Synergistic effect of environmental food pollutants: pesticides and marine biotoxins

Emerging marine biotoxins such as ciguatoxins and pyrethroid compounds, widely used in agriculture, are independently treated as environmental toxicants. Their maximum residue levels in food components are set without considering their possible synergistic effects as consequence of their interaction with the same cellular target. There is an absolute lack of data on the possible combined cellular effects that biological and chemical pollutants, may have. Nowadays, an increasing presence of ciguatoxins in European Coasts has been reported and these toxins can affect human health. Similarly, the increasing use of phytosanitary products for control of food plagues has raised exponentially during the last decades due to climate change. The lack of data and regulation evaluating the combined effect of environmental pollutants with the same molecular target led us to analyse their in vitro effects. In this work, the effects of ciguatoxins and pyrethroids in human sodium channels were investigated. The results presented in this study indicate that both types of compounds have a profound synergistic effect in voltage-dependent sodium channels. These food pollutants act by decreasing the maximum peak inward sodium currents and hyperpolarizing the sodium channels activation, effects that are boosted by the simultaneous presence of both compounds. A fact that highlights the need to re-evaluate their limits in feedstock as well as their potential in vivo toxicity considering that they act on the same cellular target. Moreover, this work sets the cellular basis to further apply this type of studies to other water and food pollutants that may act synergistically and thus implement the corresponding regulatory limits taking into account its presence in a healthy dietThe research leading to these results has received funding from the following FEDER cofounded grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01). From Ministerio de Ciencia e Innovación IISCIII/PI19/001248 and PID 2020-11262RB-C21. From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOXS

Determination of the toxicity equivalency factors for ciguatoxins using human sodium channels

Ciguatoxins (CTXs) which are produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa and share a ladder-shaped polyether structure, are causative compounds of one of the most frequent foodborne illness disease known as ciguatera fish poisoning (CFP). CFP was initially found in tropical and subtropical areas but nowadays the dinoflagellates producers of ciguatoxins had spread to European coasts. Therefore, this raises the need of establishing toxicity equivalency factors for the different compounds that can contribute to ciguatera fish poisoning, since biological methods have been replaced by analytical techniques. Thus, in this work, the effects of six compounds causative of ciguatera, on their main target, the human voltage-gated sodium channels have been analyzed for the first time. The results presented here led to the conclusion that the order of potency was CTX1B, CTX3B, CTX4A, gambierol, gambierone and MTX3. Furthermore, the data indicate that the activation voltage of sodium channels is more sensitive to detect ciguatoxins than their effect on the peak sodium current amplitudeThe research leading to these results has received funding from the following FEDER cofunded-grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01). From Ministerio de Ciencia e Innovación IISCIII/PI19/001248, PID 2020-11262RB-C21. From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOXS

Introducing ATR-FTIR Spectroscopy through Analysis of Acetaminophen Drugs: Practical Lessons for Interdisciplinary and Progressive Learning for Undergraduate Students.

Infrared (IR) spectroscopy is a vibrational spectroscopic technique useful in chemical, pharmaceutical, and forensic sciences. It is essential to identify chemicals for reasons spanning from scientific research and academic practices to quality control in companies. However, in some university degrees, graduate students do not get the proficiency to optimize the experimental parameters to obtain the best IR spectra; to correlate the IR spectral bands with the molecular vibrations (chemical elucidation); to have some criteria for any substance identification (especially relevant in quality control to recognize counterfeit); and to apply chemometrics for comparing, visualizing, and classifying the IR spectra. This work presents an experimental laboratory practice for an introductory teaching of the IR instrumental conditions in the identification of substances based on visual spectra comparison and statistical analysis and matching. Then, the selected IR conditions are applied to different commercial drugs, in the solid state or insolution, mostly composed of acetaminophen. Finally, the students apply chemometrics analysis to the IR data. This practice was designed for the training in a chemistry subject for undergraduate students of the chemistry, pharmacy, or forensics degrees, among others related to science, medical, food, or technological sciences.Instituto Universitario de Investigación en Ciencias Policiales (IUICP

A Practical Beginner's Guide to Raman microscopy

Raman microscopy is a highly suitable and well settled down analytical techniquefor qualitative determination of chemical substances. However, many universityundergraduate chemical degrees do not incorporate its practical training in theexperimental laboratory practises that constitute their curricula. For this reason,this work aimed at designing a practical beginner's guide to Raman microscopyuseful for undergraduate students, teachers or practitioners who need to use it forthe first time. After a brief explanation of the main concepts about Ramanmicroscopy, the methodology development and results interpretation are mainlyexplained using paracetamol (acetaminophen) drugs as example. In addition, thisguide presents an application to the identification of different components withina mixture, which shows the instrumental potential and how to use it effectively.Finally, acetaminophen, ascorbic acid, and sucrose were positively detected usingRaman microscopy on a commercial drug whose major component wasacetaminophen. In fact, the guide shows the detection and unequivocalidentification of different components in the mixtures, even for those lowconcentration components (5-10 % mass ratio). This work clearly proposesdifferent pragmatic criteria at the laboratory for identifying substances in mixturesto promote an easy implementation of the Raman microscopy technique

Electrophoretic fingerprinting of benzodiazepine tablets in spike drinks

Over the last few years, there has been an increase in the reports of drug-facilitated crimes. The list of drugs associated with these crimes is extensive and benzodiazepines constitute one of the groups of substances more commonly used. The sedative properties, which characterize benzodiazepines, are enhanced when such drugs are combined with alcohol, being more attractive for committing these types of crimes. In this work, a capillary electrophoresis method was applied to the analysis of 63 different samples of club drinks spiked with benzodiazepine tablets. The resulting electropherograms were processed and analyzed with the chemometric multivariate techniques: principal component analysis (PCA) and soft independent modeling of class analogies (SIMCA) classification. The PCA results allowed a clear differentiation of each drug class in a 3D plot. In addition, the SIMCA classification model (5% significance level) showed that eight out of nine test samples were automatically assigned by software to their proper sample class. The conflicting sample was correctly classified in the Coomans? plot (95% confidence). This novel approach based on the comparison of electrophoretic profiles of spiked drinks by chemometric tools allows determining the benzodiazepine used for drink spiking without the use of drug standards.Moreover, it provides an opportunity for the forensic laboratories to incorporate the identification capability provided by the electrophoretic fingerprinting of benzodiazepine solutions in existing or new databases

In Vivo Evaluation of the Chronic Oral Toxicity of the Marine Toxin Palytoxin

Palytoxin (PLTX) is one of the most poisonous substances known to date and considered as an emergent toxin in Europe. Palytoxin binds to the Na+-K+ ATPase, converting the enzyme in a permeant cation channel. This toxin is known for causing human fatal intoxications associated with the consumption of contaminated fish and crustaceans such as crabs, groupers, mackerel, and parrotfish. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Different reports have previously explored the acute oral toxicity of PLTX in mice. Although the presence of palytoxin in marine products is currently not regulated in Europe, the European Food Safety Authority expressed its opinion on PLTX and demanded assessment for chronic toxicity studies of this potent marine toxin. In this study, the chronic toxicity of palytoxin was evaluated after oral administration to mice by gavage during a 28-day period. After chronic exposure of mice to the toxin, a lethal dose 50 (LD50) of 0.44 µg/kg of PLTX and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 µg/kg for repeated daily oral administration of PLTX were determined. These results indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods, pointing out the need to further reevaluate the levels of this compound in marine productsThe research leading to these results has received funding from the following European Regional Development Fund (FEDER) cofounded grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, 2017 GRC GI-1682 (ED431C 2017/01). From European Union POCTEP 0161-Nanoeaters-1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069- EMERTOXS

Detection of Cyclic Imine Toxins in Dietary Supplements of Green Lipped Mussels (Perna canaliculus) and in Shellfish Mytilus chilensis

Seafood represents a significant part of the human staple diet. In the recent years, the identification of emerging lipophilic marine toxins has increased, leading to the potential for consumers to be intoxicated by these toxins. In the present work, we investigate the presence of lipophilic marine toxins (both regulated and emerging) in commercial seafood products from non-European locations, including mussels Mytilus chilensis from Chile, clams Tawerea gayi and Metetrix lyrate from the Southeast Pacific and Vietnam, and food supplements based on mussels formulations of Perna canaliculus from New Zealand. All these products were purchased from European Union markets and they were analyzed by UPLC-MS/MS. Results showed the presence of the emerging pinnatoxin-G in mussels Mytilus chilensis at levels up to 5.2 µg/kg and azaspiracid-2 and pectenotoxin-2 in clams Tawera gayi up to 4.33 µg/kg and 10.88 µg/kg, respectively. This study confirms the presence of pinnatoxins in Chile, one of the major mussel producers worldwide. Chromatograms showed the presence of 13-desmethyl spirolide C in dietary supplements in the range of 33.2–97.9 µg/kg after an extraction with water and methanol from 0.39 g of the green lipped mussels powder. As far as we know, this constitutes the first time that an emerging cyclic imine toxin in dietary supplements is reported. Identifying new matrix, locations, and understanding emerging toxin distribution area are important for preventing the risks of spreading and contamination linked to these compoundsAndrea Boente-Juncal and Celia Costas are recipients of fellowships from Ministerio de Educación, Cultura y Deporte, Spain (FPU16/07129 and FPU18/05681). Paz Otero is recipient of Postdoctoral Funding from the Ministerio de Ciencia, Innovación y Universidades, Spain (IJCI-2016-27774)S

Folic Acid Homeostasis and Its Pathways Related to Hepatic Oxidation in Adolescent Rats Exposed to Binge Drinking

Chronic ethanol consumption and liver disease are intimately related to folic acid (FA) homeostasis. Despite the fact that FA decreases lipid oxidation, its mechanisms are not yet well elucidated. Lately, adolescents have been practising binge drinking (BD), consisting of the intake of a high amount of alcohol in a short time; this is a particularly pro-oxidant form of consumption. The aim of this study is to examine, for the first time, FA homeostasis in BD adolescent rats and its antioxidant properties in the liver. We used adolescent rats, including control rats and rats exposed to an intermittent intraperitoneal BD model, supplemented with or without FA. Renal FA reabsorption and renal FA deposits were increased in BD rats; hepatic deposits were decreased, and heart and serum levels remained unaffected. This depletion in the liver was accompanied by higher transaminase levels; an imbalance in the antioxidant endogenous enzymatic system; lipid and protein oxidation; a decrease in glutathione (GSH) levels; hyper-homocysteinemia (HHcy); an increase in NADPH oxidase (NOX) 1 and NOX4 enzymes; an increase in caspase 9 and 3; and a decrease in the anti-apoptotic metallopeptidase inhibitor 1. Furthermore, BD exposure increased the expression of uncoupled endothelial nitric oxide synthase (eNOS) by increasing reactive nitrogen species generation and the nitration of tyrosine proteins. When FA was administered, hepatic FA levels returned to normal levels; transaminase and lipid and protein oxidation also decreased. Its antioxidant activity was due, in part, to the modulation of superoxide dismutase activity, GSH synthesis and NOX1, NOX4 and caspase expression. FA reduced HHcy and increased the expression of coupled eNOS by increasing tetrahydrobiopterin expression, avoiding nitrosative stress. In conclusion, FA homeostasis and its antioxidant properties are affected in BD adolescent rats, making it clear that this vitamin plays an important role in the oxidative, nitrosative and apoptotic hepatic damage generated by acute ethanol exposure. For this, FA supplementation becomes a potential BD therapy for adolescents, preventing future acute alcohol-related harms.Junta de Andalucía CTS-19

How Safe Is Safe for Marine Toxins Monitoring?

Current regulation for marine toxins requires a monitoring method based on mass spectrometric analysis. This method is pre-targeted, hence after searching for pre-assigned masses, it identifies those compounds that were pre-defined with available calibrants. Therefore, the scope for detecting novel toxins which are not included in the monitoring protocol are very limited. In addition to this, there is a poor comprehension of the toxicity of some marine toxin groups. Also, the validity of the current approach is questioned by the lack of sufficient calibrants, and by the insufficient coverage by current legislation of the toxins reported to be present in shellfish. As an example, tetrodotoxin, palytoxin analogs, or cyclic imines are mentioned as indicators of gaps in the system that require a solid comprehension to assure consumers are protectedThe research leading to these results has received funding from the following FEDER cofunded-grants. From Centro Desarrollo Tecnológico e Industrial (CDTI), supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA—Research Executive Agency (FP7/2007–2013) under grant agreement 312184 PHARMASEAS