Echis ocellatus venom-induced sperm functional deficits, pro-apoptotic and inflammatory activities in male reproductive organs in rats: antagonistic role of kaempferol.

BACKGROUND: Echis ocellatus envenoming is potentially toxic initiating clinical damages on male reproductive system. Kaempferol is a therapeutic agent with neutralizing potentials on snake venom toxins. This study investigated the antagonistic effect of kaempferol on E. ocellatus venom (EoV)-induced reproductive toxicities. METHODS: Fifty adult male rats were sorted at random into five groups of ten rats for this study. The control rats were allotted to group 1, while rats in groups 2-5 were injected with 0.22 mg/kg bw (LD50) of EoV intraperitoneally. Rats in group 2 were not treated while groups 3-5 rats were treated with serum antivenom (0.2 ml), and 4 and 8 mg/kg bw of kaempferol post envenoming, respectively. RESULTS: EoV actuated reproductive toxicity, significantly decreased sperm parameters, and enhanced inflammatory, oxidative stress, and apoptotic biomarkers in reproductive organs of untreated envenomed rats. However, treatment with kaempferol alleviated the venom-induced reproductive disorders with a dose dependent effect. Kaempferol significantly increased the testicular weight, organo-somatic index, sperm parameters, and normalized the levels of serum luteinizing hormone, testosterone, and follicle stimulating hormone. Kaempferol ameliorated testicular and epididymal oxidative stress as evidenced by significant decrease in malondialdehyde (MDA) levels, enhancement of reduced glutathione (GSH) levels, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities. The inflammatory biomarkers; nitric oxide (NO) levels and myeloperoxidase activity (MPO), and apoptotic biomarkers; caspase 3 and caspase 9 activities were substantially suppressed in the testis and epididymis of envenomed rats treated with kaempferol. CONCLUSION: Results revealed kaempferol as a potential remedial agent against reproductive toxicity that could manifest post-viper envenoming.

Experimental production and efficacy testing of mono-specific antibodies against the venom of carpet viper (Echis ocellatus) from savannah Nigeria.

Echis ocellatus is one of the commonest snakes responsible for envenomation in Nigeria. Antivenom is the only effective treatment, but the country suffers from a limited supply of effective antivenom. This study therefore aimed to explore the feasibility of effective, mono-specific antibodies production through immunization in rabbits using the venom of Echis ocellatus from Nigeria. The World Health Organization guide on antivenom production was employed in the immunization and the resultant antibodies were purified using protein A agarose column chromatography. Antibody titer reached a high plateau by 2-month immunization, and SDS PAGE of the sera suggests the presence of intact immunoglobulins accompanied with the heavy (50 kDa) and light (25 kDa) chains. The venom has an intravenous LD50 of 0.35 mg/kg in mice, and the venom lethality at a challenge dose of 2 LD50 was effectively neutralized by the antibodies with a potency value of 0.83 mg venom per g antibodies. The antibodies also neutralized the procoagulant activity of the venom with an effective dose (ED) of 13 ± 0.66 µl, supporting its use for hemotoxic envenomation. The study establishes the feasibility of developing effective, mono-specific antibodies against the Nigerian Carpet viper.

First report of clinically significant bites due to Platyceps ventromaculatus (GRAY, 1834) from Thar Desert region, Rajasthan, India.

BACKGROUND: Platyceps ventromaculatus is a non-front-fanged colubrid snake of unknown medical significance. In this study, we highlight the clinical manifestations and outcomes of P. ventromaculatus bites. We also emphasise the need to create awareness among clinicians and the public for its potential to be confused with serious venomous bites such as Echis carinatus sochureki. METHODS: This series is part of an ongoing observational clinical study from our tertiary care hospital in Jodhpur, India on the profile and outcomes of snakebite envenoming. Data was collected after approval from Institute Ethics Committee. The date and time of the bite, geographical location, type of human-snake conflict, time-to-reach a healthcare facility, antivenom used (dose), and outcomes were recorded. We retrospectively examined our clinical data for images suggestive of P. ventromaculatus and present the clinical details of these patients. The photographs were identified utilising taxonomic keys for species identification. RESULTS: A total of four images and three patients with bites due to P. ventromaculatus were identified. The clinical effects included mild local erythema, pain, transient local bleeding, and edema. All bites occurred during daylight hours, 2 during agricultural activities, and one at home. Twenty-minute Whole Blood Clotting Test was persistently prolonged for 12 h after the bite in one patient. All patients were treated symptomatically, observed at the emergency department, and discharged within 24 h. None of the patients received antivenom. CONCLUSIONS: To our knowledge, P. ventromaculatus has so far not been reported to result in envenoming or medically significant bites. This study highlights that Platyceps bite can present with clinically significant local and possibly systemic findings that may lead to confusion with saw-scaled viper (Echis) envenoming. Clinicians must receive appropriate training so as to be aware and recognize regional snake species that do not require antivenom so as to avoid unnecessary antivenom administration.

Preparation, Characterization, and Anticancer Activity Assessment of Chitosan/TPP Nanoparticles Loaded with Echis carinatus Venom.

AIMS AND BACKGROUND: Echis carinatus venom is a toxic substance naturally produced by special glands in this snake species. Alongside various toxic properties, this venom has been used for its therapeutic effects, which are applicable in treating various cancers (liver, breast, etc.). OBJECTIVE: Nanotechnology-based drug delivery systems are suitable for protecting Echis carinatus venom against destruction and unwanted absorption. They can manage its controlled transfer and absorption, significantly reducing side effects. METHODS: In the present study, chitosan nanoparticles were prepared using the ionotropic gelation method with emulsion cross-linking. The venom's encapsulation efficiency, loading capacity, and release rate were calculated at certain time points. Moreover, the nanoparticles' optimal formulation and cytotoxic effects were determined using the MTT assay. RESULTS: The optimized nanoparticle formulation increases cell death induction in various cancerous cell lines. Moreover, chitosan nanoparticles loaded with Echis carinatus venom had a significant rate of cytotoxicity against cancer cells. CONCLUSION: It is proposed that this formulation may act as a suitable candidate for more extensive assessments of cancer treatment using nanotechnology-based drug delivery systems.

Systematic review and meta-analysis on the efficacy of Indian polyvalent antivenom against the Indian snakes of clinical significance.

Snakebite in India is a severe problem as it causes a mortality rate of 58,000 and a disability rate of 140,000 every year which is the highest among any other country. Antivenom is the primary therapy for snakebite, and its manufacturing techniques have essentially stayed unaltered for over a century. Indian polyvalent antivenom, a scientifically validated medicine for treating the toxic effects of snakebites, is available against the venom of the so-called Big Four snakes namely Spectacled cobra (Naja naja), Saw-scaled viper (Echis carinatus), Russell's viper (Daboia russelli) and the Common krait (Bungarus caeruleus), responsible for majority of the deaths in India. India hosts many other species of snakes, including cobras, kraits, saw-scaled vipers, sea snakes, and pit vipers, responsible for clinically severe envenomation. Neutralization strategy has been applied to access the efficacy of antivenoms, crucial for reducing snake bite deaths and disabilities. This review aims to conduct a systematic review and meta-analysis on the neutralization efficiency of the Polyvalent Antivenom (PAV) and focus on the factors that may contribute to the poor recognition of the antivenom towards the venom toxins. Reports focusing on the investigation of antivenom efficacy were searched and collected from several databases. Preclinical studies that reported the neutralization efficacy of the commercial antivenom against the medically important snakes of India were included. The articles were screened based on the inclusion criteria and 8 studies were shortlisted for meta-analysis. Pooled proportion was calculated for the antivenom efficacy reported by the studies and was found to be statistically significant with a 95% confidence interval. The heterogenicity in the venom toxicity and neutralization potency of the antivenom was evident in the overall estimate (proportion) and individual data. We provide comprehensive evidence on antivenom efficacy against medically important snakes from various parts of India which may aid in identifying the gaps in snake envenomation therapy and the need for novel potentially improved treatment of snakebites.

Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights.

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

Immunoreactivity and neutralization efficacy of Pakistani Viper Antivenom (PVAV) against venoms of Saw-scaled Vipers (Echis carinatus subspp.) and Western Russell's Vipers (Daboia russelii) from the Indian subcontinent.

Snakebite envenoming (SBE) is a priority Neglected Tropical Disease listed by the World Health Organization. South Asia is heavily affected, and virtually all countries in the region import polyvalent antivenom products from India for clinical use. The imported antivenoms, however, have suboptimal effectiveness due to geographical venom variation. Recently, a domestic bivalent product, named Pakistani Viper Antivenom (PVAV) has been developed specifically for Pakistani vipers, Echis carinatus sochureki and Daboia russelii. As a bivalent viperid antivenom, it is unknown yet if PVAV exhibits higher immunological binding and neutralization activities against viper venoms from distant locales compared with polyvalent antivenoms manufactured in India. This study thus examined the preclinical efficacy of PVAV against venoms of Western Russell's Vipers and Saw-scaled Viper subspecies from selected locales in the Indian subcontinent. PVAV generally outperformed the commonly used VINS polyvalent antivenom (VPAV, manufactured in India) in binding toward venoms, and showed superior or comparable neutralization efficacy against the venom procoagulant and hemorrhagic effects of Saw-scaled Vipers as well as Russell's Vipers from Pakistan and Sri Lanka. Based on normalized potency values, PVAV is far more potent than VPAV in neutralizing the lethality of all viper venoms, except that of the Indian Russell's Viper. The study shows conserved antigenicity of toxins responsible for major toxicity across these viperid venoms, and suggests the feasible production of a viper-specific antivenom with higher potency and broader geographical utility for the region.

Biochemistry of the Thrombin-Like Enzyme and Its Purification from Iranian Echis Carinatus Snake Venom: Its Interaction with Platelet Receptors.

Snake venoms are rich in valuable substances that have medical potential in the diagnosis and treatment of hemostatic diseases. The present paper was aimed at the purification and functional characterization basis of a thrombin-like enzyme and its role in the functioning of the coagulation cascade and platelet aggregation pathway. A thrombin-like serine protease was purified from the Iranian Echis carinatus venom (TLIECV), employing a one-step chromatographic procedure. This peptide was collected in high yield and purity by a single chromatographic step using RP-HPLC equipped with a C18 column. This peptide showed a 3000 Da molecular weight in gel-electrophoresis. Evidence in the SDS-PAGE gel has confirmed high recovery of fraction in optimal terms. Subsequently, this peptide was identified via its intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Multiple sequence alignments were performed by ClustalW, the Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of the peptide. The enzyme possessed fibrinogenolytic, procoagulant, and aggregation inducer properties. Moreover, the SDS-PAGE (12%) was applied to examine fibrinogenolytic function. The purified enzyme degraded the Aα chain of fibrinogen while the Bß and γ chains were not digested. According to that, the deficient human plasma in factor X and normal human plasma were also coagulated by TLIECV, it takes part in the common and intrinsic routes of the coagulation cascade. These findings proved that TLIECV is a serine protease identical to procoagulant thrombin-like snake venom proteases; however, it specifically releases the Aα chain of bovine fibrinogen. Because of its function to make up for the deficiency of factor X and its platelet aggregation inducer property, TLIECV could be considered a molecular impact to reveal the hemostasis mechanisms.

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry.

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom. These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.