Identification of mycotoxins in yogurt samples using an optimized QuEChERS extraction and UHPLC-MS/MS detection.

Yogurt, a milk-derived product, is susceptible to mycotoxin contamination. While various methods have been developed for the analysis of dairy products, only a few have been specifically validated for yogurt. In addition, these methods are primarily focus on detecting aflatoxins and zearalenone. This study aimed to conduct a preliminary investigation into the presence of regulated, emerging, and modified mycotoxins in natural and oat yogurts available in the Spanish market. For this, a QuEChERS-based extraction method was optimized and then validated to detect and quantify 32 mycotoxins using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The method was in-house validated for the analysis of natural and oat yogurt in terms of linearity, matrix effect, sensitivity, accuracy, and precision. Satisfactory performance characteristics were achieved; for most of the analytes, LOQs were lower than 2 ng/g, and recoveries ranged from 60 to 110% with a precision, expressed as the relative standard deviation of the recovery, lower than 15%. Subsequently, the validated method was applied to analyze commercial yogurt samples, revealing a notable incidence of beauvericin and enniatins, with some analogues found in up to 100% of the samples. Alternariol methyl ether was also frequently found, appearing in 50% of the samples. Additionally, the study identified regulated toxins such as fumonisins, ochratoxin A , and HT-2 toxin. These results provide new incidence data in yogurt, raising concerns about potential health risks for consumers.

Magnetic nanostructured agents for the mitigation of mycotoxins and cyanotoxins in the food chain.

Natural toxins, such as mycotoxins and cyanotoxins, can contaminate food and feed, leading to toxicity in humans and animals. This study focused on using nine magnetic nanostructured agents to remove the main types of toxins. Initially, the efficacy of these materials was evaluated in water solutions, revealing that composites with sizes below 3 mm, containing magnetite, activated carbon, esterified pectin, and sodium alginate, removed up to 90% of mycotoxins and cyanotoxins with an adsorption of 873 ng/g. The application of the nanostructures was then assessed in beer, milk, Distillers Dried Grains with Solubles and water contaminated with cyanobacteria. The presence of matrix slightly decreases the adsorption capacity for some toxins. The maximum toxin removal capacity was calculated with cyanotoxins, composites achieved a removal of up to 0.12 mg/g, while nanocomposites (15 µm) reached 36.6 mg/g. Therefore, these findings point out the potential for using nanotechnology in addressing natural toxins contamination.

Nanostructured Magnetic Particles for Removing Cyanotoxins: Assessing Effectiveness and Toxicity In Vitro.

The rise in cyanobacterial blooms due to eutrophication and climate change has increased cyanotoxin presence in water. Most current water treatment plants do not effectively remove these toxins, posing a potential risk to public health. This study introduces a water treatment approach using nanostructured beads containing magnetic nanoparticles (MNPs) for easy removal from liquid suspension, coated with different adsorbent materials to eliminate cyanotoxins. Thirteen particle types were produced using activated carbon, CMK-3 mesoporous carbon, graphene, chitosan, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidised cellulose nanofibers (TOCNF), esterified pectin, and calcined lignin as an adsorbent component. The particles' effectiveness for detoxification of microcystin-LR (MC-LR), cylindrospermopsin (CYN), and anatoxin-A (ATX-A) was assessed in an aqueous solution. Two particle compositions presented the best adsorption characteristics for the most common cyanotoxins. In the conditions tested, mesoporous carbon nanostructured particles, P1-CMK3, provide good removal of MC-LR and Merck-activated carbon nanostructured particles, P9-MAC, can remove ATX-A and CYN with high and fair efficacy, respectively. Additionally, in vitro toxicity of water treated with each particle type was evaluated in cultured cell lines, revealing no alteration of viability in human renal, neuronal, hepatic, and intestinal cells. Although further research is needed to fully characterise this new water treatment approach, it appears to be a safe, practical, and effective method for eliminating cyanotoxins from water.

Enniatins A1 and B1 Modulate Calcium Flux through Alternative Pathways beyond Mitochondria.

Enniatins (ENNs) A1 and B1, previously considered ionophores, are emerging mycotoxins with effects on Ca2+ homeostasis. However, their exact mechanism of action remains unclear. This study investigated how these toxins affect Ca2+ flux in SH-SY5Y cells. ENN A1 induced Ca2+ influx through store-operated channels (SOC). The mitochondrial uncoupler FCCP reduced this influx, suggesting that the mitochondrial status influences the toxin effect. Conversely, ENN B1 did not affect SOC but acted on another Ca2+ channel, as shown when nickel, which directly blocks the Ca2+ channel pore, is added. Mitochondrial function also influenced the effects of ENN B1, as treatment with FCCP reduced toxin-induced Ca2+ depletion and uptake. In addition, both ENNs altered mitochondrial function by producing the opening of the mitochondrial permeability transition pore. This study describes for the first time that ENN A1 and B1 are not Ca2+ ionophores and suggests a different mechanism of action for each toxin.

Antioxidant micropeptins from a Microcoleus autumnalis-dominated benthic cyanobacterial mat from Western Ireland.

Exploring the chemical diversity present in cyanobacterial mats increasingly frequent in fresh and marine waters is imperative for both evaluating risks associated with these diverse biofilms and their potential for biodiscovery. During a project aimed at the study of the (eco)toxicity of benthic cyanobacteria blooming in some lakes of the West of Ireland, three previously undescribed ahp-cyclodepsipeptides micropeptin LOF941 (1), micropeptin LOF925 (2) and micropeptin LOF953 (3) were isolated from the Microcoleus autumnalis-dominated benthic cyanobacterial biofilm collected from the shore of Lough O'Flynn, Co. Roscommon, Ireland. Their structures remain consistent in their amino acid sequence with the presence of an unusual methionine, and differ by their exocyclic side chains. The planar structures of the previously undescribed micropeptins were elucidated by 1D and 2D NMR and HRESIMS analyses, and their 3D configurations assessed by ROESY NMR and Marfey's analyses. The three isolated compounds showed no cytotoxic effects and all three compounds were shown to exhibit antioxidant properties, with 1 showing the highest bioactivity. Additionally, several micropeptin analogues are proposed from the methanolic fraction of the biofilm extract by UHPLC-HRESIMS/MS analysis and molecular networking. Notably, the known cyanotoxins anatoxin-a and dihydroanatoxin-a were annotated in the molecular network therefore raising issues about the toxicity of this cyanobacterial mat.

Psammaplin A and Its Analogs Attenuate Oxidative Stress in Neuronal Cells through Peroxisome Proliferator-Activated Receptor γ Activation.

Psammaplins are sulfur containing bromotyrosine alkaloids that have shown antitumor activity through the inhibition of class I histone deacetylases (HDACs). The cytotoxic properties of psammaplin A (1), the parent compound, are related to peroxisome proliferator-activated receptor γ (PPARγ) activation, but the mechanism of action of its analogs psammaplin K (2) and bisaprasin (3) has not been elucidated. In this study, the protective effects against oxidative stress of compounds 1-3, isolated from the sponge Aplysinella rhax, were evaluated in SH-SY5Y cells. The compounds improved cell survival, recovered glutathione (GSH) content, and reduced reactive oxygen species (ROS) release at nanomolar concentrations. Psammaplins restored mitochondrial membrane potential by blocking mitochondrial permeability transition pore opening and reducing cyclophilin D expression. This effect was mediated by the capacity of 1-3 to activate PPARγ, enhancing gene expression of the antioxidant enzymes catalase, nuclear factor E2-related factor 2 (Nrf2), and glutathione peroxidase. Finally, HDAC3 activity was reduced by 1-3 under oxidative stress conditions. This work is the first description of the neuroprotective activity of 1 at low concentrations and the mechanism of action of 2 and 3. Moreover, it links for the first time the previously described effects of 1 in HDAC3 and PPARγ signaling, opening a new research field for the therapeutic potential of this compound family.

First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot.

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.

Analytical and functional profiles of paralytic shellfish toxins extracted from Semele proficua and Senilia senilis from Angola.

Paralytic shellfish poisoning is a foodborne illness that typically derive from the consumption of shellfish contaminated with saxitoxin-group of toxins produced by dinoflagellates of the genus Gymnodinium, Alexandrium and Pyrodinium. N-sulfocarbamoyl, carbamate and dicarbamoyl are the most abundant. In 2007 and 2008 some episodes of PSP occurred in Angola where there is not monitoring program for shellfish contamination with marine biotoxins. Therefore, ten samples extracted from Semele proficua from Luanda Bay and Senilia senilis from Mussulo Bay, were analyzed by HPLC finding saxitoxin, decarbamoylsaxitoxin and other three compounds that have an unusual profile different to the known hydrophilic PSP toxins were found in different amounts and combinations. These new compounds were not autofluorescent, and they presented much stronger response after peroxide oxidation than after periodate oxidation. The compounds appear as peaks eluted at 2.5 and 5.6 min after periodate oxidation and 8.2 min after peroxide oxidation. Electrophysiological studies revealed that none of the three unknown compounds had effect at cellular level by decreasing the maximum peak inward sodium currents by blocking voltage-gated sodium channels. Thus, not contributing to PSP intoxication. The presence in all samples of saxitoxin-group compounds poses a risk to human health and remarks the need to further explore the presence of new compounds that contaminate seafood, investigating their activity and developing monitoring programs.

Synergistic Effect of Brevetoxin BTX-3 and Ciguatoxin CTX3C in Human Voltage-Gated Nav1.6 Sodium Channels.

Emerging marine biotoxins such as ciguatoxins and brevetoxins have been widely and independently studied as food pollutants. Their maximum levels in food components were set without considering their possible synergistic effects as consequence of their coexistence in seafood and their action at the same cellular target. The absolute lack of data and regulations of the possible combined effects that both marine biotoxins may have raised the need to analyze their direct in vitro effects using electrophysiology techniques. The results presented in this study indicate that ciguatoxins and brevetoxins had a synergistic effect on human Nav1.6 voltage-gated sodium channels by hyperpolarizing their activation and inactivation states. The results presented here indicate that brevetoxin 3 (BTX-3) acts as partial agonist of human sodium channels, while ciguatoxin 3C (CTX3C) was a full agonist, explaining the differences in the effect of each toxin in the channel. Therefore, this work sets the cellular basis to further apply this type of studies to other food toxicants that may act synergistically and thus implement the corresponding regulatory limits considering their coexistence and the risks to human and animal health derived from it.

Acute toxicology report of the emerging marine biotoxin Brevetoxin 3 in mice: Food safety implications.

Brevetoxins (PbTxs) are emerging marine toxins that can lead to Neurotoxic Shellfish Poisoning in humans by the ingestion of contaminated seafood. Recent reports on brevetoxin detection in shellfish in regions where it has not been described before, arise the need of updated guidelines to ensure seafood consumers safety. Our aim was to provide toxicological data for brevetoxin 3 (PbTx3) by assessing oral toxicity in mice and comparing it with intraperitoneal administration. We followed an Up-and-Down procedure administering PbTx3 to mice and registering clinical signs, neuromuscular function, histopathology, and blood changes. Neuromuscular dysfunction like seizures and ataxia, as well as loss of limb strength were observed at 6 h. Performance and clinical signs largely improved at 24 h, time at which no blood biochemical or histological alterations were detected independently of the administration route. However, PbTx3 oral administration results in lower toxicity than intraperitoneal administration. Mortality was only observed at 4000 µg/kg bw PbTx3 administered via oral, but we still found toxicity clinical signs at low toxin doses. We could stablish an oral Lowest-Observable-Adverse-Effect-Level for PbTx3 of 100 µg/kg bw and an oral No-Observable-Adverse-Effect-Level of 10 µg/kg bw in mice. The data here reported should be considered in the evaluation of risks of PbTxs for human health.

Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection.

The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels.

Cyclophilin B serum levels present variations across the menstrual cycle.

Cyclophilins are a family of chaperones involved in inflammation and cell death. Cyclophilin B is released by inflammatory cells and acts through the receptor CD147, affecting matrix metalloproteases release, whilst cyclophilin D participates in hypoxia-induced apoptosis. Previous studies related hormones like estradiol or prolactin to these proteins, however, their blood concentrations across the menstrual cycle have not been determined. In this work, eleven healthy women (BMI: 21.8 kg/m2) were monitored during a single menstrual cycle, making blood extractions at follicular, periovulatory and mid-luteal phases. Hormone and cyclophilin levels were determined in each phase. Statistical differences were determined by repeated measures ANOVA and estimated marginal means tests, or by Friedman and Dunn-Bonferroni tests for parametric and non-parametric variables, respectively. Bivariate correlations were evaluated with the Spearman coefficient. Cyclophilin B concentrations presented significant differences during the menstrual cycle (p = 0.012). The highest levels of this protein were found at follicular extraction, followed by a decrease at periovulatory phase and a slight increase at mid-luteal phase. Cyclophilin D showed the same profile, although statistical significance was not reached. This immunophilin exhibited a positive correlation with luteinizing hormone at periovulatory phase (r = 0.743, p = 0.009) and with follicle stimulating hormone at mid-luteal phase (r = 0.633, p = 0.036). This is the first study describing the changes in cyclophilin B concentrations across the menstrual cycle, as well as the association of luteinizing and follicle stimulating hormones with cyclophilin D. These results suggest a role of these proteins in the cyclic inflammatory events that affect female reproductive system that should be explored.

Proteomic and toxicological analysis of the response of dinoflagellate Alexandrium catenella to changes in NaNO3 concentration.

Dinoflagellates of the genus Alexandrium cause Harmful Algal Blooms (HABs) in coastal waters worldwide, damaging marine environments, aquaculture, and human health. They synthesize potent neurotoxic alkaloids known as PSTs (i.e., Paralytic Shellfish Toxins), the etiological agents of PSP (i.e., Paralytic Shellfish Poisoning). In recent decades, the eutrophication of coastal waters with inorganic nitrogen (e.g., nitrate, nitrite, and ammonia) has increased the frequency and scale of HABs. PSTs concentrations within Alexandrium cells can increase by up to 76% after a nitrogen enrichment event; however, the mechanisms that underlie their biosynthesis in dinoflagellates remains unclear. This study combines mass spectrometry, bioinformatics, and toxicology and investigates the expression profiles of PSTs in Alexandrium catenella grown in 0.4, 0.9 and 1.3 mM NaNO3. Pathway analysis of protein expression revealed that tRNA amino acylation, glycolysis, TCA cycle and pigment biosynthesis were upregulated in 0.4 mM and downregulated in 1.3 mM NaNO3 compared to those grown in 0.9 mM NaNO3. Conversely, ATP synthesis, photosynthesis and arginine biosynthesis were downregulated in 0.4 mM and upregulated in 1.3 mM NaNO3. Additionally, the expression of proteins involved in PST biosynthesis (sxtA, sxtG, sxtV, sxtW and sxtZ) and overall PST production like STX, NEO, C1, C2, GTX1-6 and dcGTX2 was higher at lower nitrate concentrations. Therefore, increased nitrogen concentrations increase protein synthesis, photosynthesis, and energy metabolism and decrease enzyme expression in PST biosynthesis and production. This research provides new clues about how the changes in the nitrate concentration can modulate different metabolic pathways and the expression of PST biosynthesis in toxigenic dinoflagellates.

Cyclophilins modify their profile depending on the organ or tissue in a murine inflammatory model.

Inflammation is the leading subjacent cause of many chronic diseases. Despite several studies in the last decades, the molecular mechanism involving its pathophysiology is not fully known. Recently, the implication of cyclophilins in inflammatory-based diseases has been demonstrated. However, the main role of cyclophilins in these processes remains elusive. Hence, a mouse model of systemic inflammation was used to better understand the relationship between cyclophilins and their tissue distribution. To induce inflammation, mice were fed with high-fat diet for 10 weeks. In these conditions, serum levels of interleukins 2 and 6, tumour necrosis factor-α, interferon-ϒ, and the monocyte chemoattractant protein 1 were elevated, evidencing a systemic inflammatory state. Then, in this inflammatory model, cyclophilins and CD147 profiles in the aorta, liver, and kidney were studied. The results demonstrate that, upon inflammatory conditions, cyclophilins A and C expression levels were increased in the aorta. Cyclophilins A and D were augmented in the liver, meanwhile, cyclophilins B and C were diminished. In the kidney, cyclophilins B and C levels were elevated. Furthermore, CD147 receptor was also increased in the aorta, liver, and kidney. In addition, when cyclophilin A was modulated, serum levels of inflammatory mediators were decreased, indicating a reduction in systemic inflammation. Besides, the expression levels of cyclophilin A and CD147 were also reduced in the aorta and liver, when cyclophilin A was modulated. Therefore, these results suggest that each cyclophilin has a different profile depending on the tissue, under inflammatory conditions.

Association of cyclophilins and cardiovascular risk factors in coronary artery disease.

Cyclophilins are chaperone proteins that play important roles in signal transduction. Among them, cyclophilins A, B, C, and D were widely associated with inflammation and cardiovascular diseases. Cyclophilins A and C have been proposed as coronary artery disease biomarkers. However, less is known about their relationship with cardiovascular risk factors. Therefore, this study aimed to determine the association between cyclophilin A, B, C, and D and cardiovascular risk factors in coronary artery disease. Serum levels of cyclophilins were measured in 167 subjects (subdivided according to cardiovascular risk factors presence). This study reveals that cyclophilin A and C are elevated in patients regardless of the risk factors presence. Moreover, cyclophilin B is elevated in male patients with hypertension, type 2 diabetes, or high glucose levels. In addition, cyclophilins A, B, and C were significantly correlated with cardiovascular risk factors, but only cyclophilin B was associated with type 2 diabetes. The multivariate analysis strengthens the predictive value for coronary artery disease presence of cyclophilin A (>8.2 ng/mL) and cyclophilin C (>17.5 pg/mL) along with the cardiovascular risk factors tobacco, hypertension, dyslipidemia, and high glucose and cholesterol levels. Moreover, the risk of coronary artery disease is increased in presence of cyclophilin B levels above 63.26 pg/mL and with hypertension or dyslipidemia in male patients. Consequently, cyclophilins A and C serum levels are reinforced as useful coronary artery disease biomarkers, meanwhile, cyclophilin B is a valuable biomarker in the male population when patients are also suffering from hypertension or dyslipidemia.

Detection of mycotoxins in cheese using an optimized analytical method based on a QuEChERS extraction and UHPLC-MS/MS quantification.

Mycotoxins can produce toxic effects on humans; hence, it is of high importance to determine their presence in food products. This work presents a reliable method for the quantification of 32 mycotoxins in cheese. The analysis procedure was optimized based on a QuEChERS extraction process and the ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) detection. The analysis method was validated for four cheese varieties (emmental, blue, brie and camembert) in terms of linearity, sensitivity, matrix effect, accuracy and precision. Satisfactory precision and accuracy values were achieved, with recoveries above 70% for most mycotoxins. The developed method was applied to the analysis of 38 commercial cheese samples. A high occurrence of beauvericin and enniatins were found, ranging from 31% for enniatin A to 100% for enniatin B. The ochratoxin A was detected in three samples at concentrations that may pose a risk to human health.

Synergistic effect of environmental food pollutants: Pesticides and marine biotoxins.

Emerging marine biotoxins such as ciguatoxins and pyrethroid compounds, widely used in agriculture, are independently treated as environmental toxicants. Their maximum residue levels in food components are set without considering their possible synergistic effects as consequence of their interaction with the same cellular target. There is an absolute lack of data on the possible combined cellular effects that biological and chemical pollutants, may have. Nowadays, an increasing presence of ciguatoxins in European Coasts has been reported and these toxins can affect human health. Similarly, the increasing use of phytosanitary products for control of food plagues has raised exponentially during the last decades due to climate change. The lack of data and regulation evaluating the combined effect of environmental pollutants with the same molecular target led us to analyse their in vitro effects. In this work, the effects of ciguatoxins and pyrethroids in human sodium channels were investigated. The results presented in this study indicate that both types of compounds have a profound synergistic effect in voltage-dependent sodium channels. These food pollutants act by decreasing the maximum peak inward sodium currents and hyperpolarizing the sodium channels activation, effects that are boosted by the simultaneous presence of both compounds. A fact that highlights the need to re-evaluate their limits in feedstock as well as their potential in vivo toxicity considering that they act on the same cellular target. Moreover, this work sets the cellular basis to further apply this type of studies to other water and food pollutants that may act synergistically and thus implement the corresponding regulatory limits taking into account its presence in a healthy diet.

Extracellular cyclophilins A and C induce dysfunction of pancreatic microendothelial cells.

Extracellular cyclophilins (eCyps) A and B are chemotactic mediators in several illnesses in which inflammation plays an important role such as diabetes and cardiovascular diseases. Recently, eCypC has been reported as a potential biomarker for coronary artery disease but its effect in endothelium has not been determined. Moreover, there is a lack of studies with all these proteins in the same model, which makes difficult a direct comparison of their effects. In this work, MS1 pancreatic microendothelial cells were treated with eCyps A, B and C and their impact on endothelial function was analysed. eCyps A and C stimulated the release of IL-6 and MCP-1 and increased the expression of the receptor CD147, but eCypB did not affect these pro-inflammatory markers. Moreover, eCypC activated the translocation of NFkB-p65 to the nucleus. All these effects were reversed by pre-treatment with cyclosporine A. eCyps also produced endothelial dysfunction, as evidenced by the decrease in eNOS activation. Finally, the crosstalk among eCyps addition and their protein and gene expression was evaluated. eCypA generated a depletion in its protein and gene levels, whilst eCyps B and C upregulated their own protein expression. Moreover, each eCyp altered the intracellular expression of other Cyps, including cyclophilin D. This work is the first report of eCyps influence on iCyps expression, as well as the first description of eCypC as an activator of CD147 receptor and a mediator of endothelial dysfunction, which points to a potential role of this protein in vascular complications associated to diabetes.

Intestinal secretory mechanisms in Okadaic acid induced diarrhoea.

Okadaic acid (OA) is an important marine lipophilic phycotoxin responsible for diarrhetic shellfish poisoning (DSP). This toxin inhibits protein phosphatases (PPs) like PP2A and PP1, though, this action does not explain OA-induced toxicity and symptoms. Intestinal epithelia comprise the defence barrier against external agents where transport of fluid and electrolytes from and to the lumen is a tightly regulated process. In some intoxications this balance becomes dysregulated appearing diarrhoea. Therefore, we evaluated diarrhoea in orally OA-treated mice as well as in mice pre-treated with several doses of cyproheptadine (CPH) and then treated with OA at different times. We assessed stools electrolytes and ultrastructural alteration of the intestine, particularly evaluating tight and adherens junctions. We detected increased chloride and sodium faecal concentrations in the OA-exposed group, suggesting a secretory diarrhoea. Pre-treatment with CPH maintains chloride concentration in values similar to control mice. Intestinal cytomorphological alterations were observed for OA mice, whereas CPH pre-treatment attenuated OA-induced damage in proximal colon and jejunum at 2 h. Conversely, tight junctions' distance was only affected by OA in jejunum at the moment diarrhoea occurred. In this study we found cellular mechanisms by which OA induced diarrhoea revealing the complex toxicity of this compound.

Bromoditerpenes from the Red Seaweed Sphaerococcus coronopifolius as Potential Cytotoxic Agents and Proteasome Inhibitors and Related Mechanisms of Action.

Seaweeds are a great source of compounds with cytotoxic properties with the potential to be used as anticancer agents. This study evaluated the cytotoxic and proteasome inhibitory activities of 12R-hydroxy-bromosphaerol, 12S-hydroxy-bromosphaerol, and bromosphaerol isolated from Sphaerococcus coronopifolius. The cytotoxicity was evaluated on malignant cell lines (A549, CACO-2, HCT-15, MCF-7, NCI-H226, PC-3, SH-SY5Y, and SK-MEL-28) using the MTT and LDH assays. The ability of compounds to stimulate the production of hydrogen peroxide (H2O2) and to induce mitochondrial dysfunction, the externalization of phosphatidylserine, Caspase-9 activity, and changes in nuclear morphology was also studied on MCF-7 cells. The ability to induce DNA damage was also studied on L929 fibroblasts. The proteasome inhibitory activity was estimated through molecular docking studies. The compounds exhibited IC50 values between 15.35 and 53.34 µM. 12R-hydroxy-bromosphaerol and 12S-hydroxy-bromosphaerol increased the H2O2 levels on MCF-7 cells, and bromosphaerol induced DNA damage on fibroblasts. All compounds promoted a depolarization of mitochondrial membrane potential, Caspase-9 activity, and nuclear condensation and fragmentation. The compounds have been shown to interact with the chymotrypsin-like catalytic site through molecular docking studies; however, only 12S-hydroxy-bromosphaerol evidenced interaction with ALA20 and SER169, key residues of the proteasome catalytic mechanism. Further studies should be outlined to deeply characterize and understand the potential of those bromoditerpenes for anticancer therapeutics.