Detection of cylindrospermopsin and its decomposition products in raw and cooked fish (Oreochromis niloticus) by analytical pyrolysis (Py-GC/MS)

The presence of the toxin cylindrospermopsin is increasingly frequent in samples from different ecosystems and it is a serious problem both at environmental level and for animal and human health. To be able to prevent CYN exposure risk, it is important to have suitable analytical methods, but also quick and economical ones. Analytical pyrolysis coupled to GC/MS (Py-GC/MS) represents an important alternative for the rapid detection, characterization or “fingerprinting” of different materials. However, it has been less studied with cyanotoxins up to date. The present work aims to investigate: 1) the suitability of Py-GC/MS for detection of CYN and its decomposition products in raw and cooked fish samples before consumption and 2) the influence of the different cooking methods on the presence of different CYN degradation products detected by Py-GC/MS. For first time, these results present that Py-GC/MS could be a rapid and economical alternative for the detection and monitoring of CYN and its degradation products (DP. m/z 290.1, 169.1 and 336.2) in raw or cooked fish. Moreover, the changes induced in CYN and DP by cooking could be amenable and detected by Py-GC/MS.Ministerio de Economía y Competitividad AGL2015-64558-R, CGL2016-78937-

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. © 2017 by the authors. Licensee MDPI, Basel, Switzerland.This work was supported by the the Ministerio de Economía y Competitividad of Spain (AGL2015-64558-R, MINECO/FEDER, UE), by the FCT Project—UID/Multi/04423/2013, and by the Structured Program of R&D&I INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR), funded by the Northern Regional Operational Program (NORTE2020) through the European Regional Development Fund (ERDF). Alexandre Campos work is supported by a post-doctoral grant (SFRH/BPD/103683/2014) from Foundation for Science and Technology (FCT, Lisbon, Portugal)

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.Funding: This research was funded by the SPANISH MINISTERIO DE ECONOMÍA Y COMPETITIVIDAD (AGL2015-64558-R, MINECO/FEDER, UE); by the FPI grant number BES-2016-078773 awarded to Leticia Díez-Quijada Jiménez; by the FCT project UID/Multi/04423/2013, and the post-doctoral grant (SFRH/BPD/103683/2014) from FCT awarded to Alexandre Campos. Acknowledgments: Spanish Ministerio de Economía y Competitividad for the project AGL2015-64558-R, MINECO/FEDER, UE, and for the grant FPI (BES-2016-078773) awarded to Leticia Díez-Quijada Jiménez. CIIMAR members acknowledge FCT project UID/Multi/04423/2013 and the post-doctoral grant (SFRH/BPD/103683/2014) from FCT awarded to Alexandre Campos

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Detección de cilindrospermopsina a través de sus productos de descomposición en músculo de pescado crudo y cocinado: utilidad de la pirólisis analítica (Py-GC/MS)

Póster con 7 figuras, 1 tabla presentado en el 2º Congreso Iberoamericano y 6º Ibérico de Cianotoxinas 3-5 junio 2019 MurciaDebido a la capacidad que tiene la citotoxina cilindrospermopsina (CYN) de acumularse en diversos organismos acuáticos, como los peces, es importante disponer de métodos analíticos adecuados que permitan su detección en muestras de pescado contaminado con la toxina. El objetivo de este trabajo fue poner a punto un método de pirólisis analítica (Py-GC/MS) con el que detectar CYN y algunos de sus fragmentos de descomposición en músculo de escado crudo y cocinado. Músculos de tilapia (Oreochromis niloticus) (4g; n=5) contaminados con 50 ng CYN/g peso seco fueron cocinados 2 min por distintas técnicas (microondas, asado, hervido o vapor). Se tomó como control positivo músculo con CYN sin cocinar y como control negativo músculo sin CYN y no cocinado. Todas las muestras fueron congeladas (-80°C) y posteriormente liofilizadas hasta análisis. Las muestras se pirolizaron durante 1 minuto a 350 °C en ausencia de oxígeno en un pirolizador de doble disparo acoplado a un sistema de cromatografía de gases con detector selectivo de masas. La Py-GC/MS directa permitió la detección de CYN (PM 416) en músculo de pescado contaminado a un tiempo de retención de 24,2 min, así como de 3 posibles fragmentos de descomposición característicos de la toxina con PM 290 (15,9 min), 169 (22,4 min) y 336 (25,25 min). Se observaron además variaciones en la abundancia relativa de cada fragmento según el tipo de cocinado, siendo característico el aumento del fragmento con PM 336 tras el cocinado en microondas. El fragmento con PM 290 se observó cuando el pescado fue cocinado por técnicas que no implican agua. De manera general, las técnicas de cocinado que conllevan el empleo de agua (especialmente el hervido), mostraron una abundancia relativa menor tanto de CYN, como de los distintos fragmentos, lo que sugiere la pérdida de la toxina a través del agua del cocinado.Ministerio de Economía y Competitividad (AGL2015-64558-R y CGL2016-78937-R, MINECO/FEDER, UE)

Pirólisis analítica (Py-GC/MS) para la determinación de cilindrospermopsina en músculo de pescado cocinado

Póster presentado en el XXIII Congreso Español de Toxicología y VII Iberoamericano. 26- 28 Junio 2019La cianotoxina cilindrospermopsina (CYN) es una sustancia hepatotóxica cada vez más frecuente a nivel mundial que puede acumularse en una gran variedad de animales acuáticos y transmitirse fácilmente lo largo de la cadena alimentaria. Por ello, es importante disponer de métodos analíticos adecuados que permitan su detección en muestras de pescado contaminado, tanto crudo como ya cocinado. El objetivo de este trabajo es optimizar un método basado en pirólisis analítica para su aplicación en la detección de CYN y sus productos de descomposición en carne de pescado cruda y cocinada. Para ello, utilizamos músculo de tilapia (Oreochromis niloticus) en filetes (4 g, n=5) a los que se les inyectó CYN pura (50 ng CYN/g peso seco) y se cocinaron durante 2 min mediante diferentes técnicas como el asado, microondas, hervido o vapor. Se mantuvo un grupo sin cocinar como control positivo y otro sin CYN y sin cocinar como control negativo. Todas las muestras se congelaron (-80°C) y se liofilizaron hasta ser analizadas. La Py-GC/MS se llevó a cabo en un pirolizador de doble disparo acoplado a un sistema de cromatografía de gases. Las muestras de pescado liofilizado (1,8-2,9 mg) se situaron en un crisol de pirólisis y se sometieron a 350 °C durante 1 min en ausencia de oxígeno. En las condiciones cromatográficas utilizadas la Py-GC/MS directa permitió la detección de la CYN (PM 416,1) en músculo de pescado contaminado a un tiempo de retención de 24,24 min, así como de 3 fragmentos de descomposición característicos con PM 336,2; 169,1 y 290,1 a 25,25 min, 22,45 min y 15,92 min, respectivamente. Además, se observa una variación en la abundancia relativa de los fragmentos en función del tipo de cocinado. De manera general, las técnicas de cocinado que conllevaban el uso de agua (especialmente el hervido), mostraron una abundancia relativa menor tanto de CYN, como de los distintos fragmentos, lo que parece indicar una pérdida de la toxina a través del agua del cocinado.Ministerio de Economía y Competitividad (AGL2015-64558-R y CGL2016-78937-R, MINECO/FEDER, UE).N

Analytical Pyrolysis of fish (Oreochromis niloticus) muscle. Effect of different cooking method

Póster (P-FA-34 ) presentado en la XVIII Reunión de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA 2018), Granada, del 2 al 4 de Octubre de 2018.In this communication a detailed analytical pyrolysis of tilapia fish (Oreochromis niloticus) muscle is described. The fish, supplied by Valenciana de Acuicultura (fish hatchery of Valencia, Spain), were acclimatized in the laboratory and for 15 days in two aquariums (8 individuals/aquarium) with 96 L of tap-water at a constant temperature (21 ± 2°C). Fish were fed daily (0.3 g/day) with commercial fish food only (Dibaq S.L., Segovia, Spain). After acclimation, were dissected and each muscle sample was cut into approximately 4 g portions. Fish muscle samples were cooked for 2 min by boiling, steaming, microwaving and broiling. Briefly, for boiling and steaming, the fish muscle was introduced into the pot or onto the food steamer, respectively, with cool water, heated to boiling (100°C) and continued to boil for 2 min. A conventional household microwave oven (Samsung M17-13, 300W, 2450 MHz) was used for microwaving, and samples were broiled in Teflon pans for both sides of the fillet. A non-cooked fish muscle fillet was used as control group. The assays were always carried out by quintuplicate (n=5). All samples were kept at -80°C until freeze dry (Cryodos 80, Telstar, Tarrasa, Spain). The Py-GC/MS was performed using a double-shot pyrolyzer (Frontier Laboratories, model 2020i, Fukushima, Japan) attached to a GC system (Agilent Technologies, Palo Alto, CA,. USA, model 6890N). The muscle samples (2 mg freeze-dry tissue) were placed in crucible deactivated steel pyrolysis capsules and introduced into a preheated micro-furnace at (350 °C) for 1 min. The volatile pyrolysates were then directly injected into the GC/MS for analysis. The gas chromatograph was equipped with a low polar-fused silica (5%-phenyl-methylpolysiloxane) capillary column (Agilent J&W HP-5ms Ultra Inert, of 30 m × 250 μm × 0.25 μm film thickness. The oven temperature was held at 50 °C for 1 min and then increased to 100 °C at 30 °C min-1, from 100 °C to 300 °C at 10 °C min-1, and stabilized at 300 °C for 10 min, with a total analysis time of 32 min. The carrier gas was He at a controlled flow of 1 mL min-1. The detector consisted of a mass selective detector (Agilent Technologies, Palo Alto, CA. USA, model 5973N) and mass spectra were acquired at 70 eV ionizing energy. Compound assignment was achieved by single-ion monitoring (SIM) for the major homologous series and by comparison with published data reported in the literature or stored in digital NIST 14 (Maryland, USA) and Wiley 7 (Weinheim, Germany) libraries. In a first analytical step, a detailed pyrolysis fingerprint of raw fish muscle tissue is produced and the effect of the pyrolysis temperature from 150 to 550 ºC in 100 ºC increments is studied in both, i) applying each temperature to a different sample or ii) sequentially (multi-shot) applying each temperature to the same sample. In a second phase, after stablishing an optimum pyrolysis temperature of 350 ºC for 1 minute, the effect of the different cooking methods (boil, steam,microwave and broil cooking) in the fish muscle pyrolyzates was studied.Projects CGL2016-78937-R and AGL2015-64558-R co-financed by FEDER Funds. Desiré Monis and Alba Carmona for technical assistance.N

Detection of cylindrospermopsin and its decomposition products in raw and cooked fish (Oreochromis niloticus) by analytical pyrolysis (Py-GC/MS)

10 páginas.- 8 figuras.- 3 tablas.- referencias.- Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2019.125469The presence of the toxin cylindrospermopsin is increasingly frequent in samples from different ecosystems and it is a serious problem both at environmental level and for animal and human health. To be able to prevent CYN exposure risk, it is important to have suitable analytical methods, but also quick and economical ones. Analytical pyrolysis coupled to GC/MS (Py-GC/MS) represents an important alternative for the rapid detection, characterization or “fingerprinting” of different materials. However, it has been less studied with cyanotoxins up to date. The present work aims to investigate: 1) the suitability of Py-GC/MS for detection of CYN and its decomposition products in raw and cooked fish samples before consumption and 2) the influence of the different cooking methods on the presence of different CYN degradation products detected by Py-GC/MS. For first time, these results present that Py-GC/MS could be a rapid and economical alternative for the detection and monitoring of CYN and its degradation products (DP. m/z 290.1, 169.1 and 336.2) in raw or cooked fish. Moreover, the changes induced in CYN and DP by cooking could be amenable and detected by Py-GC/MS.The authors wish to thank Ministerio de Economía y Competitividad, Spain (MINECO) Projects AGL2015-64558-R and CGL2016-78937-R, co-funded with FEDER, UE fundsPeer reviewe

Effects of Chrysosporum (Aphanizomenon) ovalisporum extracts containing cylindrospermopsin on growth, photosynthetic capacity, and mineral content of carrots (Daucus carota)

Natural toxins produced by freshwater cyanobacteria, such as cylindrospermopsin, have been regarded as an emergent environmental threat. Despite the risks for food safety, the impact of these water contaminants in agriculture is not yet fully understood. Carrots (Daucus carota) are root vegetables, extensively consumed worldwide with great importance for human nourishment and economy. It is, therefore, important to evaluate the possible effects of using water contaminated with cyanotoxins on carrot cultivation. The aim of this work was to investigate cylindrospermopsin effects on D. carota grown in soil and irrigated for 30 days, with a Chrysosporum ovalisporum extract containing environmentally relevant concentrations of cylindrospermopsin (10 and 50 μg/L). The parameters evaluated were plant growth, photosynthetic capacity, and nutritional value (mineral content) in roots of carrots, as these are the edible parts of this plant crop. The results show that, exposure to cylindrospermopsin did not have a clear negative effect on growth or photosynthesis of D. carota, even leading to an increase of both parameters. However, alterations in mineral contents were detected after exposure to crude extracts of C. ovalisporum containing cylindrospermopsin. A general decline was observed for most minerals (Ca, Mg, Na, Fe, Mn, Zn, Mo, and P), although an increase was shown in the case of K and Cu, pointing to a possible interference of the cyanobacterial extract in mineral uptake. This study is the first to evaluate the effects of C. ovalisporum extracts on a root vegetable, however, more research is necessary to understand the effects of this toxin in environmentally relevant scenarios. © 2016, Springer Science+Business Media New York.This work was partially funded by Funda????o para a Ci??ncia e Tecnologia (FCT), under the framework of the project UID/Multi/04423/2013, by Ministerio de Ciencia e Innovaci??n of Spain under the project AGL2009-10026, and by Junta de Andaluc??a under the project P09-AGR-4672. Remedios Guzm??n's work is supported by the Spanish Ministerio de Educaci??n fellowship. Alexandre Campos work is supported by a post-doctoral grant (SFRH/BPD/103683/2014) from FCT

Detection of carvacrol in biological tissues by analytical pyrolysis

Abstracts of the 52nd Congress of the European Societies of Toxicology (EUROTOX) Fibes Congress Center Seville, Spain, 04th-07th September 2016Oregano essential oil is being included in new packaging materials due to its bioactive properties. Carvacrol, main compound of this essential oil, with antioxidant and antimicrobial properties, is intended to be used in food packaging. According to the recommendation of the EFSA for the genotoxic assessment of substances in food, the in vivo negative results are acceptable when direct or indirect evidence supportive of exposure of the target tissues have been demonstrated. Previous studies reported negative results for comet assay in stomach and liver of rats orally exposed to carvacrol (81, 256 and 810 mg/kg bw). Hence, in this work, direct pyrolysis–gas chromatography–mass spectrometry (Py–GC/MS) analysis was performed using a double-shot pyrolyzer attached to a GC/MS system to ensure the exposure of such tissues to carvacrol. Results showed the presence of carvacrol in stomach and liver in all of the concentrations assayed. Moreover, pyrolysis technique showed clear and distinct dose–response relationships depending on the tissue evaluated. However, the ingestion of concentrations over 256 mg/kg bw in the liver, exhibited no-linear relation. This does not relate to the ratios predicted by the linear model found in the stomach. In this regard, the differences observed in those curves may be related to carvacrol metabolism in rats and the possible occurrence of saturation mechanisms limiting an excess of carvacrol metabolization/presence in liver. Therefore, our results indicate that Py–GC/MS is a valuable tool to evaluate the exposure of biological tissues to carvacrol avoiding sample pre-treatmenEuropean Soc Toxicol u projects AGL2012-38357-C02-0 y CGL2012-38655-C04-01, co-financed by FEDERN