Riesgos tóxicos por cianotoxinas en peces de consumo : optimización de métodos de análisis, potenciales mecanismos de toxicidad y tratamiento

Las cianobacterias han ido adquiriendo gran importancia a lo largo de los años debido a su capacidad de formar floraciones o ¿blooms¿, las cuales pueden llegar a ser tóxicas debido a la producción de cianotoxinas, por lo que se han convertido en una preocupación a nivel mundial en materia de contaminación ambiental, toxicológica, sanitaria y económica, ya que pueden afectar tanto a animales y plantas como a seres humanos. De todas las cianotoxinas, y dado que son las únicas que han causado fenómenos masivos de intoxicación en poblaciones humanas, merecen especial atención las Microcistinas (MCs) y la Cilindrospermopsina (CYN), constituyendo esta última el principal objeto de estudio de la presente Tesis Doctoral. Por todo ello, decidimos realizar una serie de estudios para investigar, en primer lugar, los efectos que pueden tener diferentes técnicas usuales de cocinado en las concentraciones de MCs en el pescado contaminado destinado al consumo humano. En el caso de la CYN, en primer lugar, nos pareció importante la puesta a punto y validación de métodos analíticos para cuantificar la toxina en diferentes matrices, y posteriormente decidimos profundizar en su mecanismo de toxicidad y sus efectos tóxicos, así como estudiar alternativas que nos permitieran contrarrestar los daños producidos por dicha toxina. Asimismo, también estudiamos su distribución en tejidos de peces contaminados. Todo ello en una de las especies acuáticas de consumo humano que pueden verse afectadas por esta toxina en su hábitat natural, como es el pez tilapia (Oreochromis niloticus). Por otra parte, también se ha abordado, mediante proteómica, el estudio del repertorio de Glutatión-S-transferasas (GSTs) en branquias de bivalvos, y los efectos de la CYN sobre plantas de consumo humano, como son las zanahorias (Daucus carota), expuestas a la toxina mediante el agua de riego. Una de las vías de exposición a MCs más importantes es la vía oral. Además, las MCs pueden bioacumularse en organismos acuáticos, pudiendo ser transferidas a lo largo de la cadena trófica hasta los escalones más altos, y llegando así a los seres humanos, por lo que el consumo de estos animales acuáticos contaminados representa un riesgo potencial para la salud humana. Así, una correcta evaluación de riesgos de la exposición humana a MCs a través de los alimentos requiere de un detallado conocimiento acerca de la influencia del procesado y cocinado del alimento sobre estas toxinas. Es por ello que nos propusimos investigar los posibles cambios en la concentración de MCs libres en músculo de tilapias (Oreochromis niloticus) (porción comestible) a las que se les añadió una mezcla de MCs puras (MC-LR, MC-RR y MC-YR), conteniendo 1,5 µg/mL de cada toxina, y sometidas posteriormente a dos técnicas de cocinado habituales, como son el horno microondas y el hervido en dos modalidades. Se ha comprobado que ambas técnicas de cocinado son capaces de reducir los niveles de MCs libres en el pescado (Oreochromis niloticus) en un rango entre 25-50%, siendo mayor con el hervido continuo en el caso de la MC-RR. Concretamente, el hervido continuo ha demostrado ser el más eficaz, debido tal vez al mayor tiempo de permanencia de la muestra en el agua. Asimismo, tras detectar MCs en el agua de hervido, mostramos que existe un riesgo adicional inherente a la utilización de esta agua para hacer sopas de pescado. En general, estas técnicas podrían ser consideradas como medidas de control de estas toxinas en la dieta tras el consumo humano de los alimentos cocinados. Los resultados de este experimento han dado lugar a la siguiente publicación: EFFECTS OF THERMAL TREATMENTS DURING COOKING, MICROWAVE OVEN AND BOILING, ON THE UNCONJUGATED MICROCYSTIN CONCENTRATION IN MUSCLE OF FISH (OREOCHROMIS NILOTICUS). (Guzmán-Guillén y col., 2011. Food and Chemical Toxicology 49, 2060-2067). La monitorización de CYN en medios contaminados por cianobacterias es de gran importancia para evaluar los riesgos que supone su exposición para la salud y el medioambiente. Las técnicas analíticas empleadas para detectar y cuantificar esta toxina, tanto en aguas como en otras matrices, son de diverso fundamento y complejidad (ELISA, LC-DAD, LC-MS y LC-MS/MS). Sin embargo, no todos estos métodos ofrecen la misma sensibilidad. La LC-MS/MS ha sido elegida como el método ideal y de referencia para detectar y cuantificar pequeñas cantidades de toxina en muestras de agua (Eaglesham y col., 1999; Bogialli y col., 2006; Oehrle y col., 2010) y en muestras más complejas, como tejidos de peces (Gallo y col., 2009) o muestras de suero y orina humanas (Foss y Aubel, 2013). Por ello nos propusimos poner a punto y validar diferentes métodos cuantitativos, incluyendo ensayos de robustez, para la determinación de CYN en distintas matrices (aguas, cultivos de cianobacterias y tejidos de peces contaminados con CYN) por LC-MS/MS. Con el empleo de cartuchos de carbón grafitizado se obtuvieron resultados fiables en la extracción de CYN a partir de aguas y de cultivos de cianobacterias, usando una mezcla acidificada de diclorometano:metanol como disolvente. En el caso de los tejidos de peces (hígado y músculo), mediante un sistema SPE de doble cartucho (uno de C18 y uno de carbón grafitizado) conseguimos extractos limpios que permitieron la recuperación de la toxina en un alto porcentaje (80-110%). Por tanto, los métodos que proponemos han sido adecuadamente validados: con amplios rangos de concentraciones de CYN, límites de detección y cuantificación aceptables (que en el caso del agua, permiten la determinación de la toxina por debajo del valor guía propuesto por la OMS de 1 µg/L), recuperaciones en el intervalo 80-110%, y valores adecuados de precisión según la guía AOAC (Association of Official Analytical Chemists). Asimismo, la combinación de factores variables en la metodología demostró la robustez de los métodos propuestos para poder ser reproducidos bajo diferentes condiciones sin sufrir variaciones significativas en los resultados. Los métodos desarrollados en esta Tesis Doctoral pudieron ser aplicados con éxito y rapidez posteriormente para el análisis de CYN en las diferentes matrices estudiadas (aguas y matrices biológicas), permitiendo un control de la exposición, degradación, transferencia y bioacumulación de la CYN.Premio Extraordinario de Doctorado U

DietaryL-Carnitine Prevents HistopathologicalChanges in Tilapia (Oreochromis Niloticus)Exposed to Cylindrospermopsin

Cylindrospermopsin (CYN) is a cytotoxin highly water-soluble, which is easily taken up by several aquatic organisms. CYN acts as a potent protein and glutathione synthesis inhibitor, as well as inducing genotoxicity, oxidative stress, and histopathological alterations. This is the first study reporting the protective effect of a l-carnitine (LC) pretreatment (400 or 880 mg LC/kg bw fish/day, for 21 days) on the histopathological alterations induced by pure CYN or Aphanizomenon ovalisporum lyophilized cells (400 µg CYN/kg bw fish) in liver, kidney, heart, intestines, and gills of tilapia (Oreochromis niloticus) acutely exposed to the toxin by oral route. The main histopathological changes induced by CYN were disorganized parenchyma with presence of glycogen and lipids in the cytoplasm (liver), glomerulonephritis, glomerular atrophy, and dilatation of Bowman's capsule (kidney), myofibrolysis, loss of myofibrils, with edema and hemorrhage (heart), intestinal villi with necrotic enterocytes and partial loss of microvilli (gastrointestinal tract), and hyperemia and hemorrhage (gills). LC pretreatment was able to totally prevent those CYN-induced alterations from 400 mg LC/kg bw fish/day in almost all organs, except in the heart, where 880 mg LC/kg bw fish/day were needed. In addition, the morphometric study indicated that LC managed to recover totally the affectation in the cross sections of the proximal and distal convoluted tubules in CYN-exposed fish

Influence of cooking (microwaving and broiling) on cylindrospermopsin concentration in muscle of nile tilapia (oreochromis niloticus) and characterization of decomposition products

Cylindrospermopsin (CYN) has become increasingly important as a freshwater algal toxin, showing cytotoxic effects. This toxin is able to bioaccumulate in freshwater food webs, representing a serious human health problem. Normally, fish is cooked before consumption, and CYN concentration can be altered. For the first time, the effects of microwaving and broiling for 1 and 2 min on CYN concentration and its decomposition products in fish muscle (Oreochromis niloticus) contaminated in the laboratory were investigated, using UPLC-MS/MS and Orbitrap. The results show that cooking the fish reduced unconjugated CYN levels by 11, 10 and 15% after microwaving for 1 and 2 min, and broiling for 2 min, respectively, compared to control fish. Different CYN decomposition products with m/z 416.1234 (7-epi-CYN) and m/z 336.16663 (diasteroisomers C-3A, C-3C, C-3D, C-3E, C-3F) are generated in fish samples submitted to cooking. Based on the relative abundance of the decomposition products, the possible degradation pathways taking place by microwaving may be through the formation of 7-epi-CYN and m/z 336.16663 compounds, whereas in the case of broiling the last route is the only one observed in this study. The influence of cooking and the toxicity characterization of the degradation products generated in CYN-contaminated fish are of importance for more realistic risk evaluation related to their consumption

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

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-

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

Changes on cylindrospermopsin concentration and characterization of decomposition products in fish muscle (Oreochromis niloticus) by boiling and steaming

The occurrence of cylindrospermopsin (CYN) in toxic blooms of cyanobacteria is increasing in both frequency and distribution over the world. CYN persistence in water potentiates its accumulation in a wide range of aquatic animals destined to human consumption representing a serious problem. The aim of this work was to study for the first time the influence of cooking (boiling and steaming) for 1 or 2 min, on the stability of CYN in muscle of fish (Oreochromis niloticus) contaminated under laboratory conditions, also analyzing the waters in which samples were cooked. CYN was extracted and quantification was carried out by a developed and validated UPLC-MS/MS method. The results show that concentrations of CYN in fish are dependent on the cooking method, being the steaming for 2 min the most effective in reducing CYN levels (26%), followed by boiling for 2 min (18%), and significant differences have been found between the two periods assayed (1 min vs. 2 min). CYN was also detected in waters in which fish muscles were cooked in the range 0.10–0.28 μg/L. Moreover, characteristic decomposition products depending on the type of cooking were detected for the first time among the results of these treatments. The present findings emphasize the need for further studies to evaluate the influence of cooking in the presence of CYN in fish for a more realistic risk evaluation for the human health.Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE

Bioaccessibility and decomposition of cylindrospermopsin in vegetablesmatrices after the application of anin vitrodigestion model

Research on the human exposure to Cylindrospermopsin (CYN) via consumption of contaminated food is of great interest for risk assessment purposes. The aim of this work is to evaluate for the first time the CYN bioaccessibility in contaminated vegetables (uncooked lettuce and spinach, and boiled spinach) after an in vitro digestion model, including the salivar, gastric and duodenal phases and, colonic fermentation under lactic acid bacteria. The results obtained showed that the digestion processes are able to diminish CYN levels, mainly in the colonic phase, especially in combination with the boiling treatment, decreasing CYN levels in a significant way. Moreover, the potential decomposition products in a pure CYN solution and in CYN-contaminated vegetables were evaluated using UHPLC-MS/MS Orbitrap. Under the conditions assayed, only two diastereoisomers of the same fragment with m/z 292.09617 have been detected in all the analysed samples, with the exception of digested vegetables. Therefore, in terms of risk assessment, the digestion seems to play an important role in reducing the final bioaccesibility of CYN, and the consumption of cooked vegetables (spinach) would be safer in comparison to raw vegetables.Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE