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  • 43 Reads
Activation of anion channels in human cells after long term exposure to the marine toxin azaspiracid
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Azaspiracids (AZAs) comprise a group of marine toxins first documented in the Netherlands after ingestion of contaminated mussels, harvested in Ireland coasts, by the end of the last century [1-3]. Azaspiracids are known to be produced by dinoflagellates belonging to the genera Azadinium and Amphidoma [4]. In recent years, part of the research on marine toxins effects on human health have focused on their chronic effects. The presence of azaspiracid in fishery products has been regulated in Europe establishing a limit of 160 μg kg-1 AZA equivalents [5]. Since then, several acute in vitro studies were undertaken to elucidate their mechanism of action, but the results obtained showed great controversy regarding the possible cellular targets of AZAs that could contribute to the symptomatology elicited in humans after ingestion of contaminated fishery products. Our group has recently described that these toxins partially blocked sodium entry into the cells and caused cytoskeletal alterations [6], however the effect of these toxins on ion channels remains almost completely unexplored. Therefore, the main aim of our study was to gain more insight on the effects of azaspiracids on ionic homeostasis and cell volume regulation [7]. Thus, electrophysiological effects of nanomolar concentrations of azaspiracids (50 nM) after a 15-20 h exposition of human embryonic kidney cells (HEK293) which express the human Nav1.7 alpha subunit of the sodium channel were determined. Here, using electrophysiological techniques combined with several pharmacological approaches, we demonstrated that AZA-1 elicited a significant increase in anion efflux that could account for the pathophysiology observed in human intoxications.

References

  1. McMahon, T. Winter toxicity of unknown aetiology in mussels. Harmful Algae News 1996, 14, 2.
  2. Ofuji, K.; Satake, M.; McMahon, T.; Silke, J.; James, K.J.; Naoki, H.; Oshima, Y.; Yasumoto, T. Two analogs of azaspiracid isolated from mussels, Mytilus edulis, involved in human intoxication in Ireland. Nat Toxins 1999, 7, 99-102.
  3. Satake, M.; Ofuji, K.; Naoki, H.; James, K.J.; Furey, A.; McMahon, T.; Silke, J.; Yasumoto, T. Azaspiracid, a New Marine Toxin Having Unique Spiro Ring Assemblies, Isolated from Irish Mussels, Mytilus edulis. Journal of the American Chemical Society 1998, 120, 9967-9968.
  4. Wietkamp, S.; Krock, B.; Clarke, D.; Voß, D.; Salas, R.; Kilcoyne, J.; Tillmann, U. Distribution and abundance of azaspiracid-producing dinophyte species and their toxins in North Atlantic and North Sea waters in summer 2018. PLOS ONE 2020, 15, e0235015.
  5. European Commission. Regulation of the European Parliament and of the Council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption, 854/2004/EC. In Official Journal, 2004; Vol. 50.
  6. Boente-Juncal, A.; Raposo-García, S.; Costas, C.; Louzao, M.C.; Vale, C.; Botana, L.M. Partial Blockade of Human Voltage-Dependent Sodium Channels by the Marine Toxins Azaspiracids. Chemical research in toxicology 2020, 10.1021/acs.chemrestox.0c00216.
  7. Vale, C.; Nicolaou, K.C.; Frederick, M.O.; Vieytes, M.R.; Botana, L.M. Cell volume decrease as a link between azaspiracid-induced cytotoxicity and c-Jun-N-terminal kinase activation in cultured neurons. Toxicol Sci 2010, 113, 158-168.

  • Open access
  • 30 Reads
Functional characterization of a novel rCRiSP (recombinant cysteine-rich secretory protein) from Crotalus oreganus helleri
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Snake venom is a highly complex and diverse cocktail of different proteins and peptides that cause a wide range of biological disturbances in an envenomated victim. While many snake venom toxins have been comprehensively characterized, other toxins such as cysteine-rich secretory proteins (CRiSPs) remain largely unexplored. CRiSPs are ubiquitous non-enzymatic toxins found in many species of snakes worldwide. Several CRiSPs isolated from Asian and Australian snake venoms have been shown to inhibit ion channel/smooth muscle contraction. We have recently reported that Hellerin, a svCRiSP that we have isolated from the venom of the Southern Pacific Rattlesnake, C. oreganus helleri, directly increases vascular permeability in vivo and in vitro. These observations may be parallel to Bj-CRP’s local effects, a CRiSP isolated from the venom of Bothrops jararaca, that has been shown to induce profound inflammatory responses in local tissues through the recruitment of neutrophils and the production of IL-6. To shed new light on svCRiSPs’ molecular targets and inflammatory responses, a recombinant CRiSP from C. o. helleri (named rHellerin) was cloned and tested for vascular and cellular permeability and pro-inflammatory responses. rHellerin was able to induce vascular leakage in vivo and cellular permeability similar to that of native CRiSP. rHellerin was also able to induce increased production of the cytokines IL-8 and IL-6 in human blood and lymphatic endothelial cells. These findings can provide a straightforward method of obtaining biologically viable svCRiSPs identical to the native form, which can accelerate research into further understanding the molecular biology of svCRiSPs by elucidating functionally active residues and subsequent molecular targets/interactions. rHellerin can represent potential in the development of new therapeutic strategies to prevent death and disability from snakebite.

  • Open access
  • 28 Reads
Evaluation of in vitro muscle regeneration after myonecrosis induced by Bothrops alternatus and Bothrops diporus venoms from Northeastern Argentina
Luciano Fusco, Andrea Van de Velde, Laura Leiva, Soledad Bustillo
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

The majority of snakebites in northeastern Argentina are caused by Bothrops alternatus (yarará grande) and Bothrops diporus (yarará chica), reptiles that belong to the Viperidae family. The specific treatment of these ophidian envenomations is serotherapy with antivenoms that ensures a rapid distribution of antibodies and controls the systemic alterations but not always the local damage in the bite site where traces of venom are capable to preclude a successful regenerative response.

In this work, we explore the characteristics of muscle tissue during the critical period after Bothrops alternatus or Bothrops diporus venom injection and their potential inhibitory effect on muscle differentiation using an in vitro study model.

Groups of CF-1 mice were injected intramuscularly in the right gastrocnemius with 50 µg of B. alternatus or B. diporus venoms. Control mice received PBS under identical conditions. Briefly, animals were sacrificed after 0, 1, 3, 24 and 168 hours, muscles were dissected out and placed in liquid nitrogen for pulverization and filtration through 0.22 µm membranes. Venom proteins present in these homogenates were quantified by the ELISA method and analyzed by Western Blotting. Myoblast cells (C2C12 cell line) were exposed for 24h to muscle homogenates and the less cytotoxic ones were used for myogenesis evaluation.

Results evidenced that the amount of both venoms in muscle homogenates decreased over time, with even traces of venom (5-13 µg/mL) being observed 168h after inoculations. No significant differences were detected between B. alternatus and B. diporus venom treatments. Identification by immunoblotting showed typical venom protein bands with molecular masses between 20 and 100 kDa for B. alternatus and 14 and 100 kDa for B. diporus whose intensities gradually decreased with time. An intense band of ~60 kDa, characteristic of metalloproteases, was mainly visualized even after 7 days of both treatments.

In addition, less cytotoxic muscle homogenates (above 85% of myoblast viability corresponding to 24 and 168 h incubation times) were used for myogenesis assay. Control showed mature myotubes formation after 72h but a complete lack of myoblast fusion occurred when myogenic cells were incubated with muscle homogenates from mice injected with bothropic venoms.

These preliminary findings suggest that a possible local treatment, complementary to serotherapy, could improve the prognosis of snakebite poisonings by accelerating muscle regeneration processes.

  • Open access
  • 29 Reads
Genetic responses and aflatoxin inhibition during interaction between aflatoxigenic and non-aflatoxigenic Aspergillus flavus
Rebecca Sweany, Brian Mack, Geromy Moore, Matthew Gilbert, Jeffrey Cary, Kanniah Rajasekaran, Kenneth Damann Jr.
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Aflatoxin is a carcinogenic mycotoxin produced by Aspergillus flavus in corn. Non-aflatoxigenic A. flavus isolates are applied to corn fields as a biocontrol to reduce aflatoxin contamination. Direct contact or touch between aflatoxigenic and non-aflatoxigenic isolates dramatically reduces aflatoxin production. To understand the mechanism of touch inhibition, a high-throughput RNA-seq study was conducted to examine gene expression during their interaction. Non-aflatoxigenic strain KD17 and aflatoxigenic strain KD53 were grown separately and in co-culture for 30 and 72 h. Toxin production was high in the aflatoxigenic monoculture and negligible in co-cultures. When grown separately, the toxigenic strain represented 7% and 33% of the combined biomass at 30 and 72 h, respectively. However, only 3% of the sequence reads uniquely aligned to the aflatoxigenic strain during co-culture, indicating growth and/or gene expression of the aflatoxigenic strain was inhibited in response to the non-aflatoxigenic strain. Few reads aligned to the aflatoxin gene cluster during co-culture. Eighteen genes expressed during mono-culture of the non-aflatoxigenic strain were further up-regulated during co-culture, indicating a response to contact. Of those genes, seven belong to a putative secondary metabolite cluster, suggesting a potentially inhibitory compound is produced. Taken together, these results suggest that non-aflatoxigenic strains inhibit growth and aflatoxin biosynthetic gene cluster expression in aflatoxin-producing strains. In addition, other secondary metabolite genes are upregulated during biocontrol interaction. This study demonstrates a potential role of inhibitory secondary metabolites in the biocontrol mechanism and deserves further exploration to improve biocontrol formulations.

  • Open access
  • 43 Reads
Exploration of the biological effects of a basic Phospholipase A2 from Agkistrodon piscivorus piscivorus venom
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Background: Phospholipases A2 (PLA2s) are found in abundance in many North American snake species. They are responsible for a wide array of pharmacological effects on tissues both locally and systemically. In A.p.piscivorus (A.p.p.), these toxins make up a significant portion of venom constituents. A basic PLA2 was recently isolated through reverse phase HPLC and identified as a D-49 PLA2 (A.p.p PLA2). After testing activities on an in vivo model, the release of pro-inflammatory mediators, systemic myotoxicity, and hemolytic effects were observed. This study aims to explore the hematological, myotoxic and pro-inflammatory activities of this toxin using in vitro models.

Methods: Whole blood was used to test hemolytic activity of A.p.p PLA2 in vitro and, through the SONOCLOT analyzer, the effects on the hemostatic system. Moreover, we tested cell viability, expression of cell activation molecules, and cell damage markers on human umbilical vein endothelial cells (HUVEC) representing the vascular system and a myoblast cell line (C2C12) as a muscle model. Cells were incubated with A.p.p PLA2 over different times. Cell viability was tested using MTT assay, the expression and release of pro-inflammatory and hemostatic mediators were done using flow cytometry and ELISA. Muscle damage was detected evaluating creatine kinase (CK) release.

Discussion/ Conclusion: It was observed that A.p.p PLA2 caused significant hemolytic activity and substantial changes in the coagulation system and mild changes in platelet function in whole human blood. Likewise, this toxin altered cell viability in C2C12 but not in HUVEC. Endothelial cells were also activated when incubated both at 3 h and 24 h. Additionally, C2C12 released IL-6 and CK, which are markers of cell damage. This data can be used for further experimentation to characterize enzymes belonging to this family to produce specialized antivenoms that target PLA2s and their biological activities.

  • Open access
  • 24 Reads
Use of neostigmine - atropine plus antivenom in the experimental envenomation by Micrurus venom. Preliminary results.
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Venoms of most elapids are neurotoxic being their most important components alpha-neurotoxins and PLA2s. The treatment with neostigmine and atropine(NA) has been suggested to revert the toxicity of nicotinic toxins. The usefulness of an alternative tool is very important due to the lack of antivenom for some Elapids like Micrurus(M.), due the scarcity of specific antivenoms (AV). We assayed in rescue experiments (mice challenged with mortal doses) the usefulness of the combination neostigmine–atropine (NA) alone or combined with AV. The venoms of Naja (N.) kaouthia, M. altirostris, M. pyrrhocryptus and M. surinamensis. The Antivenom used were therapeutic anti-Micrurus and experimental anti-Naja siamensis antivenoms. Despite that all the cases received a single dose of 20 μg atropine + 2.5μg neostigmine by i.p. route delayed the time of death (p<0.05), no good protection was observed using only this treatment. In the other hand, only high doses of AV achieve some level of protection. Nevertheless the combination of NA plus AV, reduced the mortality, as well as the dose of antivenom required for protection in all the cases regarding these treatments used alone. In the case of M. altirostris venom, the protection using NA was from 0 to 20% and that using 50 μl of AV ranged from 0 to 60%, while using the combined treatment the protection was from 80 to 100% (p 0.046 and 0,02 regarding AV or NA alone). In the other cases an improvement was observed regarding the uses of NA alone, AV alone (250 μl) or their combination. In the case of N. kaoutia= 0%, 20%, 40% respectively, M. pyrrhocryptus= 0%, 60%, 100% and M. surinamensis= 0%, 0 to 20%, 40-80%. These preliminary results suggest the utility of this combination for the treatment of these envenomations, which could be helpful to reduce the dose of AV.

  • Open access
  • 49 Reads
Paraspecific neutralization of the venom form adults and young Crotalus atrox by paraspecific South American Antivenoms.
Adolfo de Roodt, Marcela Desio, Laura Lanari, Néstor Lago, Fernando Morón Goñi, Daniel Dozoretz, Leandro Calderón, Pablo Regner, Vanessa de Oliveira, Carlos Damin
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Crotalus atrox is one of the species of venomous snakes most commonly found in herpetological collections around the world and it is usually commercialized in the black market. Several collections have specimens but lack of the specific antivenom. We tested the toxicity of venom of adults and young snakes (2 to 3 years old) specimens of C. atrox in captivity and the para-specific neutralization provided by the antivenoms most used in Argentina. The i.p. lethal potency of the venoms were 100(95-105) μg and 43(42-45) μg20g mouse and the indirect hemolytic activity was 7.9 (6.7-9.2) μg and 9.0(8.3-9.9) for adults and juvenile venoms. Despite the adult´s venoms lower lethal potency, these venoms were more difficult to neutralize, around 1.5 ml of antibothropic Antivenom (AB) were necessaries to neutralize 1 mg of venom in contrast to 0.54 ml required to neutralize young´s venoms. The neutralization by the Anticrotalic (AC) antivenom was despicable. The dose of AB required for the neutralization 5.0 LD50 of young snakes was in the range of those required for the neutralization of the specific venoms, nevertheless the dose required to neutralize venom from adults was 6 fold higher. The experiments using 2LD50 as challenge dose, showed similar results. The indirect hemolysis caused by both venoms was similarly neutralized by AB (p<0.05) while the AC did not show neutralizing activity. The myotoxicity determined by the increase of creatinquinase or by histopathology, was neutralized by both antivenoms, possibly due to the presence of myotoxins like K49 phospholipases present in this venom. Although the paraspecificity of AB has a potential use as treatment, especially in young snakes bites, the doses required in adult attacks are high. Despite AB seems to be useful for emergencies, these results suggest advantages in using specific antivenom for the treatment of these snakebites.

  • Open access
  • 58 Reads
Analogous venom peptides acting on different pathways: a study of Bicarinalin and U9-MYRTX-Tb1a from T. bicarinatum venom
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

For decades venom were studied for various applications as agriculture, therapeutics or as pharmacological tools. Currently, six venom-derived drugs and one venom-derived insecticide are available on market. Ant venoms exhibit a high diversity of peptides, similarly to other arthropod venoms. The first studies shown many in vitro and in vivo biological effects such as anti-microbial, anti-inflammatory, anti-viral or even ion channel modulators. Our study focused on M-MYRTX-Tb1a (Bicarinalin) and U9-MYRTX-Tb1a, two peptides from Tetramorium bicarinatum venom exhibiting similar sequences. Bicarinalin is an amphipatic α-helical peptide, which was found to forms pores in cell membranes. As the sequence of U9-MYRTX-Tb1a suggests a similar structure with identical physico-chemical properties, we hypothesized a similar biological function. In this study we investigated the biological effect of U9-MYRTX-Tb1a. We tested the two peptides on Drosophila melanogaster embryonic cells to evaluate their cytotoxicity. As predicted, the Drosophila cells lysis was observed with the addition of both peptides. However, the cell morphology after peptide incubation and the time-effect was different between U9-MYRTX-Tb1a and Bicarinalin suggesting different mechanisms of action. First, incubation with U9-MYRTX-Tb1a leads to cell growing, and blebs forming with a potential condensation of the nucleus. Then, the cytotoxicity effect of Bicarinalin is faster than the U9-MYRTX-Tb1a. These first results, suggest that pro-apoptotic and/or autophagic pathways could be involved in biological activity of U9-MYRTX-Tb1a. By confirming this hypothesis, we could update the first peptide from ant venom with a pro-apoptotic and/or autophagic effect and the various associated applications.

  • Open access
  • 37 Reads
Biological activities of phosphodiesterase from Crotalus durissus terrificus venom
Luciano Fusco, David Hernández, Stephen Hyslop, Laura Leiva
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Phosphodiesterases (PDEs) are an enzymes family that hydrolyze phosphodiester bonds sequentially from the 3´ terminus of polynucleotides to produce 5’-mononucleotides. Historically, snake venom PDEs have been widely used in sequencing and structural studies of nucleic acids. In contrast, the potential pharmacological activities of these enzymes are poorly understood and their role in envenomation remains unclear. Previosuly, we isolated and preliminary characterized a PDE from C. d. terrificus (CDT) venom, demonstrating that is capable of hidrolizing ATP, ADP, AMP and DNA. Here, we evaluated the edema-forming activity and locomotory behavior induced by CDT-PDE. The enzyme was purified through two chromatographic steps (Sephadex G-75 and HiTrap Q-FF). The CDT-PDE activity was tested by chromogenic reaction with sodium salt of bis (p-nitrophenyl phosphate). Groups of five mice were subplantar injected in the right hind foot with 1 µg of purified PDE or a mixture of PDE (1 μg) and ADP (50 nmol) that was co-injected or adenosine (50 nmol) or ADP (50 nmol) or PBS. Edema was measured as the increase in paw thickness using low-pressure spring calipers at various intervals (0.5, 1, 3, and 6 h). At the end of the experiment, the hind feet were removed and processed for histological analysis. Locomotory behavior was assessed in an open-field test Each mouse (n=6) received an i.p. injection of PDE or PBS. Mice not injected with PDE or PBS were used as controls. The results indicate that PDE from CDT venom from northeastern Argentina is edematogenic and causes an inflammatory infiltrate. Besides, PDE-CDT reduced the locomotor activity in the initial minutes after injection. Results all indicate that PDE exhibits pharmacological activities that they deserve to be studied in further detail. Further investigations are required to assess the contribution of this enzyme to the systemic manifestations associated with envenomation by this species.

  • Open access
  • 195 Reads
Linear Scorpion Peptides: An unexplored pool for peptide hydrogels
Constantinos Avraamides, Spiridoula Diavoli, Ariana Robertson, Manos Vlasiou, Elena Mourelatou, Christos Petrou, Yiannis Sarigiannis
Published: 13 January 2021 by MDPI in 1st International Electronic Conference on Toxins session Poster

Scorpions during their long evolutionary existence on the planet, more than 400 million years, managed to develop series of venom peptides that display diverse biological activities and pharmacological functions. Scorpion venom peptides are generally classified into two main groups: the disulfide bridged peptides (DBPs) which usually target membrane bound ion channels and then non-disulfide bridged peptides (NDBPs), a smaller group with multifunctional activities.

Our current study focuses on the short (13-19 amino acids) antimicrobial linear scorpion peptides. Many of these peptides contain sections, ranging from short to long, of identical amino acid sequences. Most of them display a net positive charge of 1 or 2, exhibit isoelectric point at pH 9-10, have a broad range of hydrophobicity and grand average of hydropathy (GRAVY). These features allow these peptides to be attracted toward the negatively charged phospholipid head groups of the lipid membranes of target cells, a force driven by electrostatic interactions.

Here we present the synthesis of Mucroporin peptide, a 17 amino acid linear peptide isolated from the venom of Lychas Mucronatus, and its synthetic analogs. Mucroporin exhibits positive charge 1 due to a Lysine at the C-terminus site of the peptide. The rest of the peptides are mainly aliphatic, leucine, glycine or isoleucine. A series of synthetic analogs are designed, synthesized, purified, and characterized with liquid chromatography - mass spectrometry techniques (LC-PDA-MS) and nuclear magnetic resonance spectroscopy (NMR). Mucroporin as well as its synthetic analogs are tested with various techniques for their ability to form hydrogels under several conditions.

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