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

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.Ministerio de Economía y Competitividad AGL2015-64558-

Arcillas modificadas en la industria alimentaria: ¿Seguridad o riesgo?

Junta de Andalucía AGR5969Ministerio de Ciencia e Innovación AGL2010-2121

Characterisation and antimicrobial activity of active polypropylene films containing oregano essential oil and Allium

Cooked ham is more prone to spoilage than other meat products, making preservation a key step in its commercialisation. One of the most promising preservation strategies is the use of active packaging. Oregano essential oil (OEO) and Proallium® (an Allium extract) have previously been shown to be useful in polylactic acid (PLA)-active films for ready-to-eat salads. The present work aims to study the suitability of polypropylene (PP) films containing OEO and Proallium® in the preservation of cooked ham. Concerning the technological features of the studied material, no significant changes in the mechanical or optical properties of PP films containing the active substances were recorded in comparison to the PP film without extracts. However, films containing both active substances were more flexible than the control film and less strong, highlighting the plasticisation effect of the natural extracts. Moreover, physical properties changed when active substances were added to the film. Incorporation of 4% Proallium® affected the transparency of the film to a higher extent compared to 8% OEO, undergoing decreases in transparency of 40% and 45%, respectively. Moreover, only the film containing the highest amount of OEO (8%) significantly decreased the thickness. Both active substances showed antibacterial properties; however, Proallium®-active films seemed to be more effective against Brochothrix thermosphacta than PP films containing OEO, with all percentages of Proallium® killing the bacterial population present in the ham after 60 days. In addition, materials containing the lowest Proallium® content exhibited higher acceptability by consumers in the sensory analyses with 63–100% willing to purchase, better even than the control package (56–89%). In fact, 2% of Proallium® obtained the best results in the odour study performed by the panellists.Junta de Andalucía (AGR7252)Ministerio de Ciencia e Innovación y Fondos FEDER (AGL2012-38357-C02-01

Pirolisis analítica como método de detección de carvacrol en tejidos biológicos.

El aceite esencial de orégano (Origanum vulgare L. virens) (OEO) es uno de los extractos naturales más interesantes para la industria alimentaria por sus propiedades antioxidantes y antimicrobianas. El carvacrol, su compuesto mayoritario, es uno de los compuestos fenolicos que le confiere estas propiedades al aceite. Para evaluar la seguridad de estas sustancias, la Autoridad Europea de Seguridad Alimentaria (EFSA) exige una serie de ensayos tanto in vitro como in vivo que estudien la capacidad genotóxica de las mismas. Para poder evaluar in vivo la genotoxicidad de carvacrol mediante la combinación del test de micronúcleos (MN) y del ensayo cometa, es necesario confirmar la exposición de los tejidos a esta sustancia. En este sentido, en el presente trabajo se evaluó mediante técnicas de pirólisis analítica (Py-CG/MS) la presencia de carvacrol en estómago e hígado de ratas Wistar expuestas por vía oral a distintas dosis de carvacrol (81, 256, 810 mg/ kg). Los resultados muestran por primera vez la presencia del compuesto en todos los órganos analizados. Por ello, demostramos que la técnica de pirólisis analítica es útil para obtener información sobre la exposición de los tejidos biológicos al agente activo.Los autores agradecen al Ministerio de Ciencia e Innovación (AGL2012-38357-C02-01 cofinanciado con fondos FEDER y CGL2012-38655-C04-01) y a la Junta de Andalucía (AGR- 7252) la financiación de este proyecto. M Llana-Ruiz-Cabello agradece a la Junta de Andalucía por su beca predoctoral y NT Jiménez-Morillo por su beca FPI (BES-2013-062573).Peer Reviewe

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

Detection of the monoterpene carvacrol in mammal tissues by analytical pyrolysis (Py‐GC/MS)

Póster presentado en el la XVI Reunión Científica de la Sociedad Española de Cromatografía y Técnicas Afines (SECyTA2016) P‐NP‐1Eds: González-Pérez, José Antonio.-- Almendros Martín, Gonzalo.-- González-Vila, Francisco Javier.-- Rosa Arranz, José M. de laResponding to consumer demands on minimal processing and preservative‐free products, the use of essential oils (EOs) to extend shelf life of foods is on the spot in the food industry [1]. Carvacrol, main compound of Oregano EO, is registered as a flavouring in Europe; however, its use for other applications, such as active food packaging, may require higher concentrations and there is an increasing concern regarding exposure. Because of this, the European Food Safety Authority (EFSA) requires additional genotoxic studies data of substances which could be incorporated into food packaging like carvacrol. Here a detailed analytical pyrolysis (Py‐GC/MS) study is conducted as a complement to in vivo genotoxicity studies. Analytical pyrolysis was the technique chosen to search for carvacrol directly in viscera and to confirm that the compound effectively reached target tissues from orally exposed (0, 81, 256 or 810 mg carvacrol/kg bw, calculated according to carvacrol Maximum Tolerated Dose (MTD)) young adult male Wistar rats strain RjHan:WI*. Doses were prepared in corn oil at a final volume of 1 mL and during the treatment period, clinical signs, body weight, and food and water consumption were recorded daily. Rat stomach and liver composite samples were selected for pyrolysis and preserved at ‐80ºC until lyophilisation (Testal Cryodos, Madrid). Direct pyrolysis was performed in a double‐shot pyrolyzer (Frontier Lab 2020i) attached to a GC/MS system (Agilent 6890N + 973MSD). Detailed chromatographic conditions can be found in [2]. In short, lyophilized tissue (stomach and liver) were thoroughly homogenized and samples introduced (0.5 mg) into a preheated micro‐furnace at 500 ºC for 1 min and evolved gases transferred to the GC/MS for analysis. Compounds assignment was via single‐ion monitoring and by comparison with published and stored (NIST and Wiley libraries) data. In a previous study, it was determined that pyrolysis of carvacrol does not to produce major effects on its chemical structure and therefore was considered an adequate technique to detect the presence of the monoterpene in animal tissues. The analytical pyrolysis of target tissues of control rats was negative to any sign of carvacrol even when searching for the specific mass fragments (m/z 135 and 150). However, carvacrol was clearly detected in the tissues of rats treated at all doses. Furthermore, when normalizing the chromatograms to a common peak, a clear dose response was obtained. A conspicuous difference was found in the dose‐response curves between stomach and liver; whereas a direct lineal correlation could be drawn from the former, the response for the latter was best fit to a quadratic equation model. 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. als received humane care.[1] M. Llana‐Ruiz‐Cabello, S. Pichardo, A. Baños, C. Nuñez, J.M. Bermúdez, E. Guillamón, S. Aucejo, A.M. Cameán, Food Sci. Technol‐LEB 64 (2015) 1354‐1361 [2] M. Llana‐Ruíz‐Cabello, S. Pichardo, N.T. Jiménez‐Morillo, J.M. Bermúdez, S. Aucejo. F.J. González‐Vila, A.M. Cameán, J.A. González‐Pérez (2016). J. Sci. Food Agr. 96 (2016) 3207‐3212 (*) The Ethics Committee on Animal Experimentation of the University of Sevilla approved the in vivo experiments. Moreover, in compliance with the Directive 2010/63/EU for the protection of animals used for scientific purposes all animN