The potential endocrine-disrupting properties of cyanotoxins, such as microcystin-LR (MC-LR) and cylindrospermopsin (CYN), are of concern due to their increasing occurrence, insufficient body of research, and potential impact on human health. Thus, an in silico procedure to assess the potential ability of MC-LR and CYN to form stable complexes with the estrogen receptor (ER) and the androgen receptor (AR) was performed for the first time. For that purpose, the molecular docking of both toxins was carried out. Ligand structures were generated in ChemDraw and subsequently docked against four distinct crystal structures of the ER and additional crystal structures of the AR. AutoDock was employed for docking to predict the potential agonist or antagonist activity of the toxins. The molecular docking of MC-LR and CYN with ER conformations revealed potential binding interactions. MC-LR exhibited a stronger affinity for the agonist conformations (1ERE: -7.1 kcal/mol; 3ERD: -8.2 kcal/mol) compared to the antagonist conformations (1ERR: -6.4 kcal/mol; 3ERT: -7.8 kcal/mol). Specific interactions in the agonist conformations involved arginine nitrogen with ASP-321 and GLU-323. Similarly, CYN displayed a preference for agonist conformations (1ERE: -6.7 kcal/mol; 3ERD: -7.0 kcal/mol) over antagonist conformations (1ERR: -6.6 kcal/mol; 3ERT: -6.1 kcal/mol). Key interactions in 1ERE involved the CYN guanidine group with SER-305, ALA-307, and ASP-369. In the case of AR, MC-LR showed a binding energy of -7.2 kcal/mol, with the leucine nitrogen interacting with PRO-682. CYN exhibited a higher affinity (-8.2 kcal/mol), with interactions involving the guanidine group with GLN-798. Taking this into account, computational docking analyses indicated possible binding interactions between these toxins and both receptors.

Acknowledgement: The authors would like to thank the MICIU/AEI/10.13039/501100011033 project; project number PID2019-104890RB-I00. A.C.-R. acknowledges the Spanish MICINN for the predoctoral grant awarded (PRE-2020-094412).

Introduction: Survivin, a protein regulating mitosis, is overexpressed in all cancers. Unlike its expression during G2 and mitosis in healthy cells, survivin in cancer cells is active throughout the interphase, localizing in the nucleus, cytosol, and mitochondria. Mitochondrial survivin has been shown to enhance phosphatidylserine (PS) decarboxylase activity, increasing the concentration of non-bilayer phosphatidylethanolamine (PE), a feature phospholipid in the inner mitochondrial membrane (IMM) that facilitates oxidative phosphorylation by promoting polymorphic transitions and proton absorption to facilitate mitochondrial ATP synthesis. By increasing PE through decarboxylation of PS, survivin may remodel IMM architecture, stimulating cancer development. To investigate the interplay of PE and PS in polymorphic transitions in IMM, this study uses cobra venom cytotoxin II (CTII), which, at low concentrations, promotes lipid polymorphism. By examining lipid polymorphism, membrane permeability and proton absorptivity, we aim to shed light on PE and PS roles in IMM functionality in cancer.

Methods: Phospholipid liposomes made of either PE or PS were prepared by ultrasonic radiation of phospholipid aqueous dispersions. The effects of CTII interaction with liposomes on membrane polymorphism and permeability were assessed using the ¹H-NMR spectroscopy and the [Cu(H₂O)₂(NH₃)₄]²⁺ complex ion spectrophotometry. Proton absorption by membranes in the absence and presence of CTII was quantified by measuring pH differences in an aqueous buffer with and without liposomes.

Results: CTII induced the formation of non-bilayer structures in both PE and PS membranes. In PS membranes, non-bilayer structures increased membrane permeability, whereas the inverted lipid micelles induced by CTII in PE membranes did not affect membrane permeability. Furthermore, CTII-treated PE membranes demonstrated superior proton absorption compared to CTII-treated PS membranes.

Conclusions: Superior proton absorption and the inverted micelles formation triggered by CTII in PE membranes—without compromising membrane permeability—may represent key features of the IMM that enhance ATP production, thereby supporting the accelerated proliferation of cancer cells.

Cylindrospermopsin (CYN) and microcystin-LR (MC-LR) are cyanotoxins of significant concern due to their widespread occurrence and toxic potential. Among exposure routes, ingestion through contaminated water and food is particularly relevant. Although CYN and MC-LR are primarily classified as cytotoxin and hepatotoxin, respectively, both cyanotoxins have shown potential neurotoxic effects. In addition, a recent study has reported CYN metabolites in the brain of orally exposed rats. However, to our knowledge, the combined impact of CYN and MC-LR on the brain remains unexplored. This study aimed to identify CYN, MC-LR, and their potential metabolites in the brain of Wistar rats after oral exposure to dose combinations of pure CYN and MC-LR (7.5+75, 23.7+237, and 75+750 µg/kg body weight) by ultra-high performance liquid chromatography coupled to a tandem mass spectrometry system (UHPLC-MS/MS). Furthermore, several oxidative stress biomarkers were assessed, including lipid peroxidation (LPO) levels and superoxide dismutase (SOD) and catalase (CAT) activities. Although CYN and MC-LR were not detected in brain tissue, up to nine potential CYN metabolites and eight MC-LR metabolites produced by different metabolic pathways were identified. Moreover, sex-dependent differences were observed in oxidative stress biomarkers. In males, exposure to the intermediate dose (23.7 + 237 µg/kg) significantly increased LPO levels (1.3-fold) and SOD (1.3-fold) and CAT (1.6-fold) activities compared to controls, while in females only changes in LPO levels were observed, where a significant increase was observed at all doses tested. These findings suggest that oral exposure to CYN+MC-LR mixtures may cause neurotoxic effects in rats and highlight the importance of considering sex as a biological variable in cyanotoxins toxicological assessment.

Acknowledgement - Project PID2023-147444OB-I00 funded by MICIU/AEI /10.13039/501100011033 and FEDER, UE. Plata-Calzado thanks the Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía for awarding the predoctoral contract (PREDOC_00447) in the Predoctores 2021 grant.

Introduction:

Voltage-gated potassium channels Kv1.1 and Kv1.2 are key players of neuronal excitability. The Kv1.3 channel mediates the immune response of T lymphocytes. Many peptide toxins are blockers of Kv1 channels and help to study channel functioning under normal and pathological conditions. New pharmacology-relevant techniques are needed to search for advanced blockers and to characterize their properties for potential drug development.

Methods:

Plasmids encoding Kv1 subunits fused with the fluorescent protein mKate2 and those encoding hongotoxin 1 (HgTx1) and agitoxin 2 (AgTx2) fused with GFP were obtained. Recombinant fluorescently labeled and unlabeled peptide blockers including Ce-peptides from Centruroides elegans scorpion venom were produced. Interactions between blockers and Kv1 channels expressed in mammalian cells were investigated with confocal microscopy and the whole-cell patch clamp technique.

Results:

The concept of creating analytical cellular systems (ACSs) for the study of Kv1.1, Kv1.2, and Kv1.3 channels, their fluorescent ligands, and peptide blockers using confocal microscopy has been successfully implemented. Point mutations that enhanced the membrane expression of fully functional channels were introduced.

High-affinity fluorescent ligands (synthetic Atto488-HgTx1 and recombinant HgTx1-GFP and AgTx2-GFP) were developed. Using competitive binding of labeled and unlabeled ligands, the apparent dissociation constants of complexes between unlabeled blockers and Kv1 channels were determined. Peptides Ce1 and Ce4 were found to be the most selective and potent peptide blockers of the Kv1.2 channel compared to channels Kv1.1 and Kv1.3.

Conclusions:

The proposed concept of ACSs is universal and can be applied to various potassium channels. Peptides Ce1 and Ce4 can be useful in the study of Kv1.2-mediated currents in neurons, and they can also be used to construct fluorescent ligands selective for Kv1.2.

Acknowledgements: The work was supported by the Russian Science Foundation (project 22-14-00406).

Paralytic shellfish toxins can be quantified in bivalve molluscs by the AOAC-2005 method. This method is quite sensitive for some toxins, while other toxins suffer from strong matrix effects or fluorescence partitioning by multiple oxidation products, reducing their sensitivity. Metals were assessed as candidate catalysts to improve the oxidation reactions instead of the oyster matrix modifier preconized in this method. The N1-H sub-group of toxins exhibited increased fluorescence upon doping with iron sulphate. The increase was inversely proportional to the fluorescence reduction caused by matrix suppression. Iron acted as a catalyst, lowering the activation energy of the reaction, which otherwise required heating to achieve a similar boost in fluorescence yield.
The fluorescence of the N1-hydroxyl sub-group of toxins GTX1+4 and GTX6 increased with zinc but decreased with iron. When doped with a metal solution that had passed in fraction 2 of the carboxylic acid partitioning, both metals reduced the fluorescence, while the eluent of fraction 2 (NaCL 0.5 M) enhanced it. As metals are retained by COOH cartridges, sulfonic acid leachables might contribute to this reduction. The reduction observed for GTX1+4 was primarily due to the decrease in the proportion of the secondary peak over the primary oxidation peak, which can be reversed by doping with an oyster matrix. Nickel chloride was able to replace the oyster modifier effectively in all five bivalve matrices tested, while zinc chloride was not similarly effective. For dcNEO spiked in several bivalve matrices, adding an oyster modifier derived from Magallana gigas caused a 16-19% reduction in fluorescence. Doping with several metals (Ni, Zn, alkaline, and alkaline earth metals) could not achieve the same fluorescence as doping with 0.1 mM acetic acid. In this case, the matrix modifier preconized in the method was not adequate.

The assessment of human dietary exposure to mycotoxins is a crucial component of food safety strategies. This study aimed to evaluate the exposure of the Croatian population to aflatoxin B₁ (AFB₁) and its precursor sterigmatocystin (STC)—an emerging mycotoxin—through the consumption of traditional meat products (TMPs). These products are not thermally processed, and their surfaces are colonized by moulds during the ripening process, which can produce mycotoxins. Additional contamination sources include spices used in their production or a carry-over effect in meat from animals fed with contaminated feed. A total of 280 TMP samples available on the Croatian market were analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), with detection limits of 0.03 µg/kg for AFB₁ and 0.02 µg/kg for STC. AFB₁, a known human carcinogen, was not detected in any sample, indicating that exposure to AFB₁ through TMPs is negligible. STC, classified as a possible human carcinogen, was detected in eight sausage samples and four samples of dry-cured TMPs. Dietary exposure assessment was conducted using national consumption data and average STC concentrations, applying lower bound (LB), middle bound (MB), and upper bound (UB) scenarios for values below the detection limit. The maximum estimated daily exposure was 0.292 ng/kg body weight (bw), which is well below the European Food Safety Authority's (EFSA) threshold of toxicological concern (TTC) for potentially genotoxic compounds (2.5 ng/kg bw/day). Although the prevalence of mycotoxins in TMPs was low, continuous monitoring is recommended due to the variability of contamination sources and influencing factors, in order to track long-term exposure trends in the population.