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.

Correction: González-Rellán et al. Anatomy of the Palmar Region of the Carpus of the Dog. Animals 2022, 12, 1573.

In the original publication [...].

Anatomy of the Palmar Region of the Carpus of the Dog.

The palmar region of the canine carpus is anatomically complex, and the information found in the literature about its anatomy is inconsistent. The aims of this prospective, descriptive, anatomic study were (1) the clarification and (2) the description of the precise anatomic composition of the palmar region of the canine carpus, with special reference to the canalis carpi. For this study, 92 cadaveric specimens were obtained from 46 dogs that had died for reasons unrelated to this study. Of these, 43 medium-to-large-breed dogs were randomly selected for the dissection of transverse slices of the carpus. Samples of the flexor retinaculum and flexor carpi radialis tendon and surrounding tissues were taken for complementary histology. For additional histology of the palmar structures in their anatomical position, three small breed dogs were randomly selected for obtaining transverse slices. The anatomic characteristics of the components of the palmar region of the canine carpus were qualitatively described, with special attention to the following structures: flexor retinaculum, flexor carpi radialis muscle, arteria and vena mediana, nervus medianus, interflexorius muscle, flexor digitorum profundus muscle, canalis carpi, and arteria and nervus ulnaris. The findings from this study provide reference information about the anatomy of the palmar region of the canine carpus.

Structural, morphometric and immunohistochemical study of the rabbit accessory olfactory bulb.

The accessory olfactory bulb (AOB) is the first neural integrative centre of the vomeronasal system (VNS), which is associated primarily with the detection of semiochemicals. Although the rabbit is used as a model for the study of chemocommunication, these studies are hampered by the lack of knowledge regarding the topography, lamination, and neurochemical properties of the rabbit AOB. To fill this gap, we have employed histological stainings: lectin labelling with Ulex europaeus (UEA-I), Bandeiraea simplicifolia (BSI-B4), and Lycopersicon esculentum (LEA) agglutinins, and a range of immunohistochemical markers. Anti-G proteins Gαi2/Gαo, not previously studied in the rabbit AOB, are expressed following an antero-posterior zonal pattern. This places Lagomorpha among the small groups of mammals that conserve a double-path vomeronasal reception. Antibodies against olfactory marker protein (OMP), growth-associated protein-43 (GAP-43), glutaminase (GLS), microtubule-associated protein-2 (MAP-2), glial fibrillary-acidic protein (GFAP), calbindin (CB), and calretinin (CR) characterise the strata and the principal components of the BOA, demonstrating several singular features of the rabbit AOB. This diversity is accentuated by the presence of a unique organisation: four neuronal clusters in the accessory bulbar white matter, two of them not previously characterised in any species (the γ and δ groups). Our morphometric study of the AOB has found significant differences between sexes in the numerical density of principal cells, with larger values in females, a pattern completely opposite to that found in rats. In summary, the rabbit possesses a highly developed AOB, with many specific features that highlight the significant role played by chemocommunication among this species.

Morphological and immunohistochemical study of the rabbit vomeronasal organ.

The characterization of the rabbit mammary pheromone, which is sensed by the main olfactory system, has made this species a unique model for the study of pheromonal communication in mammals. This discovery has brought attention to the global understanding of chemosensory communication in this species. Chemocommunication is mediated by two distinct organs located in the nasal cavity, the main olfactory epithelium and the vomeronasal organ (VNO). However, there is a lack of knowledge about the vomeronasal system in rabbits. To understand the role of this system, an exhaustive anatomical and histological study of the rabbit VNO was performed. The rabbit VNO was studied macroscopically by light microscopy, and by histochemical and immunohistochemical techniques. We employed specific histological staining techniques (periodic acid-Schiff, Alcian blue, Gallego's trichrome), confocal autofluorescence, histochemical labelling with the lectin Ulex europaeus agglutinin (UEA-I), and immunohistochemical studies of the expression of the Gαi2 and Gαo proteins and olfactory marker protein. The opening of the vomeronasal duct into the nasal cavity and its indirect communication with the oral cavity through a functional nasopalatine duct was demonstrated by classical dissection and microdissection. In a series of transverse histological sections, special attention was paid to the general distribution of the various soft-tissue components of this organ (duct, glands, connective tissue, blood vessels and nerves) and to the nature of the capsule of the organ. Among the main morphological features that distinguish the rabbit VNO, the presence of a double envelope, which is bony externally and cartilaginous internally, and highly developed venous sinuses stand out. This observation indicates the crucial role played in this species by the pumping mechanism that introduces chemical signals into the vomeronasal duct. The functional properties of the organ are also confirmed by the presence of a well-developed neuroepithelium and profuse glandular tissue that is positive for neutral mucopolysaccharides. The role of glycoconjugates was assessed by the identification of the α1-2 fucose glycan system in the neuroepithelium of the VNO employing UEA-I lectin. The pattern of labelling, which was concentrated around the commissures of the sensory epithelium and more diffuse in the central segments, is different from that found in most mammals studied. According to the expression of G-proteins, two pathways have been described in the VNOs of mammals: neuroreceptor cells expressing the Gαi2 protein (associated with vomeronasal receptor type 1); and cells expressing Gαo (associated with vomeronasal receptor type 2). The latter pathway is absent in most mammals studied. The expression of both G-protein families in the rabbit VNO places Lagomorpha together with rodents and insectivores in a small group of mammals belonging to the two-path model. These findings support the notion that the rabbit possesses a highly developed VNO, with many specific morphological features, which highlights the significance of chemocommunication in this species.

Characterization of the dinophysistoxin-2 acute oral toxicity in mice to define the Toxicity Equivalency Factor.

Ingestion of shellfish with dinophysistoxin-2 (DTX2) can lead to diarrheic shellfish poisoning (DSP). The official control method of DSP toxins in seafood is the liquid chromatography-mass spectrometry analysis (LC-MS). However in order to calculate the total toxicity of shellfish, the concentration of each compound must be multiplied by individual Toxicity Equivalency Factor (TEF). Considering that TEFs caused some controversy and the scarce information about DTX2 toxicity, the aim of this study was to characterize the oral toxicity of DTX2 in mice. A 4-Level Up and Down Procedure allowed the characterization of DTX2 effects and the estimation of DTX2 oral TEF based on determination of the lethal dose 50 (LD50). DTX2 passed the gastrointestinal barrier and was detected in urine and feces. Acute toxicity symptoms include diarrhea and motionless, however anatomopathology study and ultrastructural images restricted the toxin effects to the gastrointestinal tract. Nevertheless enterocytes microvilli and tight junctions were not altered, disconnecting DTX2 diarrheic effects from paracellular epithelial permeability. This is the first report of DTX2 oral LD50 (2262 µg/kg BW) indicating that its TEF is about 0.4. This result suggests reevaluation of the present TEFs for the DSP toxins to better determine the actual risk to seafood consumers.

Tetracyclic Truncated Analogue of the Marine Toxin Gambierol Modifies NMDA, Tau, and Amyloid ß Expression in Mice Brains: Implications in AD Pathology.

Gambierol and its two, tetra- and heptacyclic, analogues have been previously proved as promising molecules for the modulation of Alzheimer's disease (AD) hallmarks in primary cortical neurons of 3xTg-AD fetuses. In this work, the effect of the tetracyclic analogue of gambierol was tested in vivo in 3xTg-AD mice (10 months old) after 1 month of weekly treatment with 50 µg/kg. Adverse effects were not reported throughout the whole treatment period and no pathological signs were observed for the analyzed organs. The compound was found in brain samples after intraperitoneal injection. The tetracyclic analogue of gambierol elicited a decrease of amyloid ß1-42 levels and a dose-dependent inhibition of ß-secretase enzyme-1 activity. Moreover, this compound also reduced the phosphorylation of tau at the 181 and 159/163 residues with an increase of the inactive isoform of the glycogen synthase kinase-3ß. In accordance with our in vitro neuronal model, this compound produced a reduction in the N2A subunit of the N-methyl-d-aspartate (NMDA) receptor. The combined effect of this compound on amyloid ß1-42 and tau phosphorylation represents a multitarget therapeutic approach for AD which might be more effective for this multifactorial and complex neurodegenerative disease than the current treatments.

Spongionella Secondary Metabolites, Promising Modulators of Immune Response through CD147 Receptor Modulation.

The modulation of the immune system can have multiple applications such as cancer treatment, and a wide type of processes involving inflammation where the potent chemotactic agent cyclophilin A (Cyp A) is implicated. The Porifera phylum, in which Spongionella is encompassed, is the main producer of marine bioactive compounds. Four secondary metabolites obtained from Spongionella (Gracilin H, A, L, and Tetrahydroaplysulphurin-1) were described to hit Cyp A and to block the release of inflammation mediators. Based on these results, some role of Spongionella compounds on other steps of the signaling pathway mediated by this chemotactic agent can be hypothesized. In the present paper, we studied the effect of these four compounds on the surface membrane CD147 receptor expression, on the extracellular levels of Cyp A and on the ability to migrate of concanavalin (Con A)-activated T lymphocytes. Similar to a well-known immunosuppressive agent cyclosporine A (CsA), Gracilin H, A, L, and tetrahydroaplysulphurin-1 were able to reduce the CD147 membrane expression and to block the release of Cyp A to the medium. Besides, by using Cyp A as chemotactic agent, T cell migration was inhibited when cells were previously incubated with Gracilin A and Gracilin L. These positive results lead us to test the in vivo effect of Gracilin H and L in a mouse ear delayed hypersensitive reaction. Thus, both compounds efficiently reduce the ear swelling as well as the inflammatory cell infiltration. These results provide more evidences for their potential therapeutic application in immune-related diseases of Spongionella compounds.

Heart Alterations after Domoic Acid Administration in Rats.

Domoic acid (DA) is one of the best known marine toxins, causative of important neurotoxic alterations. DA effects are documented both in wildlife and experimental assays, showing that this toxin causes severe injuries principally in the hippocampal area. In the present study we have addressed the long-term toxicological effects (30 days) of DA intraperitoneal administration in rats. Different histological techniques were employed in order to study DA toxicity in heart, an organ which has not been thoroughly studied after DA intoxication to date. The presence of DA was detected by immunohistochemical assays, and cellular alterations were observed both by optical and transmission electron microscopy. Although histological staining methods did not provide any observable tissue damage, transmission electron microscopy showed several injuries: a moderate lysis of myofibrils and loss of mitochondrial conformation. This is the first time the association between heart damage and the presence of the toxin has been observed.

Oral toxicity of okadaic acid in mice: study of lethality, organ damage, distribution and effects on detoxifying gene expression.

In vivo, after administration by gavage to mice and rats, okadaic acid has been reported to produce lesions in liver, small intestine and forestomach. Because several reports differ in the damage detected in different organs, and on okadaic acid distribution after consumption, we determined the toxicity of this compound after oral administration to mice. After 24 hours, histopathological examination showed necrotic foci and lipid vacuoles in the livers of intoxicated animals. By immunohistochemical analysis, we detected this toxin in the liver and kidneys of intoxicated animals. Okadaic acid induces oxidative stress and can be activated in vitro into reactive compounds by the post-mitochondrial S9 fraction, so we studied the okadaic effect on the gene expression of antioxidant and phase II detoxifying enzymes in liver. We observed a downregulation in the expression of these enzymes and a reduction of protein expression of catalase and superoxide dismutase 1 in intoxicated animals.

Pharmacokinetic and toxicological data of spirolides after oral and intraperitoneal administration.

Spirolides are a kind of marine toxins included in the cyclic imine toxin group and produced by the dinoflagellate Alexandrium ostenfeldii. This study shows for the first time a complete and detailed description about the symptoms observed in mice when these toxins were intraperitoneal (i.p.) administered. It is also compared the i.p. toxicity of 13-desmethyl spirolide C (13-desMeC), 13,19-didesMeC (13,19-didesMeC) and 20-methyl spirolide G (20-Me-G) in experiments performed with highly purified toxins. The bioassay indicates that 13-desMeC and 13,19-didesMeC are extremely toxic compounds which have a LD(50) of 27.9µg/kg and 32.2µg/kg, respectively. However, when 20-MeG was i.p administrated with dose up 63.5µg/kg, no deaths were recorded. In order to evaluate the oral toxicity, spirolides were administered by gastric intubation into mice. Then, samples of blood, urine and faeces were collected and analyzed by liquid chromatography-mass spectrometry tandem (LC-MS/MS) technique. Spirolides appear in blood at 15min and in urine after 1h of being toxin administered. In summary, in this paper, it is provided new data about the toxicity, absorption, and excretion of spirolides in mouse. So far, little information is available on this item but necessary for spirolide regulation in the European Union (EU).

Diversity of the vomeronasal system in mammals: the singularities of the sheep model.

The enormous morphological diversity and heterogeneity of the vomeronasal system (VNS) in mammals--as well as its complete absence in some cases--complicates the extrapolation of data from one species to another, making any physiological and functional conclusions valid for the whole Mammalian Class difficult and risky to draw. Some highly-evolved macrosmatic mammals, like sheep, have been previously used in interesting behavioral studies concerning the main and accessory olfactory systems. However, in this species, certain crucial morphological peculiarities have not until now been considered. Following histological, histochemical and immunohistochemical procedures, we have studied the vomeronasal organ (VNO) and the accessory olfactory bulb (AOB) of adult sheep. We have determined: (1) that all structures which classically define the VNO in mammals are present and well developed, providing the morphological basis for functional activity. (2) that, conversely, there is only a scant population of scattered mitral/tufted cells. One morphological consequence of both details is that the strata of the AOB in adult sheep are not as sharply defined as in other species; moreover, the small number of the mitral/tufted cells in the AOB may imply that the VNS of adult sheep is not capable of functioning in the way a well-developed VNS does in other species. (3) the zone to zone projection from the apical and basal sensory epithelium of the VNO to the anterior and posterior part of the AOB, respectively, typical in rodents, lagomorphs and marsupials, is not present in adult sheep.

General organization of the perinatal and adult accessory olfactory bulb in mice.

The vomeronasal system is currently a topical issue since the dual functional specificity, vomeronasal system-pheromones, has recently been questioned. Irrespective of the tools used to put such specificity in doubt, the diversity of the anatomy of the system itself in the animal kingdom is probably of more importance than has previously been considered. It has to be pointed out that a true vomeronasal system is integrated by the vomeronasal organ, the accessory olfactory bulb, and the so-called vomeronasal amygdala. Therefore, it seems reasonable to establish the corresponding differences between a well-developed vomeronasal system and other areas of the nasal cavity in which putative olfactory receptors, perhaps present in other kinds of mammals, may be able to detect pheromones and to process them. In consequence, a solid pattern for one such system in one particular species needs to be chosen. Here we report on an analysis of the general morphological characteristics of the accessory olfactory bulb in mice, a species commonly used in the study of the vomeronasal system, during growth and in adults. Our results indicate that the critical period for the formation of this structure comprises the stages between the first and the fifth day after birth, when the stratification of the bulb, the peculiarities of each type of cell, and the final building of glomeruli are completed. In addition, our data suggest that the conventional plexiform layers of the main olfactory bulb are not present in the accessory bulb.

Placenta: regularization of neovascularization. Microvascularization pattern of the rabbit term placenta.

Mammalian blastocyst produces membranes that gradually attach in the endometrial epithelium and establish a close relationship between fetal and maternal circulatory systems for physiologic exchange. This fact results in the formation of a combined organ, the placenta. Placentation includes extensive neovascularization in maternal and embryonic placental tissues. Recent studies, particularly of genetically altered mice, have greatly improved our understanding of the molecular basis of the development of the vascular system. Numerous factors have been implicated in the regulation of the neovascularization process of the placenta. The aim of this article is to highlight briefly the pattern circulation of the rabbit term placenta as an example of the microvascularization of hemochorial placenta typical of humans and to summarize the main factors responsible for modulating the growth of decidual placental vessels and the vasculogenesis of the basal plate.