Impact of Gastrointestinal Digestion In Vitro Procedure on the Characterization and Cytotoxicity of Reduced Graphene Oxide.

The growing interest in graphene derivatives is a result of their variety of applications in many fields. Due to their use, the oral route could be a potential way of entrance for the general population. This work assesses the biotransformation of reduced graphene oxide (rGO) after an in vitro digestion procedure (mouth, gastric, intestinal, and colon digestion), and its toxic effects in different cell models (HepG2, Caco-2, and 3D intestinal model). The characterization of rGO digestas evidenced the agglomeration of samples during the in vitro gastrointestinal (g.i.) digestion. Internalization of rGO was only evident in Caco-2 cells exposed to the colonic phase and no cellular defects were observed. Digestas of rGO did not produce remarkable cytotoxicity in any of the experimental models employed at the tested concentrations (up to 200 µg/mL), neither an inflammatory response. Undigested rGO has shown cytotoxic effects in Caco-2 cells, therefore these results suggest that the digestion process could prevent the systemic toxic effects of rGO. However, additional studies are necessary to clarify the interaction of rGO with the g.i. tract and its biocompatibility profile.

In Vitro Mutagenic and Genotoxic Assessment of Anatoxin-a Alone and in Combination with Cylindrospermopsin.

Anatoxin-a (ATX-a) is a cyanobacterial toxin whose occurrence has been reported worldwide and has attracted increasing scientific interest due to its toxicity. Moreover, in nature, ATX-a usually appears together with other cyanotoxins, such as cylindrospermopsin (CYN), so possible interaction phenomena could happen and should be considered for risk assessment purposes. For this reason, the aim of this work was to explore the potential mutagenicity and genotoxicity of pure ATX-a and an ATX-a/CYN mixture using a battery of in vitro assays, including the bacterial reverse-mutation assay in Salmonella typhimurium (OECD 471) and the micronucleus test (MN) (OECD 487) on L5178Y Tk+/- cells. The results showed that ATX-a was not mutagenic either alone or in combination with CYN under the conditions tested. Nevertheless, genotoxic effects were observed for both ATX-a and its mixture with CYN following the in vitro MN assay. The genotoxicity exhibited by ATX-a was only observed in the absence of S9 mix, whereas in the cyanotoxin mixture the concentration-dependent genotoxicity of ATX-a/CYN in vitro was observed only in the presence of S9. Thus, the toxicity induced by cyanotoxin mixtures may vary from that produced by toxins alone, and consequently more studies are necessary in order to perform more realistic risk assessments.

Effect of cold food storage techniques on the contents of Microcystins and Cylindrospermopsin in leaves of spinach (Spinacia oleracea) and lettuce (Lactuca sativa).

The presence of Cylindrospermopsin (CYN) and Microcystins (MCs) in vegetables is considered as a significant worldwide toxicological risk. Thus, this work aims to assess for the first time the impact of refrigeration (4 °C) and freezing (-20 °C) on the levels of CYN, MCs and their mixtures (CYN + MCs) in lettuce and spinach. Samples were spiked with 750 µg cyanotoxins/g dry weight (d.w.). Several storage conditions were studied: refrigeration after 24, 48 h and 7 days, and freezing for 7 days, 1 and 3 months. Cyanotoxin concentrations were determined by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). For CYN, refrigeration at 48 h and 7 days was effective to decrease its concentrations up to 26% and 32%, respectively, in spinach. For MCs, refrigeration was only effective in lettuce compared to spinach, showing an important decrease of 80.3% MC-LR and 85.1% MC-YR. In spinach, CYN was stable after 3 months freezing, whereas MC contents were still reduced up to 44%. Overall, cyanotoxins were less stable in the mixture compared to individual toxins for both processes, and the effect of these storage techniques were toxin and food-specific. Further studies of cyanotoxins in foods are required for evaluating the risk for humans.

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.

Immunomodulatory Effects of Pure Cylindrospermopsin in Rats Orally Exposed for 28 Days.

Cylindrospermopsin (CYN) is a ubiquitous cyanotoxin showing increasing incidence worldwide. CYN has been classified as a cytotoxin and, among its toxic effects, its immunotoxicity is scarcely studied. This work investigates for the first time the influence of oral CYN exposure (18.75; 37.5 and 75 µg/kg b.w./day, for 28 days) on the mRNA expression of selected interleukin (IL) genes (IL-1ß, IL-2, IL-6, Tumor Necrosis Factor alpha (TNF-α), Interferon gamma (IFN-γ)) in the thymus and the spleen of male and female rats, by quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, their serum levels were also measured by a multiplex-bead-based immunoassay, and a histopathological study was performed. CYN produced immunomodulation mainly in the thymus of rats exposed to 75 µg CYN/kg b.w./day in both sexes. However, in the spleen only IL-1ß and IL-2 (males), and TNF-α and IFN-γ (females) expression was modified after CYN exposure. Only female rats exposed to 18.75 µg CYN/kg b.w./day showed a significant decrease in TNF-α serum levels. There were no significant differences in the weight or histopathology in the organs studied. Further research is needed to obtain a deeper view of the molecular mechanisms involved in CYN immunotoxicity and its consequences on long-term exposures.

Influence of refrigeration and freezing in Microcystins and Cylindrospermopsin concentrations on fish muscle of tilapia (Oreochromis niloticus) and tench (Tinca tinca).

The consumption of fish contaminated with cyanotoxins is an important public health issue due to their potential adverse effects. The aim of this study was to assess the influence of refrigeration (4 °C) and freezing (-20 °C) on the concentration of Cylindrospermopsin (CYN), Microcystins (MCs) and their combination in tilapia (Oreochromis niloticus) and tench (Tinca tinca). Fish muscle were spiked with a stock solution of each toxin to reach 750 µg/g dry weight (d.w.). Three different periods of time were investigated for each treatment: 24 h, 48 h and 7 days for refrigeration, and 24 h, 7 days and 1 month for freezing. Samples were extracted and quantified by Ultra Performance Liquid Chromatography - Tandem Mass Spectrometry (UPLC-MS/MS). The results showed that freezing for 1 month produced highest decreases of these toxins in both species in comparison to refrigeration, being CYN the most stable cyanotoxin. Moreover, MCs are more stable to storage processes in the mixtures than alone, and fish species is a factor to take into account in their stability. These findings highlight the need to assess the influence of food storage processes on the presence of cyanotoxins in fish species for a more realistic human health risk assessment.

Immunotoxic Effects Induced by Microcystins and Cylindrospermopsin: A Review.

Cyanotoxin occurrence is gaining importance due to anthropogenic activities, climate change and eutrophication. Among them, Microcystins (MCs) and Cylindrospermopsin (CYN) are the most frequently studied due to their ubiquity and toxicity. Although MCs are primary classified as hepatotoxins and CYN as a cytotoxin, they have been shown to induce deleterious effects in a wide range of organs. However, their effects on the immune system are as yet scarcely investigated. Thus, to know the impact of cyanotoxins on the immune system, due to its importance in organisms' homeostasis, is considered of interest. A review of the scientific literature dealing with the immunotoxicity of MCs and CYN has been performed, and both in vitro and in vivo studies have been considered. Results have confirmed the scarcity of reports on the topic, particularly for CYN. Decreased cell viability, apoptosis or altered functions of immune cells, and changed levels and mRNA expression of cytokines are among the most common effects reported. Underlying mechanisms, however, are still not yet fully elucidated. Further research is needed in order to have a full picture of cyanotoxin immunotoxicity.

Evaluation of toxic effects induced by repeated exposure to Cylindrospermopsin in rats using a 28-day feeding study.

Cylindrospermopsin (CYN) is a toxin with a world-wide increasing occurrence. It can induce toxic effects both in humans and the environment, and toxicity studies are needed to complete its toxicological profile. In this sense, in vivo oral toxicity studies with pure CYN are scarce. The aim of this work was to perform a repeated dose 28-day oral study in rats following the OECD guideline 407 to provide information on health hazard likely to arise from this kind of exposure. Male and female Sprague-Dawley rats were dosed with 18.75, 37.5 and 75 µg CYN/kg b.w./day. After the study period, no clinical signs or mortality and no significant differences in final body weight, body weight gain and total feed intake in both sexes were observed. Only in females some biochemical parameters (triglycerides (TRIG) levels and aspartate aminotransferase (AST) activity) as well as changes in the weight of organs (absolute liver weight values, relative kidney/body weight ratios or relative liver weight/brain weight ratios) were altered, but without toxicological relevance. Histopathological analysis revealed a very mild affectation of liver and kidney in rats. These results suggest the need to perform longer oral toxicity studies to define the potential consequences of long term CYN exposure.

Genotoxic Effects of Cylindrospermopsin, Microcystin-LR and Their Binary Mixture in Human Hepatocellular Carcinoma (HepG2) Cell Line.

Simultaneous occurrence of cylindrospermopsin (CYN) and microcystin-LR (MCLR) has been reported in the aquatic environment and thus human exposure to such mixtures is possible. As data on the combined effects of CYN/MCLR are scarce, we aimed to investigate the adverse effects related to genotoxic activities induced by CYN (0.125, 0.25 and 0.5 µg/mL) and MCLR (1 µg/mL) as single compounds and their combinations in HepG2 cells after 24 and 72 h exposure. CYN and CYN/MCLR induced DNA double-strand breaks after 72 h exposure, while cell cycle analysis revealed that CYN and CYN/MCLR arrested HepG2 cells in G0/G1 phase. Moreover, CYN and the combination with MCLR upregulated CYP1A1 and target genes involved in DNA-damage response (CDKN1A, GADD45A). Altogether, the results showed that after 72 h exposure genotoxic activity of CYN/MCLR mixture was comparable to the one of pure CYN. On the contrary, MCLR (1 µg/mL) had no effect on the viability of cells and had no influence on cell division. It did not induce DNA damage and did not deregulate studied genes after prolonged exposure. The outcomes of the study confirm the importance of investigating the combined effects of several toxins as the effects can differ from those induced by single compounds.

Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats.

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 µg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

Physiological and Metabolic Responses of Marine Mussels Exposed to Toxic Cyanobacteria Microcystis aeruginosa and Chrysosporum ovalisporum.

Toxic cyanobacterial blooms are a major contaminant in inland aquatic ecosystems. Furthermore, toxic blooms are carried downstream by rivers and waterways to estuarine and coastal ecosystems. Concerning marine and estuarine animal species, very little is known about how these species are affected by the exposure to freshwater cyanobacteria and cyanotoxins. So far, most of the knowledge has been gathered from freshwater bivalve molluscs. This work aimed to infer the sensitivity of the marine mussel Mytilus galloprovincialis to single as well as mixed toxic cyanobacterial cultures and the underlying molecular responses mediated by toxic cyanobacteria. For this purpose, a mussel exposure experiment was outlined with two toxic cyanobacteria species, Microcystis aeruginosa and Chrysosporum ovalisporum at 1 × 105 cells/mL, resembling a natural cyanobacteria bloom. The estimated amount of toxins produced by M. aeruginosa and C. ovalisporum were respectively 0.023 pg/cell of microcystin-LR (MC-LR) and 7.854 pg/cell of cylindrospermopsin (CYN). After 15 days of exposure to single and mixed cyanobacteria, a depuration phase followed, during which mussels were fed only non-toxic microalga Parachlorella kessleri. The results showed that the marine mussel is able to filter toxic cyanobacteria at a rate equal or higher than the non-toxic microalga P. kessleri. Filtration rates observed after 15 days of feeding toxic microalgae were 1773.04 mL/ind.h (for M. aeruginosa), 2151.83 mL/ind.h (for C. ovalisporum), 1673.29 mL/ind.h (for the mixture of the 2 cyanobacteria) and 2539.25 mL/ind.h (for the non-toxic P. kessleri). Filtering toxic microalgae in combination resulted in the accumulation of 14.17 ng/g dw MC-LR and 92.08 ng/g dw CYN. Other physiological and biochemical endpoints (dry weight, byssus production, total protein and glycogen) measured in this work did not change significantly in the groups exposed to toxic cyanobacteria with regard to control group, suggesting that mussels were not affected with the toxic microalgae. Nevertheless, proteomics revealed changes in metabolism of mussels related to diet, specially evident in those fed on combined cyanobacteria. Changes in metabolic pathways related with protein folding and stabilization, cytoskeleton structure, and gene transcription/translation were observed after exposure and feeding toxic cyanobacteria. These changes occur in vital metabolic processes and may contribute to protect mussels from toxic effects of the toxins MC-LR and CYN.

In vivo genotoxicity evaluation of cylindrospermopsin in rats using a combined micronucleus and comet assay.

Cylindrospermopsin (CYN) is a potent cyanotoxin recognized as an emerging human threat due to its cytotoxicity and potential carcinogenicity. Although the genotoxicity of CYN has been extensively studied in vitro, limited data are available on its in vivo genotoxicity. The aim of this study was to evaluate the in vivo genotoxicity of pure CYN (7.5-75 µg/kg body weight) after oral exposure of rats through a combined assay of the micronucleus test (MN) in bone marrow, and the standard and modified comet assay in stomach, liver and blood. Also, histopathological changes in stomach and liver were evaluated. Positive results in the MN test were observed in bone marrow in the exposed rats at all the tested concentrations. However, the comet assay revealed that CYN did not induce DNA strand breaks nor oxidative DNA damage in any of the tissues investigated. Finally, histopathological changes were observed in stomach and liver (7.5-75 µg/kg) in intoxicated rats. These results could indicate that CYN is able to induce irritation in stomach before its biotransformation in rats orally exposed, and genotoxicity in bone marrow.

In Vitro Mutagenic and Genotoxic Assessment of a Mixture of the Cyanotoxins Microcystin-LR and Cylindrospermopsin.

The co-occurrence of various cyanobacterial toxins can potentially induce toxic effects different than those observed for single cyanotoxins, as interaction phenomena cannot be discarded. Moreover, mixtures are a more probable exposure scenario. However, toxicological information on the topic is still scarce. Taking into account the important role of mutagenicity and genotoxicity in the risk evaluation framework, the objective of this study was to assess the mutagenic and genotoxic potential of mixtures of two of the most relevant cyanotoxins, Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN), using the battery of in vitro tests recommended by the European Food Safety Authority (EFSA) for food contaminants. Mixtures of 1:10 CYN/MC-LR (CYN concentration in the range 0.04-2.5 µg/mL) were used to perform the bacterial reverse-mutation assay (Ames test) in Salmonella typhimurium, the mammalian cell micronucleus (MN) test and the mouse lymphoma thymidine-kinase assay (MLA) on L5178YTk± cells, while Caco-2 cells were used for the standard and enzyme-modified comet assays. The exposure periods ranged between 4 and 72 h depending on the assay. The genotoxicity of the mixture was observed only in the MN test with S9 metabolic fraction, similar to the results previously reported for CYN individually. These results indicate that cyanobacterial mixtures require a specific (geno)toxicity evaluation as their effects cannot be extrapolated from those of the individual cyanotoxins.

Occurrence and toxicity of microcystin congeners other than MC-LR and MC-RR: A review.

The occurrence of cyanobacterial toxins is being increasingly reported. This is a reason for concern as they can induce toxic effects both in humans and in the environment. Among them, microcystins (MCs) are the best described and most diverse group of cyanobacterial toxins, and MC-LR and MC-RR are the congeners most widely investigated. However, the number of MC variants has also increased in recent years. Some of these minority variants have been shown to have a different toxicokinetic and toxicodynamic profile, but research focused on them is still limited. Moreover, in some water bodies these minority variants can be the predominant toxins. Nonetheless, MC-LR is the only one used for risk evaluation purposes at present. In order to contribute to more realistic risk assessments in the future, the aim of this review was to compile the available information in the scientific literature regarding the occurrence and concentration of minority MCs in water and food samples, and their toxic effects. The data retrieved demonstrate the congener-specific toxicity of MCs, as well as many data gaps in relation to analytical or mechanistic aspects, among others. Therefore, further research is needed to improve the toxicological characterization of these toxins and the exposure scenarios.

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.

New Method for Simultaneous Determination of Microcystins and Cylindrospermopsin in Vegetable Matrices by SPE-UPLC-MS/MS.

Cyanotoxins are a large group of noxious metabolites with different chemical structure and mechanisms of action, with a worldwide distribution, producing effects in animals, humans, and crop plants. When cyanotoxin-contaminated waters are used for the irrigation of edible vegetables, humans can be in contact with these toxins through the food chain. In this work, a method for the simultaneous detection of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR), and Cylindrospermopsin (CYN) in lettuce has been optimized and validated, using a dual solid phase extraction (SPE) system for toxin extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Results showed linear ranges (5⁻50 ng g-1 f.w.), low values for limit of detection (LOD) (0.06⁻0.42 ng g-1 f.w.), and limit of quantification (LOQ) (0.16⁻0.91 ng g-1 f.w.), acceptable recoveries (41⁻93%), and %RSDIP values for the four toxins. The method proved to be robust for the three variables tested. Finally, it was successfully applied to detect these cyanotoxins in edible vegetables exposed to cyanobacterial extracts under laboratory conditions, and it could be useful for monitoring these toxins in edible vegetables for better exposure estimation in terms of risk assessment.

Validation of a Method for Cylindrospermopsin Determination in Vegetables: Application to Real Samples Such as Lettuce (Lactuca sativa L.).

Reports on the occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) have increased worldwide because of CYN toxic effects in humans and animals. If contaminated waters are used for plant irrigation, these could represent a possible CYN exposure route for humans. For the first time, a method employing solid phase extraction and quantification by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) of CYN was optimized in vegetables matrices such as lettuce (Lactuca sativa). The validated method showed a linear range, from 5 to 500 ng CYN g-1 of fresh weight (f.w.), and detection and quantitation limits (LOD and LOQ) of 0.22 and 0.42 ng CYN g-1 f.w., respectively. The mean recoveries ranged between 85 and 104%, and the intermediate precision from 12.7 to 14.7%. The method showed to be robust for the three different variables tested. Moreover, it was successfully applied to quantify CYN in edible lettuce leaves exposed to CYN-contaminated water (10 µg L-1), showing that the tolerable daily intake (TDI) in the case of CYN could be exceeded in elderly high consumers. The validated method showed good results in terms of sensitivity, precision, accuracy, and robustness for CYN determination in leaf vegetables such as lettuce. More studies are needed in order to prevent the risks associated with the consumption of CYN-contaminated vegetables.