An alternative method for comparing hemorrhagic activity of snake venoms following one of the 3R's principles.

Venom-induced hemorrhage analysis usually is performed by Minimum Hemorrhagic Dose (MHD), however a similar method can be used to compare venoms with fewer laboratory animals. Our work compared the MHD of five different venoms, with the size of hemorrhagic spot, finding good correlations in the results. Considering the 3Rs principle, we propose the use of the hemorrhagic spot method to compare hemorrhagic activity of snake venoms, rather than using the MHD method, since the first one needs 5 times less animals than the other.

Fibrinogenolytic potential of venoms of medically important Brazilian snakes.

One of the main clinical manifestations presented by victims of snake bite envenoming are coagulation disorders. Considering that fibrinogen is a key molecule for crosslinked fibrin clot formation, the objective of this work was the quantitative analysis of the fibrinogenolytic activity of snakes of medical importance in Brazil and neutralization by specific antivenom. For this, pools of three genera of medical importance (Bothrops, Crotalus and Lachesis) that are used for the production of antivenom were used, and three pools of species of the genus Bothrops that are not part of the pool for the production of antivenom. The Lachesis pool had the highest fibrinogenolytic activity, even demonstrating partial cleavage (42.9 % consumption) of the fibrinogen gamma chain. The Bothrops genus venom pools have shown subtle variations between them. The Crotalus pool, despite not showing total cleavage of any fibrinogen chain, began cleavage of fibrinogen by the beta chain. The specific antivenoms used were able to delay the cleavage of fibrinogen in all the venoms used, which could be the first step towards implementing previous in vitro tests to analyze the quality of the batches of antivenoms produced, thus potentially reducing the use of animals used in this process.

Analysis of the genus B othrops snake venom: An inter and intraspecific comparative study.

The genus Bothrops are considered Category 1 of medical importance by the World Health Organization, responsible for approximately 85 % of snakebites occurring throughout Brazil. Main factors determining snake venom variations can be genetics, diet, gender, geographic distribution, age, or even seasonality. In this study, we compared the composition of protein profile, biochemical activities, and immunorecognition of toxins present in the venom of eight adults of Bothrops species (B. alternatus, B. atrox, B. jararaca, B. jararacussu, B. leucurus, B. moojeni, B. neuwiedi and B. pauloensis). The following methods were used to analyze the venoms: protein dosage; electrophoresis in polyacrylamide gel containing SDS; High Performance Liquid Chromatography - Reverse Phase; enzymatic activities, western blotting and Enzyme Linked Immuno Sorbent Assay. The results show inter and intraspecific differences in the electrophoretic profile. LAAO and PLA2 activities, in general, were higher in males than females and proteolytic activity was higher in females than males. The bothropic antivenom produced by Instituto Butantan recognized most of the protein bands in all Bothrops species analyzed, with only the regions between 37 and 25 kDa presenting lower intensity. A notable variability in the chromatograms was observed. Bothrops venom demonstrated inter-intraspecific disparities in protein composition and biochemical activity.

Seasonality in Crotalus durissus venom.

Rattlesnakes belonging to the genus Crotalus are widely distributed throughout the Americas. In Brazil, symptoms commonly associated with envenomation by Crotalus durissus collilineatus include myalgia, rhabdomyolysis, renal failure, neurotoxicity, and progressive paralysis, which are related to the protein composition of this venom. Snake venom composition exhibits compositional variability that may reflect geographic distribution, age, captivity, diet, sex, and even individual genetics. Although seasonality is also considered a possible source of variation, there are few reports of such variability in snake venom. In this work, venoms of the same eight C. durissus collilineatus were extracted every three months for two years, to analyze seasonal changes in composition and activities. To this end, venom composition was analyzed by protein quantification, SDS-PAGE, and HPLC, and the LAAO, PLA2 and coagulant activities were measured. Venoms of these C. d. collilineatus showed minor seasonal differences in venom activities and no composition differences were found. LAAO and coagulant activities displayed a pattern of seasonal change, while PLA2 activity seemed to have no seasonality tendency. Also, there are sexual differences, in which males seem to be more stable than females in regard to some activities. Individual variability occurs even in seasonal variation of activities, highlighting the importance of controlling circumstances of venom extraction before comparing results between groups of snakes.

Proteomics and life-history variability of Endogenous Phospholipases A2 Inhibitors (PLIs) in Bothrops jararaca plasma.

In Brazil, the genus Bothrops is responsible for most ophidian accidents. Snake venoms have a wide variety of proteins and peptides exhibiting a broad repertoire of pharmacological and toxic effects that elicit systemic injury and characteristic local effects. The snakes' natural resistance to envenomation caused by the presence of inhibitory compounds on their plasma have been extensively studied. However, the presence of these inhibitors in different developmental stages is yet to be further discussed. The aim of this study was to evaluate the ontogeny of Bothrops jararaca plasma inhibitor composition and, to this end, plasma samples of B. jararaca were obtained from different developmental stages (neonates, youngs, and adults) and sexes (female and male). SDS-PAGE, Western blotting, affinity chromatography, and mass spectrometry were performed to analyze the protein profile and interaction between B. jararaca plasma and venom proteins. In addition, the presence of γBjPLI, a PLA2 inhibitor previously identified and characterized in B. jararaca serum, was confirmed by Western blotting. According to our results, 9-17% of plasma proteins were capable of binding to venom proteins in the three developmental stages. The presence of different endogenous inhibitors and, more specifically, different PLA2 inhibitor (PLI) classes and antihemorrhagic factors were confirmed in specimens of B. jararaca from newborn by mass spectrometry. For the first time, the αPLI and ßPLI were detected in B. jararaca plasma, although low or no ontogenetic and sexual correlation were found. The γPLI were more abundant in adult female, than in neonate and young female, but similar to neonate, young and adult male according to the results of mass spectrometry analysis. Our results suggest that there are proteins in the plasma of these animals that can help counteract the effects of self-envenomation from birth.

Analysis of the genus Bothrops snake venom: an inter and intraspecific comparative study

The genus Bothrops are considered Category 1 of medical importance by the World Health Organization, responsible for approximately 85 % of snakebites occurring throughout Brazil. Main factors determining snake venom variations can be genetics, diet, gender, geographic distribution, age, or even seasonality. In this study, we compared the composition of protein profile, biochemical activities, and immunorecognition of toxins present in the venom of eight adults of Bothrops species (B. alternatus, B. atrox, B. jararaca, B. jararacussu, B. leucurus, B. moojeni, B. neuwiedi and B. pauloensis). The following methods were used to analyze the venoms: protein dosage; electrophoresis in polyacrylamide gel containing SDS; High Performance Liquid Chromatography – Reverse Phase; enzymatic activities, western blotting and Enzyme Linked Immuno Sorbent Assay. The results show inter and intraspecific differences in the electrophoretic profile. LAAO and PLA2 activities, in general, were higher in males than females and proteolytic activity was higher in females than males. The bothropic antivenom produced by Instituto Butantan recognized most of the protein bands in all Bothrops species analyzed, with only the regions between 37 and 25 kDa presenting lower intensity. A notable variability in the chromatograms was observed. Bothrops venom demonstrated inter-intraspecific disparities in protein composition and biochemical activity.

Fibrinogenolytic potential of venoms of medically important Brazilian snakes

One of the main clinical manifestations presented by victims of snake bite envenoming are coagulation disorders. Considering that fibrinogen is a key molecule for crosslinked fibrin clot formation, the objective of this work was the quantitative analysis of the fibrinogenolytic activity of snakes of medical importance in Brazil and neutralization by specific antivenom. For this, pools of three genera of medical importance (Bothrops, Crotalus and Lachesis) that are used for the production of antivenom were used, and three pools of species of the genus Bothrops that are not part of the pool for the production of antivenom. The Lachesis pool had the highest fibrinogenolytic activity, even demonstrating partial cleavage (42.9 % consumption) of the fibrinogen gamma chain. The Bothrops genus venom pools have shown subtle variations between them. The Crotalus pool, despite not showing total cleavage of any fibrinogen chain, began cleavage of fibrinogen by the beta chain. The specific antivenoms used were able to delay the cleavage of fibrinogen in all the venoms used, which could be the first step towards implementing previous in vitro tests to analyze the quality of the batches of antivenoms produced, thus potentially reducing the use of animals used in this process.

Red-on-yellow queen bio-layer interferometry reveals functional diversity within micrurus venoms and toxin resistance in prey species

Snakes in the family Elapidae largely produce venoms rich in three-fnger toxins (3FTx) that bind to the α1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specifc mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specifcity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan→serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Red‑on‑yellow queen: bio‑layer interferometry reveals functional diversity within Micrurus venoms and toxin resistance in prey species

Snakes in the family Elapidae largely produce venoms rich in three-fnger toxins (3FTx) that bind to the α1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specifc mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specifcity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan→serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Seasonality in Crotalus durissus venom

Rattlesnakes belonging to the genus Crotalus are widely distributed throughout the Americas. In Brazil, symptoms commonly associated with envenomation by Crotalus durissus collilineatus include myalgia, rhabdomyolysis, renal failure, neurotoxicity, and progressive paralysis, which are related to the protein composition of this venom. Snake venom composition exhibits compositional variability that may reflect geographic distribution, age, captivity, diet, sex, and even individual genetics. Although seasonality is also considered a possible source of variation, there are few reports of such variability in snake venom. In this work, venoms of the same eight C. durissus collilineatus were extracted every three months for two years, to analyze seasonal changes in composition and activities. To this end, venom composition was analyzed by protein quantification, SDS-PAGE, and HPLC, and the LAAO, PLA2 and coagulant activities were measured. Venoms of these C. d. collilineatus showed minor seasonal differences in venom activities and no composition differences were found. LAAO and coagulant activities displayed a pattern of seasonal change, while PLA2 activity seemed to have no seasonality tendency. Also, there are sexual differences, in which males seem to be more stable than females in regard to some activities. Individual variability occurs even in seasonal variation of activities, highlighting the importance of controlling circumstances of venom extraction before comparing results between groups of snakes.

Proteomics and life-history variability of Endogenous Phospholipases A2 Inhibitors (PLIs) in Bothrops jararaca plasma

In Brazil, the genus Bothrops is responsible for most ophidian accidents. Snake venoms have a wide variety of proteins and peptides exhibiting a broad repertoire of pharmacological and toxic effects that elicit systemic injury and characteristic local effects. The snakes’ natural resistance to envenomation caused by the presence of inhibitory compounds on their plasma have been extensively studied. However, the presence of these inhibitors in different developmental stages is yet to be further discussed. The aim of this study was to evaluate the ontogeny of Bothrops jararaca plasma inhibitor composition and, to this end, plasma samples of B. jararaca were obtained from different developmental stages (neonates, youngs, and adults) and sexes (female and male). SDS-PAGE, Western blotting, affinity chromatography, and mass spectrometry were performed to analyze the protein profile and interaction between B. jararaca plasma and venom proteins. In addition, the presence of γBjPLI, a PLA2 inhibitor previously identified and characterized in B. jararaca serum, was confirmed by Western blotting. According to our results, 9–17% of plasma proteins were capable of binding to venom proteins in the three developmental stages. The presence of different endogenous inhibitors and, more specifically, different PLA2 inhibitor (PLI) classes and antihemorrhagic factors were confirmed in specimens of B. jararaca from newborn by mass spectrometry. For the first time, the αPLI and βPLI were detected in B. jararaca plasma, although low or no ontogenetic and sexual correlation were found. The γPLI were more abundant in adult female, than in neonate and young female, but similar to neonate, young and adult male according to the results of mass spectrometry analysis. Our results suggest that there are proteins in the plasma of these animals that can help counteract the effects of self-envenomation from birth.

Biochemical and toxicological profiles of venoms from an adult female South American bushmaster (Lachesis muta rhombeata) and her offspring

In this work, we compared the biochemical and toxicological profiles of venoms from an adult female specimen of Lachesis muta rhombeata (South American bushmaster) and her seven offspring born in captivity, based on SDS-PAGE, RP-HPLC, enzymatic, coagulant, and hemorrhagic assays. Although adult and juvenile venoms showed comparable SDS-PAGE profiles, juveniles lacked some chromatographic peaks compared with adult venom. Adult venom had higher proteolytic (caseinolytic) activity than juvenile venoms (p < 0.05), but there were no significant inter-venom variations in the esterase, PLA2, phosphodiesterase and L-amino acid oxidase (LAAO) activities, although the latter activity was highly variable among the venoms. Juveniles displayed higher coagulant activity on human plasma, with a minimum coagulant dose ∼42% lower than the adult venom (p < 0.05), but there were no age-related differences in thrombin-like activity. Adult venom was more fibrinogenolytic (based on the rate of fibrinogen chain degradation) and hemorrhagic than juvenile venoms (p < 0.05). The effective dose of Bothrops/Lachesis antivenom (produced by the Instituto Butantan) needed to neutralize the coagulant activity was ∼57% greater for juvenile venoms (p < 0.05), whereas antivenom did not attenuate the thrombin-like activity of juvenile and adult venoms. Antivenom significantly reduced the hemorrhagic activity of adult venom (400 μg/kg, i. d.), but not that of juvenile venoms. Overall, these data indicate a compositional and functional ontogenetic shift in L. m. rhombeata venom.

Children and Snakebite: Snake Venom Effects on Adult and Paediatric Plasma.

Snakebite is a globally neglected tropical disease, with coagulation disturbances being the primary pathology of many deadly snake venoms. Age-related differences in human plasma have been abundantly reported, yet the effect that these differences pose regarding snakebite is largely unknown. We tested for differences in coagulotoxic effects (via clotting time) of multiple snake venoms upon healthy human adult (18+) and paediatric (median 3.3 years old) plasma in vivo and compared these effects to the time it takes the plasmas to clot without the addition of venom (the spontaneous clotting time). We tested venoms from 15 medically significant snake species (from 13 genera) from around the world with various mechanisms of coagulotoxic actions, across the three broad categories of procoagulant, pseudo-procoagulant, and anticoagulant, to identify any differences between the two plasmas in their relative pathophysiological vulnerability to snakebite. One procoagulant venom (Daboia russelii, Russell's Viper) produced significantly greater potency on paediatric plasma compared with adult plasma. In contrast, the two anticoagulant venoms (Pseudechis australis, Mulga Snake; and Bitis cornuta, Many-horned Adder) were significantly more potent on adult plasma. All other procoagulant venoms and all pseudo-procoagulant venoms displayed similar potency across both plasmas. Our preliminary results may inform future studies on the effect of snake venoms upon plasmas from different age demographics and hope to reduce the burden of snakebite upon society.

The antineoplastic potential of crotoxin isolated from Crotalus durissus terrificus snake venom on oral squamous cell carcinoma.

This study investigated the antineoplastic effects of crotoxin isolated from snake venom of the South American Crotalus durissus terrificus in oral cancer cell lines and in an animal model of chemically induced oral cancer. We analyzed cell viability and death, clonogenic formation, DNA fragmentation, migration assay, and gene expression of MMP2, MMP9, COL1A1, and CASP3. In the animal model, after induction of oral cancer by 4-nitroquinoline-1-oxide carcinogen, mice were treated with crotoxin to investigate its effects on tumor development in tongue and oral mucosa. Crotoxin inhibited cell proliferation, viability, colony formation, and migration, favoring cell death. Furthermore, crotoxin increased caspase-3 expression, decreased Ki-67 protein and mRNA expression of MMP2, MMP9, and COL1A1. Mice treated with crotoxin at 10 µg/kg did not alter biochemical parameters total cholesterol, very-low-density lipoprotein, high-density lipoprotein, liver transaminases, glycemia, creatinine, and urea. Crotoxin treatment significantly reduced the frequency of oral squamous cell carcinoma lesions by 50%. Thus, this study highlights crotoxin as a promising chemotherapeutic substance, considering its effects on controlling the neoplastic cell population, reducing cell migration, and inhibiting tumor development. Clinical studies are necessary to understand better the impact of crotoxin as a potential adjuvant therapeutic agent for oral cancer patients.

Unveiling the peptidome diversity of Lachesis muta snake venom: discovery of novel fragments of metalloproteinase, l-amino acid oxidase, and bradykinin potentiating peptides

Snake venoms are known to be major sources of peptides with different pharmacological properties. In this study, we comprehensively explored the venom peptidomes of three specimens of Lachesis muta, the largest venomous snake in South America, using mass spectrometry techniques. The analysis revealed 19 main chromatographic peaks common to all specimens. A total of 151 peptides were identified, including 69 from a metalloproteinase, 58 from the BPP-CNP precursor, and 24 from a l-amino acid oxidase. To our knowledge, 126 of these peptides were reported for the first time in this work, including a new SVMP-derived peptide fragment, Lm-10a. Our findings highlight the dynamic nature of toxin maturation in snake venoms, driven by proteolytic processing, post-translational modifications, and cryptide formation.

Children and snakebite: snake venom effects on adult and paediatric plasma

Snakebite is a globally neglected tropical disease, with coagulation disturbances being the primary pathology of many deadly snake venoms. Age-related differences in human plasma have been abundantly reported, yet the effect that these differences pose regarding snakebite is largely unknown. We tested for differences in coagulotoxic effects (via clotting time) of multiple snake venoms upon healthy human adult (18+) and paediatric (median 3.3 years old) plasma in vivo and compared these effects to the time it takes the plasmas to clot without the addition of venom (the spontaneous clotting time). We tested venoms from 15 medically significant snake species (from 13 genera) from around the world with various mechanisms of coagulotoxic actions, across the three broad categories of procoagulant, pseudo-procoagulant, and anticoagulant, to identify any differences between the two plasmas in their relative pathophysiological vulnerability to snakebite. One procoagulant venom (Daboia russelii, Russell’s Viper) produced significantly greater potency on paediatric plasma compared with adult plasma. In contrast, the two anticoagulant venoms (Pseudechis australis, Mulga Snake; and Bitis cornuta, Many-horned Adder) were significantly more potent on adult plasma. All other procoagulant venoms and all pseudo-procoagulant venoms displayed similar potency across both plasmas. Our preliminary results may inform future studies on the effect of snake venoms upon plasmas from different age demographics and hope to reduce the burden of snakebite upon society.

The antineoplastic potential of crotoxin isolated from Crotalus durissus terrificus snake venom on oral squamous cell carcinoma

This study investigated the antineoplastic effects of crotoxin isolated from snake venom of the South American Crotalus durissus terrificus in oral cancer cell lines and in an animal model of chemically induced oral cancer. We analyzed cell viability and death, clonogenic formation, DNA fragmentation, migration assay, and gene expression of MMP2, MMP9, COL1A1, and CASP3. In the animal model, after induction of oral cancer by 4-nitroquinoline-1-oxide carcinogen, mice were treated with crotoxin to investigate its effects on tumor development in tongue and oral mucosa. Crotoxin inhibited cell proliferation, viability, colony formation, and migration, favoring cell death. Furthermore, crotoxin increased caspase-3 expression, decreased Ki-67 protein and mRNA expression of MMP2, MMP9, and COL1A1. Mice treated with crotoxin at 10 μg/kg did not alter biochemical parameters total cholesterol, very-low-density lipoprotein, high-density lipoprotein, liver transaminases, glycemia, creatinine, and urea. Crotoxin treatment significantly reduced the frequency of oral squamous cell carcinoma lesions by 50%. Thus, this study highlights crotoxin as a promising chemotherapeutic substance, considering its effects on controlling the neoplastic cell population, reducing cell migration, and inhibiting tumor development. Clinical studies are necessary to understand better the impact of crotoxin as a potential adjuvant therapeutic agent for oral cancer patients.

Analyzing the influence of age and sex in Bothrops pauloensis snake venom.

Considerable heterogeneity and ontogenetic changes in venom composition have already been observed in different species of snakes within the Viperidae family. Since the venom of young and adult can cause distinct pathological effects and because the antivenom may be less effective in neutralizing envenoming by young snakes compared to adults, it is of paramount importance to understand the ontogenetic variation of snake venom. Thus, the present study aimed to analyze and compare the venom of Bothrops pauloensis snakes, searching for possible influences of ontogeny and sex in their biochemical and biological aspects. The venom of younger individuals was more complex in relation to high molecular mass proteins, with a greater abundance of metalloproteinases, while adults showed a greater abundance of medium and low molecular mass proteins, such as phospholipases A2 (PLA2), C-type lectins and serine proteases. The antivenom showed better immunorecognition towards the venom of adult snakes than younger ones, in addition to a deficiency in the recognition of medium molecular mass proteins, suggesting the need for an improvement in the antivenom. Younger snakes showed higher coagulant, caseinolytic, and hemorrhagic activity, while adult snakes showed higher L-amino acid oxidase (LAAO) activity and acted faster in lethality. Differences between males and females were observed mainly in the rate of loss of coagulant activity, change in PLA2 activity and lethality action time. Furthermore, considering only the adult groups, males showed a higher LAAO and thrombin-like activity, while females showed a higher caseinolytic and hyaluronidase activity. With the results obtained in this work, it was possible to conclude that there is an ontogenetic variation in the composition and some activities of the B. pauloensis snake venom, in addition to differences between the venom of males and females, reinforcing that there is an intraspecific variation that may result in different symptoms in their envenoming and, consequently, differences in the response to treatment with the antivenom.