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Background: Sulforaphane (SFN) is a dietary isothiocyanate, derived from glucoraphanin, present in cruciferous vegetables belonging to the Brassica genus. It is a biologically active phytochemical that acts as a nuclear factor erythroid 2-related factor 2 (Nrf2) inducer. Thus, it has been reported to have multiple protective functions including anticancer responses and protection against a toxic agent's action., Purpose: The present work systematically reviewed and synthesised the protective properties of sulforaphane against a toxic agent. This review reveals the mechanism of the action of SFN in each organ or system., Methods: The PRISMA guideline was followed in this sequence: researched literature, organised retrieved documents, abstracted relevant information, assessed study quality and bias, synthesised data, and prepared a comprehensive report. Searches were conducted on Science Direct and PubMed using the keywords "Sulforaphane" AND ("protective effects" OR "protection against")., Results: Reports showed that liver and the nervous system are the target organs on which attention was focused, and this might be due to the key role of oxidative stress in liver and neurodegenerative diseases. However, protective activities have also been demonstrated in the lungs, heart, immune system, kidneys, and endocrine system. SFN exerts its protective effects by activating the Nrf2 pathway, which enhances antioxidant defenses and reduces oxidative stress. It also suppresses inflammation by decreasing interleukin production. Moreover, SFN inhibits apoptosis by preventing caspase 3 cleavage and increasing Bcl2 levels. Overall, SFN demonstrates multifaceted mechanisms to counteract the adverse effects of toxic agents., Conclusion: SFN has potential clinical applications as a chemoprotective agent. Nevertheless, more studies are necessary to set the safe doses of SFN in humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Anatoxin-a (ATX-a) is a potent neurotoxin produced by several species of cyanobacteria whose exposure can have direct consequences, including neurological disorders and death. The increasing prevalence of harmful cyanobacterial blooms makes the detection and reliable assessment of ATX-a levels essential to prevent the risk associated with public health. Therefore, the aim of this review is to compile the analytical methods developed to date for the detection and quantification of ATX-a levels alone and in mixtures with other cyanotoxins and their suitability. A classification of the analytical methods available is fundamental to make an appropriate choice according to the type of sample, the equipment available, and the required sensitivity and specificity for each specific purpose. The most widely used detection technique for the quantification of this toxin is liquid chromatography-tandem mass spectrometry (LC-MS/MS). The analytical methods reviewed herein focus mainly on water and cyanobacterial samples, so the need for validated analytical methods in more complex matrices (vegetables and fish) for the determination of ATX-a to assess dietary exposure to this toxin is evidenced. There is currently a trend towards the validation of multitoxin methods as opposed to single-ATX-a determination methods, which corresponds to the real situation of cyanotoxins' confluence in nature., Competing Interests: The authors declare no conflicts of interest.

Anatoxin-a (ATX-a) is a cyanobacterial toxin whose occurrence has been reported worldwide and has attracted increasing scientific interest due to its toxicity. Moreover, in nature, ATX-a usually appears together with other cyanotoxins, such as cylindrospermopsin (CYN), so possible interaction phenomena could happen and should be considered for risk assessment purposes. For this reason, the aim of this work was to explore the potential mutagenicity and genotoxicity of pure ATX-a and an ATX-a/CYN mixture using a battery of in vitro assays, including the bacterial reverse-mutation assay in Salmonella typhimurium (OECD 471) and the micronucleus test (MN) (OECD 487) on L5178Y Tk +/- cells. The results showed that ATX-a was not mutagenic either alone or in combination with CYN under the conditions tested. Nevertheless, genotoxic effects were observed for both ATX-a and its mixture with CYN following the in vitro MN assay. The genotoxicity exhibited by ATX-a was only observed in the absence of S9 mix, whereas in the cyanotoxin mixture the concentration-dependent genotoxicity of ATX-a/CYN in vitro was observed only in the presence of S9. Thus, the toxicity induced by cyanotoxin mixtures may vary from that produced by toxins alone, and consequently more studies are necessary in order to perform more realistic risk assessments.

The incidence and interest of cyanobacteria are increasing nowadays because they are able to produce some toxic secondary metabolites known as cyanotoxins. Among them, the presence of cylindrospermopsin (CYN) is especially relevant, as it seems to cause damage at different levels in the organisms: the nervous system being the one most recently reported. Usually, the effects of the cyanotoxins are studied, but not those exerted by cyanobacterial biomass. The aim of the present study was to assess the cytotoxicity and oxidative stress generation of one cyanobacterial extract of R. raciborskii non-containing CYN (CYN-), and compare its effects with those exerted by a cyanobacterial extract of C. ovalisporum containing CYN (CYN+) in the human neuroblastoma SH-SY5Y cell line. Moreover, the analytical characterization of potential cyanotoxins and their metabolites that are present in both extracts of these cultures was also carried out using Ultrahigh Performance Liquid Chromatography-Mass Spectrometry, in tandem (UHPLC-MS/MS). The results show a reduction of cell viability concentration- and time-dependently after 24 and 48 h of exposure with CYN+ being five times more toxic than CYN-. Furthermore, the reactive oxygen species (ROS) increased with time (0-24 h) and CYN concentration (0-1.11 µg/mL). However, this rise was only obtained after the highest concentrations and times of exposure to CYN-, while this extract also caused a decrease in reduced glutathione (GSH) levels, which might be an indication of the compensation of the oxidative stress response. This study is the first one performed in vitro comparing the effects of CYN+ and CYN-, which highlights the importance of studying toxic features in their natural scenario.

Due to climate change and anthropogenic activities, the levels of pollution of aquatic and terrestrial environments have increased in the last decades. In this sense, the rise of cyanobacterial blooms, which release secondary metabolites with toxic properties, and the global use of pesticides for agricultural purposes have a negative impact on ecosystems. Thus, it would be interesting to study the concomitance of both types of toxicants in the same sample, since it is possible that they appear together. The aim of the present work was to state the effects of the interaction between the cyanotoxin cylindrospermopsin and the pesticide chlorpyrifos in differentiated SH-SY5Y neuronal cells to assess how they could affect the nervous system. To this end, cytotoxicity, morphological, and acetylcholinesterase activity studies were performed during 24 and 48 h. The results revealed a concentration-dependent decrease in viability and interaction between both toxicants, together with clear signs of apoptosis and necrosis induction. In this sense, different stages on the differentiation process would lead to differences in the toxicity exerted by the compounds both isolated as in combination, which it is not observed in non-differentiated cells. Additionally, the acetylcholinesterase activity appeared not to be affected, which is a clear difference compared to non-differentiated cells. These results show the importance of studying not only the toxicants themselves, but also in combination, to assess their possible effects in a more realistic scenario., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

The presence of Cylindrospermopsin (CYN) and Microcystins (MCs) in vegetables is considered as a significant worldwide toxicological risk. Thus, this work aims to assess for the first time the impact of refrigeration (4 °C) and freezing (-20 °C) on the levels of CYN, MCs and their mixtures (CYN + MCs) in lettuce and spinach. Samples were spiked with 750 μg cyanotoxins/g dry weight (d.w.). Several storage conditions were studied: refrigeration after 24, 48 h and 7 days, and freezing for 7 days, 1 and 3 months. Cyanotoxin concentrations were determined by Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS). For CYN, refrigeration at 48 h and 7 days was effective to decrease its concentrations up to 26% and 32%, respectively, in spinach. For MCs, refrigeration was only effective in lettuce compared to spinach, showing an important decrease of 80.3% MC-LR and 85.1% MC-YR. In spinach, CYN was stable after 3 months freezing, whereas MC contents were still reduced up to 44%. Overall, cyanotoxins were less stable in the mixture compared to individual toxins for both processes, and the effect of these storage techniques were toxin and food-specific. Further studies of cyanotoxins in foods are required for evaluating the risk for humans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Organosulfur compounds (OSCs) are secondary metabolites produced by different Allium species which present important biological activities such as antimicrobial, antioxidant, anti-inflammatory antidiabetic, anticarcinogenic, antispasmodic, etc. In recent years, their use has been promoted in the agri-food industry as a substitute for synthetic preservatives, increasing potential accumulative exposure to consumers. Before their application in the food industry, it is necessary to pass a safety assessment as specified by the European Food Safety Authority (EFSA). This work reviews the scientific literature on OSCs regarding their in vitro toxicity evaluation following PRISMA guidelines for systematic reviews. Four electronic research databases were searched (Web of Science, Scopus, Science Database and PubMed) and a total of 43 works were selected according to predeterminate inclusion and exclusion criteria. Different data items and the risk of bias for each study were included. Currently, there are very few in vitro studies focused on investigating the potential toxicity of OSCs. Most research studies aimed to evaluate the cytotoxicity of OSCs to elucidate their antiproliferative effects focusing on their therapeutic aspects using cancer cell lines as the main experimental model. The results showed that diallyl disulfide (DADS) is the compound most studied, followed by diallyl trisulfide (DATS), diallyl sulfide (DAS), Allicin and Ajoene. Only 4 studies have been performed specifically to explore the safety of OSCs for agri-food applications, and genotoxicity studies are limited. More toxicity studies of OSCs are necessary to ensure consumers safety and should mainly be focused on the evaluation of genotoxicity and long-term toxicity effects., Competing Interests: The authors declare no conflict of interest. The co-authors Dr. Alberto Baños and Dr. Enrique Guillamón affiliated to DMC Research Center declare that they have no conflict of interest.

Cylindrospermopsin (CYN) is a cyanotoxin whose incidence has been increasing in the last decades. Due to its capacity to exert damage at different levels of the organism, it is considered a cytotoxin. Although the main target organ is the liver, recent studies indicate that CYN has potential toxic effects on the nervous system, both in vitro and in vivo. Thus, the aim of the present work was to study the effects of this cyanotoxin on neuronal viability and synaptic integrity in murine primary cultures of neurons exposed to environmentally relevant concentrations (0-1 µg/mL CYN) for 12, 24, and 48 h. The results demonstrate a concentration- and time-dependent decrease in cell viability; no cytotoxicity was detected after exposure to the cyanotoxin for 12 h, while all of the concentrations assayed decreased this parameter after 48 h. Furthermore, CYN was also demonstrated to exert damage at the synaptic level in a murine primary neuronal culture in a concentration- and time-dependent manner. These data highlight the importance of studying the neurotoxic properties of this cyanotoxin in different experimental models.

The consumption of fish contaminated with cyanotoxins is an important public health issue due to their potential adverse effects. The aim of this study was to assess the influence of refrigeration (4 °C) and freezing (-20 °C) on the concentration of Cylindrospermopsin (CYN), Microcystins (MCs) and their combination in tilapia (Oreochromis niloticus) and tench (Tinca tinca). Fish muscle were spiked with a stock solution of each toxin to reach 750 μg/g dry weight (d.w.). Three different periods of time were investigated for each treatment: 24 h, 48 h and 7 days for refrigeration, and 24 h, 7 days and 1 month for freezing. Samples were extracted and quantified by Ultra Performance Liquid Chromatography - Tandem Mass Spectrometry (UPLC-MS/MS). The results showed that freezing for 1 month produced highest decreases of these toxins in both species in comparison to refrigeration, being CYN the most stable cyanotoxin. Moreover, MCs are more stable to storage processes in the mixtures than alone, and fish species is a factor to take into account in their stability. These findings highlight the need to assess the influence of food storage processes on the presence of cyanotoxins in fish species for a more realistic human health risk assessment., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Propyl-propanethiosulfinate (PTS) is a component of Allium essential oils. This organosulfur molecule can be used as a feed additive to decrease the appearance of bacterial resistances caused by the residues of antibiotics. In previous in vitro genotoxicity studies, contradictory results were reported for PTS. In this work, the in vivo genotoxicity of PTS in male and female rats was assessed for the first time, following OECD (Organisation for Economic Co-operation and Development) guidelines. After oral administration (doses: 5.5, 17.4, and 55.0 mg/kg PTS body weight), a combination of the micronucleus (MN) assay (OECD 474) in bone marrow and the standard and enzyme-modified comet assay (OECD 489) was performed. After necropsy, histopathological studies were also carried out. The results did not show the in vivo genotoxicity of PTS at any doses assayed, revealed by the absence of increased MN, and DNA strand breaks or oxidative DNA damage in the standard and enzyme-modified comet assays. The histopathological study revealed that only the highest dose tested (55.0 mg/kg) in the liver and all dose groups in the stomach presented minimal pathological lesions in the organs studied. Consequently, the present work confirms that PTS is not genotoxic at the doses assayed, and it is a promising natural alternative to synthetic preservatives and antibiotics in animal feed.

Stilbenes are secondary metabolites of great interest produced by many plant species due to their important bioactive properties. These phytochemicals have become of increasing interest in the wine industry as a natural alternative to sulphur dioxide, which has been associated with human health risks. However, there is still little toxicological information on stilbenes and the results thus far have been contradictory. Considering the key role of genotoxicity in risk assessment and the need to offer safe products in the market, the aim of this study was to assess the mutagenic and genotoxic potential of a stilbene extract with 99% purity (ST-99 extract). A complete series of different in vitro tests (Ames test, micronucleus (MN) test, and standard and enzyme-modified comet assays) was performed before its use as a preservative in wines. The ST-99 extract induces a significant increase of binucleated cells with micronuclei only in presence of the metabolic fraction S9 at the highest concentration assayed. Neither the Ames test nor the comet assay revealed the extract's genotoxic potential. Further studies are necessary, including in vivo assays, to ensure consumer safety before it can be used., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The application of stilbenes in the food industry is being considered because of their biological activities. Piceatannol, pterostilbene and ε-viniferin have awakened the industry's interest. However, before they can be commercialized, we must first guarantee their safety for consumers. The present work reviews the toxicological studies performed with these stilbenes. A wide variety of studies has demonstrated their cytotoxic effects in both cancer and non-cancerous cell lines. In contrast, although DNA damage was detected by some authors, in vitro genotoxic studies on the effects of piceatannol, pterostilbene, and ε-viniferin remain scarce. None of the three reviewed substances have been evaluated using the in vitro tests required by the European Food Safety Authority (EFSA) as the first step in genotoxicity testing. We did not find any study on the toxic effects of these stilbenes in vivo. Thus, more studies are needed to confirm their safe use before they can be authorized as additive in the food industry.

Genotoxic data of substances that could be used as food additives are required by the European Food Safety Authority. In this sense, the use of an extract from grapevine shoots containing a stilbene richness of 99% (ST-99), due to its antioxidant and antibacterial activities, has been proposed as an alternative to sulfur dioxide in wine. The aim of this work was to study, for the first time, the in vivo genotoxic effects produced in rats orally exposed to 90, 180, or 360 mg ST-99/kg body weight at 0, 24, and 45 h. The combination of micronucleus assay in bone marrow (OECD 474) and standard (OECD 489) and enzyme-modified comet assay was used to determine the genotoxicity on cells isolated from stomach, liver, and blood of exposed animals. The ST-99 revealed no in vivo genotoxicity. These results were corroborated by analytical studies that confirm the presence of stilbenes and their metabolites in plasma and tissues. Moreover, to complete these findings, a histopathological study was performed under light microscopy in liver and stomach showing only slight modifications in both organs at the highest concentration used. The present work confirms that this extract is not genotoxic presenting a good profile for its potential application as a preservative in the wine industry.

Propyl-propane-thiosulfonate (PTSO) is one of the main organosulfur compounds present in Allium essentials oil. Different applications in the food sector have been proposed for PTSO, such as food and feed additive and as active packaging. However, the authorization of its use depends on its toxicity profile. Thus, as a part of its safety assessment, in this work a repeated dose 90-day oral toxicity study has been conducted for the first time in rats following the OECD guideline 408. PTSO was administered to groups of 10 male and 10 female rats at dose levels of 0, 14, 28, and 55 mg/kg/day. No clinical signs or mortality and no changes in body weight, food consumption and feed conversion efficiency were detected through the study. Moreover, no treatment-related changes in hematological and biochemical parameters were observed, for either sex or dose groups. The histopathology study performed revealed no differences in organ weights, and no morphological and histopathological changes were observed. Based on these results, the no-observed-adverse-effect level (NOAEL) of PTSO was judged to be ≥ 55 mg/kg/day for both sexes., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The appearance of MDR strains and the development of biofilms make Pseudomonas aeruginosa infections a therapeutic challenge. To overcome this scenario, bacteriocins have been proposed as a potential adjuvant or alternative to antibiotic treatment., Objectives: To study the activity of R-pyocins on biofilms and in a murine model of pneumonia using a high-risk clone of P. aeruginosa., Methods: The activity of R-pyocins on P. aeruginosa biofilms was tested on bacteria attached to a silicone surface, before and after biofilm formation. The effectiveness of R1-pyocin was studied in a murine model of pneumonia using ST175, a high-risk clone of P. aeruginosa., Results: R-pyocins attacked adherent bacteria, preventing biofilm formation, and penetrated into the biofilm, killing P. aeruginosa within it, resulting in a dramatic reduction in bacterial load. R1-pyocin was active in a murine model of P. aeruginosa lung infection, administered before infection as a preventive treatment, and in acute pneumonia, with efficiency higher than standard colistin treatment. In addition, this work is the first to describe histopathological lung changes after administration of R-pyocins, contributing to the resolution of P. aeruginosa pneumonia in a murine model., Conclusions: This work highlights the potential use of the R-pyocins as therapeutic agents, alone or as adjuvants, due to its effectiveness on biofilms and in a murine model of pneumonia using ST175, a high-risk clone of P. aeruginosa. It may thus be feasible to consider R-pyocins as a possible therapeutic alternative in XDR infections, where treatment alternatives are limited., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cylindrospermopsin (CYN) is a cyanotoxicant which occurrence is increasing due to climate change. Cylindrospermopsin is able to exert damage in the organism at several levels, among them, in the nervous system. Moreover, it is important to take into account that it is not usually present isolated in nature, but in combination with some other pollutants, being the case of the pesticide chlorpyrifos (CPF). Thus, the aim of the present work was to assess the effects of the interaction of CYN in combination with CPF in the human neuroblastoma cell line SH-SY5Y by evaluating cytotoxicity and mechanistic endpoints. The mixtures 0.25 + 21, 0.5 + 42, 1 + 84 μg/mL of CYN + CPF based on cytotoxicity results, were evaluated, and the isobologram method detected an antagonistic effect after 24 and 48 h of exposure. Moreover, although no alterations of reactive oxygen species were detected, a significant decrease of glutathione levels was observed after exposure to both, CPF alone and the combination, at all the concentrations and times of exposure assayed. In addition, CYN + CPF caused a marked decrease in the acetylcholinesterase activity, providing similar values to CPF alone. However, these effects were less severe than expected. All these findings, together with the morphological study results, point out that it is important to take into account the interaction of CYN with other pollutants. Further research is required to contribute to the risk assessment of CYN and other contaminants considering more realistic exposure scenarios., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Microcystins (MCs) and cylindrospermopsin (CYN) are among the most frequent toxins produced by cyanobacteria. These toxic secondary metabolites are classified as hepatotoxins and cytotoxin, respectively. Furthermore, both may present the ability to induce damage to the nervous system. In this sense, there are many studies manifesting the potential of MCs to cause neurotoxicity both in vitro and in vivo, due to their probable capacity to cross the blood-brain-barrier through organic anion transporting polypeptides. Moreover, the presence of MCs has been detected in brain of several experimental models. Among the neurological effects, histopathological brain changes, deregulation of biochemical parameters in brain (production of oxidative stress and inhibition of protein phosphatases) and behavioral alterations have been described. It is noteworthy that minority variants such as MC-LF and -LW have demonstrated to exert higher neurotoxic effects compared to the most studied congener, MC-LR. By contrast, the available studies concerning CYN-neurotoxic effects are very scarce, mostly showing inflammation and apoptosis in neural murine cell lines, oxidative stress, and alteration of the acetylcholinesterase activity in vivo. However, more studies are required in order to clarify the neurotoxic potential of both toxins, as well as their possible contribution to neurodegenerative diseases., (Copyright © 2018. Published by Elsevier B.V.)

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.

Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN) are produced by cyanobacteria. Although being considered as a hepatotoxin and a cytotoxin, respectively, different studies have revealed neurotoxic properties for both of them. The aim of the present work was to study their cytotoxic effects, alone and in combination, in the SH-SY5Y cell line. In addition, toxicity mechanisms such as oxidative stress and acetylcholinesterase (AChE) activity, and morphological studies were carried out. Results showed a cytotoxic response of the cells after their exposure to 0-100 μg/mL of MC-LR or 0-10 μg/mL CYN in both differentiated and undifferentiated cells. Thus, CYN resulted to be more toxic than MC-LR. Respect to their combination, a higher cytotoxic effect than the toxins alone in the case of undifferentiated cells, and almost a similar response to the presented by MC-LR in differentiated cells were observed. However, after analyzing this data with the isobolograms method, an antagonistic effect was mainly obtained. The oxidative stress study only showed an affectation of glutathione levels at the highest concentrations assayed of MC-LR and the combination in the undifferentiated cells. A significant increase in the AChE activity was observed after exposure to MC-LR in undifferentiated cells, and after exposure to the combination of both cyanotoxins on differentiated cells. However, CYN decreased the AChE activity only on differentiated cultures. Finally, the morphological study revealed different signs of cellular affectation, with apoptotic processes at all the concentrations assayed. Therefore, both cyanotoxins isolated and in combination, have demonstrated to cause neurotoxic effects in the SH-SY5Y cell line., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The occurrence of cyanobacterial toxins is being increasingly reported. This is a reason for concern as they can induce toxic effects both in humans and in the environment. Among them, microcystins (MCs) are the best described and most diverse group of cyanobacterial toxins, and MC-LR and MC-RR are the congeners most widely investigated. However, the number of MC variants has also increased in recent years. Some of these minority variants have been shown to have a different toxicokinetic and toxicodynamic profile, but research focused on them is still limited. Moreover, in some water bodies these minority variants can be the predominant toxins. Nonetheless, MC-LR is the only one used for risk evaluation purposes at present. In order to contribute to more realistic risk assessments in the future, the aim of this review was to compile the available information in the scientific literature regarding the occurrence and concentration of minority MCs in water and food samples, and their toxic effects. The data retrieved demonstrate the congener-specific toxicity of MCs, as well as many data gaps in relation to analytical or mechanistic aspects, among others. Therefore, further research is needed to improve the toxicological characterization of these toxins and the exposure scenarios., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Cylindrospermopsin (CYN) is a cyanobacterial toxin with an increasing world-wide occurrence. The main route of human exposure is through the ingestion of contaminated food and water. The European Food Safety Authority has identified the need to further characterize the toxicological profile of cyanotoxins and in this regard the genotoxicity is a key toxicological effect. The data available in the scientific literature show contradictory results. Thus, the aim of this study was to investigate the mutagenic and genotoxic effects of pure CYN using a battery of different in vitro assays including: the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test) (0-10 μg/mL), the mammalian cell micronucleus (MN) test (0-1.35 μg/mL and 0-2 μg/mL in absence or presence of S9 fraction, respectively) and the mouse lymphoma thymidine-kinase assay (MLA)(0-0.675 μg/mL) on L5178YTk ± cells, and the standard and enzyme-modified comet assays (0-2.5 μg/mL) on Caco-2 cells. Positive results were obtained only when the metabolic fraction S9 was employed in the MN test, suggesting pro-genotoxic properties of CYN. Also, DNA damage was not mediated by oxidative stress as CYN did not induced changes in the modified comet assay. These data could contribute to a better risk assessment of this cyanotoxin., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: The appearance and dissemination of MDR among pathogenic bacteria has forced the search for new antimicrobials. Bacteriocins have been proposed as potential alternatives for the treatment of infections due to multiresistant strains., Objectives: To analyse the activity of R-pyocins against clinical isolates of Pseudomonas aeruginosa from patients with cystic fibrosis and other sources and evaluate them as a potential adjuvant or alternative to the current antibiotic treatment., Methods: The activity of R-pyocins against 150 strains of P. aeruginosa isolated from patients with cystic fibrosis or bacteraemia was studied through spot assay. Interactions between R-pyocins and antipseudomonal agents were quantitatively studied by the chequerboard method., Results: The proportion of P. aeruginosa isolates susceptible to R-pyocins was found to be higher in cystic fibrosis isolates compared with bacteraemia isolates (79.41% versus 50%). Moreover, no interactions were found between common antipseudomonal agents and R-pyocin susceptibility, except for the ST175 high-risk clone., Conclusions: Our results highlight the possibility of using R-pyocins as therapeutic agents, alone or as adjuvants, against P. aeruginosa in cystic fibrosis.

Reports on the occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) have increased worldwide because of CYN toxic effects in humans and animals. If contaminated waters are used for plant irrigation, these could represent a possible CYN exposure route for humans. For the first time, a method employing solid phase extraction and quantification by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) of CYN was optimized in vegetables matrices such as lettuce ( Lactuca sativa ). The validated method showed a linear range, from 5 to 500 ng CYN g -1 of fresh weight (f.w.), and detection and quantitation limits (LOD and LOQ) of 0.22 and 0.42 ng CYN g -1 f.w., respectively. The mean recoveries ranged between 85 and 104%, and the intermediate precision from 12.7 to 14.7%. The method showed to be robust for the three different variables tested. Moreover, it was successfully applied to quantify CYN in edible lettuce leaves exposed to CYN-contaminated water (10 µg L -1 ), showing that the tolerable daily intake (TDI) in the case of CYN could be exceeded in elderly high consumers. The validated method showed good results in terms of sensitivity, precision, accuracy, and robustness for CYN determination in leaf vegetables such as lettuce. More studies are needed in order to prevent the risks associated with the consumption of CYN-contaminated vegetables.

β-Lactamases (BLs) able to hydrolyze β-lactam antibiotics and more importantly the last resort carbapenems, represent a major mechanism of resistance in Gram-negative bacteria showing multi-drug or extensively drug resistant phenotypes. The early detection of BLs responsible of resistant infections is challenging: approaches aiming at the identification of new BLs inhibitors (BLI) can thus serve as the basis for the development of highly needed diagnostic tools. Starting from benzo-[b]-thiophene-2-boronic acid (BZB), a nanomolar inhibitor of AmpC β-lactamase (K i  = 27 nM), we have identified and characterized a set of BZB analogues able to inhibit clinically-relevant β-lactamases, including AmpC, Extended-Spectrum BLs (ESBL), KPC- and OXA-type carbapenemases and metallo-β-lactamases (MBL). A multiligand set of boronic acid (BA) β-lactamase inhibitors was obtained using covalent molecular modeling, synthetic chemistry, enzyme kinetics and antibacterial susceptibility testing. Data confirmed the possibility to discriminate between clinically-relevant β-lactamases on the basis of their inhibition profile. Interestingly, this work also allowed the identification of potent KPC-2 and NDM-1 inhibitors able to potentiate the activity of cefotaxime (CTX) and ceftazidime (CAZ) against resistant clinical isolates (MIC reduction, 32-fold). Our results open the way to the potential use of our set of compounds as a diagnostic tool for the sensitive detection of clinically-relevant β-lactamases.

Cylindrospermopsin (CYN) has become increasingly important as a freshwater algal toxin, showing cytotoxic effects. This toxin is able to bioaccumulate in freshwater food webs, representing a serious human health problem. Normally, fish is cooked before consumption, and CYN concentration can be altered. For the first time, the effects of microwaving and broiling for 1 and 2 min on CYN concentration and its decomposition products in fish muscle ( Oreochromis niloticus ) contaminated in the laboratory were investigated, using UPLC-MS/MS and Orbitrap. The results show that cooking the fish reduced unconjugated CYN levels by 11, 10 and 15% after microwaving for 1 and 2 min, and broiling for 2 min, respectively, compared to control fish. Different CYN decomposition products with m / z 416.1234 (7-epi-CYN) and m / z 336.16663 (diasteroisomers C-3A, C-3C, C-3D, C-3E, C-3F) are generated in fish samples submitted to cooking. Based on the relative abundance of the decomposition products, the possible degradation pathways taking place by microwaving may be through the formation of 7-epi-CYN and m / z 336.16663 compounds, whereas in the case of broiling the last route is the only one observed in this study. The influence of cooking and the toxicity characterization of the degradation products generated in CYN-contaminated fish are of importance for more realistic risk evaluation related to their consumption.

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., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS-MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS-MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature., Competing Interests: The authors declare no competing financial interests.

Garlic (Allium sativum) and onion (Allium cepa) are being used in the food industry as flavoring but also for their antimicrobial activities. These activities are mainly derived from the organosulfur compounds (OSCs). Propyl propane thiosulfinate (PTS) is an OSC with potential use in the active packaging, but its safety should be guaranteed before being commercialized. The aim of this work was to investigate for the first time the cytotoxicity of PTS as well as its in vitro mutagenic/genotoxic potential using the following battery of genotoxicity tests:(1)the bacterial reverse-mutation assay in S. typhimurium (Ames test, OECD 471, 1997); (2) the micronucleus test (MN, OECD 487, 2016); (3) the mouse lymphoma thymidine-kinase assay (MLA, OECD 476, 2015), and (4) the comet assay (standard and modified with restriction enzymes). The results revealed that PTS was not mutagenic neither in the Ames test nor in MLA. However, genotoxic effects were recorded in the MN test on mammalian cells (L5178YTk +/- cells) after PTS exposure at the highest concentration tested (17.25 μM) without S9, and also its metabolites (+S9, from 20 μM). Moreover, in the comet assay, PTS induced DNA breaks damage in Caco-2 cells at the highest concentration tested (280 μM) but it did not induce oxidative DNA damage., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Genotoxic data of substances which could be incorporated into food packaging are required by the European Food Safety Authority. Due to its antioxidant and antibacterial properties carvacrol is one of these compounds. This work aims to study for the first time the in vivo genotoxic effects produced in rats orally exposed to 81, 256 or 810 mg cavacrol/kg body weight (bw) at 0, 24 and 45 h. A combination of the micronucleus assay (OECD 474) in bone marrow and the standard (OECD 489) and enzyme-modified comet assay was used to determine the genotoxicity on cells isolated from stomach and liver of exposed animals. In addition, a histopathological study was performed on the assayed tissues, and also in the lungs due to the volatility of carvacrol. Direct analytical pyrolysis was used to search for carvacrol in viscera and to ensure that the compound reaches stomach and liver cells. Results from MN-comet assay revealed that carvacrol (81-810 mg/kg bw) did not induce in vivo genotoxicity or oxidative DNA damage in any of the tissues investigated. Moreover, no histopathological changes were observed. Altogether, these results suggest lack of genotoxicity of carvacrol and therefore its good profile for its potential application as food preservative., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Components of Allium species have antimicrobial and antioxidant properties. A commercial Allium sp. extract (Proallium AP(®)), of which the main constituent is propyl thiosulphinate oxide (PTSO), is being used in the development of active food packaging. In previous in vitro genotoxicity studies, PTSO, in the presence of metabolic activation, increased the appearance of micronuclei (MN). We assessed the genotoxicity PTSO in rats following oral administration (doses: 5.5, 17.4, and 55mg/kg). The comet assay in liver and stomach (OECD 489) and the MN assay in bone marrow (OECD 474) were carried out. After necropsy, histopathological examinations of the liver and the stomach were performed. The results revealed no in vivo genotoxicity and the histopathological analysis showed only slight modifications, such as increased glycogen storage in the liver and a degenerative process in stomach, with vacuolization of cell membranes, only at the highest dose. Therefore, the present work confirms that this compound is not genotoxic and could be considered as a natural alternative to synthetic preservatives used in the food packaging industry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Essential oils from onion (Allium cepa L.), garlic (Allium sativum L.), and their main components, such as propyl thiosulfinate oxide (PTSO) are being intended for active packaging with the purpose of maintaining and extending food product quality and shelf life. The present work aims to assess for the first time the potential mutagenicity/genotoxicity of PTSO (0-50 µM) using the following battery of genotoxicity tests: (1) the bacterial reverse-mutation assay in Salmonella typhimurium (Ames test, OECD 471); (2) the micronucleus test (OECD 487) (MN) and (3) the mouse lymphoma thymidine-kinase assay (OECD 476) (MLA) on L5178YTk(+/-), cells; and (4) the comet assay (with and without Endo III and FPG enzymes) on Caco-2 cells. The results revealed that PTSO was not mutagenic in the Ames test, however it was mutagenic in the MLA assay after 24 h of treatment (2.5-20 µM). The parent compound did not induce MN on mammalian cells; however, its metabolites (in the presence S9) produced positive results (from 15 µM). Data from the comet assay indicated that PTSO did not induce DNA breaks or oxidative DNA damage. Further in vivo genotoxicity tests are needed to confirm its safety before it is used as active additive in food packaging., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Modern food packaging has made great advances as result of global trends and consumer preferences, which are oriented to obtain improved food quality and safety. In this regard, clay minerals, and mainly Montmorillonite (Mt) are attracting considerable interest in food packaging because of the improvements developed in mechanical and barrier properties. Hence, the present work aim to assess the toxicity of four Montmorillonite-based clay minerals, an unmodified clay, Cloisite®Na+ (CNa+), and three modified Mt clays: Cloisite®30B (C30B), a commercial clay, and Clay1 and Clay2, two novel modified organoclays developed by the Packaging, Transport, & Logistics Research Institute (ITENE). First, the cytotoxic effects were studied in the Human Umbilical Vein Endothelial Cells (HUVEC). In addition, the potential mutagenicity of the clays was evaluated by the Ames test. Clay1 did not induce any cytotoxic effects in HUVEC, although it exhibited potential mutagenicity in TA98 Salmonella typhimurium strain. In contrast, Clay2 produced cytotoxicity in endothelial cells but no mutagenicity was recorded. However, CNa+ was not cytotoxic neither mutagenic. And finally, C30B showed positive results in both assays. Therefore, results showed that clay minerals have a different toxicity profile and a case by case toxicity evaluation is required., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Essential oils (EOs) and their main constituent compounds have been extensively investigated due to their application in the food industry for improving the shelf life of perishable products. Although they are still not available for use in food packaging in the market in Europe, considerable research in this field has been carried out recently. The safety of these EOs should be guaranteed before being commercialized. The aim of this work was to review the scientific publications, with a primary focus on the last 10 years, with respect to different in vitro toxicological aspects, mainly focussed on mutagenicity/genotoxicity. In general, fewer genotoxic studies have been reported on EOs in comparison to their main components, and most of them did not show mutagenic activity. However, more studies are needed in this field since the guidelines of the European Food Safety Authority have not always been followed accurately. The mutagenic/genotoxic activities of these substances have been related to metabolic activation. Therefore, in vivo tests are required to confirm the absence of genotoxic effects. Considering the great variability of the EOs and their main compounds, a case-by-case evaluation is needed to assure their safe use in food packaging., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Currently, antimicrobial additives derived from essential oils (Eos) extracted from plants or spices, such as Origanum vulgare, are used in food packaging. Thymol and carvacrol, the major EO compounds of O. vulgare, have demonstrated their potential use as active additives. These new applications use high concentrations, thereby increasing the concern regarding their toxicological profile and especially their genotoxic risk. The aim of this work was to investigate the potential in vitro genotoxicity of thymol (0-250 μM) and carvacrol (0-2500 μM) at equivalent doses to those used in food packaging. The micronucleus (MN) test and the mouse lymphoma (MLA) assay on L5178Y/Tk(±) mouse lymphoma cells were used. The negative results for thymol with the MN with and without the S9 fraction and also with the MLA assay reinforce the view that this compound is not genotoxic in mammalian cells. However, carvacrol presented slight genotoxic effects, but only in the MN test at the highest concentration assayed (700 μM) and in the absence of metabolic activation. The lack of genotoxic response in the MLA assay after 4 and 24h of exposure indicates a low genotoxic potential for carvacrol. Alternatively, the general negative findings observed in both assays suggest that the MN results of carvacrol are marginal data without biological relevance. These results can be useful to identify the appropriate concentrations of these substances to be used as additives in food packaging., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria, such as Aphanizomenon ovalisporum. CYN is a tricyclic alkaloid known for its ability to inhibit both protein and glutathione synthesis, and the alteration of different oxidative stress biomarkers in mammals and vertebrates. Although the liver and kidney appear to be the main CYN targets for this toxin, it also affects other organs. In fish, there is no evidence about the neurotoxicity of CYN yet. In the present study, we aimed to study the potential neurotoxicity of CYN, based on the measure of Acetylcholinesterase (AChE) activity, lipid peroxidation (LPO) levels and histopathological studies in brain of tilapia (Oreochromis niloticus) subchronically exposed to repeated concentrations of 10μg CYN/L by immersion in an A.ovalisporum culture for 14 days. The results showed significant inhibition of AChE activity and increases in LPO levels, as well as relevant histopathological alterations in the brain of fish (O. niloticus) subchronically exposed to the toxin. Moreover, we also investigated the potential recovery of these parameters by subjecting the fish to two depuration periods (3 and 7 days) in clean uncontaminated water, showing a recovery of the biochemical parameters since 3 days of depuration, and being necessary 7 days to recover the histopathological changes. In order to support these results, CYN was detected and quantified by enzyme-linked immunosorbent assay (ELISA) in brain of all the exposed fish and the effects of the depuration periods were also observed. Based on these results, it was demonstrated for the first time the neurotoxicity of CYN and its presence in brain of tilapia fish subchronically exposed to CYN., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The acute toxicity of cylindrospermopsin (CYN) has been established in rodents, based on diverse intraperitoneal an oral exposure studies and more recently in fish. But no data have been reported in fish after subchronic exposure to cyanobacterial cells containing this cyanotoxin, so far. In this work, tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels of CYN (10 or 100 μg CYN L(-1)) and deoxy-cylindrospermopsin (deoxy-CYN) (0.46 or 4.6 μg deoxy-CYN L(-1)), during two different exposure times: 7 or 14 d. This is the first study showing damage in the liver, kidney, hearth, intestines, and gills of tilapia after subchronic exposure to cyanobacterial cells at environmental relevant concentrations. The major histological changes observed were degenerative processes and steatosis in the liver, membranous glomerulopathy in the kidney, myofibrolysis and edema in the heart, necrotic enteritis in the gastrointestinal tract, and hyperemic processes in gill lamellae and microhemorrhages. Moreover, these histopathological findings confirm that the extent of damage is related to the CYN concentration and length of exposure. Results from the morphometric study indicated that the average of nuclear diameter of hepatocytes and cross-sections of proximal and distal convoluted tubules are useful to evaluate the damage induced by CYN in the main targets of toxicity., (© 2013 Wiley Periodicals, Inc.)

Edible members of the Allium family are widely used since they exhibit antioxidant and antibacterial related to the organosulphur compounds. One the most promising use of Allium species, hence, onion essential oil, could be in the packaging food industry. The present work aims to assess the safety of two organosulphur compounds present in onion essential oil; dipropyl disulphide, dipropyl sulphide and their mixture. For this purpose, cytotoxicity, reactive oxygen species and glutathione contents, and ultrastructural cellular damages were studied in the human intestinal cells, Caco-2, exposed to these organosulphur compounds. Moreover, their potential mutagenicity was also assessed. The results revealed no significant adverse effects. Additionally, reactive oxygen species scavenger activity was observed for both compounds. Therefore, they could be a good natural alternative to other synthetic antioxidant and antibacterial substances used in the food industry., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Currently, direct antimicrobial and antioxidant additives derived from essential oils are used in food packaging and are perceived by consumers as low-health-risk compounds. In this study, we investigated the potential mutagenicity and genotoxicity of carvacrol and thymol, major compounds in several essential oils, using the Ames Salmonella test and the alkaline, Endo III- and formamidopyrimidine glycosylase (FPG)-modified comet assays, respectively. Thymol did not show any mutagenic activity at any concentration assayed (0-250 μM), whereas carvacrol exhibited mutagenic potential, displaying greater activity in presence of the metabolic fraction (29-460 μM). The genotoxic effects were evaluated in the human colon carcinoma cell line Caco-2, and the standard comet assay revealed that neither carvacrol (0-460 μM) nor thymol (0-250 μM) had any affects at 24 and 48 h. The FPG-modified comet assay showed that the highest concentration of carvacrol (460 μM) caused DNA damage, indicating damage to the purine bases. These results should be used to identify the appropriate concentrations of carvacrol and thymol as additives in food packaging. Moreover, further studies are necessary to explore the safety and/or the toxicity mechanisms of these compounds., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The hepatotoxin cylindrospermopsin (CYN) is produced by freshwater cyanobacteria becoming an emerging threat for human health. Methods for the rapid determination of CYN in environmental samples are needed. Conventional analytical pyrolysis (Py-GC/MS) and thermally-assisted hydrolysis and methylation (TCh-GC/MS) were used to study a CYN standard, two Aphanizomenon ovalisporum cultures (CYN+) and one culture of Cylindrospermopsis raciborskii (CYN-). A micro-furnace pyrolyzer was used directly attached to a GC/MS system fitted with a 30 m × 250 μm × 0.25 μm film thickness column (14% cyanopropyl phenyl, 86% dimethyl polysiloxane pahase composition). Oven temperature was held at 50 °C for 1 min and 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 using helium (1 mL min(-1)) as carrier gas. Pyrolysis at 500 °C yield over 70 compounds with 20 specific for CYN+ samples. Two peaks containing a diagnostic fragment (m/z 194) were found at 25.0 and 28.9 min only in CYN+ samples. Fewer peaks with limited diagnostic value were released after TCh-GC/MS, including breakdown products and TMAH adducts. A compound was detected that may correspond to the CYN molecule (MW 415 Da) thermoevaporation product after the loss of SO3 (MW 80 Da). This TCh-GC/MS peak (m/z 336) together with the fragments obtained by conventional Py-GC/MS (m/z 194) are diagnostic ions with potential use for the direct detection of CYN toxin in environmental samples at last with an estimated 5 ppm detection threshold., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Cylindrospermopsin (CYN) is a cytotoxic polyketide-derived alkaloid produced by several freshwater cyanobacterial species. It is now considered the second most studied cyanotoxin worldwide. Among the toxic mechanisms suggested for CYN pathogenicity are inhibition of protein and glutathione synthesis, genotoxicity by DNA fragmentation, and oxidative stress. The study of depuration of cyanobacterial toxins by aquatic organisms, particularly by fish, is important for fish economy and public health, but in the case of CYN is practically nonexistent. In this work, we investigated the efficiency of two distinct depuration periods, 3 or 7d, in a clean environment, as a mean of restoring the levels of several oxidative stress biomarkers in tilapia (Oreochromis niloticus) subchronically exposed to CYN by immersion in an Aphanizomenon ovalisporum culture (by adding 10 μg CYN/L every two days during 14 d). Lipid peroxidation (LPO) and DNA oxidation returned to normal values after 7d of depuration, whereas the time needed for restoring of the oxidatively damaged proteins was longer. Superoxide dismutase (SOD) and gamma-glutamyl-cysteine-synthetase (γ-GCS) activities recovered after just 3d of depuration, while catalase (CAT) activity needed up to 7d to return to control values. Ratio of reduced glutathione to oxidized glutathione (GSH/GSSG) returned to control levels after 7d of depuration in both organs. These results validate the depuration process as a very effective practice for detoxification in fish contaminated with these toxins., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The cyanobacterial toxin Cylindrospermopsin (CYN), a potent protein synthesis inhibitor, is increasingly being found in freshwater bodies infested by cyanobacterial blooms worldwide. Moreover, it has been reported to be implicated in human intoxications and animal mortality. Recently, the alteration of the activity and gene expression of some glutathione related enzymes in tilapias (Oreochromis niloticus) exposed to a single dose of CYN has been reported. However, little is known about the effects induced by repeated doses of this toxin in tilapias exposed by immersion and the potential reversion of these biochemical alterations after two different depuration periods (3 or 7 days). In the present study, tilapias were exposed by immersion to repeated doses of a CYN-containing culture of Aphanizomenon ovalisporum during 14 days, and then were subjected to depuration periods (3 or 7 days) in clean water in order to examine the potential reversion of the effects observed. The activity and relative mRNA expression by real-time polymerase chain reaction (PCR) of the antioxidant enzymes glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST), and also the sGST protein abundance by Western blot analysis were evaluated in liver and kidney of fish. Results showed significant alterations in most of the parameters evaluated and their recovery after 3 days (GPx activity, sGST relative abundance) or 7 days (GPx gene expression, sGST activity). These findings not only confirm the oxidative stress effects produced in fish by cyanobacterial cells containing CYN, but also show the effectiveness of depuration processes in mitigating the CYN-containing culture toxic effects.

Cyanobacteria are able to produce several metabolites that have toxic effects on humans and animals. Among these cyanotoxins, the hepatotoxic microcystins (MC) occur frequently. The intracellular MC content produced by two strains of Microcystis aeruginosa, PCC7806 and PCC7820, and its production kinetics during the culture time were studied in order to elucidate the conditions that favour the growth and proliferation of these toxic strains. Intracellular MC concentrations measured by liquid chromatography (LC) coupled to electrospray ionization mass spectrometer (MS) were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) anti-Adda and protein phosphatase 2A (PP2A) inhibition assays. It has been demonstrated there are discrepancies in the quantification of MC content when comparing ELISA and LC-MS results. However, a good correlation has been obtained between PP2A inhibition assay and LC-MS. Three MC were identified using LC-MS in the PCC7806 strain: MC-LR, demethylated MC-LR and a new variant detected for the first time in this strain, [L-MeSer(7)] MC-LR. In PCC7820, MC-LR, D-Asp(3)-MCLR, Dglu(OCH3)-MCLR, MC-LY, MC-LW and MC-LF were identificated. The major one was MC-LR in both strains, representing 81 and 79% of total MC, respectively. The total MC content in M. aeruginosa PCC7820 was almost three-fold higher than in PCC7806 extracts.

Cylindrospermopsin (CYN) is a cytotoxic cyanotoxin produced by several species of freshwater cyanobacteria (i.e., Aphanizomenon ovalisporum). CYN is a tricyclic alkaloid combined with a guanidine moiety. It is well known that CYN inhibits both protein and glutathione synthesis, and also induces genotoxicity and the alteration of different oxidative stress biomarkers. Although the liver and kidney appear to be the main target organs for this toxin based on previous studies, CYN also affects other organs. In the present study, we studied the distribution of CYN in fish (Oreochromis niloticus) under two different exposure scenarios using immunohistochemical (IHC) techniques. In the first method, fish were exposed acutely by intraperitoneal injection or by gavage to 200 µg pure CYN/Kg body weight (bw), and euthanized after 24 h or five days of exposure. In the second method, fish were exposed by immersion to lyophilized A. ovalisporum CYN-producing cells using two concentration levels (10 or 100 µg/L) for two different exposure times (7 or 14 days). The IHC was carried out in liver, kidney, intestine, and gills of fish. Results demonstrated a similar pattern of CYN distribution in both experimental methods. The organ that presented the most immunopositive results was the liver, followed by the kidney, intestine, and gills. Moreover, the immunolabeling signal intensified with increasing time in both assays, confirming the delayed toxicity of CYN, and also with the increment of the dose, as it is shown in the sub-chronic assay. Thus, IHC is shown to be a valuable technique to study CYN distribution in these organisms.

In the present study a new method has been developed and validated for detecting free microcystins (MCs) (MC-RR, MC-LR and MC-YR) by liquid chromatography-mass spectrometry (LC-MS) in the cyprinid Tinca tinca and in the crayfish Procambarus clarkii collected from three ponds in Extremadura (Spain) where the presence of the cyanobacteria species Microcystis aeruginosa and Anabaena spiroides has been confirmed. Once the method had been validated, free MCs were determined in fish (tench, T. tinca) and crayfish from different ponds in order to understand how they are bioaccumulated through the food web. MCs were not detected in any of the fish samples analyzed. It was confirmed that P. clarkii accumulated MCs in their tissues without losing their organoleptic characteristics, with MC-LR (2.3-18.1 μg MC-LR/g body weight) being the predominant MC variant detected in all the crayfish samples. MC-RR was measured in 50% of the samples analyzed, ranging between 1.4 and 7.8 μg MC-RR/g body weight and no MC-YR was detected. The results indicated that crayfish can accumulate free MCs in higher quantities than tench that live in ponds contaminated by toxic cyanobacteria species, and emphasized the need for regular monitoring if the health risks associated with their consumption are to be avoided., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cylindrospermopsin (CYN) is one of the most important cyanotoxins in terms of both human health and environmental quality and is produced by several different species of cyanobacteria, including Aphanizomenon ovalisporum. The principal mechanisms of action of CYN involve inhibition of protein and glutathione synthesis. In addition, CYN-mediated genotoxicity results from DNA fragmentation. The results of both in vivo and in vitro studies suggest that oxidative stress also plays a significant role in CYN pathogenesis in fish. We investigated the protective effects of l-carnitine (LC) pre-treatment on A. ovalisporum-induced oxidative stress in cells containing CYN and deoxy-CYN, or pure standard CYN, in tilapia (Oreochromis niloticus) that had been acutely exposed via oral administration. Various oxidative stress markers, including lipid peroxidation (LPO), protein oxidation, DNA oxidation, and the ratio of reduced glutathione to oxidised glutathione (GSH/GSSG), and the activities of NADPH oxidase, superoxide dismutase (SOD), catalase (CAT), and gamma-glutamyl-cysteine synthetase (γ-GCS), were evaluated in the livers and kidneys of fish in the absence and presence of 400 or 880mgLC/kgfish/day during a 21 day period prior to CYN-intoxication. The results of our study demonstrated for the first time the beneficial antioxidant effects of LC dietary supplementation on oxidative stress status in fish. No pro-oxidant effects were detected at any of the LC doses assayed, suggesting that LC is a chemoprotectant that reduces hepatic and renal oxidative stress and may be effective when used for the prophylaxis and treatment of CYN-related intoxication in fish., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cylindrospermopsin (CYN) is a potent cyanobacterial cytotoxin produced by certain freshwater cyanobacteria. Structurally, it is an alkaloid with a tricyclic guanidine moiety combined with hydroxymethyluracil. It has proved to be a potent inhibitor of protein synthesis, and to deplete hepatic glutathione. Recently, some studies have shown that CYN produces changes in some oxidative stress biomarkers in fish acutely exposed to pure CYN by oral and intraperitoneal (i.p.) routes. In the present study tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels, 10 or 100 μg CYN L(-1), during two different exposure times: 7 and 14 d. Fish were sacrificed and liver and kidney were extracted. The oxidative status of fish was evaluated by analyzing in both organs the following biomarkers: lipid peroxidation (LPO), protein oxidation, DNA oxidation, reduced-oxidized glutathione ratio (GSH/GSSG), and changes in the activity of Glutathione-S-transferase (GST), Glutathione Peroxidase (GPx), Superoxide dismutase (SOD), Catalase (CAT), and γ-Glutamyl-cysteine synthetase (GCS). In general, major changes were observed in tilapia treated with 100 μg CYN L(-1) after 14 d of exposure. However, some endpoints were altered at the lowest concentration assayed only after 7d of exposure, such as DNA oxidation and γ-GCS in kidney, and CAT and GSH/GSSG decrease in the liver and kidney. The kidney was the most affected organ. These findings confirm that the oxidative stress play a role in the pathogenicity induced by CYN in this fish species, and the results obtained could be useful for future ecotoxicological risks assessment studies, for the protection of fish and aquatic ecosystems. To our knowledge this is the first study dealing with the oxidative stress changes induced by cyanobacterial cells containing CYN and its derivative deoxy-CYN on fish exposed sub-chronically under laboratory conditions., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A new method for determining dissolved cylindrospermopsin (CYN) in waters using solid-phase extraction (SPE) with graphitized carbon cartridges and quantification by liquid chromatography coupled with tandem mass spectrometry is described and discussed. The method has been suitably validated: the linear range covered is from 0.900 to 125 µg CYN/L. Limits of detection and quantification were 0.5 and 0.9 µg CYN/L, respectively, and allow CYN determination at concentrations below the guideline proposed of 1 µg CYN/L in natural waters. The method exhibits mean recoveries from 83 to 95%, and intermediate precision (relative standard deviation (%)) values from 5 to 12%, ensuring adequacy against the Association of Official Analytical Chemists guidelines. The method is robust against the following three influential factors considered in the cleanup stage: the batch of the graphitized carbon cartridges, the flow rate of the water sample through the cartridge, and the final redissolved water volume after SPE treatment. The method has been successfully applied to detection and quantification of CYN in water samples from aquaria of a toxicological in vivo laboratory experiment., (Copyright © 2012 SETAC.)

Cylindrospermopsin (CYN) is a toxin produced by various cyanobacteria species. Fish can be exposed to this cyanotoxin in their natural environments and in aquaculture ponds, and toxic effects can be derived. The present study investigated the effects of dietary N-acetylcysteine (NAC) on the oxidative stress induced by pure CYN and CYN from lyophilized cells of Aphanizomenon ovalisporum in tilapia (Oreochromis niloticus). Fish were pretreated with 0, 22, and 45 mg NAC/fish/d for a week, and on day seven, they received a single dose of 200 µg/kg CYN and were killed after 24 h. Oxidative biomarkers evaluated included lipid peroxidation, protein oxidation, glutathione (GSH)/oxidized glutathione (GSSG) ratio, activity of the enzyme γ-glutamylcysteine synthetase, and activity and gene expression of glutathione-S-transferase and glutathione peroxidase. Results showed that CYN induced oxidative stress as evidenced by the increase of lipid peroxidation and protein oxidation, the decrease in GSH/GSSG, and the alteration of the enzymatic activities assayed. Moreover, exposure to cyanobacterial cells containing CYN induced higher toxic effects in comparison to pure CYN. N-acetylcysteine supplementation was effective at reducing the toxicity induced by CYN, particularly at the highest dose employed, with a recovery of some of the biomarkers assayed to basal levels. Therefore, NAC can be considered a useful chemoprotectant that reduces hepatic and renal oxidative stress in the prophylaxis and treatment of CYN-related intoxication in fish., (Copyright © 2012 SETAC.)