Evaluación de la genotoxicidad de un compuesto del extracto proallium ap® mediante el ensayo de micronucleos con potencial uso en envases alimentarios

Los aceites esenciales (AEs) que proceden del género Allium spp, como es el caso del ajo (Allium sativum) y la cebolla (Allium cepa) se caracterizan por poseer numerosas propiedades, destacando sus actividades antioxidante y antimicrobianas, las cuales se les atribuye a sus compuestos organosulfurados. Estas características están siendo utilizadas por la industria para el desarrollo de nuevos envases activos que mejoren y conserven las características del alimento así como su vida útil. Antes de aprobar su uso, estas sustancias deben ser evaluadas a través de distintos ensayos toxicológicos que confirmen su seguridad. En este sentido, y siguiendo las recomendaciones de la EFSA (European Food Safety Authority) para la evaluación genotóxica de sustancias que van a estar en contacto con el alimento, el objetivo de este trabajo es evaluar por primera vez el potencial genotóxico de Propil Propano Tiosulfinato (PTS) presente en el extracto comercial Proallium AP®. El PTS se evaluó in vitro en la línea celular L5178Tk± de células de linfoma de ratón a través del ensayo de micronúcleos (MN) (0-25 μM) siguiendo el protocolo de la OECD 487. Se evaluó durante 24h en ausencia de S9 y durante 4h en presencia de S9. Los resultados obtenidos, muestran como el PTS en ausencia de S9 produce un leve pero significativo incremento en la frecuencia de MN en células binucleadas a la concentración de 17,25 μM. Sin embargo, en presencia de la fracción microsómica, el compuesto estudiado no actúa como agente clastogénico o aneugénico. No obstante, es necesario completar estos estudios con otros ensayos in vitro que confirmen la seguridad del PTS. En caso de obtener resultados contradictorios, y siguiendo las recomendaciones de las autoridades europeas sería necesario realizar la evaluación del PTS in vivo, para confirmar su seguridad antes de autorizar su uso en el envase alimentario.Universidad de Sevilla. Grado en Farmaci

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.España Ministerio de Economía y Competitividad AGL2015-64558-

In vitro assessment of cyanotoxins bioaccessibility in raw and cooked mussels

The oral route by ingestion of water and food contaminated with cyanotoxins is the main route of exposure to these toxins. This study addresses for the first time the bioaccessibility of some of the most common Microcystins (MC-LR, MC-RR and MC-YR) and Cylindrospermopsin (CYN) simultaneously in raw and steamed mussels spiked at 250 ng/g fresh weight of each cyanotoxin, after an in vitro digestion, including the salivary (incubation with artificial saliva, 30s), gastric (with pepsin, 2h, pH 2), duodenal (with pancreatin and bile salts, 2h, pH 6.5) and colonic phases (with lactic-acid bacteria, 48h, pH 7.2). The results obtained suggest that the potential absorption of these cyanotoxins by consumption of contaminated mussels is lower than expected. After the total effect of cooking and digestion, the mean bioaccessibility levels recorded were 24.65% (CYN), 31.51% (MC-RR), 17.51% (MC-YR) and 13.20% (MC-LR). Moreover, toxins were transferred to the steaming waters at 3.77 ± 0.24 μg L−1 CYN, 2.29 ± 0.13 μg L−1 MC-LR, 6.60 ± 0.25 μg L−1 MC-RR and 3.83 ± 0.22 μg L−1 MC-YR. These bioaccessibility results should be considered for a more accurate risk assessment related to these cyanotoxins in mussels, including the fact that the steaming waters could also represent a risk after human consumption.Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE

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.Ministerio de Ciencia e Innovación (PID2019-104890RB-I00 MICIN/ AEI/10.13039/ 501100011033

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.Ministerio de Ciencia, Innovación y Universidades PID2019-104890RB-I00Ministerio de Economia, Industria y Competitividad - FPI grant number BES-2016-07877

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.España MINECO AGL2015-64558-

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 CYNPortuguese Science Foundation and under the Projects MOREBIVALVES (PTDC/ASP-PES/31762/2017) and UID/Multi/04423/2013NORTE 2020, Portugal 2020 and the European Union through the ERDF, and by FCT. Moreover, Project AGL2015-64558-RMINECO/FEDER, UE, and the grant FPI (BES-2016–078773

Validation of a method for cylindrospermopsin determination in vegetables: application to real samples such as lettuce (Lactuca sativa L.)

Ministerio de Economía y Competitividad AGL2015-64558-R, MINECO/FEDER, U

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.Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE

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.Fondo Europeo de Desarrollo Regional US-1259106, P18-RT1993Junta de Andalucía POSTDOC_21_0013