 10.1016/0014-5793(90)80245-E 848 Malbrouck C, Kestemont P. Effects of microcystins on fish. Environ Toxicol Chem. 849 2006; 25:72–86. Doi: 10.1897/05-029R.1 850 McLellan NL, Manderville RA. Toxic mechanisms of microcystins in mammals. 851 Toxicol Res (Camb). 2017; 6:391–405. Doi: 10.1039/c7tx00043j 852 Menezes C, Alverca E, Dias E, Sam-Bento F, Pereira P. Involvement of endoplasmic 853 reticulum and autophagy in microcystin-LR toxicity in Vero-E6 and HepG2 cell 854 lines. Toxicol In Vitro. 2013; 27:138–48. Doi: 10.1016/j.tiv.2012.09.009 ACCEPTED MANUSCRIPT 37 855 Meng G, Sun Y, Fu W, Guo Z, Xu L. Microcystin-LR induces cytoskeleton system 856 reorganization through hyperphosphorylation of tau and HSP27 via PP2A 857 inhibition and subsequent activation of the p38 MAPK signaling pathway in 858 neuroendocrine (PC12) cells. Toxicology. 2011; 290:219–29. Doi: 859 10.1016/j.tox.2011.09.085 860 Meng G, Liu J, Lin S, Guo Z, Xu L. Microcystin-LR-caused ROS generation involved 861 in p38 activation and tau hyperphosphorylation in neuroendocrine (PC12) cells. 862 Environ Toxicol. 2013; 30:366-74. Doi: 10.1002/tox.21914 863 Mello FD, Braidy N, Marçal H, Guillemin G, Nabavi SM, Neilan BA. Mechanisms and 864 Effects Posed by Neurotoxic Products of Cyanobacteria/Microbial 865 Eukaryotes/Dinoflagellates in Algae Blooms: a Review. Neurotox Res. 2018; 866 33(1):153–67. Doi: 10.1007/s12640-017-9780-3 867 Oehrle SA, Southwell B, Westrick J. Detection of various freshwater cyanobacterial 868 toxins using ultra-performance liquid chromatography tandem mass 869 spectrometry. Toxicon. 2010; 55:965–72. Doi:10.1016/j.toxicon.2009.10.001 870 Park SE, Kim ND, Yoo YH. Acetylcholinesterase plays a pivotal role in apoptosome 871 formation. Cancer Res. 2004; 64:2652–5. Doi:10.1158/0008-5472.CAN-04- 872 0649 873 Pichardo S, Jos A, Zurita JL, Salguero M, Camean AM, Repetto G. The use of the fish 874 cell lines RTG-2 and PLHC-1 to compare the toxic effects produced by 875 microcystins LR and RR. Toxicol In Vitro. 2005; 19:865–73. Doi: 876 10.1016/j.tiv.2005.06.016 ACCEPTED MANUSCRIPT 38 877 Pichardo S, Cameán AM, Jos A. In vitro toxicological assessment of 878 cylindrospermopsin: A review. Toxins (Basel). 2017; 9(12). Doi: 879 10.3390/toxins9120402 880 Pouria S, De Andrade A, Barbosa J, Cavalcanti RL, Barreto VTS, Ward CJ. Fatal 881 microcystin intoxication in haemodialysis unit in Caruaru, Brazil. Lancet. 1998; 882 352:21–6. Doi: 10.1016/S0140-6736(97)12285-1 883 Puerto M, Pichardo S, Jos Á, Prieto AI, Sevilla E, Frías JE, Cameán AM. Differential 884 oxidative stress responses to pure Microcystin-LR and Microcystin-containing 885 and non-containing cyanobacterial crude extracts on Caco-2 cells. Toxicon. 886 2010; 55:514–22. Doi: 10.1016/j.toxicon.2009.10.003 887 Puerto M, Gutiérrez-Praena D, Prieto AI, Pichardo S, Jos A, Miguel-Carrasco JL, 888 Vázquez CM, Cameán AM. Subchronic effects of cyanobacterial cells on the 889 transcription of antioxidant enzyme genes in tilapia (Oreochromis niloticus). 890 Ecotoxicology. 2011; 20:479–90. Doi: 10.1007/s10646-011-0600-x 891 Puerto M, Prieto AI, Maisanaba S, Gutiérrez-Praena D, Mellado-García P, Jos Á, et al. 892 Mutagenic and genotoxic potential of pure Cylindrospermopsin by a battery of 893 in vitro tests. Food Chem Toxicol. 2018; 121:413–22. Doi: 894 10.1016/j.fct.2018.09.013 895 Qian H, Liu G, Lu T, Sun L. Developmental neurotoxicity of Microcystis aeruginosa in 896 the early life stages of zebrafish. Ecotoxicol Environ Saf. 2018; 151:35–41. Doi: 897 10.1016/j.ecoenv.2017.12.059 898 Qiu T, Xie P, Liu Y, Li G, Xiong Q, Hao L, et al. The profound effects of microcystin 899 on cardiac antioxidant enzymes, mitochondrial function and cardiac toxicity in 900 rat. Toxicology. 2009; 257:86–94. Doi: 10.1016/j.tox.2008.12.012 ACCEPTED MANUSCRIPT 39 901 Ruiz MJ, Macáková P, Juan-García A, Font G. Cytotoxic effects of mycotoxin 902 combinations in mammalian kidney cells. Food Chem Toxicol. 2011a; 903 49(10):2718–24. Doi: 10.1016/j.fct.2011.07.021 904 Ruiz MJ, Franzova P, Juan-García A, Font G. Toxicological interactions between the 905 mycotoxins beauvericin, deoxynivalenol and T-2 toxin in CHO-K1 cells in vitro. 906 Toxicon. 2011b; 58:315–26. Doi: 10.1016/j.toxicon.2011.07.015 907 Rozman KB, Jurič DM, Šuput D. Selective cytotoxicity of microcystins LR, LW and LF 908 in rat astrocytes. Toxicol Lett. 2017; 265:1–8. Doi: 10.1016/j.toxlet.2016.11.008 909 Santillo MF, Liu Y. A fluorescence assay for measuring acetylcholinesterase activity in 910 rat blood and a human neuroblastoma cell line (SH-SY5Y). J Pharmacol Toxicol 911 Methods. 2015; 76:15–22. Doi: 10.1016/j.vascn.2015.07.002 912 Schembri M a, Neilan B a, Saint CP. Identification of genes implicated in toxin 913 production in the cyanobacterium Cylindrospermopsis raciborskii. Environ 914 Toxicol. 2001; 16:413–21. Doi: 10.1002/tox.1051 915 Schneider L, Giordano S, Zelickson BR, Johnson MS, Benavides GA, Ouyang X, 916 Fineberg N, Darley-Usmar VM, Zhang J. Differentiation of SH-SY5Y cells to a 917 neuronal phenotype changes cellular bioenergetics and the response to oxidative 918 stress. Free Rad Biol Med. 2011; 51:2007-17. Doi: 919 10.1016/j.freeradbiomed.2011.08.030 920 Shaw GR, Sukenik A, Livne A, Chiswell RK, Smith MJ, Seawright AA, Norris RL, 921 Eaglesham GK, Moore MR. Blooms of the cylindrospermopsin containing 922 cyanobacterium, Aphanizomenon ovalisporum (Fofti), in newly constructed 923 lakes, Queensland, Australia. Environ Toxicol. 1999; 14:167–77. Doi: 924 10.1002/(SICI)1522-7278(199902)14:1<167::AID-TOX22>3.0.CO;2-O ACCEPTED MANUSCRIPT 40 925 Sivonen K and Jones G. Cyanobacterial toxins. In: Chorus, I. and Bartram, J., Eds., 926 Toxic cyanobacteria in water: a guide to their public health consequences, 927 monitoring, and management, E & FN Spon. 1999, 41-111. 928 Spoof L, Catherine A. Appendix 3, in: Meriluto J, Spoof L, Codd GA. Handbook of 929 cyanobacterial monitoring and cyanotoxin analysis. 2017; 526-537. 930 Svirčev ZB, Tokodi N, Drobac D, Codd GA. Cyanobacteria in aquatic ecosystems in 931 Serbia: Effects on water quality, human health and biodiversity. Syst Biodivers. 932 2014; 12:261–70. Doi: 10.1080/14772000.2014.921254 933 Tait SWG, Green DR. Mitochondrial regulation of cell death. Cold Spring Harb 934 Perspect Biol. 2013; 5:1-15. Doi: 10.1101/cshperspect.a008706 935 Takser L, Benachour N, Husk B, Cabana H, Gris D. Cyanotoxins at low doses induce 936 apoptosis and inflammatory effects in murine brain cells: Potential implications 937 for neurodegenerative diseases. Toxicol Reports. 2016; 3:180–9. Doi: 938 10.1016/j.toxrep.2015.12.008 939 Tatay E, Meca G, Font G, Ruiz MJ. Interactive effects of zearalenone and its 940 metabolites on cytotoxicity and metabolization in ovarian CHO-K1 cells. 941 Toxicol In Vitro. 2014; 28:95–103. Doi: 10.1016/j.tiv.2013.06.025 942 Terao K, Ohmori S, Igarashi K, Ohtani I, Watanabe MF, Harada KI, Ito E, Watanabe 943 M. Electron microscopic studies on experimental poisoning in mice induced by 944 cylindrospermopsin isolated from blue-green algae Umezakia natans. Toxicon. 945 1994; 32:833–43. Doi: 10.1016/0041-0101(94)90008-6 ACCEPTED MANUSCRIPT electronic microscopy of differentiated SH-SY5Y cells exposed to 22.5 µg/mL MC-LR. Cells with irregular shape presenting nuclei (N) with chromatin condensation (Chr). Bar=2 µm (G). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 11.25 µg/mL MC-LR. Numerous dilated endoplasmic reticuli (arrows) with protein condensation (arowheads). Bar=25 µm (H). Transmission electronic microscopy of differentiated SH-SY5Y cells exposed to 11.25 µg/mL MC-LR. Presence of mitochondria (Mit), protein condensation (arrowheads) and lipidic vacuoles (Lp). Bar=2 µm (I, J). Figure 13. Morphology of undifferentiated SH-SY5Y cells after 24 h of exposure to CYN. Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 1 µg/mL CYN. Detention of cellular growth and decrease of cell number caused by cellular death by apoptosis. Apoptotic bodies (arrows). Heterochromatin in apoptotic nucleus (arrowhead). Bar=20µm (A, B). Transmission electronic microscopy of SHSY5Y cells exposed to 1 µg/mL CYN. Fractionated cytoplasm and formation of apoptotic bodies (arrows). Condensed chromatin in the inner face of the nucleolar membrane (asterisks). Fibrillar component of the nucleoulus (n). Nucleus (N). Bar=2µm (C). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.5 µg/mL CYN. No evidence of morphological alterations. Bar=20µm (D). Transmission electronic microscopy of SH-SY5Y cells exposed to 0.5 and 0.25 µg/mL CYN. Euchromatic nuclei with irregular shape (N). Cytoplasm with numerous free ribosomes (asterisk). Mitochondria (arrows). Bar=2µm (E). Figure 14. Morphology of differentiated SH-SY5Y cells after 24 h of exposure to CYN. Phase-contrast microscopy of cells exposed to 0.3 µg/mL CYN. Rounded cells (arrows) with clear signs of cell death. Bar=100 µm (A). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.3 µg/mL CYN. Presence of numerous apoptotic bodies (arrows) as clear sign of cell death. Presence of dilated endoplasmic reticulum (arrowheads). Bar=25 µm (B). Transmission electronic microscopy of SHSY5Y cells exposed to 0.3 µg/mL CYN. Condensed chromatin in the inner face of the nucleolar membrane (Chr). Bar=2µm (C). Phase-contrast microscopy of cells exposed to 0.15 µg/mL CYN. Rounded cells (arrows) with clear signs of cell death. Bar=100 µm (D). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.15 µg/mL CYN. Presence of dilated endoplasmic reticulum (arrowheads) and lipidic vacuoles (arrows). Bar=25 µm (E). Transmission electronic microscopy of SH-SY5Y ACCEPTED MANUSCRIPT cells exposed to 0.15 µg/mL CYN. Condensed chromatin (Chr) in apoptotic nucleus (N). Bar=2µm (F). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 0.075 µg/mL CYN. Presence of numerous apoptotic bodies (arrows) as clear sign of cell death. Bar=25 µm (G). Transmission electronic microscopy of SHSY5Y cells exposed to 0.075 µg/mL CYN. Euchromatic nuclei with irregular shape (N). Dilate endoplasmic reticulum (ER) and lipidic vacuoles (Lp). Bar=2 µm (H). Figure 15. Morphology of undifferentiated SH-SY5Y cells after 24 h of exposure to MC-LR + CYN. Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 37 µg/mL MC-LR + 1 µg/mL CYN. Intense cellular death by apoptosis. Heterochromatin accumulations in apoptotic nuclei (arrows). Cellular debris as apoptotic bodies (arrowheads). Bar=20µm (A). Transmission electronic microscopy of SH-SY5Y cells exposed to 37 µg/mL MC-LR + 1 µg/mL CYN. Heterochromatin condensation in the inner face of the nuclear membrane (asterisks). Nucleolar segregation with the fibrillary component (n). Apoptotic bodies formation (arrows). Autophagy vacuoles in the cytoplasm (arrowheads). Nucleus (N). Bar=2µm (B). Semithin sections of SH-SY5Y cells stained with toluidine blue. Cells exposed to 18.5 µg/mL MC-LR + 0.5 µg/mL CYN. Blister formation in cellular surface (arrows). Apoptotic bodies (arrowheads). Bar=20µm (C, D). Figure 16. Morphology of differentiated SH-SY5Y cells after 24 h of exposure to MCLR + CYN. Transmission electronic microscopy of SH-SY5Y cells exposed to 45 µg/mL MC-LR + 0.3 µg/mL CYN. Apoptotic nucleus (N) with heterochromatin condensation (Chr). Lipidic degeneration (Lp). Dilated endoplasmic reticulum (ER) with protein condensation (arrowheads). Pre-apoptotic bodies (arrows). Heterophagosome (Het). Nucleoulus (n) with the presence of a nuclear body (NB). Bar=2 µm (A, B, C, D). Semithin sections of cells culture were stained with toluidine blue. Cells exposed to 45 µg/mL MC-LR + 0.3 µg/mL CYN. Clear formation of apoptotic bodies (arrowheads). Bar=25 µm (E). Cells exposed to 22.5 µg/mL MC-LR + 0.15 µg/mL CYN and 11.25 µg/mL MC-LR + 0.075 µg/mL CYN. No significant alterations observed. Bar=25 µm (F, G). ACCEPTED MANUSCRIPT Highlights 1. Microcystin-LR and cylindrospermopsin induced cytotoxicity in SH-SY5Y cells. 2. Both cyanotoxins presented an antagonistic effect when in combination. 3. The acetylcholinesterase activity vary with the exposure to the toxins. 4. Apoptosis was the main cell death mechanism observed by microscopy. ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT MTS assay NR assay PC assay Toxin Cell type Time (h) EC50 values (µg/mL) Significant results (from) (µg/mL) EC50 values (µg/mL) Significant results (from) (µg/mL) EC50 values (µg/mL) Significant results (from) (µg/mL) 24 36.21 ± 1.89 20 67.69 ± 1.65 40 >100 40 Undifferentiated 48 20.80 ± 2.08 20 24.27 ± 0.45 20 29.58 ± 2.70 20 24 44.30 ± 0.91 20 - - >100 20 MC-LR Differentiated 48 37.01 ± 1.71 20 - - >100 40 24 0.87 ± 0.13 0.2 2.26 ± 0.29 0.5 4.37 ± 1.34 0.75 Undifferentiated 48 0.32 ± 0.08 0.2 1.27 ± 0.27 0.4 2.01 ± 0.29 0.4 24 0.30 ± 0.05 0.1 - - >10 2.5 CYN Differentiated 48 0.53 ± 0.02 0.1 - - >10 1 Table 1. Cytotoxicity results of undifferentiated and differentiated SH-SY5Y cells exposed to MC-LR and CYN. Results are expressed as mean ± s.d. ACCEPTED MANUSCRIPT CI values (undifferentiated cells) Cyanotoxin Time (hours) Dm (µg/mL) m r CI50 CI75 CI90 MC-LR 24 44.02 5.20 0.86603 48 19.13 3.51 0.99320 CYN 24 1.01 2.20 0.98604 48 0.58 1.66 0.97832 MC-LR + CYN 24 29.30 5.44 0.96064 1.45 Ant 1.24 Ant 1.08 Add 48 16.17 4.25 0.99509 1.60 Ant 1.31 Ant 1.09 Add Table 2. The parameter m, Dm and r are the antilog of x-intercept, the slope and the linear correlation coefficient of the median-effect plot, which signifies the shape of the doseeffect curve, the potency (IC50), and the conformity of the data to the mass-action law, respectively. Dm and m values are used for calculating the CI value (CI< 1, indicates synergism (Syn); CI = 1, indicates additive effect (Add); CI > 1, indicates antagonism (Ant). IC50, IC75 and IC90 are the doses required to inhibit proliferation 50, 75 and 90%, respectively. CalcuSyn software provide automatically the IC50, IC75 and IC90 values. ACCEPTED MANUSCRIPT CI values (differentiated cells) Cyanotoxin Time (hours) Dm (µg/mL) m r CI50 CI75 CI90 MC-LR 24 30.17 4.14 0.96974 48 28.09 4.06 0.97042 CYN 24 0.25 2.32 0.96974 48 0.23 2.53 0.96292 MC-LR + CYN 24 27.32 2.37 0.99939 1.65 Ant 1.84 Ant 2.08 Ant 48 26.50 3.00 0.99411 1.72 Ant 1.77 Ant 1.83 Ant Table 3. The parameter m, Dm and r are the antilog of x-intercept, the slope and the linear correlation coefficient of the median-effect plot, which signifies the shape of the doseeffect curve, the potency (IC50), and the conformity of the data to the mass-action law, respectively. Dm and m values are used for calculating the CI value (CI< 1, indicates synergism (Syn); CI = 1, indicates additive effect (Add); CI > 1, indicates antagonism (Ant). IC50, IC75 and IC90 are the doses required to inhibit proliferation 50, 75 and 90%, respectively. CalcuSyn software provide automatically the IC50, IC75 and IC90 values.