Glutamine ameliorates Bungarus multicinctus venom-induced lung and heart injury through HSP70: NF-κB p65 and P53/PUMA signaling pathways involved

Background: Bungarus multicinctus is one of the most dangerous venomous snakes prone to cardiopulmonary damage with extremely high mortality. In our previous work, we found that glutamine (Gln) and glutamine synthetase (GS) in pig serum were significantly reduced after Bungarus multicinctus bite. In the present study, to explore whether there is a link between the pathogenesis of cardiopulmonary injury and Gln metabolic changes induced by Bungarus multicinctus venom. We investigated the effect of Gln supplementation on the lung and heart function after snakebite. Methods: We supplemented different concentrations of Gln to mice that were envenomated by Bungarus multicinctus to observe the biological behavior, survival rate, hematological and pathological changes. Gln was supplemented immediately or one hour after the venom injection, and then changes in Gln metabolism were analyzed. Subsequently, to further explore the protective mechanism of glutamine on tissue damage, we measured the expression of heat-shock protein70 (HSP70), NF-κB P65, P53/PUMA by western blotting and real-time polymerase in the lung and heart. Results: Gln supplementation delayed the envenoming symptoms, reduced mortality, and alleviated the histopathological changes in the heart and lung of mice bitten by Bungarus multicinctus. Additionally, Gln increased the activity of glutamine synthetase (GS), glutamate dehydrogenase (GDH) and glutaminase (GLS) in serum. It also balanced the transporter SLC7A11 expression in heart and lung tissues. Bungarus multicinctus venom induced the NF-κB nuclear translocation in the lung, while the HO-1 expression was suppressed. At the same time, venom activated the P53/PUMA signaling pathway and the BAX expression in the heart. Gln treatment reversed the above phenomenon and increased HSP70 expression. Conclusion: Gln alleviated the glutamine metabolism disorder and cardiopulmonary damage caused by Bungarus multicinctus venom. It may protect lungs and heart against venom by promoting the expression of HSP70, inhibiting the activation of NF-κB and P53/PUMA, thereby delaying the process of snake venom and reducing mortality. The present results indicate that Gln could be a potential treatment for Bungarus multicinctus bite.

Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Abstract Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.

Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.(AU)

Maintenance of venomous snakes in captivity for venom production at Butantan Institute from 1908 to the present: a scoping history

Maintenance of snakes at Butantan Institute started in the last century, intending to produce a different antivenom serum to reduce death caused by snakebites. Through a successful campaign coordinated by Vital Brazil, farmers sent venomous snakes to Butantan Institute by the railway lines with no cost. From 1908 to 1962, the snakes were kept in an outdoor serpentarium, where venom extraction was performed every 15 days. During this period, the snake average survival was 15 days. In 1963, the snakes were transferred to an adapted building, currently called Laboratory of Herpetology (LH), to be maintained in an intensive system. Although the periodicity of venom extraction remained the same, animal average survival increased to two months. With the severe serum crisis in 1983, the Ministry of Health financed remodeling for the three public antivenom producers, and with this support, the LH could be improved. Air conditioning and exhausting systems were installed in the rooms, besides the settlement of critical hygienic-sanitary managements to increase the welfare of snakes. In the early 1990s, snake survival was ten months. Over the years to the present day, several improvements have been made in the intensive serpentarium, as the establishment of two quarantines, feeding with thawed rodents, an interval of two months between venom extraction routines, and monitoring of snake health through laboratory tests. With these new protocols, average snake survival increased significantly, being eight years for the genus Bothrops, ten years for genus Crotalus and Lachesis, and four years for the genus Micrurus. Aiming the production of venoms of good quality, respect for good management practices is essential for the maintenance of snakes in captivity. New techniques and efficient management must always be sought to improve animal welfare, the quality of the venom produced, and the safety of those working directly with the venomous snakes.(AU)

Maintenance of venomous snakes in captivity for venom production at Butantan Institute from 1908 to the present: a scoping history

Maintenance of snakes at Butantan Institute started in the last century, intending to produce a different antivenom serum to reduce death caused by snakebites. Through a successful campaign coordinated by Vital Brazil, farmers sent venomous snakes to Butantan Institute by the railway lines with no cost. From 1908 to 1962, the snakes were kept in an outdoor serpentarium, where venom extraction was performed every 15 days. During this period, the snake average survival was 15 days. In 1963, the snakes were transferred to an adapted building, currently called Laboratory of Herpetology (LH), to be maintained in an intensive system. Although the periodicity of venom extraction remained the same, animal average survival increased to two months. With the severe serum crisis in 1983, the Ministry of Health financed remodeling for the three public antivenom producers, and with this support, the LH could be improved. Air conditioning and exhausting systems were installed in the rooms, besides the settlement of critical hygienic-sanitary managements to increase the welfare of snakes. In the early 1990s, snake survival was ten months. Over the years to the present day, several improvements have been made in the intensive serpentarium, as the establishment of two quarantines, feeding with thawed rodents, an interval of two months between venom extraction routines, and monitoring of snake health through laboratory tests. With these new protocols, average snake survival increased significantly, being eight years for the genus Bothrops, ten years for genus Crotalus and Lachesis, and four years for the genus Micrurus. Aiming the production of venoms of good quality, respect for good management practices is essential for the maintenance of snakes in captivity. New techniques and efficient management must always be sought to improve animal welfare, the quality of the venom produced, and the safety of those working directly with the venomous snakes.(AU)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

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)

De novo venom gland transcriptomics of Calliophis bivirgata flaviceps: uncovering the complexity of toxins from the Malayan blue coral snake

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)

King Cobra and snakebite envenomation: on the natural history, human-snake relationship and medical importance of Ophiophagus hannah

King Cobra (Ophiophagus hannah) has a significant place in many cultures, and is a medically important venomous snake in the world. Envenomation by this snake is highly lethal, manifested mainly by neurotoxicity and local tissue damage. King Cobra may be part of a larger species complex, and is widely distributed across Southeast Asia, southern China, northern and eastern regions as well as the Western Ghats of India, indicating potential geographical variation in venom composition. There is, however, only one species-specific King Cobra antivenom available worldwide that is produced in Thailand, using venom from the snake of Thai origin. Issues relating to the management of King Cobra envenomation (e.g., variation in the composition and toxicity of the venom, limited availability and efficacy of antivenom), and challenges faced in the research of venom (in particular proteomics), are rarely addressed. This article reviews the natural history and sociocultural importance of King Cobra, cases of snakebite envenomation caused by this species, current practice of management (preclinical and clinical), and major toxinological studies of the venom with a focus on venom proteomics, toxicity and neutralization. Unfortunately, epidemiological data of King Cobra bite is scarce, and venom proteomes reported in various studies revealed marked discrepancies in details. Challenges, such as inconsistency in snake venom sampling, varying methodology of proteomic analysis, lack of mechanistic and antivenomic studies, and controversy surrounding antivenom use in treating King Cobra envenomation are herein discussed. Future directions are proposed, including the effort to establish a standard, comprehensive Pan-Asian proteomic database of King Cobra venom, from which the venom variation can be determined. Research should be undertaken to characterize the toxin antigenicity, and to develop an antivenom with improved efficacy and wider geographical utility. The endeavors are aligned with the WHO´s roadmap that aims to reduce the disease burden of snakebite by 50% before 2030.(AU)

Epidemiological and clinical aspects of snakebites in the upper Juruá River region, western Brazilian Amazonia / Aspectos clínicos e epidemiológicos de acidentes ofídicos na região do Alto Juruá, oeste da Amazônia brasileira

This study addresses the clinical and epidemiological aspects of envenoming cases resulting from snakebites treated at a hospital in Cruzeiro do Sul, in the upper Juruá River region, western Brazilian Amazonia. The specific identity of snakes that caused the envenomings was inferred (a) from the diagnosis of patient symptoms and signs upon hospital admission, (b) by enzyme immunoassay for detection of Bothrops atrox and Lachesis muta venom from serum samples taken from patients before antivenom therapy, or (c) by direct identification of the snake, when it was brought along to the hospital or photographed. There were 133 snakebites (76.2 cases per 100,000 inhabitants) registered during one year (July 2017 to June 2018). Most snakebites (88.7%) were caused by Bothrops spp., and the rest by non-venomous snakes or dry bites. Snakebites tended to occur more often during the rainy season, coinciding with the period of greater reproductive activity of the snakes and greater availability of their prey. In addition, the increase in the water level of rivers and lakes during the rainy season tends to concentrate snakes in dry places and, thus, to increase encounters with humans. Information campaigns on prevention and first aid, specially among the most vulnerable groups (indigenous people, farmers, and children and teenagers in rural areas), and the importance of using protective equipment (boots, leggings, leather gloves) in certain high risk activities (e.g. agriculture and extractivism in forests) are fundamental for the reduction of snakebite morbidity.(AU)

Este estudo aborda os aspectos clínicos e epidemiológicos dos envenenamentos ofídicos tratados em um hospital em Cruzeiro do Sul, Acre, resultantes de acidentes que ocorreram na região do Alto Juruá, no oeste da Amazônia brasileira. A identidade específica das serpentes que causaram os envenenamentos foi inferida (a) pelos sinais e sintomas apresentados pelo paciente na admissão hospitalar, (b) por imunoensaio enzimático para detecção de veneno de Bothrops atrox e Lachesis muta em amostras de soro retiradas de pacientes antes da soroterapia, e (c) pela identificação direta da serpente, quando esta foi levada para o hospital ou fotografada. Houve 133 casos (76,2 casos por 100.000 habitantes) de acidentes ofídicos registrados durante um ano (julho 2017 a junho 2018). A maioria das picadas de serpentes (88,7%) foi causada por Bothrops spp., e o restante por espécies não peçonhentas ou picadas secas. Os acidentes ofídicos tenderam a ocorrer com maior frequência durante a estação chuvosa, coincidindo com o período de maior atividade reprodutiva das serpentes e maior disponibilidade de suas presas. Além disso, o aumento dos níveis de rios e lagos pode fazer com que esses animais procurem locais mais secos, aumentando a frequência de encontro com seres humanos. Campanhas educativas sobre prevenção e primeiros socorros, principalmente entre os grupos mais vulneráveis (indígenas, agricultores, crianças e adolescentes em áreas rurais), e sobre a importância da utilização de equipamentos de proteção (botas, perneiras, luvas de couro) em determinadas atividades de maior risco (e.g., agricultura e extrativismo em florestas), são fundamentais para reduzir a morbidade de picadas de serpentes.(AU)

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

Abstract Background: Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America. Methods: PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature. Results: The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia. Conclusion: A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than to sbγPLIs from non-venomous species from Asia. The physiological role played by sbγPLIs in non-venomous snake species remains to be understood. Further investigation is needed.(AU)

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Abstract Background: Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods: The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results: The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion: These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)

Crotamine in Crotalus durissus: distribution according to subspecies and geographic origin, in captivity or nature

Background:Crotalus durissus is considered one of the most important species of venomous snakes in Brazil, due to the high mortality of its snakebites. The venom of Crotalus durissus contains four main toxins: crotoxin, convulxin, gyroxin and crotamine. Venoms can vary in their crotamine content, being crotamine-negative or -positive. This heterogeneity is of great importance for producing antivenom, due to their different mechanisms of action. The possibility that antivenom produced by Butantan Institute might have a different immunorecognition capacity between crotamine-negative and crotamine-positive C. durissus venoms instigated us to investigate the differences between these two venom groups. Methods:The presence of crotamine was analyzed by SDS-PAGE, western blotting and ELISA, whereas comparison between the two types of venoms was carried out through HPLC, mass spectrometry analysis as well as assessment of antivenom lethality and efficacy. Results:The results showed a variation in the presence of crotamine among the subspecies and the geographic origin of snakes from nature, but not in captive snakes. Regarding differences between crotamine-positive and -negative venoms, some exclusive proteins are found in each pool and the crotamine-negative pool presented more phospholipase A2 than crotamine-positive pool. This variation could affect the time to death, but the lethal and effective dose were not affected. Conclusion:These differences between venom pools indicate the importance of using both, crotamine-positive and crotamine-negative venoms, to produce the antivenom.(AU)

Identification and characterization of the first endogenous phospholipase A2 inhibitor from a non-venomous tropical snake, Boa constrictor (Serpentes: Boidae)

Background:Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America.Methods:PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature.Results:The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia.Conclusion:A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than...(AU)

Cytotoxic and anticancer properties of the Malaysian mangrove pit viper (Trimeresurus purpureomaculatus) venom and its disintegrin (purpureomaculin)

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)

Cytotoxic and anticancer properties of the Malaysian mangrove pit viper (Trimeresurus purpureomaculatus) venom and its disintegrin (purpureomaculin)

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)

Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase

Background:Lachesis muta rhombeata is one of the venomous snakes of medical importance in Brazil whose envenoming is characterized by local and systemic effects which may produce even shock and death. Its venom is mainly comprised of serine and metalloproteinases, phospholipases A2 and bradykinin-potentiating peptides. Based on a previously reported fractionation of L. m. rhombeata venom (LmrV), we decided to perform a subproteome analysis of its major fraction and investigated a novel component present in this venom.Methods:LmrV was fractionated through molecular exclusion chromatography and the main fraction (S5) was submitted to fibrinogenolytic activity assay and fractionated by reversed-phase chromatography. The N-terminal sequences of the subfractions eluted from reversed-phase chromatography were determined by automated Edman degradation. Enzyme activity of LmrSP-4 was evaluated upon chromogenic substrates for thrombin (S-2238), plasma kallikrein (S-2302), plasmin and streptokinase-activated plasminogen (S-2251) and Factor Xa (S-2222) and upon fibrinogen. All assays were carried out in the presence or absence of possible inhibitors. The fluorescence resonance energy transfer substrate Abz-KLRSSKQ-EDDnp was used to determine the optimal conditions for LmrSP-4 activity. Molecular mass of LmrSP-4 was determined by MALDI-TOF and digested peptides after trypsin and Glu-C treatments were analyzed by high resolution MS/MS using different fragmentation modes.Results:Fraction S5 showed strong proteolytic activity upon fibrinogen. Its fractionation by reversed-phase chromatography gave rise to 6 main fractions (S5C1-S5C6). S5C1-S5C5 fractions correspond to serine proteinases whereas S5C6 represents a C-type lectin. S5C4 (named LmrSP-4) had its N-terminal determined by Edman degradation up to the 53rd amino acid residue and was chosen for characterization studies. LmrSP-4 is a fibrinogenolytic...(AU)

Divergences between the Brazilian national information systems for recording deaths from venomous animals

This paper aims to highlight and analyze discrepancies in reporting of deaths due to venomous animals in Brazil, from 2001 to 2015, between two national information systems: The Notifiable Diseases Information System (Sistema de Informação de Agravos de Notificação - SINAN) and the Mortality Information System (Sistema de Informações sobre Mortalidade - SIM). Methods: Descriptive and comparative study of the SINAN and SIM information systems, was conducted via the following steps: collecting the death notices from SINAN and SIM; constructing tables and comparative graphics; and, only in scorpion sting fatalities, analyzing the distribution of deaths by age group as described in the specialized literature. Results: While SINAN identifies strong growth in the number of deaths from scorpion stings, SIM shows greater increase in the number of reported deaths from bee stings, especially in the South and Southeast regions. Notably, bees are the sole etiological agent that received more reports in SIM than in SINAN for every year in the period studied. The age-group distribution of the data on deaths from scorpion stings reinforced the indication of problems occurring in their registration in SINAN, especially since 2007, which may have an effect on analyses based on these data. Conclusion: Comparative analysis of these databases permits identification of important differences between profiles presented by these systems, which have equal relevance for Brazil as a whole and for its regions. These differences may influence the construction of various scenarios.(AU)

Venomous animals of state of Piauí: epidemiology of accidents and list of medical importance species / Animais peçonhentos do estado do Piauí: epidemiologia dos acidentes e lista de espécies de importância médica

Os casos de envenenamentos aumentaram consideravelmente no Brasil, no entanto, para o estado do Piauí apenas dois estudos foram realizados até o momento. Aqui, investigamos, através dos formulários de notificação, as características epidemiológicas dos casos de envenenamentos ocorridos durante 11 anos na macrorregião de Picos, estado do Piauí, Nordeste do Brasil. Além disso, também verificamos se as espécies registradas nos formulários de notificação correspondem às espécies que ocorrem na região. Registramos 1.249 casos de envenenamentos, sendo 41% causados por escorpiões, 35% causados por serpentes e 24% causados por aranhas. Na maioria dos casos (aracnídeos = 99%, escorpiões = 93%, serpentes = 52%) não houve a identificação da espécie causadora do acidente. A maioria das vítimas dos acidentes (66%) eram indivíduos do sexo masculino entre 18 e 24 anos de áreas rurais, picadas durante atividades de campo na estação chuvosa (janeiro a março). Os locais de lesão mais comuns foram os membros inferiores (37%) e a maioria (53%) dos acidentes foi do tipo leve. Este é o primeiro estudo que reporta o perfil epidemiológico de uma série de envenenamentos (incluindo aracnídeos e serpentes) durante 11 anos de ocorrência no estado do Piauí. Além disso, também apresentamos a primeira lista de espécies de aracnídeos e serpentes de importância médica para o estado. Nossos resultados demonstram que houve uma alta incidência de acidentes por animais peçonhentos na região centro-sul do estado, a maioria dos registros não foi notificada no SINAN, a maioria dos registros não apresentou a identificação das espécies responsáveis pelo acidente, e há registros cujas espécies foram identificadas incorretamente.

Cases of poisoning have increased considerably in Brazil, however for state of Piauí only two studies were carried out so far. Here, we investigated, through the notification forms, the epidemiological characteristics of cases of poisoning that occurred during 11 years in a macro-region of Picos, state of Piauí, Northeastern Brazil. Furthermore, we also check whether the species recorded in the notification forms correspond to the species that occur in the region. We recorded 1.249 cases of envenomations, being 41% caused by scorpions, 35% caused by snakes and 24% caused by spiders. Most cases (arachnids = 99%, scorpions = 93%, snakes = 52%) there was no identification of species causing the accident. Most of the victims of the accidents (66%) were male individuals between 18 and 24 years from rural areas, stung during field activities in the rainy season (January-March). The most common injury sites were the lower limbs (37%) and the majority (53%) of the accidents was the mild type. This is the first study that reports the epidemiological profile of a series of envenomations (including arachnids and snakes) during 11 years of records in the state of Piauí. Furthermore, we also present the first list of species of arachnids and snakes of medical importance to the state. Our results demonstrate that there was a high incidence of accidents by venomous animals in the Center-South region of the state, most of the records was not notified in SINAN, most records does not present the identification of the species responsible for the accident, and there are records whose species was incorrectly identified.

Venomous animals of state of Piauí: epidemiology of accidents and list of medical importance species / Animais peçonhentos do estado do Piauí: epidemiologia dos acidentes e lista de espécies de importância médica

Os casos de envenenamentos aumentaram consideravelmente no Brasil, no entanto, para o estado do Piauí apenas dois estudos foram realizados até o momento. Aqui, investigamos, através dos formulários de notificação, as características epidemiológicas dos casos de envenenamentos ocorridos durante 11 anos na macrorregião de Picos, estado do Piauí, Nordeste do Brasil. Além disso, também verificamos se as espécies registradas nos formulários de notificação correspondem às espécies que ocorrem na região. Registramos 1.249 casos de envenenamentos, sendo 41% causados por escorpiões, 35% causados por serpentes e 24% causados por aranhas. Na maioria dos casos (aracnídeos = 99%, escorpiões = 93%, serpentes = 52%) não houve a identificação da espécie causadora do acidente. A maioria das vítimas dos acidentes (66%) eram indivíduos do sexo masculino entre 18 e 24 anos de áreas rurais, picadas durante atividades de campo na estação chuvosa (janeiro a março). Os locais de lesão mais comuns foram os membros inferiores (37%) e a maioria (53%) dos acidentes foi do tipo leve. Este é o primeiro estudo que reporta o perfil epidemiológico de uma série de envenenamentos (incluindo aracnídeos e serpentes) durante 11 anos de ocorrência no estado do Piauí. Além disso, também apresentamos a primeira lista de espécies de aracnídeos e serpentes de importância médica para o estado. Nossos resultados demonstram que houve uma alta incidência de acidentes por animais peçonhentos na região centro-sul do estado, a maioria dos registros não foi notificada no SINAN, a maioria dos registros não apresentou a identificação das espécies responsáveis pelo acidente, e há registros cujas espécies foram identificadas incorretamente.(AU)

Cases of poisoning have increased considerably in Brazil, however for state of Piauí only two studies were carried out so far. Here, we investigated, through the notification forms, the epidemiological characteristics of cases of poisoning that occurred during 11 years in a macro-region of Picos, state of Piauí, Northeastern Brazil. Furthermore, we also check whether the species recorded in the notification forms correspond to the species that occur in the region. We recorded 1.249 cases of envenomations, being 41% caused by scorpions, 35% caused by snakes and 24% caused by spiders. Most cases (arachnids = 99%, scorpions = 93%, snakes = 52%) there was no identification of species causing the accident. Most of the victims of the accidents (66%) were male individuals between 18 and 24 years from rural areas, stung during field activities in the rainy season (January-March). The most common injury sites were the lower limbs (37%) and the majority (53%) of the accidents was the mild type. This is the first study that reports the epidemiological profile of a series of envenomations (including arachnids and snakes) during 11 years of records in the state of Piauí. Furthermore, we also present the first list of species of arachnids and snakes of medical importance to the state. Our results demonstrate that there was a high incidence of accidents by venomous animals in the Center-South region of the state, most of the records was not notified in SINAN, most records does not present the identification of the species responsible for the accident, and there are records whose species was incorrectly identified.(AU)

Acidentes botrópicos em cães / Bothrops snakebites in dogs

Background: Snakebites occur frequently among humans and animals. In Brazil, about 23,000 cases of snakebite involving humans were recorded in 2016, 11% of them caused by venomous snakes. In the region of Cuiabá, capital of MatoGrosso, the highest occurrence is of snakes of the genus Bothrops (81%), followed by Crotalus (4%). Bothrops venom hasproteolytic, coagulant, and hemorrhagic effects, whereas Crotalus venom is neurotoxic, myotoxic and nephrotoxic. Thispaper reports on three cases of Bothrops snakebites in dogs treated at the Veterinary Hospital of the Federal Universityof Mato Grosso in 2017.Cases: Three dogs were treated showing clinical signs of snakebite, possibly by Bothrops, since their owners reportedthat these are the snakes most frequently found where they live. This information is consistent with the known geographicdistribution of snakes of the genus Bothrops in the state of Mato Grosso, Brazil. In the first report (Case 11), the patientwas bitten on two different occasions in a five-month interval, and died presumably due to anaphylactic shock causedby the venom injected by a second snakebite (Case 12). In both episodes, the animal presented a swollen face and neck,dyspnea and eye bleeding, and these signs were more severe in the second episode. Case 2 was a dog with severe neckand chest swelling and pain. The owner of this dog reported that he had taken his dog for a walk in the countryside, alongwith the dog of Case 1, and had returned home with both dogs about three hours before the consultation. Case 3 was aRottweiler living at a small family farm located 67 km from Cuiabá. The animal presented slight swelling and pain in theleft forelimb, and puncture wounds on the lower limb. Neutrophilic leukocytosis was observed in cases 1 and 3. In addition, Case 12 (the second episode of Case 1) presented thrombocytopenia and increased prothrombin time and activatedpartial...