Snake Venom Toxicity Research Articles

Lethality associated with snake venom exposure can be predicted by temperature drop in Swiss mice

Establishing humane endpoints to minimize animal suffering in studies on snake venom toxicity and antivenom potency tests is crucial. Our findings reveal that Swiss mice exhibit early temperature drop following exposure to different snake venoms and combinations of venoms and antivenoms, predicting later mortality. Evaluating temperature we can identify within 3 h post-inoculation, the animals that will not survive in a period of 48 h. Implementing temperature as a criterion would significantly reduce animal suffering in these studies without compromising the outcomes.

Inhibition of Bungarus caeruleus snake venom toxicity by Citrus reticulata methanolic extract and in-silico analysis of possible binding modes

Inhibition of Bungarus caeruleus snake venom toxicity by Citrus reticulata methanolic extract and in-silico analysis of possible binding modes

Acute Toxicity Test Of The Green Viper Snake (Trimeresurus albolabris), Macroscopic Description Of The Kidney And Liver Of Mice (Mus musculus)

According to WHO (2010), the Trimeresurus albolabris snake is a venomous snake with a high number of bite cases in Indonesia. Information on the character and level of toxicity of snake venom in Indonesia, including Trimeresurus spp. snakes, is still minimal. This study aims to determine the LD50 value (lethal dose 50), clinical symptoms, and to determine the macroscopic appearance of the liver and kidneys of mice (Mus musculus) after being injected by a the white-lipped pit viper (Trimeresurus albolabris) venom. The LD50 value of Trimeresurus albolabris venom injected subcutaneously into mice was 5,09 mg/kgBW. Clinical symptoms that appear increased movement activity, increased respiratory rate, lethargy, decreased activity, and reduced grooming. Macroscopic appearance of the liver that looks abnormal is found (1) color changes in the form of pale, pale yellowish, and red spots (petechiae). (2) changes in surface structure found only one liver that is not flat. (3) changes in consistency in the form of brittle easily. In the kidney that looks abnormal, found (1) changes in color in the form of pale. (2) changes in surface structure were only found in one kidney in the form of nodules. (3) changes in consistency in the form of brittle easily and soft.

Assessment of quality and pre-clinical efficacy of a newly developed polyvalent antivenom against the medically important snakes of Sri Lanka

Snake envenomation is a severe problem in Sri Lanka (SL) and Indian polyvalent antivenom (PAV) is mostly used for treating snakebite albeit due to geographical variation in venom composition, Indian PAV shows poor efficacy in neutralizing the lethality and toxicity of venom from the same species of snakes in SL. Therefore, the quality and in vivo venom neutralization potency of a country-specific PAV produced against the venom of the five most medically important snakes of SL (Daboia russelii, Echis carinatus, Hypnale hypnale, Naja naja, Bungarus caeruleus) was assessed. LC-MS/MS analysis of two batches of PAV showed the presence of 88.7–97.2% IgG and traces of other plasma proteins. The tested PAVs contained minor amounts of undigested IgG and F(ab′)2 aggregates, showed complement activation, were devoid of IgE, endotoxin, and content of preservative was below the threshold level. Immunological cross-reactivity and in vitro neutralization of enzymatic activities, pharmacological properties demonstrated superior efficacy of SL PAV compared to Indian PAV against SL snake venoms. The in vivo neutralization study showed that the tested PAVs are potent to neutralize the lethality and venom-induced toxicity of SL snake venoms. Therefore, our study suggests that introduction of SL-specific PAV will improve snakebite management in SL.

Synergistic effect of flavonoids combined with antivenom on neutralisation of Naja naja venom

Objective: To evaluate the neutralizing effects of flavonoids on snake venom toxicity by stand-alone and combinatorial approaches. Methods: Synthetic flavonoids were assessed, either individually or in combination with antivenom, for their neutralization of phospholipase A2 (PLA2), protease, antioxidant (DPPH) assay and hemotoxic activity. Molecular docking studies were performed to understand possible binding of flavonoids with Naja naja venom PLA2. In vivo studies were carried out to confirm the neutralisation effects using a mouse model. Moreover, inhibition of PLA2 was monitored using combinatorial approaches. Results: Among the flavonoids used, quercetin and naringenin inhibited PLA2 (56% and 45%), protease (71% and 64%), DPPH scavenging (69.0% and 77.5%) and hemotoxic (70%) activities. Molecular docking studies indicated that the flavonoids bind to the substrate-binding site of PLA2 (Cys44 and Tyr63). In vivo studies showed a reduction in the venom toxicity level in the presence of naringenin. Additionally, combinatorial studies using the mixture of flavonoid and anti-venom revealed the possibility of synergistic effect (up to 32% enhancement) in neutralising the venom enzymes. Conclusions: These flavonoids can be used as additives for the treatment of snake bites, which may exert synergistic effects in combination with antivenom and decrease the post-therapeutic effects caused by excessive use of antivenom.

Inhibition of Snake Venom Enzymes and Antivenom Adjuvant Effects of Azadirachta indica A. Juss. (Meliaceae) Leaf Extracts

Snake venom enzymes are the key substances involved in snake venom toxicity. Thus, inactivating these enzymes is generally considered to be the fundamental step in the management of snakebite. Conventionally, snakebite envenomation is treated parenterally with serum-based antivenins, and adjuvants to these antivenins are required for maximum protection of victims. Hence, this research was aimed at evaluating the inhibitory effect of Azadirachta indica leaf extracts on Naja nigricollis Reinhardt venom enzymes and screens for their antivenom adjuvant effects. A. indica leaf was collected, authenticated and extracted using 95% methanol followed by fractionation using hexane and ethyl acetate. The venom enzymes inhibition assays was evaluated using in vitro methods, while, adjuvant effect was screened using Albino rats. The results revealed that both the hexane and the ethyl acetate fractions showed capability of inhibiting the venom enzymes significantly (P<0.05) when compared with the venom controls in varying degrees of efficacies. For the adjuvant effect, no significant effect (P>0.05) of the venom at the administered dose was observed on bleeding time, clotting time, defibrinogenating and haemorrhagic effects compared to the normal control. However, the size of necrotic lesion and the percentage haemolysis were significantly (P<0.05) higher in the venom control rats. Both the hexane and the ethyl acetate fractions significantly mitigated these effects in the treated animals. The degree of protection was about 3 folds more than when the antivenin was used alone. Finally, these findings would be of importance in the area of drug development with a view to actualizing the substitution or enhancing the effect of conventional snakebite therapeutic options.

Toxicity and cross-neutralization of snake venoms from two lesser-known arboreal pit vipers in Southeast Asia: Trimeresurus wiroti and Trimeresurus puniceus

The lesser-known Sundaic lance-headed pit vipers Trimeresurus wiroti (Malaysia) and Trimeresurus puniceus (Indonesia) contribute to the disease burden of snakebite envenomation in Southeast Asia, but their venom toxicity and neutralization remain insufficiently investigated. This study demonstrated that both venoms were procoagulant (involving thrombin-like activity), hemorrhagic, and lethal to mice, with T. wiroti venom being more lethal (LD50 = 0.78 μg/g c.f. 1.21 μg/g). The hetero-specific antivenom from Thailand, Green Pit Viper Antivenom (GPVAV, raised against Trimeresurus albolabris) cross-reacted with T. wiroti and T. puniceus venoms with a higher efficacy of immunological binding activity for the latter. The antivenom was also effective in cross-neutralizing the procoagulant, hemorrhagic and lethal effects of the venoms. In lethality neutralization, GPVAV showed a potency of 0.79–1.05 mg venom per mL antivenom, corresponding to the complete neutralization of approximately 8–10 mg venom per unit vial of antivenom for T. wiroti and T. puniceus venoms. Taken together, it was inferred that T. wiroti, T. puniceus, and T. albolabris venoms share common toxin epitopes, thus enabling the cross-neutralization observed. These findings suggest that GPVAV may be potentially useful in the management of envenomation by T. wiroti and T. puniceus while awaiting clinical trial and validation.

Hepatoprotective Effect of Azadirachta indica Leaf Fractionated Extracts against Snake Venom Toxicity on Albino Rats

Hepatoprotective Effect of Azadirachta indica Leaf Fractionated Extracts against Snake Venom Toxicity on Albino Rats

Amplification of Snake Venom Toxicity by Endogenous Signaling Pathways.

The active components of snake venoms encompass a complex and variable mixture of proteins that produce a diverse, but largely stereotypical, range of pharmacologic effects and toxicities. Venom protein diversity and host susceptibilities determine the relative contributions of five main pathologies: neuromuscular dysfunction, inflammation, coagulopathy, cell/organ injury, and disruption of homeostatic mechanisms of normal physiology. In this review, we describe how snakebite is not only a condition mediated directly by venom, but by the amplification of signals dysregulating inflammation, coagulation, neurotransmission, and cell survival. Although venom proteins are diverse, the majority of important pathologic events following envenoming follow from a small group of enzyme-like activities and the actions of small toxic peptides. This review focuses on two of the most important enzymatic activities: snake venom phospholipases (svPLA2) and snake venom metalloproteases (svMP). These two enzyme classes are adept at enabling venom to recruit homologous endogenous signaling systems with sufficient magnitude and duration to produce and amplify cell injury beyond what would be expected from the direct impact of a whole venom dose. This magnification produces many of the most acutely important consequences of envenoming as well as chronic sequelae. Snake venom PLA2s and MPs enzymes recruit prey analogs of similar activity. The transduction mechanisms that recruit endogenous responses include arachidonic acid, intracellular calcium, cytokines, bioactive peptides, and possibly dimerization of venom and prey protein homologs. Despite years of investigation, the precise mechanism of svPLA2-induced neuromuscular paralysis remains incomplete. Based on recent studies, paralysis results from a self-amplifying cycle of endogenous PLA2 activation, arachidonic acid, increases in intracellular Ca2+ and nicotinic receptor deactivation. When prolonged, synaptic suppression supports the degeneration of the synapse. Interaction between endothelium-damaging MPs, sPLA2s and hyaluronidases enhance venom spread, accentuating venom-induced neurotoxicity, inflammation, coagulopathy and tissue injury. Improving snakebite treatment requires new tools to understand direct and indirect effects of envenoming. Homologous PLA2 and MP activities in both venoms and prey/snakebite victim provide molecular targets for non-antibody, small molecule agents for dissecting mechanisms of venom toxicity. Importantly, these tools enable the separation of venom-specific and prey-specific pathological responses to venom.

Metabolites from the citrus extracts inhibit the activity of selected proteins in Indian Cobra (Naja naja) venom

Ethnopharmacological relevanceSnakebite is a severe problem in many parts of the world, specifically in tropical and subtropical regions. A range of medicinal plant extracts are administered for treating snake bite. Of the many common plants, extracts of Citrus species have been documented to be used for treating snake bite and have been shown to decrease the snake venom toxicity. AimThe aim of the current work is to evaluate the utility of citrus peel extracts (Citrus aurantium L. and Citrus reticulate Blanco) in the management of Indian cobra envenomation. Materials and methodsPeels of citrus species were evaluated for their phospholipase A2, protease and haemolytic inhibition properties. The phytochemicals present in the extract were inferred using GC-MS. In-vivo studies, using mice model, were done to confirm the inhibitory effect of the extracts. Molecular docking was used to understand the possible binding modes of selected phytochemicals to snake venom phospholipase. ResultsCitrus peel extracts are rich in polyphenols, flavonoids and tannins. The methanolic extract of Citrus aurantium L. and Citrus reticulate Blanco inhibits phospholipase (75%), protease (71%) and hemolysis (80%) activity of the venom. GC-MS analyses indicate the presence of β-sitosterol, n-hexadecanoic acid, eicosanoic acid, and flavone in both the extracts. In addition, C. reticulate extract contains α-tocopherol and squalene. Molecular docking revealed that α-tocopherol, spiro [androst-5-ene-17,1′-cyclobutan]-2′-one,3-hydroxy-(3β,17β)- and β-sitosterol acetate bind with moderate affinity to the catalytic site of phospholipase A2. ConclusionThe present study provides new molecular insight and scientific evidence on the utility of the methanolic extracts of citrus peels to neutralize the venom toxins of Naja naja.

VIABILITY OF VERO E6 CELLS EXPOSED TO THE Bothrops leucurus SNAKE CRUDE VENOM

Primary and continuous cell culture systems are a good in vitro alternative for the quantitative assessment of snake venom toxicity. There are still few studies evaluating the cellular response against Bothrops leucurus venom (BlV). This study aimed to evaluate the cytotoxic effect of crude BlV on culture of VERO E6 cells. Neutral Red incorporation (NR) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) tests were performed for viable cells, and Trypan Blue (TB) dye exclusion test was performed for non-viable cells. The differences between the groups were evaluated by ANOVA-One-Way and Dunnet through the program Graph Pad 5.0 with significance of 5%. The 50% cytotoxic concentration (CC50) of BlV was 7.86 μg/mL. There was a trend of dose-dependent reduction on cell viability in all assays evaluated. However, MTT and NR tests were more sensitive for the evaluation of BlV cytotoxicity on the VERO E6 cell line.

Protective Effect of Azadirachta indica Leaf Fractionated Extracts on Renal and Haematological Indices against Snake Venom Toxicity in Albino Rats

Protective Effect of Azadirachta indica Leaf Fractionated Extracts on Renal and Haematological Indices against Snake Venom Toxicity in Albino Rats

Snake Venom Disintegrins: An Overview of their Interaction with Integrins.

Disintegrins are non-enzymatic proteins that interfere on cell-cell interactions and signal transduction, contributing to the toxicity of snake venoms and play an essential role in envenomations. Most of their pharmacological and toxic effects are the result of the interaction of these molecules with cell surface ligands, which has been widely described and studied. These proteins may act on platelets, leading to hemorrhage, and may also induce apoptosis and cytotoxicity, which highlights a high pharmacological potential for the development of thrombolytic and antitumor agents. Additionally, these molecules interfere with the functions of integrins by altering various cellular processes such as migration, adhesion and proliferation. This review gathers information on functional characteristics of disintegrins isolated from snake venoms, emphasizing a comprehensive view of the possibility of direct use of these molecules in the development of new drugs, or even indirectly as structural models.

Analgesic effect of morphine and tramadol in standard toxicity assays in mice injected with venom of the snake Bothrops asper

Routine laboratory animal tests necessary to assess the toxicity of snake venoms and the preclinical neutralizing ability of antivenoms and other inhibitory substances induce significant pain and distress. This has prompted initiatives to introduce the routine use of analgesia. In this study, the analgesic effect of morphine and tramadol was assessed in tests assessing the lethal, hemorrhagic, myotoxic and edema-forming activities of the venom of the viperid snake Bothrops asper. The Mouse Grimace Scale (MGS) and mouse-exploration activity were used to assess pain and its inhibition by the analgesics. Results demonstrate that tests assessing lethality and myotoxicity induce higher levels of pain than assays quantifying hemorrhagic and edema-forming activities. Our observations also indicate that pretreatment of mice with both analgesics, at the doses used, were similarly effective in reducing the MGS magnitude and increase mouse-exploration activity after the administration of B. asper venom. Moreover, the analgesic effect of both drugs was more evident in the myotoxic and lethality assays. Combined with previous observations showing that these analgesics do not alter the extent of toxic effects induced by B. asper venom, our results strongly indicate that the use of analgesia (using either morphine or tramadol) should be considered in the routine assessment of venom toxicity and antivenom efficacy.

Protective effects of a natural herbal compound quercetin against snake venom-induced hepatic and renal toxicities in rats

Echis pyramidum is a highly poisonous viper snake. Previous studies have shown acute phase hepatic and renal toxicities of Echis pyramidum venom (EPV) in rats. This study reports the protective effects of a natural herbal compound quercetin (QRC) on EPV-induced hepatic and renal toxicities in rats. A singly injection of EPV (4.76 mg/kg) caused significant increase in serum biomarkers of liver and kidney function. Pre-treatment of QRC (10 mg/kg) significantly reduced the toxic effects of EPV on functional impairment in liver and kidneys of rats. Administration of QRC also reversed EPV-induced increase in lipid peroxidation and decrease in total thiols. The histopathology of liver showed fat accumulation, focal degeneration and cytoplasmic vacuolation of hepatocytes in EPV treated rats. EPV also caused renal tubular dilation and focal atrophy of glomerular tufts in rat kidneys. Administration of QRC prevented EPV-induced structural tissue damage in liver and kidneys of rats. In conclusion, QRC significantly inhibited the acute phase toxic effects of EPV on liver and kidneys of rats by preventing the oxidative stress in these organs. QRC is also known for its anti-inflammatory, anti-edema, anti-hemorrhagic and PLA2-inhibitory properties and therefore may be regarded as a multi-action antidote against snake venom toxicity.

Preclinical Evaluation of the Efficacy of Antivenoms for Snakebite Envenoming: State-of-the-Art and Challenges Ahead.

Animal-derived antivenoms constitute the mainstay in the therapy of snakebite envenoming. The efficacy of antivenoms to neutralize toxicity of medically-relevant snake venoms has to be demonstrated through meticulous preclinical testing before their introduction into the clinical setting. The gold standard in the preclinical assessment and quality control of antivenoms is the neutralization of venom-induced lethality. In addition, depending on the pathophysiological profile of snake venoms, the neutralization of other toxic activities has to be evaluated, such as hemorrhagic, myotoxic, edema-forming, dermonecrotic, in vitro coagulant, and defibrinogenating effects. There is a need to develop laboratory assays to evaluate neutralization of other relevant venom activities. The concept of the 3Rs (Replacement, Reduction, and Refinement) in Toxinology is of utmost importance, and some advances have been performed in their implementation. A significant leap forward in the study of the immunological reactivity of antivenoms against venoms has been the development of “antivenomics”, which brings the analytical power of mass spectrometry to the evaluation of antivenoms. International partnerships are required to assess the preclinical efficacy of antivenoms against snake venoms in different regions of the world in order to have a detailed knowledge on the neutralizing profile of these immunotherapeutics.

English

In this study, we verify the possible activity of Artemisia herba-alba and A. campestris (Asteraceae) essential oil against the snake venoms. The leaves of A. campestris has been used in the folk medicine against snake moisture. The aerial part extracts of both plants have been tested for their action against snake venom. Interaction of A. campestris (E1) and A. herba alba (E2) essential oils with proteins extracted from snake venom has been tested. Lethality in mice, inflammation and cytotoxicity induced by Cerastes cerastes venoms were significantly inhibited by the mixture of different amounts of E1 and E2 and the venom. However, both extracts showed neutralization of the venoms hemolytic activity which is more important with E2. Chemical characterization of E1 and E2 revealed there is an important amount of polyphenols and tannins which are known by their bio-active effect. The result could lead as to think that these components have an action by inhibiting the toxicity of snake venoms in vitro. We can conclude therefore that the use of the essential oil from the two Artemisia species have a curative effect on the snake venom. These results confirmed the traditional use of A. campestris and we discovered a similar effect of A. herba-alba. No data have been reported till now about the essential oil curative effect of both species. © 2016 Friends Science Publishers

The Role of Calcium on the Active Site of Snake Venom Phospholipase A2: Molecular Dynamics Simulations

Snake venoms are rich in phospholipase A2 (PLA2) and their hydrolysis of cell membrane phospholipids explains the role of the enzyme in venom toxicity. Calcium is known to plays important role at the active site of PLA2 during catalysis. In this study, molecular dynamics simulations of free PLA2 and calcium bound PLA2 were carried out using GROMACS 4.5.5 to evaluate the role of calcium in PLA2 catalysis. The results showed that calcium induced formation of helical structures between Arg62 - Lys66, Asn107 - Tyr111 and Asp114 - Cys119 in PLA2 which with time disappeared through the formation and opening of loops. Calcium induced atomistic movements and conformational changes in snake venom PLA2 which led to the formation of a widened cleft at the active site of calcium bound PLA2 when compared with free PLA2. This could lead to a better binding and accommodation of substrate, thus enhancing catalysis. This study confirms the role of calcium towards the action of PLA2 in snake venom toxicity and could provide useful information for the design of small molecules that can function as PLA2 inhibitors.

Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution.

This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 μg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic β-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.

IgE antibodies, FcεRIα, and IgE-mediated local anaphylaxis can limit snake venom toxicity

Type 2 cytokine-related immune responses associated with development of antigen-specific IgE antibodies can contribute to pathology in patients with allergic diseases and to fatal anaphylaxis. However, recent findings in mice indicate that IgE also can enhance defense against honeybee venom. We tested whether IgE antibodies, IgE-dependent effector mechanisms, and a local anaphylactic reaction to an unrelated antigen can enhance defense against Russell viper venom (RVV) and determined whether such responses can be influenced by immunization protocol or mouse strain. We compared the resistance of RVV-immunized wild-type, IgE-deficient, and Fcer1a-deficient mice after injection of a potentially lethal dose of RVV. A single prior exposure to RVV enhanced the ability of wild-type mice, but not mice lacking IgE or functional FcεRI, to survive challenge with a potentially lethal amount of RVV. Moreover, IgE-dependent local passive cutaneous anaphylaxis in response to challenge with an antigen not naturally present in RVV significantly enhanced resistance to the venom. Finally, we observed different effects on resistance to RVV or honeybee venom in BALB/c versus C57BL/6 mice that had received a second exposure to that venom before challenge with a high dose of that venom. These observations illustrate the potential benefit of IgE-dependent effector mechanisms in acquired host defense against venoms. The extent to which type 2 immune responses against venoms can decrease pathology associated with envenomation seems to be influenced by the type of venom, the frequency of venom exposure, and the genetic background of the host.