Freshwater algal toxins: Monitoring and toxicity profile

Ministerio de Ciencia e Innovación PID2019-104890RB-I00, MICINN/FEDER, U

In vitro toxicological assessment of cylindrospermopsin: A review

Cylindrospermopsin (CYN) is a cyanobacterial toxin that is gaining importance, owing to its increasing expansion worldwide and the increased frequency of its blooms. CYN mainly targets the liver, but also involves other organs. Various mechanisms have been associated with its toxicity, such as protein synthesis inhibition, oxidative stress, etc. However, its toxic effects are not yet fully elucidated and additional data for hazard characterization purposes are required. In this regard, in vitro methods can play an important role, owing to their advantages in comparison to in vivo trials. The aim of this work was to compile and evaluate the in vitro data dealing with CYN available in the scientific literature, focusing on its toxicokinetics and its main toxicity mechanisms. This analysis would be useful to identify research needs and data gaps in order to complete knowledge about the toxicity profile of CYN. For example, it has been shown that research on various aspects, such as new emerging toxicity effects, the toxicity of analogs, or the potential interaction of CYN with other cyanotoxins, among others, is still very scarce. New in vitro studies are therefore welcome.Ministerio de Economía y Competitividad AGL2015-64558-

Analysis of the Use of Cylindrospermopsin and/or Microcystin-Contaminated Water in the Growth, Mineral Content, and Contamination of Spinacia oleracea and Lactuca sativa

Cyanobacteria and cyanotoxins constitute a serious environmental and human health problem. Moreover, concerns are raised with the use of contaminated water in agriculture and vegetable production as this can lead to food contamination and human exposure to toxins as well as impairment in crop development and productivity. The objective of this work was to assess the susceptibility of two green vegetables, spinach and lettuce, to the cyanotoxins microcystin (MC) and cylindrospermopsin (CYN), individually and in mixture. The study consisted of growing both vegetables in hydroponics, under controlled conditions, for 21 days in nutrient medium doped with MC or CYN at 10 μg/L and 50 μg/L, or CYN/MC mixture at 5 + 5 μg/L and 25 + 25 μg/L. Extracts from M. aeruginosa and C. ovalisporum were used as sources of toxins. The study revealed growth inhibition of the aerial part (Leaves) in both species when treated with 50µg/L of MC, CYN and CYN/MC mixture. MC showed to be more harmful to plant growth than CYN. Moreover spinach leaves growth was inhibited by both 5 + 5 and 25 + 25 µg/L CYN/MC mixtures, whereas lettuce leaves growth was inhibited only by 25 + 25 µg/L CYN/MC mixture. Overall, growth data evidence increased sensitivity of spinach to cyanotoxins in comparison to lettuce. On the other hand, plants exposed to CYN/MC mixture showed differential accumulation of CYN and MC. In addition, CYN, but not MC, was translocated from the roots to the leaves. CYN and MC affected the levels of minerals particularly in plant roots. The elements most affected were Ca, K and Mg. However, in leaves K was the mineral that was affected by exposure to cyanotoxinsEspaña, MINECO/FEDER Project AGL2015-64558-

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-

Hazard characterization of graphene nanomaterials in the frame of their food risk assessment: A review

Different applications have been suggested for graphene nanomaterials (GFNs) in the food and feed chain. However, it is necessary to perform a risk assessment before they become market-ready, and when consumer exposure is demonstrated. For this purpose, the European Food Safety Authority (EFSA) has published a guidance that has been recently updated. In this sense, the aim of this study is to identify and characterise toxicological hazards related to GFNs after oral exposure. Thus, existing scientific literature in relation to in vitro degradation studies, in vitro and in vivo genotoxicity, toxicokinetics data, in vivo oral studies, and other in-depth studies such as effects on the microbiome has been revised. The obtained results showed that the investigations performed up to now did not follow internationally agreed-upon test guidelines. Moreover, GFNs seemed to resist gastrointestinal digestion and were able to be absorbed, distributed, and excreted, inducing toxic effects at different levels, including genotoxicity. Also, dose has an important role as it has been reported that low doses are more toxic than high doses because GFNs tend to aggregate in the digestive system, changing the internal exposure scenario. Thus, further studies including a thorough toxicological evaluation are required to protect consumer's safety.Junta de Andalucía US-1259106, P18-RT-199

Toxic Effects Produced by Anatoxin-a under Laboratory Conditions: A Review

The presence of cyanotoxins and its bioaccumulation in the food chain is an increasingly common problem worldwide. Despite the toxic effects produced by Anatoxin-a (ATX-a), this neurotoxin has been less studied compared to microcystins (MCs) and cylindrospermopsin (CYN). Studies conducted under laboratory conditions are of particular interest because these provide information which are directly related to the effects produced by the toxin. Currently, the World Health Organization (WHO) considers the ATX-a toxicological database inadequate to support the publication of a formal guideline reference value. Therefore, the aim of the present work is to compile all of the in vitro and in vivo toxicological studies performed so far and to identify potential data gaps. Results show that the number of reports is increasing in recent years. However, more in vitro studies are needed, mainly in standardized neuronal cell lines. Regarding in vivo studies, very few of them reflect conditions occurring in nature and further studies with longer periods of oral exposure would be of interest. Moreover, additional toxicological aspects of great interest such as mutagenicity, genotoxicity, immunotoxicity and alteration of hormonal balance need to be studied in depth.España Ministerio de Ciencia e Innovación PID2019-104890RBI00/AEI/10.13039/50110001103

Evaluation of the development of communication and analysis skills in practicum students of pharmacy

El equipo de Prácticas Tuteladas de la Facultad de Farmacia de la Universidad de Sevilla, en su compromiso de adecuación al Espacio Europeo de Educación Superior del Practicum de la licenciatura, viene desarrollando desde el curso académico 2008-2009 un Proyecto de Innovación y Mejora Docente dentro del I Plan Propio de Docencia de esta Universidad. Su objetivo es impulsar cambios en la metodología docente donde se de un mayor protagonismo al aprendizaje del estudiante, durante el cual, debe adquirir nuevas habilidades, destrezas, conocimientos, conductas o valores como resultado del estudio, la experiencia, la instrucción y la observación. En este sentido, los alumnos deben adquirir la habilidad de analizar el aprendizaje recibido, reconociendo sus limitaciones y deficiencias, así como sus destrezas, y aprender a expresar de forma oral, escrita y sintéticamente sus conocimientos y capacidades. Los profesores definieron los criterios de evaluación tanto de la actividad escrita como oral. Así se analizaron aspectos relativos a la construcción del conocimiento como capacidad de análisis, síntesis, creatividad y pensamiento crítico, la presentación de la memoria e iniciativa y originalidad mostrada por el alumnado. Respecto a la expresión oral del trabajo desarrollado, los alumnos expusieron una de las competencias específicas definidas para la asignatura ante el equipo de Prácticas Tuteladas, apoyándose en una presentación en power point. Para la evaluación de esta actividad se valoraron además aspectos como claridad de ideas, riqueza de vocabulario y selección del lenguaje adecuado y diseño de la presentaciónThe team responsible for teaching the course “Prácticas Tuteladas” at the Degree of Pharmacy, University of Seville, in its commitment to adapting to the European Space for Higher Education , has been developing since the 2008-2009 academic year an Innovation Project of Teaching Improvement that belongs to the I Teaching Plan of the University. It aims to promote changes in teaching methodology giving a stronger role to the student learning process, in which he must acquire new skills, abilities, knowledge, behaviours, or values as a result of the study, experience, training and observation. In this sense, students must acquire the ability to analyze the learning received, recognizing their limitations and weaknesses and their skills and learn to express orally, written and synthetically their knowledge and skills. The teachers previously defined the criteria for the evaluation of both written and oral activitiesTeacher assessed the acquisition of different skills such as analysis capacity, synthesis, creativity and critical thinking, quality of the report and initiative and originality displayed by the students. Regarding to the oral presentation of the work developed, the students gave a seminar about one of the competencies developed to the academic tutors using a power point presentation. For the evaluation of this activity other aspects such as clarity of ideas, vocabulary and appropriate terminology and presentation design were also evaluate

Potential Oestrogenic Effects (Following the OECD Test Guideline 440) and Thyroid Dysfunction Induced by Pure Cyanotoxins (Microcystin-LR, Cylindrospermopsin) in Rats

Potential endocrine-disrupting properties of cyanotoxins, such as microcystin-LR (MC-LR) and cylindrospermopsin (CYN) are of concern due to their increasing occurrence, the scarcity of reports on the topic (particularly for CYN) and the impact of human's health at different levels. Thus, this work performed for the first time the uterotrophic bioassay in rats, following the Organization for Economic Cooperation and Development (OECD) Test Guideline 440, to explore the oestrogenic properties of CYN and MC-LR (75, 150, 300 μg/kg b.w./day) in ovariectomized (OVX) rats. Results revealed neither changes in the wet and blotted uterus weights nor in the morphometric study of uteri. Moreover, among the steroid hormones analysed in serum, the most remarkable effect was the dose-dependent increase in progesterone (P) levels in rats exposed to MC-LR. Additionally, a histopathology study of thyroids and serum levels of thyroids hormones were determined. Tissue affectation (follicular hypertrophy, exfoliated epithelium, hyperplasia) was observed, as well as increased T3 and T4 levels in rats exposed to both toxins. Taken together, these results point out that CYN and MC-LR are not oestrogenic compounds at the conditions tested in the uterotrophic assay in OVX rats, but, however, thyroid disruption effects cannot be discarded.Ministerio de Ciencia e Innovación PRE 2020-094412Junta de Andalucía P18-RTJ-195

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-

In Vitro Safety Assessment of Reduced Graphene Oxide in Human Monocytes and T Cells.

Considering the increase in the use of graphene derivatives in different fields, the environmental and human exposure to these materials is likely, and the potential consequences are not fully elucidated. This study is focused on the human immune system, as this plays a key role in the organism's homeostasis. In this sense, the cytotoxicity response of reduced graphene oxide (rGO) was investigated in monocytes (THP-1) and human T cells (Jurkat). A mean effective concentration (EC50-24 h) of 121.45 ± 11.39 μg/mL and 207.51 ± 21.67 μg/mL for cytotoxicity was obtained in THP-1 and Jurkat cells, respectively. rGO decreased THP-1 monocytes differentiation at the highest concentration after 48 h of exposure. Regarding the inflammatory response at genetic level, rGO upregulated IL-6 in THP-1 and all cytokines tested in Jurkat cells after 4 h of exposure. At 24 h, IL-6 upregulation was maintained, and a significant decrease of TNF-α gene expression was observed in THP-1 cells. Moreover, TNF-α, and INF-γ upregulation were maintained in Jurkat cells. With respect to the apoptosis/necrosis, gene expression was not altered in THP-1 cells, but a down regulation of BAX and BCL-2 was observed in Jurkat cells after 4 h of exposure. These genes showed values closer to negative control after 24 h. Finally, rGO did not trigger a significant release of any cytokine at any exposure time assayed. In conclusion, our data contributes to the risk assessment of this material and suggest that rGO has an impact on the immune system whose final consequences should be further investigated.Fondo Europeo de Desarrollo Regional US-1259106Junta de Andalucía P18-RT-199