Cytotoxic Effects and Oxidative Stress Produced by a Cyanobacterial Cylindrospermopsin Producer Extract versus a Cylindrospermopsin Non-Producing Extract on the Neuroblastoma SH-SY5Y Cell Line.

The incidence and interest of cyanobacteria are increasing nowadays because they are able to produce some toxic secondary metabolites known as cyanotoxins. Among them, the presence of cylindrospermopsin (CYN) is especially relevant, as it seems to cause damage at different levels in the organisms: the nervous system being the one most recently reported. Usually, the effects of the cyanotoxins are studied, but not those exerted by cyanobacterial biomass. The aim of the present study was to assess the cytotoxicity and oxidative stress generation of one cyanobacterial extract of R. raciborskii non-containing CYN (CYN-), and compare its effects with those exerted by a cyanobacterial extract of C. ovalisporum containing CYN (CYN+) in the human neuroblastoma SH-SY5Y cell line. Moreover, the analytical characterization of potential cyanotoxins and their metabolites that are present in both extracts of these cultures was also carried out using Ultrahigh Performance Liquid Chromatography-Mass Spectrometry, in tandem (UHPLC-MS/MS). The results show a reduction of cell viability concentration- and time-dependently after 24 and 48 h of exposure with CYN+ being five times more toxic than CYN-. Furthermore, the reactive oxygen species (ROS) increased with time (0-24 h) and CYN concentration (0-1.11 µg/mL). However, this rise was only obtained after the highest concentrations and times of exposure to CYN-, while this extract also caused a decrease in reduced glutathione (GSH) levels, which might be an indication of the compensation of the oxidative stress response. This study is the first one performed in vitro comparing the effects of CYN+ and CYN-, which highlights the importance of studying toxic features in their natural scenario.

Toxic effects of the cylindrospermopsin and chlorpyrifos combination on the differentiated SH-SY5Y human neuroblastoma cell line.

Due to climate change and anthropogenic activities, the levels of pollution of aquatic and terrestrial environments have increased in the last decades. In this sense, the rise of cyanobacterial blooms, which release secondary metabolites with toxic properties, and the global use of pesticides for agricultural purposes have a negative impact on ecosystems. Thus, it would be interesting to study the concomitance of both types of toxicants in the same sample, since it is possible that they appear together. The aim of the present work was to state the effects of the interaction between the cyanotoxin cylindrospermopsin and the pesticide chlorpyrifos in differentiated SH-SY5Y neuronal cells to assess how they could affect the nervous system. To this end, cytotoxicity, morphological, and acetylcholinesterase activity studies were performed during 24 and 48 h. The results revealed a concentration-dependent decrease in viability and interaction between both toxicants, together with clear signs of apoptosis and necrosis induction. In this sense, different stages on the differentiation process would lead to differences in the toxicity exerted by the compounds both isolated as in combination, which it is not observed in non-differentiated cells. Additionally, the acetylcholinesterase activity appeared not to be affected, which is a clear difference compared to non-differentiated cells. These results show the importance of studying not only the toxicants themselves, but also in combination, to assess their possible effects in a more realistic scenario.

In Vitro Toxicity Evaluation of Cyanotoxins Cylindrospermopsin and Microcystin-LR on Human Kidney HEK293 Cells.

Cyanotoxins are secondary metabolites produced by different types of cyanobacteria. Among them, Cylindrospermopsin (CYN) and Microcystins (MCs) stand out due to their wide geographical distribution and toxicity in various organs, including the kidney, which is involved in their distribution and elimination. However, the renal toxicity caused by CYN and MCs has hardly been studied. The aim of this work was to assess the cytotoxicity effects caused by CYN and MC-LR in the renal cell line HEK293, and for the first time, the influence of CYN on the gene expression of selected genes in these cells by quantitative real-time PCR (qRT-PCR). CYN caused an upregulation in the gene expression after exposure to the highest concentration (5 µg/mL) and the longest time of exposure (24 h). Moreover, shotgun proteomic analysis was used to assess the molecular responses of HEK293 cells after exposure to the individuals and combinations of CYN + MC-LR. The simultaneous exposure to both cyanotoxins caused a greater number of alterations in protein expression compared to single toxins, causing changes in the cellular, lipid and protein metabolism and in protein synthesis and transport. Further studies are needed to complete the toxicity molecular mechanisms of both CYN and MC-LR at the renal level.

Microcystin-RR: Occurrence, content in water and food and toxicological studies. A review.

Microcystins (MCs) are hepatotoxins, produced by various species of cyanobacteria, whose occurrence is increasing worldwide owing to climate change and anthropogenic activities. More than 100 variants have been reported, and among them MC-LR is the most extensively studied, but there are other MC congeners that deserve to be investigated. The need for data to characterize the toxicological profile of MC variants other than MC-LR has been identified in order to improve risk assessment in humans and wildlife. Accordingly, the aim of this study was to evaluate the information available in the scientific literature dealing with MC-RR, as this congener is the second most common cyanotoxin in the environment. The review focuses on aspects such as occurrence in water and food, and toxicity studies both in vitro and in vivo. It reveals that, although MC-RR is a real hazard with a high exposure potential in some countries, little is known yet about its specific toxicological properties that differ from those of MC-LR, and important aspects such as genotoxicity and chronic effects have not yet been sufficiently addressed.

Cytotoxic and morphological effects of microcystin-LR, cylindrospermopsin, and their combinations on the human hepatic cell line HepG2.

Cylindrospermopsin (CYN) and Microcystin-LR (MC-LR) are toxins produced by different cyanobacterial species, which are found mainly in freshwater reservoirs. Both of them can induce, separately, toxic effects in humans and wildlife. However, little is known about the toxic effects of the combined exposure, which could likely happen, taking into account the concomitant occurrence of the producers. As both cyanotoxins are well known to induce hepatic damage, the human hepatocellular HepG2 cell line was selected for the present study. Thus, the cytotoxicity of both pure cyanotoxins alone (0-5 µg/mL CYN and 0-120 µg/mL MC-LR) and in combination for 24 and 48 h was assayed, as long as the cytotoxicity of extracts from CYN-producing and nonproducing cyanobacterial species. The potential interaction of the combination was evaluated by the isobologram or Chou-Talalay's method, which provides a combination index as a quantitative measure of the two cyanotoxins interaction's degree. Moreover, a morphological study of the individual pure toxins and their combinations was also performed. Results showed that CYN was the most toxic pure cyanotoxin, being the mean effective concentrations obtained ≈4 and 90 µg/mL for CYN and MC-LR, respectively after 24 h. However, the simultaneous exposure showed an antagonistic effect. Morphologically, autophagy, at low concentrations, and apoptosis, at high concentrations were observed, with affectation of the rough endoplasmic reticulum and mitochondria. These effects were more pronounced with the combination. Therefore, it is important to assess the toxicological profile of cyanotoxins combinations in order to perform more realistic risk evaluations.

Mutagenic and genotoxic potential of pure Cylindrospermopsin by a battery of in vitro tests.

Cylindrospermopsin (CYN) is a cyanobacterial toxin with an increasing world-wide occurrence. The main route of human exposure is through the ingestion of contaminated food and water. The European Food Safety Authority has identified the need to further characterize the toxicological profile of cyanotoxins and in this regard the genotoxicity is a key toxicological effect. The data available in the scientific literature show contradictory results. Thus, the aim of this study was to investigate the mutagenic and genotoxic effects of pure CYN using a battery of different in vitro assays including: the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test) (0-10 µg/mL), the mammalian cell micronucleus (MN) test (0-1.35 µg/mL and 0-2 µg/mL in absence or presence of S9 fraction, respectively) and the mouse lymphoma thymidine-kinase assay (MLA)(0-0.675 µg/mL) on L5178YTk ±â€¯cells, and the standard and enzyme-modified comet assays (0-2.5 µg/mL) on Caco-2 cells. Positive results were obtained only when the metabolic fraction S9 was employed in the MN test, suggesting pro-genotoxic properties of CYN. Also, DNA damage was not mediated by oxidative stress as CYN did not induced changes in the modified comet assay. These data could contribute to a better risk assessment of this cyanotoxin.

In vitro toxicity evaluation of new silane-modified clays and the migration extract from a derived polymer-clay nanocomposite intended to food packaging applications.

The clay montmorillonite (Mt) is among the nanofillers more frequently used for food packaging applications. The organomodification of clays with different modifiers, such as silanes, is an important step in the preparation of improved polymer/clay materials known as nanocomposites. However, the toxicological data about these nanofillers is still scarce. In the present study, an in vitro toxicological evaluation in Caco-2 cells of two silane-modified clays based on Mt, Clay3 and Clay4 (0-250µg/ml), was performed. The cytotoxicity, cell death, genotoxicity and oxidative stress produced by both organoclays were evaluated after 24 and 48h of exposure. Moreover, the migration extracts obtained from nanocomposites of polypropylene (PP) + Clay3 and only PP were also investigated. Only Clay4 induced cytotoxicity, showing a reduction of cell viability to 63% of the control, as well as oxidative stress in a concentration-dependent manner. Regarding the PP-Clay3 migration extract, no cytotoxic effects were observed after exposure to the tested concentrations (0-100%). Moreover, significant differences in the presence of Ca, Mg and Si compared to the PP extract were obtained, although migration levels were in accordance with the food contact materials regulation. These findings indicate that a case-by-case toxicological assessment of organoclays should be performed.

Potential Use of Chemoprotectants against the Toxic Effects of Cyanotoxins: A Review.

Cyanobacterial toxins, particularly microcystins (MCs) and cylindrospermopsin (CYN), are responsible for toxic effects in humans and wildlife. In order to counteract or prevent their toxicity, various strategies have been followed, such as the potential application of chemoprotectants. A review of the main substances evaluated for this aim, as well as the doses and their influence on cyanotoxin-induced toxicity, has been performed. A search of the literature shows that research on MCs is much more abundant than research on CYN. Among chemoprotectants, antioxidant compounds are the most extensively studied, probably because it is well known that oxidative stress is one of the toxic mechanisms common to both toxins. In this group, vitamin E seems to have the strongest protectant effect for both cyanotoxins. Transport inhibitors have also been studied in the case of MCs, as CYN cellular uptake is not yet fully elucidated. Further research is needed because systematic studies are lacking. Moreover, more realistic exposure scenarios, including cyanotoxin mixtures and the concomitant use of chemoprotectants, should be considered.

Vitamin E pretreatment prevents histopathological effects in tilapia (Oreochromis niloticus) acutely exposed to cylindrospermopsin.

Cylindrospermopsin (CYN) is a cyanotoxin frequently involved in blooms with a predominantly extracellular availability, which makes it easily taken up by a variety of aquatic organisms. CYN is a potent protein and glutathione synthesis inhibitor, and also induces genotoxicity, oxidative stress and several histopathological lesions. The present study investigates the protective role of a vitamin E pretreatment (700 mg vit E/kg fish bw/day, for 7 days) on the histopathological alterations induced in different organs of tilapia (Oreochromis niloticus) acutely exposed to a single oral dose of 400 µg pure CYN/kg bw fish. The major histological changes observed were degenerative glucogenic process and loss of the hepatic structure in the liver, glomerulopathy and tubular tumefaction in the kidney, myofibrolysis and edema in the heart, catarrhal enteritis and necrosis in the gastrointestinal tract, hyperemic processes in the gill lamellae, and high basophilia, degeneration and tumefaction of granular neurons in the brain. Vitamin E pretreatment was effective in preventing or ameliorating the abovementioned alterations induced by CYN. In addition, a morphometric study indicated that the average nuclear diameter of hepatocytes, and cross-sections of proximal and distal convoluted tubules, together with the cardiac fiber and capillaries diameters represent a useful tool to evaluate the damage induced by CYN. This is the first study reporting vitamin E prevention of histopathological damage in tissues (liver, kidney, heart, gastrointestinal tract, gills and brain) of fish intoxicated with CYN. Therefore, vitamin E can be considered a useful chemoprotectant in the treatment of histopathological changes induced in CYN-intoxicated fish. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1469-1485, 2016.

Proteomic analysis of anatoxin-a acute toxicity in zebrafish reveals gender specific responses and additional mechanisms of cell stress.

Anatoxin-a is a potent neurotoxin produced by several genera of cyanobacteria. Deaths of wild and domestic animals due to anatoxin-a exposure have been reported following a toxic response that is driven by the inhibition of the acetylcholine receptors at neuromuscular junctions. The consequent neuron depolarization results in an overstimulation of the muscle cells. In order to unravel further molecular events implicated in the toxicity of anatoxin-a, a proteomic investigation was conducted. Applying two-dimensional gel electrophoresis (2DE) and MALDI-TOF mass spectrometry, we report early proteome changes in brain and muscle of zebrafish (Danio rerio) caused by acute exposure to anatoxin-a. In this regard, the test group of male and female zebrafish received an intraperitoneal (i.p.) injection of an anatoxin-a dose of 0.8µgg(-1) of fish body weight (bw) in phosphate buffered saline solution (PBS), while the control received an i.p. injection of PBS only. Five minutes after i.p. injection, brain and muscle tissues were collected, processed and analyzed with 2DE. Qualitative and quantitative analyzes of protein abundance allowed the detection of differences in the proteome of control and exposed fish groups, and between male and female fish (gender specific responses). The altered proteins play functions in carbohydrate metabolism and energy production, ATP synthesis, cell structure maintenance, cellular transport, protein folding, stress response, detoxification and protease inhibition. These changes provide additional insights relative to the toxicity of anatoxin-a in fish. Taking into account the short time of response considered (5min of response to the toxin), the changes in the proteome observed in this work are more likely to derive from fast occurring reactions in the cells. These could occur by protein activity regulation through degradation (proteolysis) and/or post-translational modifications, than from a differential regulation of gene expression, which may require more time for proteins to be synthesized and to produce changes at the proteomic level.

In vitro genotoxicity testing of carvacrol and thymol using the micronucleus and mouse lymphoma assays.

Currently, antimicrobial additives derived from essential oils (Eos) extracted from plants or spices, such as Origanum vulgare, are used in food packaging. Thymol and carvacrol, the major EO compounds of O. vulgare, have demonstrated their potential use as active additives. These new applications use high concentrations, thereby increasing the concern regarding their toxicological profile and especially their genotoxic risk. The aim of this work was to investigate the potential in vitro genotoxicity of thymol (0-250 µM) and carvacrol (0-2500 µM) at equivalent doses to those used in food packaging. The micronucleus (MN) test and the mouse lymphoma (MLA) assay on L5178Y/Tk(±) mouse lymphoma cells were used. The negative results for thymol with the MN with and without the S9 fraction and also with the MLA assay reinforce the view that this compound is not genotoxic in mammalian cells. However, carvacrol presented slight genotoxic effects, but only in the MN test at the highest concentration assayed (700 µM) and in the absence of metabolic activation. The lack of genotoxic response in the MLA assay after 4 and 24h of exposure indicates a low genotoxic potential for carvacrol. Alternatively, the general negative findings observed in both assays suggest that the MN results of carvacrol are marginal data without biological relevance. These results can be useful to identify the appropriate concentrations of these substances to be used as additives in food packaging.

Acute toxicological studies of the main organosulfur compound derived from Allium sp. intended to be used in active food packaging.

Some plant extracts have been proposed as potential alternative to the use of synthetic preservatives in the food industry. Among those, extracts from Allium species exhibit interesting antimicrobial and antioxidant properties for the food packaging industry. The present work aims to assess the usefulness and potential safety of the major organosulfur compound present in a commercial Allium sp. extract (PROALLIUM AP®), namely propyl thiosulfinate oxide (PTSO). For this purpose, its antimicrobial activity was studied in a wide range of microorganisms. Moreover, cytotoxicity and ultrastructural cellular damages caused by PTSO were studied in two human cell lines, Caco-2 and HepG2, being the colonic cells more sensitive to this compound. Finally, the protective role of PTSO against an induced oxidative situation was evaluated in the human intestinal Caco-2 cells. The results revealed damage at high concentration, although no significant adverse effects were recorded for the concentration to be used in food packaging. Moreover, the in vivo study also revealed the potential safety use at the established concentrations. In addition, the antimicrobial properties and the antioxidant role of PTSO were confirmed. Therefore, this compound could be considered as a good natural alternative to synthetic preservatives used in the food packaging industry.

In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: A review.

Essential oils (EOs) and their main constituent compounds have been extensively investigated due to their application in the food industry for improving the shelf life of perishable products. Although they are still not available for use in food packaging in the market in Europe, considerable research in this field has been carried out recently. The safety of these EOs should be guaranteed before being commercialized. The aim of this work was to review the scientific publications, with a primary focus on the last 10 years, with respect to different in vitro toxicological aspects, mainly focussed on mutagenicity/genotoxicity. In general, fewer genotoxic studies have been reported on EOs in comparison to their main components, and most of them did not show mutagenic activity. However, more studies are needed in this field since the guidelines of the European Food Safety Authority have not always been followed accurately. The mutagenic/genotoxic activities of these substances have been related to metabolic activation. Therefore, in vivo tests are required to confirm the absence of genotoxic effects. Considering the great variability of the EOs and their main compounds, a case-by-case evaluation is needed to assure their safe use in food packaging.

Toxicological evaluation of clay minerals and derived nanocomposites: a review.

Clays and clay minerals are widely used in many facets of our society. This review addresses the main clays of each phyllosilicate groups, namely, kaolinite, montmorillonite (Mt) and sepiolite, placing special emphasis on Mt and kaolinite, which are the clays that are more frequently used in food packaging, one of the applications that are currently exhibiting higher development. The improvements in the composite materials obtained from clays and polymeric matrices are remarkable and well known, but the potential toxicological effects of unmodified or modified clay minerals and derived nanocomposites are currently being investigated with increased interest. In this sense, this work focused on a review of the published reports related to the analysis of the toxicological profile of commercial and novel modified clays and derived nanocomposites. An exhaustive review of the main in vitro and in vivo toxicological studies, antimicrobial activity assessments, and the human and environmental impacts of clays and derived nanocomposites was performed. From the analysis of the scientific literature different conclusions can be derived. Thus, in vitro studies suggest that clays in general induce cytotoxicity (with dependence on the clay, concentration, experimental system, etc.) with different underlying mechanisms such as necrosis/apoptosis, oxidative stress or genotoxicity. However, most of in vivo experiments performed in rodents showed no clear evidences of systemic toxicity even at doses of 5000mg/kg. Regarding to humans, pulmonary exposure is the most frequent, and although clays are usually mixed with other minerals, they have been reported to induce pneumoconiosis per se. Oral exposure is also common both intentionally and unintentionally. Although they do not show a high toxicity through this pathway, toxic effects could be induced due to the increased or reduced exposure to mineral elements. Finally, there are few studies about the effects of clay minerals on wildlife, with laboratory trials showing contradictory outcomes. Clay minerals have different applications in the environment, thus with a strict control of the concentrations used, they can provide beneficial uses. Despite the extensive number of reports available, there is also a need of systematic in vitro-in vivo extrapolation studies, with still scarce information on toxicity biomarkers such as inmunomodulatory effects or alteration of the genetic expression. In conclusion, a case by case toxicological evaluation is required taking into account that different clays have their own toxicological profiles, their modification can change this profile, and the potential increase of the human/environmental exposure to clay minerals due to their novel applications.

In vitro pro-oxidant/antioxidant role of carvacrol, thymol and their mixture in the intestinal Caco-2 cell line.

The food industry needs to provide consumers with fresh and healthy products. In this context, food packaging plays an important role. Thus, certain essential oils are being incorporated into plastic polymers to confer better preservative properties. The oregano essential oil contains carvacrol and thymol, two important polyphenols. Considering their increasing use in active food packaging, the evaluation of their suitability and safety is of great interest. In the present work, a concentration-dependent increase in the antioxidant effects of carvacrol, thymol, and their mixture (10:1) was determined using DPPH and ABTS assays. In addition, the safety of these compounds was tested in vitro. Reactive oxygen species and glutathione levels were measured after exposing cells for 24 and 48 h to different concentrations of carvacrol, thymol and their mixture. The abilities of these compounds to protect against or revert the effects of H2O2 on cells were also studied. The results showed that oxidative stress plays a role in the damage induced by carvacrol and the mixture at high concentrations. However, at lower concentrations, both compounds and their mixture were shown, for the first time, to protect cells against the damage induced by the H2O2.

Cytotoxic and mutagenic in vitro assessment of two organosulfur compounds derived from onion to be used in the food industry.

Edible members of the Allium family are widely used since they exhibit antioxidant and antibacterial related to the organosulphur compounds. One the most promising use of Allium species, hence, onion essential oil, could be in the packaging food industry. The present work aims to assess the safety of two organosulphur compounds present in onion essential oil; dipropyl disulphide, dipropyl sulphide and their mixture. For this purpose, cytotoxicity, reactive oxygen species and glutathione contents, and ultrastructural cellular damages were studied in the human intestinal cells, Caco-2, exposed to these organosulphur compounds. Moreover, their potential mutagenicity was also assessed. The results revealed no significant adverse effects. Additionally, reactive oxygen species scavenger activity was observed for both compounds. Therefore, they could be a good natural alternative to other synthetic antioxidant and antibacterial substances used in the food industry.

Exposure of Lycopersicon esculentum to microcystin-LR: effects in the leaf proteome and toxin translocation from water to leaves and fruits.

Natural toxins such as those produced by freshwater cyanobacteria have been regarded as an emergent environmental threat. However, the impact of these water contaminants in agriculture is not yet fully understood. The aim of this work was to investigate microcystin-LR (MC-LR) toxicity in Lycopersicon esculentum and the toxin accumulation in this horticultural crop. Adult plants (2 month-old) grown in a greenhouse environment were exposed for 2 weeks to either pure MC-LR (100 µg/L) or Microcystis aeruginosa crude extracts containing 100 µg/L MC-LR. Chlorophyll fluorescence was measured, leaf proteome investigated with two-dimensional gel electrophoresis and Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF)/TOF, and toxin bioaccumulation assessed by liquid chromatography-mass spectrometry (LC-MS)/MS. Variations in several protein markers (ATP synthase subunits, Cytochrome b6-f complex iron-sulfur, oxygen-evolving enhancer proteins) highlight the decrease of the capacity of plants to synthesize ATP and to perform photosynthesis, whereas variations in other proteins (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and ribose-5-phosphate isomerase) suggest an increase of carbon fixation and decrease of carbohydrate metabolism reactions in plants exposed to pure MC-LR and cyanobacterial extracts, respectively. MC-LR was found in roots (1635.21 µg/kg fw), green tomatoes (5.15-5.41 µg/kg fw), mature tomatoes (10.52-10.83 µg/kg fw), and leaves (12,298.18 µg/kg fw). The results raise concerns relative to food safety and point to the necessity of monitoring the bioaccumulation of water toxins in agricultural systems affected by cyanotoxin contamination.

In vivo toxicity evaluation of the migration extract of an organomodified clay-poly(lactic) acid nanocomposite.

The food packaging industry is in continuous development in order to obtain more secure and stable food and beverages. The incorporation of inorganic and organic materials with plastic polymers leads to polymer composites. Among the inorganic compounds, clays such as montmorillonite (MTT) and its derivatives are of great interest due to their advantageous properties. The Technological Institute of Packaging, Transport,and Logistics (ITENE) developed a novel nanocomposite based on a poly(lactic) acid (PLA) polymer using an MMT derivative, named Clay1, as filler, to be used in the beverage industry. The improvement of the technological properties of this new material was demonstrated, but safety issues are also of concern. In the present study, a histopathological examination by optical and electron microscopy of organs from Wistar rats exposed for 90 d to a migration extract of PLA-Clay1 nanocomposite was carried out. Moreover, different clinical biochemistry, inflammation,and oxidative stress biomarkers were determined. Results showed no apparent evidence of damage, indicating that this nanocomposite has a good profile to be used in the food packaging industry, although further research is still needed.

In vivo evaluation of activities and expression of antioxidant enzymes in Wistar rats exposed for 90 days to a modified clay.

Although clays are wildly used in a range of applications, the toxicity assessment of these new materials is still scarce. In the present study, oxidative stress induced by Clay 1, a novel clay, was determined in rats after 90 d of oral exposure. The activities of antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), were examined. In addition, genetic expressions of SOD and CAT and relative protein abundance of CAT were also determined. Data showed that most of the biomarkers assayed remained unaltered. Only CAT activity, as well as its genetic and protein expressions, appeared enhanced in the kidney. Therefore, further studies are needed to clarify the relevance and consequences of these findings to ensure the safety of this clay.

Immunohistochemical approach to study cylindrospermopsin distribution in tilapia (Oreochromis niloticus) under different exposure conditions.

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria (i.e., Aphanizomenon ovalisporum). CYN is a tricyclic alkaloid combined with a guanidine moiety. It is well known that CYN inhibits both protein and glutathione synthesis, and also induces genotoxicity and the alteration of different oxidative stress biomarkers. Although the liver and kidney appear to be the main target organs for this toxin based on previous studies, CYN also affects other organs. In the present study, we studied the distribution of CYN in fish (Oreochromis niloticus) under two different exposure scenarios using immunohistochemical (IHC) techniques. In the first method, fish were exposed acutely by intraperitoneal injection or by gavage to 200 µg pure CYN/Kg body weight (bw), and euthanized after 24 h or five days of exposure. In the second method, fish were exposed by immersion to lyophilized A. ovalisporum CYN-producing cells using two concentration levels (10 or 100 µg/L) for two different exposure times (7 or 14 days). The IHC was carried out in liver, kidney, intestine, and gills of fish. Results demonstrated a similar pattern of CYN distribution in both experimental methods. The organ that presented the most immunopositive results was the liver, followed by the kidney, intestine, and gills. Moreover, the immunolabeling signal intensified with increasing time in both assays, confirming the delayed toxicity of CYN, and also with the increment of the dose, as it is shown in the sub-chronic assay. Thus, IHC is shown to be a valuable technique to study CYN distribution in these organisms.