Mycotoxins are fungal secondary metabolites naturally present in different food and feed with toxic effects to humans and animals that consume those contaminated products. Among them, ergot alkaloids (EAs), produced mainly by fungi of the Claviceps genus, as Claviceps purpurea, are present in cereals such as rye, triticale, wheat and barley. Improvements in agricultural practices have significantly reduce the risk of severe epidemic outbreaks of ergotism, however, EAs can be found in cereal-based food and feed, partially due to new cereal hybrids susceptible to C. purpurea and climate changes. The European Commission has established a maximum content of 0.5 g/kg of ergot sclerotia in unprocessed cereals (with the exception of corn and rice), but the maximum content for EAs is still under study. Moreover, other countries (as Algeria) has no legislation regarding mycotoxin contamination.

In this study, the major EAs [ergometrine (Em), ergosine (Es), ergotamine (Et), ergocornine (Eco), ergokryptine (Ekr), ergocristine (Ecr)], and their corresponding epimers [ergometrinine (Emn), ergosinine (Esn), ergotaminine (Etn), ergocorninine (Econ), ergokryptinine (Ekrn) and ergocristinine (Ecrn)] have been determined by a QuEChERS-UHPLC-MS/MS method in cereal samples from Algeria (30 samples of wheat and 30 samples of barley). Procedural calibration curves were stablished for both matrices and limits of quantification were below 3.3 μg/kg (wheat) and 3.9 μg/kg (barley). The recoveries ranged between 85 and 109%, with a matrix effect lower than 20% in most cases and precision (RSD), lower than 11%. Four barley samples were contaminated with Em and Emn, and 3 of them showed also contamination by Et, with total EAs contents ranging from 18.0 to 54.0 μg/kg. Wheat samples showed a higher contamination, with 8 positive samples: one sample was contaminated only with Em, while the rest were contaminated with 5 up to 11 EAs, with total EAs contents ranging from 6.5 to 77.4 μg/kg.

Pimelea poisoning of cattle (also known as St. George or Marree disease) is a poisoning unique to Australia and caused by inadvertent grazing of native Pimelea within pastures. The toxin responsible for the poisoning was previously isolated and identified as the novel diterpenoid orthoester simplexin, but no effective treatments for poisoned animals exist. A previous feeding trial reported that cattle fed daily with increasing low doses of simplexin showed reduced poisoning symptoms over time which suggested cattle developed resistance against the toxin, potentially via by the adaptation of rumen microorganisms. To date, there are no reports on simplexin degradation by rumen microorganisms.

This study aims to develop a microbial probiotic derived from the rumen fluid of field-exposed animals that is capable of detoxifying simplexin, thus allowing cattle to consume Pimelea with less adverse effects. Investigations are ongoing to identify rumen bacteria able to hydrolyse simplexin in in vitro mixed rumen-based anaerobic fermentations fed daily with Pimelea plant species (P. trichostachya) and to assess isolated rumen bacteria in in vitro incubation trials. Simplexin levels in both studies were analysed by ultra-performance liquid chromatography coupled with high-resolution, accurate mass spectrometry (UPLC-MS/MS) which allows simplexin quantification at ppb concentrations (ng/mL) on a Thermo Scientific Q-Exactive Orbitrap mass spectrometer. Results to date showed decreases in simplexin levels, suggestive of simplexin detoxification by rumen microorganisms. Simplexin acid hydrolysis studies were also performed to create a metabolite database to aid in future elucidation of potential simplexin degradation pathways. UPLC-MS/MS analysis based on predicted molecular formulae enabled identification of three hydrolysed simplexin products which also shared several fragment ions with simplexin. Future studies will include the identification and characterisation of simplexin metabolites from fermentation and incubation trials in which simplexin levels indicate that degradation has occurred.

Ribosome-inactivating proteins (RIPs) are N-glycosidases. They depurinate A-4324 in rat 28S ribosomal RNA in the conserved α-sarcin/ricin loop (α-SRL) and cease protein synthesis. Our group has shown that the internal peptide of the maize RIP precursor reduced the anti-HIV activity of the protein in infected macaque peripheral blood mononuclear cells (PBMC) and SHIV 89.6-infected Chinese rhesus macaque. We made use of the switch-on mechanism of maize RIP to incorporate HIV-1 protease recognition sequences to its internal inactivation region. Upon activation of this engineered maize RIP by HIV-1 protease in HIV-infected cells, the N-glycosidase activity and inhibitory effect on p24 antigen production in vitro and in infected human T cells were enhanced. This switch-on mechanism can also be applied to ricin A chain (RTA). RTA variants with HIV-1 protease recognition sequence at the C-terminus can be cleaved both in vitro and in HIV-infected cells. Furthermore, its antiviral effect was enhanced and the cytotoxicity towards uninfected cells was reduced. Our study provides a platform technology in creating protein toxin derivatives with increased pathogen-specific cytotoxicity.

Ergot alkaloids (EAs) are mycotoxins produced mainly by fungi of the Claviceps genus, as Claviceps purpurea. The fungus infects the seed heads of living plants, specially cereals, at the time of flowering replacing the developing grain or seed with specialized fungal structures known as sclerotium (or ergot body), which contains alkaloid substances. More than 50 different EAs have been identified, being the major compounds: ergometrine (Em), ergosine (Es), ergotamine (Et), ergocornine (Eco), ergokryptine (Ekr), ergocristine (Ecr), and their corresponding epimers, ergometrinine (Emn), ergosinine (Esn), ergotaminine (Etn), ergocorninine (Econ), ergokryptinine (Ekrn) and ergocristinine (Ecrn). Although the sclerotia can be mechanically removed during the harvesting process, EAs can be found in cereal-based food and feed, and their ingestion might cause adverse health effects in humans and animals. The European Commission has established a maximum content of 0.5 g/kg of ergot sclerotia in most unprocessed cereals; however, the maximum content for EAs in food is still under study. In this work, we propose the extraction and quantification of the main EAs and their epimers in different oat-based products by QuEChERS extraction followed by UHPLC-MS/MS. The recoveries ranged between 89 and 106%, with a matrix effect lower than 20% in most cases and precision (intra- and interday), expressed as relative standard deviation (RSD), lower than 15%. Procedural calibration curves were established and limits of detection and quantification were below 1.0 μg/kg and 3.2 μg/kg, respectively. Finally, 25 oat-based samples (including bran, flakes, juices, hydroalcoholic extracts, flours, tablets and grass) were analyzed. One of the samples of oat bran was contaminated with Em, Emn, Es and Esn in the range of 1.1-7.2 μg/kg, with a total content of EAs of 10.7 μg/kg.

Acknowledgements: Financial support of the Spanish Ministry of Science, Innovation and Universities (Project Ref.: RTI2018-097043-B-I00).

Ergot alkaloids (EAs) are secondary metabolites produced by fungi in the genus Claviceps that contaminate a large variety of cereals. More than 50 different EAs have been identified, being the major compounds: ergometrine (Em), ergosine (Es), ergotamine (Et), ergocornine (Eco), ergokryptine (Ekr), ergocristine (Ecr), and their corresponding epimers, ergometrinine (Emn), ergosinine (Esn), ergotaminine (Etn), ergocorninine (Econ), ergokryptinine (Ekrn) and ergocristinine (Ecrn). The ingestion of contaminated cereals might cause adverse health effects in humans and animals, as the well-known ergotism. In fact, pigs, cattle, sheep, and poultry are involved in sporadic outbreaks, although most other species are also susceptible. Their toxicity is linked to their structural similarity with dopamine, noradrenaline, adrenaline and serotonin, enabling binding to the biogenic amine receptor and the interruption of neurotransmission. European Union (EU) has established a maximum content of 1000 mg/kg of rye ergot sclerotia (Claviceps purpurea) in feed materials and compound feed containing unground cereals. Although EAs as such are still not regulated, feed industry recommends practical limits for EAs in pig feeds to reduce negative effects on health and performance. However, the absence of sclerotia does not exclude the presence of EAs. In this work, 12 EAs have been quantified in 228 feed samples intended for swine using QuEChERS as sample treatment and UHPLC-MS/MS for determination. The 12.7% of samples (29 samples) revealed contamination by at least one EA, and among contaminated samples, 65% were contaminated by more than one EA. Only 6 of 12 target EAs showed concentrations above the limit of quantification. The highest concentration was detected for Emn (with concentrations up to 145 μg/kg), while the total EA content ranged from 5.9 to 158.7 μg/kg. This study revealed scarce contamination by EAs in Spanish feed samples.

Aflatoxins are secondary metabolites produced by Aspergillus species distributed on three main sections of the genus namely section Flavi, section Ochraceorosei, and section Nidulantes. They are common contaminates of dietary staples worldwide, including cereals, oil seeds, nuts, spices, meats, dairy products, fruit juices, dried fruits, eggs, and feeds and foods derived from these products. Aflatoxins are unavoidable widespread natural contaminants of foodstuffs with serious impacts on food safety, health, agricultural and livestock productivity. Aflatoxin B₁ is the analyte with the highest toxic significance and the most potent hepatocarcinogenic among other aflatoxins, and humans may get exposed to it at any stage of life. Dietary exposure to aflatoxins is a public health concern due to their carcinogenic, acute aflatoxicosis and chronic effects, immunosuppression properties, among others.

This study focused on aflatoxin B₁ in rice grains. Rice is important staple food consumed widely, and consists of a major part of the diets for half of the world population. In general, there have been few reports on the occurrence of the aflatoxin B₁ in rice grains compared to other cereals in Africa. However, love the occurrence of the aflatoxin B₁ levels compared to other crops, is of concern because of the high consumption of rice in several countries in Africa. This study assessed aflatoxin B₁ in rice grains, occurrence, control, socioeconomic and health implications. We quantitatively determine the levels of aflatoxin B₁ content using Enzyme Linked Immunosorbent Assay method.

43.1 % of examined samples were positive in which 15.9 % for local rice and 27.2 % of imported rice, respectively, and 11.3 % of examined samples are above the maximum limit of aflatoxin B₁ in rice established by European Union. According to the manufacturer instructions, the limit of detection is 1 μg/kg (ppb) in cereals. The concentration of aflatoxin B₁ in examined samples ranged from 0 μg/kg to 3.2 μg/kg. These results are indicative of exposure of the population to aflatoxin and possible health hazard. The procedure used in this study is suitable for detection of mycotoxins at a very low concentration.

Palytoxin (PLTX) is a marine toxin that nowadays is recognized amongst the most toxic compounds isolated from natural products. Originally, the toxin was only identified in a single tidal pool of the island of Maui (Hawaii). Currently, this compound is considered as an emergent toxin in Europe and its prevalence in continental European waters has increased during the last years. The high toxicity of palytoxin is related with the binding to the Na⁺-K⁺ ATPase, converting this ubiquitously distributed enzyme in a permeant cation channel [1-3]. Several reports have shown that this toxin is responsible for human fatal intoxications, either after inhalation of toxin-containing marine aerosols or after ingestion of marine products contaminated with PLTX, such as crabs, groupers, mackerel, and parrotfish. So far, different groups have explored the acute oral toxicity of PLTX in mice however, discrepancies in the PLTX source as well as in the monitoring time for the toxic effects yielded controversial results. Although the presence of palytoxin in marine products is not yet currently regulated in Europe, the European Food Safety Authority (EFSA) expressed its opinion on PLTX toxicity and prompted the need to obtain more data regarding the in vivo toxicity of this compound [4]. Therefore, in this study, the acute and chronic toxicity of palytoxin was evaluated after oral administration of the toxin to mice either in a single dose and in a follow-up period of 96 hours or after chronic administration 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, much lower than that observed in the acute experiments, and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 µg/kg for repeated daily oral administration of PLTX were determined. Therefore, these data indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods remarking the need to further evaluate the potential teratogenic effects of this emerging marine toxin in mammals.

References

1. Artigas, P.; Gadsby, D.C. Ion occlusion/deocclusion partial reactions in individual palytoxin-modified Na⁺/K⁺ pumps. Ann N Y Acad Sci 2003, 986, 116-126.
2. Artigas, P.; Gadsby, D.C. Na⁺/K⁺-pump ligands modulate gating of palytoxin-induced ion channels. Proc Natl Acad Sci U S A 2003, 100, 501-505.
3. Artigas, P.; Gadsby, D.C. Large diameter of palytoxin-induced Na⁺/K⁺ pump channels and modulation of palytoxin interaction by Na⁺/K⁺ pump ligands. J Gen Physiol 2004, 123, 357-376.
4. EFSA. Panel on contaminants in the food chain (CONTAM). Scientific Opinion on marine biotoxins in shellfish–Palytoxin group. EFSA Journal, 2009; Vol. 7, p 1393.

Gambierdiscus species are marine dinoflagellates producers of toxins causative of a widespread human illness known as Ciguatera Fish Poisoning (CFP) which comprises gastrointestinal, neurological, and cardiovascular symptoms. Blooms of these dinoflagellates have expanded worldwide reaching even European coasts. In fact, the presence of Gambierdiscus species and the related toxins and CFP intoxications have been repetitively identified in Europe during the last decades, especially in the Canary Islands [1,2] and Madeira [3]. Besides ciguatoxins, which can cause long term neurological sequela in humans as a consequence of their permanent activation voltage-gated sodium channels [4-6], the structure of an additional ciguatoxin-related toxin named 44-methylgambierone (MTX3) has been recently elucidated [7]. Initial studies on the biological activity of 44-methylgambierone described an effect similar to that of the synthetic ciguatoxin CTX3C although of much lower potency [7]. With the aim of further explore the relative toxicities and activities of these compounds additional experiments were performed. First, the neurotoxic effect of CTX3C and MTX3 was evaluated using a human neuronal cell model based on the incubation of SH-SY5Y with ouabain and veratridine together with ciguatoxin or ciguatoxin-like compounds to evaluate their in vitro toxic potency [8]. Our data illustrate that CTX3C aggravated the ouabain and veratridine neurotoxicity but 44-methylgambierone did not resembled this effect. Additionally, while CTX3C at nanomolar concentrations hyperpolarized the activation of voltage-gated sodium channels and decreased current amplitude, 44-methylgambierone did not affect sodium currents. Moreover, oral chronic toxicity studies using daily CTX3 concentrations of 10, 32 and 100 ng/kg or MTX3 at 550 or 1760 ng/kg and an observation period of 28 days did not shown behavioral or biochemical alterations during treatment. Based on in vitro and in vivo results, the ciguatoxin-related compound 44-methylgambierone, recently identified in Gambierdiscus extracts, is less potent than CTX3C and thus indicates that the effect on human CFP symptoms may also be minor.

References

1. Estevez, P.; Sibat, M.; Leão-Martins, J.M.; Tudó, A.; Rambla-Alegre, M.; Aligizaki, K.; Diogène, J.; Gago-Martinez, A.; Hess, P. Use of Mass Spectrometry to Determine the Diversity of Toxins Produced by Gambierdiscus and Fukuyoa Species from Balearic Islands and Crete (Mediterranean Sea) and the Canary Islands (Northeast Atlantic). Toxins 2020, 12.
2. Perez-Arellano, J.L.; Luzardo, O.P.; Perez Brito, A.; Hernandez Cabrera, M.; Zumbado, M.; Carranza, C.; Angel-Moreno, A.; Dickey, R.W.; Boada, L.D. Ciguatera fish poisoning, Canary Islands. Emerg Infect Dis 2005, 11, 1981-1982.
3. Otero, P.; Pérez, S.; Alfonso, A.; Vale, C.; Rodríguez, P.; Gouveia, N.N.; Gouveia, N.; Delgado, J.; Vale, P.; Hirama, M., et al. First toxin profile of ciguateric fish in Madeira Arquipelago (Europe). Analytical chemistry 2010, 82, 6032-6039.
4. Martín, V.; Vale, C.; Hirama, M.; Yamashita, S.; Rubiolo, J.A.; Vieytes, M.R.; Botana, L.M. Synthetic ciguatoxin CTX 3C induces a rapid imbalance in neuronal excitability. Chemical research in toxicology 2015, 28, 1095-1108.
5. Martín, V.; Vale, C.; Rubiolo, J.A.; Roel, M.; Hirama, M.; Yamashita, S.; Vieytes, M.R.; Botana, L.M. Chronic ciguatoxin treatment induces synaptic scaling through voltage gated sodium channels in cortical neurons. Chemical research in toxicology 2015, 28, 1109-1119.
6. Vale, C.; Antero, A.; Martín, V. Pharmacology of ciguatoxins. Phycotoxins, Chemistry and Biochemistry; Alfonso, LBAA, Ed.; Wiley Blackwell: Hoboken, NJ, USA 2015, 23-48.
7. Boente-Juncal, A.; Álvarez, M.; Antelo, Á.; Rodríguez, I.; Calabro, K.; Vale, C.; Thomas, O.P.; Botana, L.M. Structure Elucidation and Biological Evaluation of Maitotoxin-3, a Homologue of Gambierone, from Gambierdiscus belizeanus. Toxins 2019, 11.
8. Coccini, T.; Caloni, F.; De Simone, U. Human neuronal cell based assay: A new in vitro model for toxicity evaluation of ciguatoxin. Environmental toxicology and pharmacology 2017, 52, 200-213.

Tetrodotoxin (TTX) is a very toxic compound responsible for human intoxications after ingestion of contaminated fishery products. Although TTX was initially associated mainly with human fatalities occurring in Asiatic countries[1], nowadays has expanded to other regions including European countries. In Europe, the first non-fatal human intoxications by TTX was reported more than 10 years ago after the ingestion of a Charonia lampas trumpet shell captured in the Portuguese coast and commercialized in Spain[2]. Since then, during the last decade, the presence of the TTX-containing pufferfish Lagocephalus sceleratus has been reported in European coasts, mainly in the Mediterranean Sea[3,4] with some fish tissues containing TTXs amounts as high as 2 mg/kg[5]. Moreover, an increasing concern regarding food safety has been raised after the detection of TTX in mussels, oysters and clams harvested in UK, Greece, the Netherlands[6-8] and Spain. The current European legislation in marine toxins did not yet regulated the levels of TTX in fishery products, and nowadays the presence of the toxin is only regularly monitored in the Netherlands although the European Food Safety Authority (EFSA) has recommended the level of 44 µg/kg TTXs for routine monitoring since, at this dose, no adverse effects were observed in humans. Considering initial data on the acute oral toxicity of TTX and in view of the EFSA opinion remarking the need for additional chronic toxicity studies to further reduce the uncertainty of the likely future toxin regulation. In this work, the oral chronic toxicity of TTX using doses of 25, 44, 75 and 125 µg/kg and an observation period of 28 days was explored in female mice using protocols internationally validated to test the toxicity of chemicals. The data presented here indicated that 25 and 44 µg/kg of TTX did not cause neither blood biochemical nor behavioral alterations in mice, while at the dose of 125 µg/kg kidney and heart alterations were observed under electron microscopy analysis. Therefore, the data presented here indicate that the safety TTX dose proposed by EFSA is low enough to prevent human adverse effects, while caution should be taken in the presence of higher TTX doses.

References:

1. Bane, V.; Lehane, M.; Dikshit, M.; O'Riordan, A.; Furey, A. Tetrodotoxin: chemistry, toxicity, source, distribution and detection. Toxins 2014, 6, 693-755.
2. Rodriguez, P.; Alfonso, A.; Vale, C.; Alfonso, C.; Vale, P.; Tellez, A.; Botana, L.M. First Toxicity Report of Tetrodotoxin and 5,6,11-TrideoxyTTX in the Trumpet Shell Charonia lampas lampas in Europe. Analytical Chemistry 2008, 80.
3. Rambla-Alegre, M.; Reverte, L.; Del Rio, V.; de la Iglesia, P.; Palacios, O.; Flores, C.; Caixach, J.; Campbell, K.; Elliott, C.T.; Izquierdo-Munoz, A., et al. Evaluation of tetrodotoxins in puffer fish caught along the Mediterranean coast of Spain. Toxin profile of Lagocephalus sceleratus. Environ Res 2017, 158, 1-6.
4. Katikou, P.; Georgantelis, D.; Sinouris, N.; Petsi, A.; Fotaras, T. First report on toxicity assessment of the Lessepsian migrant pufferfish Lagocephalus sceleratus (Gmelin, 1789) from European waters (Aegean Sea, Greece). Toxicon 2009, 54, 50-55.
5. Leonardo, S.; Kiparissis, S.; Rambla-Alegre, M.; Almarza, S.; Roque, A.; Andree, K.B.; Christidis, A.; Flores, C.; Caixach, J.; Campbell, K., et al. Detection of tetrodotoxins in juvenile pufferfish Lagocephalus sceleratus (Gmelin, 1789) from the North Aegean Sea (Greece) by an electrochemical magnetic bead-based immunosensing tool. Food Chem 2019, 290, 255-262.
6. Katikou, P. Public Health Risks Associated with Tetrodotoxin and Its Analogues in European Waters: Recent Advances after The EFSA Scientific Opinion. Toxins 2019, 11.
7. Turner, A.D.; Powell, A.; Schofield, A.; Lees, D.N.; Baker-Austin, C. Detection of the pufferfish toxin tetrodotoxin in European bivalves, England, 2013 to 2014. Eurosurveillance 2015, 20, 21009.
8. Vlamis, A.; Katikou, P.; Rodriguez, I.; Rey, V.; Alfonso, A.; Papazachariou, A.; Zacharaki, T.; Botana, A.M.; Botana, L.M. First Detection of Tetrodotoxin in Greek Shellfish by UPLC-MS/MS Potentially Linked to the Presence of the Dinoflagellate Prorocentrum minimum. Toxins 2015, 7, 1779-1807.

Marine biotoxins portrait a major threat to public health. Microalgae, such as diatoms or dinoflagellates, are the major producers of these compounds. Blooms of these species commonly correspond with increased toxin concentration in filter-feeding organisms, which can lead to poisoning outbreaks due to shellfish consumption. Within toxins of marine origin, we focus our work on the okadaic acid (OA) group that also includes the analogues dynophysistoxins 1 and 2. Diarrheic Shellfish Poisoning (DSP) is developed after ingestion of contaminated food with these lipophilic compounds, consisting mainly on gastrointestinal symptoms like nausea and diarrhoea. OA group of toxins inhibit protein phosphatases (PPs) with ubiquitous distribution like PP1 or PP2A. Yet, several reports raise the possibility of the phycotoxin targeting different mechanisms. A substantial variety of pathogenic stimuli trigger diarrhoea through the activation of the Enteric Nervous System (ENS). Neuropeptide Y (NPY) is a 36 aa peptide of neuronal origin known to maintain an antisecretory tone by acting on the receptors Y₁ and Y₂, both expressed along the gastrointestinal tract. Previous in vitro studies have exposed that OA downregulates NPY gene and protein expression. Moreover, the toxin is reported to cause diarrhoea within the first 2 h of treatment. Here we assess how the pro-absorptive NPY could be modifying OA-induced diarrhoea in vivo. Mice were first given NPY via intraperitoneal 15 minutes prior to OA oral gavage administration. Body weight variations, symptoms along with food and water intake were monitored for the 2 h treatment. Afterwards, anatomopathological examination took place and intestine samples were collected for transmission electron microscopy evaluation. In the presence of NPY, no delay on diarrhoea onset was observed though ultrastructural mild recovery was detected in the large intestine. Hence, it could be feasible that OA modifies NPY antisecretory tone, resulting in diarrhoea.