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Viperids of the genus Lachesis, also known as bushmasters, are capable of injecting great amounts of venom that cause severe envenomation incidents. Since phospholipases type A2 are mainly involved in edema and myonecrosis within the snakebite sites, in this work, the isolation, amino acid sequence and biochemical characterization of the first phospholipase type A2 from the venom of Lachesis acrochorda, named Lacro_PLA2, is described. Lacro_PLA2 is an acidic aspartic 49 calcium-dependent phospholipase A2 with 93% similarity to the L. stenophrys phospholipase. Lacro_PLA2 has a molecular mass of 13,969.7 Da and an experimental isoelectric point around 5.3. A combination of N-terminal Edman degradation and MS/MS spectrometry analyses revealed that Lacro_PLA2 contains 122 residues including 14 cysteines that form 7 disulfide bridges. A predicted 3D model shows a high resemblance to other viperid phospholipases. Nevertheless, immunochemical and phospholipase neutralization tests revealed a notorious level of immunorecognition of the isolated protein by two polyclonal antibodies from viperids from different genus, which suggest that Lacro_PLA2 resembles more to bothropic phospholipases. Lacro_PLA2 also showed significantly high edema activity when was injected into mice; so, it could be an alternative antigen in the development of antibodies against toxins of this group of viperids, seeking to improve commercial polyclonal antivenoms.

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In this study, we examined the potential use of N-acetyl-L-cysteine (NAC) in association with a polyvalent antivenom and as stand-alone therapy to reduce the acute local and systemic effects induced by Lachesis muta muta venom in rats. Male Wistar rats (300-350 g) were exposed to L. m. muta venom (1.5 mg/kg - i.m.) and subsequently treated with anti-Bothrops/Lachesis serum (antivenom:venom ratio 1:3 'v/w' - i.p.) and NAC (150 mg/kg - i.p.) separately or in association; the animals were monitored for 120 min to assess changes in temperature, locomotor activity, local oedema formation and the prevalence of haemorrhaging. After this time, animals were anesthetized in order to collect blood samples through intracardiac puncture and then euthanized for collecting tissue samples; the hematological-biochemical and histopathological analyses were performed through conventional methods. L. m. muta venom produced pronounced local oedema, subcutaneous haemorrhage and myonecrosis, with both antivenom and NAC successfully reducing the extent of the myonecrotic lesion when individually administered; their association also prevented the occurrence of subcutaneous haemorrhage. Venom-induced creatine kinase (CK) release was significantly prevented by NAC alone or in combination with antivenom; NAC alone failed to reduce the release of hepatotoxic (alanine aminotransferase) and nephrotoxic (creatinine) serum biomarkers induced by L. m. muta venom. Venom induced significant increase of leucocytes which was also associated with an increase of neutrophils, eosinophils and monocytes; antivenom and NAC partially reduced these alterations, with NAC alone significantly preventing the increase of eosinophils whereas neither NAC or antivenom prevented the increase in monocytes. Venom did not induce changes in the erythrogram parameters. In the absence of a suitable antivenom, NAC has the potential to reduce a number of local and systemic effects caused by L. m. muta venom.

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The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

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The Malayan blue coral snake, Calliophis bivirgata flaviceps, is a medically important venomous snake in Southeast Asia. However, the complexity and diversity of its venom genes remain little explored. Methods: To address this, we applied high-throughput next-generation sequencing to profile the venom gland cDNA libraries of C. bivirgata flaviceps. The transcriptome was de novo assembled, followed by gene annotation, multiple sequence alignment and analyses of the transcripts. Results: A total of 74 non-redundant toxin-encoding genes from 16 protein families were identified, with 31 full-length toxin transcripts. Three-finger toxins (3FTx), primarily delta-neurotoxins and cardiotoxin-like/cytotoxin-like proteins, were the most diverse and abundantly expressed. The major 3FTx (Cb_FTX01 and Cb_FTX02) are highly similar to calliotoxin, a delta-neurotoxin previously reported in the venom of C. bivirgata. This study also revealed a conserved tyrosine residue at position 4 of the cardiotoxin-like/cytotoxin-like protein genes in the species. These variants, proposed as Y-type CTX-like proteins, are similar to the H-type CTX from cobras. The substitution is conservative though, preserving a less toxic form of elapid CTX-like protein, as indicated by the lack of venom cytotoxicity in previous laboratory and clinical findings. The ecological role of these toxins, however, remains unclear. The study also uncovered unique transcripts that belong to phospholipase A2 of Groups IA and IB, and snake venom metalloproteinases of PIII subclass, which show sequence variations from those of Asiatic elapids. Conclusion: The venom gland transcriptome of C. bivirgata flaviceps from Malaysia was de novo assembled and annotated. The diversity and expression profile of toxin genes provide insights into the biological and medical importance of the species.(AU)

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SUMMARY: The Viperidae venoms are composed of a mixture of constituents with enzymatic and non-enzymatic actions, which act on ultrastructural components of cells and tissues. Here, the number of mitochondria, mitochondrial area and the number of mitochondrial cristae from adrenal glands cortex treated with snake venoms were tested after 3, 6 and 24 hours of venom injections. The mitochondria quantitative changes showed a statistically significant decrease, in the number of mitochondria past 3, 6 and 24 h. There was an increase in the mitochondrial area after 6 h, where Crotalus vegrandis venom did not present significant differences with Crotalus pifanorum or Bothrops venezuelensis venoms. After 24 h, there was an escalation of mitochondrial area in all tested venoms. The number of mitochondrial cristae after 3 h did not present important differences with the control treatment. After 6 h, the number of mitochondrial cristae initiated to decrease under the activities of the 3 venoms action, until 24 h of observation. In the qualitative observations it was possible to witness an intense damage of the mitochondria, with loss and swelling of membranes, disappearance of cristae and the appearance of myelin figures, which started at 3 h after the Crotalus and Bothrops venoms injections. These damages probably were due to cytotoxic effects of phospholipases, metalloproteases and/or other proteolytic activities present in Viperidae snake venoms, being more evident in Crotalus venoms. As far as we know, these results define a novel finding that suggest that Viperidae snake venoms are extremely toxic to mammalian mitochondria.

RESUMEN: Los venenos de Viperidae tienen acciones enzimáticas y no enzimáticas, que actúan sobre la estructura celular. Aquí se probaron, a las 3, 6 y 24 horas de la inyección del veneno, el número de mitocondrias, el área mitocondrial y el número de crestas mitocondriales de la corteza de las glándulas adrenales. Los cambios cuantitativos de las mitocondrias mostraron una disminución en el número de mitocondrias a las 3, 6 y 24 h. Hubo un aumento en el área mitocondrial a las 6 h, donde el veneno de la serpiente Crotalus vegrandis no presentó diferencias significativas con los venenos de Crotalus pifanorum o Bothrops venezuelensis. Después de 24 h, hubo un aumento del área mitocondrial en todos los venenos. El número de crestas mitocondriales a las 3 h no presentó alteraciones o diferencias importantes con el tratamiento de control. Después de 6 h, el número de crestas mitocondriales comenzó a disminuir bajo la acción de los 3 venenos, hasta las 24 h de observación. En las observaciones cualitativas se observó un daño intenso de las mitocondrias, con pérdida y edema de las membranas, desaparición de las cristae y aparición de figuras mielínicas, que comenzó a las 3 h después de las inyecciones de veneno de Crotalus y Bothrops. Estos daños se debieron factiblemente a los efectos citotóxicos de componentes proteolíticos de los venenos. Creemos que estos resultados definen un nuevo y original hallazgo, que sugiere que los venenos de serpiente Viperidae son extremadamente tóxicos para las mitocondrias de mamíferos.

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In Colombia, Lachesis acrochorda causes 2-3% of all snake envenomations. The accidents promote a high mortality rate (90%) due to blood and cardiovascular complications. Here, the effects of the snake venom of L. acrochorda (SVLa) were analyzed on human blood cells and on cardiovascular parameters of rats. SVLa induced blood coagulation, as measured by the prothrombin time test, but did not reduce the cell viability of neutrophils and platelets evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay and by the lactate dehydrogenase (LDH) enzyme assay. In fact, SVLa increased the absorbance in tests made with platelets subjected to the MTT assay. SVLa induced platelet aggregation whose magnitude was comparable to that of the positive control adenosine diphosphate (ADP), and occurred earlier with increasing SVLa concentration. Acetylsalicylic acid (ASA, a cyclooxygenase inhibitor) or clopidogrel (an ADP receptor blocker) inhibited the aggregating effect of SVLa. Inhibition of SVLa-elicited platelet aggregation also resulted from the treatment with disodium ethylenediaminetetraacetate (Na2-EDTA; metalloproteinase inhibitor) and with 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF, serine protease inhibitor). In isolated right atrium of rats, SVLa increased slightly, but significantly, the magnitude of the spontaneous contractions and, in isolated rat aorta, SVLa relaxed KCl- or phenylephrine-induced contractions. In vivo, SVLa induced hypotension and bradycardia in rats, with detection of hemorrhage in pulmonary and renal tissues. Altogether, under experimental conditions, SVLa induced blood coagulation, platelet aggregation, hypotension and bradycardia. Part of the effects presented here may be explained by the presence of snake venom metalloproteinases (SVMPs) and snake venom serine proteases (SVSPs), constituents of SVLa.

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The Asiatic pit vipers from the Trimeresurus complex are medically important venomous snakes. These pit vipers are often associated with snakebite that leads to fatal coagulopathy and tissue necrosis. The cytotoxic venoms of Trimeresurus spp.; however, hold great potential for the development of peptide-based anticancer drugs. Methods: This study investigated the cytotoxic effect of the venom from Trimeresurus purpureomaculatus, the mangrove pit viper (also known as shore pit viper) which is native in Malaysia, across a panel of human cancer cell lines from breast, lung, colon and prostate as well as the corresponding normal cell lines of each tissue. Results: The venom exhibited dose-dependent cytotoxic activities on all cell lines tested, with median inhibition concentrations (IC50) ranging from 0.42 to 6.98 µg/mL. The venom has a high selectivity index (SI = 14.54) on breast cancer cell line (MCF7), indicating that it is significantly more cytotoxic toward the cancer than to normal cell lines. Furthermore, the venom was fractionated using C18 reversed-phase high-performance liquid chromatography and the anticancer effect of each protein fraction was examined. Fraction 1 that contains a hydrophilic low molecular weight (approximately 7.5 kDa) protein was found to be the most cytotoxic and selective toward the breast cancer cell line (MCF7). The protein was identified using liquid chromatography-tandem mass spectrometry as a venom disintegrin, termed purpureomaculin in this study. Conclusion: Taken together, the findings revealed the potent and selective cytotoxicity of a disintegrin protein isolated from the Malaysian T. purpureomaculatus venom and suggested its anticancer potential in drug discovery.(AU)

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Background:Several studies have pointed out that certain snake venoms contain compounds presenting cytotoxic activities that selectively interfere with cancer cell metabolism. In this study, Pseudocerastes persicus venom and its fractions were investigated for their anticancer potential on lung cancer cells.Methods:Lung cancer cells (A549) and normal fibroblast cells (Hu02) were treated with the P. persicus venom and its HPLC fractions and the cell cytotoxic effects were analyzed using MTT and lactate dehydrogenase release assays. Apoptosis was determined in venom-treated cell cultures using caspase-3 and caspase-9 assay kits.Results:The treatment of cells with HPLC fraction 21 (25-35 kDa) of P. persicus venom resulted in high LDH release in normal fibroblast cells and high caspase-3 and caspase-9 activities in lung cancer cells. These results indicate that fraction 21 induces apoptosis in cancer cells, whereas necrosis is predominantly caused by cell death in the normal cells. Fraction 21 at the final concentration of 10 μg/mL killed approximately 60% of lung cancer cells, while in normal fibroblast cells very low cell cytotoxic effect was observed.Conclusion:HPLC fraction 21 at low concentrations displayed promising anticancer properties with apoptosis induction in the lung cancer cells. This fraction may, therefore, be considered a promising candidate for further studies.(AU)

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The European continent is inhabited by medically important venomous Viperinae snakes. Vipera ammodytes, Vipera berus, and Vipera aspis cause the greatest public health problems in Europe, but there are other equally significant snakes in specific regions of the continent. Immunotherapy is indicated for patients with systemic envenoming, of which there are approximately 4000 annual cases in Europe, and was suggested as an indication for young children and pregnant women, even if they do not have systemic symptoms. In the present study, the safety and venom-neutralizing efficacy of Inoserp Europe-a new F(ab')2 polyvalent antivenom, designed to treat envenoming by snakes in the Eurasian region-were evaluated. In accordance with World Health Organization recommendations, several quality control parameters were applied to evaluate the safety of this antivenom. The venom-neutralizing efficacy of the antivenom was evaluated in mice and the results showed it had appropriate neutralizing potency against the venoms of several species of Vipera, Montivipera, and Macrovipera. Paraspecificity of the antivenom was demonstrated as well, since it neutralized venoms of species not included in the immunization schemes and contains satisfactory levels of total proteins and F(ab')2 fragment concentration. Therefore, this new polyvalent antivenom could be effective in the treatment of snake envenoming in Europe, including Western Russia and Turkey.

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