Identification of purinergic system components in the venom of Bothrops mattogrossensis and the inhibitory effect of specioside extracted from Tabebuia aurea.

Snake bites are a severe problem in the countryside of Brazil and are usually attributed to snakes of the genera Bothrops, Crotalus, and Lachesis. Snake venom can release ectoenzymes and nucleotidases that modulate the purinergic system. In addition to serum therapy against snake poisoning, medicinal plants with anti-inflammatory activities, such as Tabebuia aurea, is empirically applied in accidents that occur in difficult-to-access areas. This study aimed was to verify the presence and activity of nucleotidases in the crude venom of Bothrops mattogrossensis (BmtV) in vitro and characterize the modulation of purinergic components, myeloid differentiation, and inflammatory/oxidative stress markers by BmtV in vivo and in vitro. Moreover, our study assessed the inhibitory activities of specioside, an iridoid isolated from Tabebuia aurea, against the effects of BmtV. Proteomic analysis of venom content and nucleotidase activity confirm the presence of ectonucleotidase-like enzymes in BmtV. In in vivo experiments, BmtV altered purinergic component expression (P2X7 receptor, CD39 and CD73), increased neutrophil numbers in peripheral blood, and elevated oxidative stress/inflammatory parameters such as lipid peroxidation and myeloperoxidase activity. BmtV also decreased viability and increased spreading index and phagocytic activity on macrophages. Specioside inhibited nucleotidase activity, restored neutrophil numbers, and mediate the oxidative/inflammatory effects produced by BmtV. We highlight the effects produced by BmtV in purinergic system components, myeloid differentiation, and inflammatory/oxidative stress parameters, while specioside reduced the main BmtV-dependent effects.

Viperidae snakes infected by mammalian-associated trypanosomatids and a free-living kinetoplastid.

Trypanosomatids have achieved significant evolutionary success in parasitizing various groups, yet reptiles remain relatively unexplored. The utilization of advanced molecular tools has revealed an increased richness of trypanosomatids in vertebrate hosts. The aim of this study was to identify the trypanosomatid species infecting Bothrops moojeni and Crotalus durissus kept in captivity from 2000 to 2022. Blood samples were obtained from 106 snakes: 73C. durissus and 33 B. moojeni. Whole blood was collected for hemoculture, blood smears and centrifugated to obtain the blood clot that had its DNA extracted and submitted to Nested PCR (18S rDNA gene) to detect Trypanosomatidae. Positive samples were quantified and submitted to both conventional (Sanger) and next generation sequencing (NGS). Cloning of the amplified PCR product was performed for only one individual of C. durissus. To exclude the possibility of local vector transmission, attempts to capture sandflies were conducted using six CDC-LT type light traps. Molecular diagnosis revealed that 34% of the snakes presented trypanosomatid DNA, 47.94% in C. durissus and 3.9% in B. moojeni. The cloning process generated four colonies identified as a new MOTU named Trypanosomatidae sp. CROT. The presence of DNA of five trypanosomatids (Trypanosoma cruzi TcII/VI, Trypanosoma sp. DID, Trypanosoma cascavelli, Trypanosomatidae sp. CROT, Leishmania infantum and Leishmania sp.) and one free-living kinetoplastid (Neobodo sp.) was revealed through NGS and confirmed by phylogenetic analysis. The haplotypic network divided the T. cascavelli sequences into two groups, 1) marsupials and snakes and 2) exclusive to marsupials. Therefore, the diversity of Kinetoplastea is still underestimated. Snakes have the ability to maintain infection with T. cruzi and L. infantum for up to 20 years and the DNA finding of Neobodo sp. in the blood of a C. durissus suggests that this genus can infect vertebrates.

Purification, Characterization and Evaluation of the Antitumoral Activity of a Phospholipase A2 from the Snake Bothrops moojeni.

Nature presents a wide range of biomolecules with pharmacological potential, including venomous animal proteins. Among the protein components from snake venoms, phospholipases (PLA2) are of great importance for the development of new anticancer compounds. Thus, we aimed to evaluate the PLA2 anticancer properties from Bothrops moojeni venom. The crude venom was purified through three chromatographic steps, monitored by enzymatic activity and SDS-PAGE (12%). The purified PLA2 denominated BmPLA2 had its molecular mass and N-terminal sequence identified by mass spectrometry and Edman degradation, respectively. BmPLA2 was assayed against human epithelial colorectal adenocarcinoma cells (Caco-2), human rhabdomyosarcoma cells (RD) and mucoepidermoid carcinoma of the lung (NCI-H292), using human fibroblast cells (MRC-5) and microglia cells (BV-2) as a cytotoxicity control. BmPLA2 presented 13,836 Da and a 24 amino acid-residue homologue with snake PLA2, which showed a 90% similarity with other Bothrops moojeni PLA2. BmPLA2 displayed an IC50 of 0.6 µM against Caco-2, and demonstrated a selectivity index of 1.85 (compared to MRC-5) and 6.33 (compared to BV-2), supporting its selectivity for cancer cells. In conclusion, we describe a new acidic phospholipase, which showed antitumor activity and is a potential candidate in the development of new biotechnological tools.

Tabebuia aurea decreases hyperalgesia and neuronal injury induced by snake venom.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia aurea (Silva Manso) Benth. & Hook. f. ex S. Moore is used as anti-inflammatory, analgesic and antiophidic in traditional medicine, though its pharmacological proprieties are still underexplored. In the bothropic envenoming, pain is a key symptom drove by an intense local inflammatory and neurotoxic event. The antivenom serum therapy is still the main treatment despite its poor local effects against pain and tissue injury. Furthermore, it is limited to ambulatorial niches, giving space for the search of new and more inclusive pharmacological approaches. AIM OF THE STUDY: evaluation of Tabebuia aurea hydroethanolic extract (HEETa) in hyperalgesia and neuronal injury induced by Bothrops mattogrossensis venom (VBm). MATERIALS AND METHODS: Stem barks from Tabebuia aurea were extracted with ethanol and water (7:3, v/v) to yield the extract HEETa. Then, HEETa was analyzed by LC-DAD-MS and its constituents were identified. Snake venoms were extracted from adult specimens of Bothrops mattogrossensis, lyophilized and kept at -20 °C until use. Male Swiss mice, weighting 20-25 g, were used to hyperalgesia (electronic von Frey), motor impairment (Rotarod test) and tissue injury evaluation (histopatology and ATF-3 immunohistochemistry). Therefore, three experimental groups were formed: VBm (1 pg, 1 ng, 0.3 µg, 1 µg, 3 and 6 µg/paw), HEETa orally (180, 540, 720, 810 or 1080 mg/kg; 10 mL/kg, 30 min prior VBm inoculation) and VBm neutralized (VBm: HEETa, 1:100 parts, respectively). In all set of experiments a control (saline group) was used. First, we made a dose-time-response course curve of VBm's induced hyperalgesia. Next, VBm maximum hyperalgesic dose was employed to perform HEETa orally dose-time-response course curve and analyses of VBm neutralized. Paw tissues for histopathology and DRGs were collected from animals inoculated with VBm maximum dose and treated with HEETa antihyperalgesic effective dose or neutralized VBm. Paws were extract two or 72 h after VBm inoculation and DRGs, in the maximum expected time expression of ATF-3 (72 h). RESULTS: From HEETa extract, glycosylated iridoids were identified, such as catalpol, minecoside, verminoside and specioside. VBm induced a time and dose dependent hyperalgesia with its highest effect seen with 3 µg/paw, 2 h after venom inoculation. HEETa effective dose (720 mg/kg) decreased significantly VBm induced hyperalgesia (3 µg/paw) with no motor impairment and signs of acute toxicity. HEETa antihyperalgesic action starts 1.5 h after VBm inoculation and lasted up until 2 h after VBm. Hyperalgesia wasn't reduced by VBm: HEETa neutralization. Histopathology revealed a large hemorragic field 2 h after VBm inoculation and an intense inflammatory infiltrate of polymorphonuclear cells at 72 h. Both HEETa orally and VBm: HEETa groups had a reduced inflammation at 72 h after VBm. Also, the venom significantly induced ATF-3 expression (35.37 ±â€¯3.25%) compared with saline group (4.18 ±â€¯0.68%) which was reduced in HEETa orally (25.87 ±â€¯2.57%) and VBm: HEETa (19.84 ±â€¯2.15%) groups. CONCLUSION: HEETa reduced the hyperalgesia and neuronal injury induced by VBm. These effects could be related to iridoid glycosides detected in HEETa and their intrinsic reported mechanism.

Aspectos clinicopatológicos e laboratoriais do envenenamento experimental por Bothrops moojeni e Bothropoides neuwiedi em ovinos / Clinicopathological and laboratory aspects of experimental poisoning by Bothrops moojeni and Bothropoides neuwiedi snake venoms in sheep

O envenenamento ofídico espontâneo, ou acidente ofídico, é descrito como causa de morte em animais domésticos. No entanto, dados concretos relativos ao gênero e espécie de serpente envolvida, à evolução do quadro clínico, e às alterações clinicopatológicas desenvolvidas, são escassos. Assim sendo, este trabalho teve como objetivo determinar as alterações clinicopatológicas e laboratoriais provocadas pelo veneno de Bothrops moojeni e Bothropoides neuwiedi em ovinos no intuito de fornecer informações adicionais referentes a acidentes ofídicos em animais de produção, auxiliando o estabelecimento do diagnóstico dessa condição. Os venenos liofilizados foram diluídos em 1 ml de solução fisiológica e administrados a quatro ovinos por via subcutânea na face direita, nas doses de 0,41mg/kg e 0,82mg/kg do veneno de B. moojeni em dois ovinos, e de 1,0mg/kg do veneno de B. neuwiedi em dois ovinos. Apenas o ovino que recebeu a menor dose (0,41mg/kg) sobreviveu, apesar de ter desenvolvido quadro clínico muito severo e semelhante aos demais. Os sinais clínicos iniciaram nos primeiros 10 minutos após a inoculação em todos os ovinos. O período de evolução variou de dois a quatro dias. O quadro clínico dos quatro ovinos caracterizou-se por apatia, acentuado aumento de volume da face, da porção ventral do pescoço e do peito, leve aumento de volume da porção proximal dos membros anteriores, tempo de sangramento aumentado, taquicardia, mucosas pálidas e grande quantidade de sangue não digerido nas fezes. Ao exame laboratorial observou-se principalmente redução das proteínas plasmáticas e aumento de creatinaquinase em todos os ovinos. À necropsia, foram observados extensos hematomas nas áreas correspondentes ao aumento de volume subcutâneo. Observaram-se petéquias, equimoses e sufusões leves a moderadas na serosa de diversos órgãos e acúmulo de sangue em meio às fezes na porção final do reto. Além de hemorragias, a principal alteração histopatológica observada foi necrose das fibras musculares esqueléticas e da parede de vasos, nas áreas próximas à inoculação do veneno. Nos ovinos deste estudo o aumento de volume, observado na face, pescoço, peito e membros, era constituído por sangue.

Spontaneous envenoming by snake bite is described as a cause of death in domestic animals. However, there are just few information about the species of snake involved, course, and clinicopathological and laboratory findings. Thus, this research aimed to determine the clinicopathological and laboratory changes induced by Bothrops moojeni and Bothropoides neuwiedi snake venoms in sheep, in order to provide additional information regarding snakebites in farm animals and to help establish the diagnosis of this condition. The lyophilized snake venoms were dissolved in 1mL saline solution and administered subcutaneously into the right face of four sheep, at doses of 0.41mg/kg and 0.82mg/kg of B. moojeni venom for two sheep, and 1.0mg/kg of B. neuwiedi venom for two other sheep. Only the sheep which had received the lowest dose (0.41mg/kg) survived, but developed severe clinical signs, similar to the others. First clinical signs were observed about 10 minutes after inoculation in all sheep. The course varied from 2 to 4 days. The clinical findings in all sheep were characterized by apathy, marked swelling of the face, the ventral neck and esternal region, and mild swelling of the proximal portion of the forelimbs, as well as increased bleeding time, tachycardia, pale mucous membranes, and large quantity of undigested blood in the intestinal lumen. Laboratory exams showed mainly a reduction in serum protein and increased creatine kinase in all sheep. At necropsy, extensive hematomas were observed in the subcutaneous tissue of the swollen areas. Also petechiae, bruises and mild to moderate hemorrhagic suffusions on the serosa of various organs, and blood within the intestinal contents of the distal rectum were observed. In addition to hemorrhages, the main histopathological changes were necrosis of skeletal muscle fibers and blood vessel walls next to the inoculation site. The swollen areas on face, neck, sternum and limbs of the sheep were due the hematomas.