Enzymatic activity and brine shrimp lethality of venom from the large brown spitting cobra (Naja ashei) and its neutralization by antivenom

doi:10.21203/rs.3.rs-20006/v4

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Enzymatic activity and brine shrimp lethality of venom from the large brown spitting cobra (Naja ashei) and its neutralization by antivenom

https://doi.org/10.21203/rs.3.rs-20006/v4

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published 06 Jul, 2020

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Objective: Naja ashei is a snake of medical importance in Kenya, Ethiopia, Somalia, Uganda, and Tanzania. Little is known about the enzymatic (snake venom phospholipases A2; svPLA2’s) and toxic (lethal) activities of Naja ashei venom and crucially, the safety and capacity of available antivenom to neutralize these effects. This study aimed to determine the enzymatic and toxic activities of Naja ashei venom and the capacity of Indian and Mexican manufactured antivenoms to neutralize these effects. The protein content of the venom and the test antivenoms were also evaluated. A 12-point log concentration-response curve (0.5-22.5 µg/mL) was generated on an agarose-egg yolk model to predict the svPLA2 activity of the venom. The toxicity profiles of the venom and antivenoms were evaluated in the brine shrimp lethality assay. Lowry’s method was used for protein estimation.

Results: Low and intermediate concentrations of the venom exhibited similar svPLA2 activities. The same was true for concentrations >15 µg/mL. Intermediate and high doses of the venom exhibited similar mortalities in brine shrimp and test antivenoms were generally non-toxic but poorly neutralized svPLA2 activity. Mexican manufactured antivenom had lower protein content but neutralized venom-induced brine shrimp lethality much more effectively than Indian manufactured antivenom.

Toxicology

Snake venom phospholipases A2

brine shrimp lethality assay

snake venom toxicity

Naja ashei

brine shrimp

Artemia salina

probit analysis

LC50

EC50

MPC50

Snakebite may be the World’s biggest hidden health crisis [1, 2]. Estimates from the World Health Organization suggest that up to 2.7 million people are envenomed by snakes yearly and close to 140,000 die [3]. Non-fatal envenoming may also result in permanent disabilities including blindness, extensive scarring, contractures, restricted mobility, and amputations [4].

Naja ashei is a category 1 snake in Kenya, Ethiopia, Somalia, and Uganda and a category 2 snake in Tanzania [5] (Figure 1). Category 1 snakes are highly venomous and result in high levels of morbidity, disability, or mortality [5]. Category 2 snakes are highly venomous, may cause morbidity, mortality, disability, or death but lack data to implicate them in snakebite [5].

Over the last decade, there has been a lot of interest in Naja ashei [6–11]. The skull structure [9], mitochondrial DNA [10], composition, antiproliferative, and antibacterial properties of Naja ashei venom have been reported [6–8, 11]. However, there has been little focus on the enzymatic, and lethal effects of this venom and the capacity of antivenoms to neutralize them. This study aimed to fill this gap by determining the enzymatic and toxic activities of Naja ashei venom and the capacity of antivenoms to neutralize them.

Snake venom and antivenom

Venom was extracted from specimens of wild-caught Naja ashei maintained at the Bioken Snake Farm in Kenya (Table S1); https://doi.org/10.6084/m9.figshare.12562055.v1. Collected venom was snap-frozen and stored at -20 °C. Reconstitution was done in phosphate-buffered saline (PBS) at the time of use. Antivenoms were sourced from hospitals in Kisumu County, Kenya. See (Table S2); https://doi.org/10.6084/m9.figshare.12562055.v1.

Animals (brine shrimp)

Brine shrimp eggs were commercially sourced from yourfishstuff (Borough of Lebanon, New Jersey, USA; Batch number; X001M8M5IZ). They were hatched at the Department of Public Health, Pharmacology, and Toxicology, University of Nairobi, and brine shrimp larvae were used for experiments.

Protein content determination of the venom and antivenoms

Lowry’s method was used [12]. An eight-point calibration curve (0.05-2 mg/mL) was developed using bovine serum albumin (BSA) as standard. Absorbance was recorded at 660 nm and the protein content of samples was inferred from the standard curve. See https://doi.org/10.6084/m9.figshare.12562136.v2

svPLA₂ activity of venom

The methods of Haberman and Hardt and Felix Silva et al were used [13, 14]. Wells were made on sterile petri dishes containing agarose egg-yolk media (1:3 v/v egg yolk: PBS+125 µL of 0.1 mM CaCl₂) prepared in a laminar flow cabinet. 10 µL of previously incubated (37 °C, one hour) and serially diluted venom was discharged into the wells and incubated for 24 hours at 50 °C. Carbol Fuchsin was used to visualize the halos which were measured by Vernier calipers. PBS was used as a negative control. Triplicate determinations were made and the least amount of venom required to elicit a 50 % svPLA₂ response (MPC₅₀) was determined by regression analysis.

Neutralization of the svPLA₂ activity of venom by antivenom

The method of Iwanaga and Suzuki 1979 was used [15]. 10 µL of a 2MPC₅₀ dose of venom was mixed with 20 µL of various doses of test antivenoms (25-400 µg/mL) in 96-well plates for 5 minutes on a microplate shaker. The plate was incubated at 37 °C for 20 minutes, 200 µL of the substrate (1.1% egg yolk suspension in 0.1 M PBS adjusted to pH 8.1 and 125 µL 0.2 mM CaCl₂) was added to all wells, incubated at 37 °C and the change in absorbance of the substrate (0 to 30 minutes) was determined spectrophotometrically at 620 nm [15]. Triplicate determinations were made and the least amount of antivenom required to reduce svPLA₂ activity by 50 % (EC₅₀) was determined by regression analysis.

Determination of the brine shrimp lethality of venom, antivenom, and controls

The method of Meyer et al was used [16]. Ten, 48-hour old brine shrimp larvae were transferred from a hatching trough to 5 mL sample vials. Aliquots (5, 50 and 500 µL) of 5 mg/mL stock solutions of the samples (venom/antivenom) were pipetted into the vials and made up to the mark using 38.5 % w/v marine salt solution to make 10, 100, and 1000 µg/mL sample concentrations respectively. PBS and vincristine sulphate were used as negative and positive controls respectively. Quintuple determinations were made and surviving larvae were counted after 24, 48, and 72 hours. LC₅₀s of samples were calculated by probit analysis. LC₅₀ was defined as the least concentration of samples which resulted in 50 % mortality of brine shrimp [17].

Neutralization of venom-induced lethality

The WHO (World Health Organization) protocol on venom neutralization by antivenoms was used with modifications [5]. Varying doses of the antivenoms (25-400 µL of 100 mg/mL) were mixed with a 2LC₅₀ dose of venom. The venom/antivenom mixtures were incubated at 37 °C for 30 minutes, added to vials containing brine shrimp larvae and surviving larvae were counted after 24, 48, and 72 hours. The median effective concentration (EC₅₀) of the antivenoms was determined by regression analysis and was defined as the minimum amount of antivenom (in µL) that was required to neutralize 1 mg of venom [5].

Statistical analysis

Venom concentrations were converted to log₁₀ (x-axis) and mean responses were converted to percentages (y-axis). The concentration of venom responsible for 50 % svPLA₂ activity (MPC₅₀) was predicted by regression analysis (SPSS v20). Mortalities were converted to probits and regressed against the log concentration of venom (MS Excel 2013) [18, 19]. Analysis of variance and Tukey’s post hoc test (p<0.05) was used to evaluate dose-dependent differences in svPLA₂ activity and brine shrimp lethality. Meyer’s and Clarkson’s criteria were used to infer the toxicity of substances tested in the brine shrimp lethality assay [16, 20].

There was no significant difference (p>0.05) in the svPLA₂ activity of venom doses ranging from 0.5-10 µg/mL (Table 1). See https://doi.org/10.6084/m9.figshare.12562163.v2. The same was true for doses > 15 µg/mL (Table 1). There was a positive linear relationship between the log concentration of venom and the %svPLA₂ activity (Figure S1); https://doi.org/10.6084/m9.figshare.12562175.v3. The correlation was significant; r(65)=0.804, p<0.05, regression equation was ŷ=26.339x+51.906, and r²=0.646; that is, 64.6% of the variance in the %svPLA₂ activity was predictable from the log concentration of venom. Based on this regression model, the MPC₅₀ was found to be 0.847 µg/mL.

There was a negative linear relationship between the log concentration of the venom+antivenom I mixture and %svPLA₂ activity (Figure S2); https://doi.org/10.6084/m9.figshare.12570617.v1. The correlation was significant r(14)=0.669, p=0.006 and regression equation was ŷ= -44.792x+154.164. Based on this regression model, 1 mL of antivenom I neutralized 0.08 µg of svPLA₂.

There was a negative linear relationship between the log concentration of the venom+antivenom II mixture and %svPLA₂ activity (Figure S3); https://doi.org/10.6084/m9.figshare.12571100.v1. The correlation was significant r(14)=0.772, p=0.001 and regression equation was ŷ= -44.706x+162.226. Based on this regression model 1 ml of antivenom II neutralized 0.05 µg of svPLA₂.

Test antivenoms were generally non-toxic (Table 2), N. ashei venom was more toxic than vincristine sulphate (positive control) after 24 hours but vincristine sulphate was more toxic than venom after 48 and 72-hours (Table 2). See also https://doi.org/10.6084/m9.figshare.12571547.v1,https://doi.org/10.6084/m9.figshare.12571283.v1, and https://doi.org/10.6084/m9.figshare.12571283.v3. There was no significant difference (p>0.05) in the % mortality of brine shrimps exposed to 100 µg/mL or 1000 µg/mL of venom after 24, 48, and 72 hours. See https://doi.org/10.6084/m9.figshare.12562199.v2.

One mL of antivenom II neutralized 0.207 mg of venom in the brine shrimp lethality assay but antivenom I was ineffective. See https://doi.org/10.6084/m9.figshare.12570620.v2. The mean protein content of the venoms and antivenoms was significantly different from each other. See https://doi.org/10.6084/m9.figshare.12573425.v1.

Naja ashei venom is yet to be included in immunizing mixtures of commercially available antivenom. In this study, we have demonstrated that Mexican and Indian manufactured antivenoms poorly neutralized the svPLA₂ activity of this venom. We have also established that only the Mexican manufactured antivenom was effective in neutralizing the toxic effects of this venom. These observations highlight the limited efficacy of imported antivenoms in neutralizing key toxins in the venom of a snake associated with many bites in East Africa [5]. The clamor for locally manufactured antivenoms seems justified [21].

In the context of African spitting cobras, Indian manufactured antivenom is indicated for Naja nigricollis and Naja haje envenomation while the Mexican manufactured antivenom is indicated for Naja nigricollis, Naja haje, Naja pallida, Naja nubiae and Naja katiensis [22]. Therefore, it may be inferred that the cross-neutralization of toxic proteins in Naja ashei venom by Mexican manufactured antivenom was because the toxicity profile of Naja ashei venom may be similar to the profile in Naja pallida, Naja nubiae, and Naja katiensis venoms but dissimilar to Naja nigricollis and Naja haje venoms.

How can the pharmacological findings in this study be explained by what is known about the composition of Naja ashei venom? Hus and colleagues indicated that the most abundant proteins in Naja ashei venom were cytotoxins (3FTxs; three-finger toxins) and svPLA₂’s[8]. Other venom proteins include 5′N-Snake venom 5′-nucleotidase; SVMPs—Snake venom metalloproteinases; CRISPs—Cysteine-rich venom proteins; CVF—Cobra venom factor; and VNGF—Venom nerve growth factor [8]. svPLA₂’s may be acidic or basic and are divided into groups IA, IIA, and IIB [23]. The fact that this study used a pH of 8.1 to run the agarose-egg yolk assay strongly suggests that the observed svPLA₂ activity was basic. This corroborates the findings of a previous study which reported that a majority of Naja ashei venom proteins were of low molecular weight and basic [8]. Group IA svPLA₂’s are primarily found in elapids, although some have been reported in colubrids [23]. Group IIA and IIB svPLA₂’s are exclusively found in viperids [24]. Since Naja ashei is an elapid, the svPLA₂ activity observed was most likely of the Group IA variety.

Naja ashei venom exhibited strong cytotoxic action in the brine shrimp lethality assay relative to vincristine sulphate (a standard cytotoxic). It is important to note that the brine shrimp lethality assay is a good predictor of cytotoxicity and has been widely used to reliably detect this phenomenon in the venom of the sea snake; Enhydrina schistosa [25], and in several venomous fish [26–29], snails [30–32], toads [33] and bees [34]. The dose and time-dependent brine-shrimp lethality observed may be a direct consequence of the non-enzymatic effects of cytotoxins i.e. paralysis, Ca²⁺ toxicity, and cell death [35]. However, it is unlikely that this observation was not supported by the enzymatic action of the basic Group IA svPLA₂’s which have been known to cause organelle toxicity, hydrolysis of the lipid environments of cell membranes, and mitochondrial membrane disruption of the respiratory muscle [35–37]. An important finding in this study was that the concentration of Naja ashei venom was not the only predictor of svPLA₂ activity. This raises a pertinent question: what other factors may be involved in predicting this activity? It was also observed that low and intermediate doses of Naja ashei venom produced similar svPLA₂ activities and there was no difference in the brine shrimp mortalities caused by intermediate and high doses of the venom. This may suggest that the activities of these toxins remain fairly constant within a narrow range of venom doses.

It was established that both antivenoms were safe in brine shrimp. The evaluation of the safety profile of the test antivenoms was important because i) snake antivenoms may cause both acute (anaphylactic/pyrogenic) and delayed (serum sickness) toxic manifestations in human envenomation [38], and ii) the safety profile was key in informing the selection of antivenom aliquots to be used in the neutralization assay.

Based on protein estimation by Lowry’s method, it was established that Indian manufactured antivenom had a higher protein content than Mexican manufactured antivenom but was ineffective in neutralizing the toxic effects of Naja ashei venom. Because both antivenoms are made up of immunoglobulin-binding fragments; F(ab)’s [22] and given the fact that Lowry’s method largely reports the aromatic acid (tyrosine and tryptophan) composition of proteins [39], it may be argued that these amino acids may not be involved in the recognition and neutralization of toxic venom proteins in Naja ashei venom.

The svPLA₂ activity and toxicity of Naja ashei venom remain fairly constant within a narrow range of venom doses. Commercially available antivenoms are generally safe but have limited efficacy in neutralizing the svPLA₂activity of Naja ashei venom. Moreover, only Mexican manufactured antivenom cross-neutralizes toxic venom proteins in Naja ashei venom. We recommend studies on the activities of other toxins in this venom and their neutralization by antivenom.

Limitations

Snake venom is a complex mixture of toxins. This study only evaluated the snake venom phospholipases A₂ activity and brine shrimp lethality of Naja ashei venom. To fully understand the capacity of antivenoms to neutralize Naja ashei venom, it may be necessary to evaluate other toxins in this venom.

svPLA₂: Snake venom phospholipase A₂; BSLA: Brine shrimp lethality assay; LC₅₀: lethal concentration responsible for 50 % mortality; MPC₅₀: The minimum phospholipase concentration responsible for 50 % response; EC₅₀: the least amount of antivenom (in µL) required to neutralize 1mg of venom; DNA: Deoxyribonucleic acid; BSA: Bovine Serum Albumin; UV: Ultraviolet; mM: Millimole; v/v: Volume by volume; µL: Microliter; µg/mL; Microgram per milliliter; mm: Millimeter; ELISA: Enzyme-linked Immunosorbent assay; PBS: phosphate-buffered saline; CaCl₂: Calcium chloride; WHO: World Health Organization; mg/mL: Milligram per milliliter; r²: Coefficient of determination; LD_50:Median lethal dose

Ethics approval and consent to participate

This study was approved by the Biosafety, Animal Use, and Ethics Committee of the Faculty of

Veterinary Medicine, University of Nairobi. REF BAUEC/2019/220 issued on 24^th April 2019.

The animals (brine shrimps; Artemia salina) used in this study were commercially sourced thus the owner’s consent was not required.

Consent for publication

Not applicable

Availability of data and material

All data generated or analyzed during this study are included in this published article. [And its Supplementary information files below].

Information on the snakes whose venom was used in this study (Table S1); https://doi.org/10.6084/m9.figshare.12562055.v1.
Details on the snake antivenom used in this study (Table S2); https://doi.org/10.6084/m9.figshare.12562055.v1.
Raw data of the absorbance values (660 nm) of different concentrations of bovine serum albumin (protein standard), Naja ashei venom, and antivenom; https://doi.org/10.6084/m9.figshare.12562136.v2
The data output from the analysis of variance (+Tukey’s post hoc test) to determine dose-dependent differences in the svPLA₂ activity of Naja ashei venom; https://doi.org/10.6084/m9.figshare.12562163.v2.
The data output from the simple linear regression analysis to determine the %vPLA₂ activity of different concentrations of Naja ashei venom (Figure S1); https://doi.org/10.6084/m9.figshare.12562175.v3.
The data output from the simple linear regression to determine the capacity of antivenom I and II to neutralize the svPLA₂ activity of Naja ashei venom (Figure S2), and (Figure S3);https://doi.org/10.6084/m9.figshare.12570617.v1,https://doi.org/10.6084/m9.figshare.12571100.v1.

The data output from probit regression analysis to determine the toxicity of Naja ashei venom, and vincristine and the safety of antivenom in brine shrimp;https://doi.org/10.6084/m9.figshare.12571547.v1,https://doi.org/10.6084/m9.figshare.12571283.v1, and https://doi.org/10.6084/m9.figshare.12571283.v3.
The data output from analysis of variance (+Tukey’s post hoc test) to determine dose-dependent differences in Naja ashei venom-induced brine shrimp lethality; https://doi.org/10.6084/m9.figshare.12562199.v2.
The data output from simple linear regression to determine the capacity of antivenom to neutralize Naja ashei venom-induced brine shrimp lethality https://doi.org/10.6084/m9.figshare.12570620.v2.
The data output from analysis of variance (+Tukey’s post hoc test) to determine the differences in the mean protein content of Naja ashei venom and antivenom I and II https://doi.org/10.6084/m9.figshare.12573425.v1.

Competing interests

The authors declare no conflict of interest

Funding

This study did not receive any external funding.

Authors’ contributions

Conceptualization: MO, FO, and JG; data curation: all authors; formal analysis: MO; investigation: all authors; methodology: MO; project administration: MO and FO, resources; all authors; software: MO; supervision; JM, JG, PM, and VO; validation: FO, JM, JG, PM, and VO; visualization: MO; writing original draft: MO: writing review and editing: all authors

Acknowledgments

The authors would like to acknowledge Ms. Claire Taylor and Mr. Boniface Momanyi of the Bioken Snake Farm (Watamu, Kenya) for providing the venom samples. We would also like to thank Ms. Vivian of the Department of Animal Physiology, the University of Nairobi who helped us in freeze-drying the venom samples and Dr. Nduhiu, Mr. Mainga, Mr. Maloba, Mr. Nderitu, Mr. Antony (BVM student) and Mr. Bett of the Department of Public Health, Pharmacology and Toxicology for their assistance in the phospholipase and brine shrimp lethality assays. Special gratitude to Dr. Nelson Odhiambo and Edna Opiyo of the Nyakach Sub County Hospital for providing the antivenom samples that were used in this study. We also wish to thank Dr. Joshua Onono and Mr. Duke Gekonge for their assistance in statistical analysis.

Snakebite: “The world’s biggest hidden health crisis” - Medical Brief. https://www.medicalbrief.co.za/archives/snakebite-worlds-biggest-hidden-health-crisis/. Accessed 22 May 2020.
The Antivenom Crisis in Africa |. http://www.snakebiteinitiative.org/?page_id=696. Accessed 14 Jul 2018.
Gutiérrez JM, Calvete JJ, Habib AG, Harrison RA, Williams DJ, Warrell DA. Snakebite envenoming. Nature reviews Disease primers. 2017;3:17063. doi:10.1038/nrdp.2017.63.
Branche C, Ozanne-Smith J, Oyebite K, Hyder AA. World report on child injury prevention. World Health Organization; 2008.
WHO. Post-ECBS version. Expert committee on biological standardization Geneva, 17 to 21 October 2016. WHO Guidelines for the Production, Control, and Regulation of Snake Antivenom Immunoglobulins. World Health Organization. 2016; October:1–138.
Petrilla V, Tomečková V, Komanický V, Lichardusová L, Šutorová M, Petrillová M, et al. Fluorescent profiling of venom-selected cobra species. Spectroscopy Letters. 2014;47:1–5.
Bocian A, Ciszkowicz E, Hus KK, Buczkowicz J, Lecka-Szlachta K, Pietrowska M, et al. Antimicrobial activity of protein fraction from Naja ashei venom against Staphylococcus epidermidis. Molecules. 2020;25.
Hus KK, Buczkowicz J, Petrilla V, Petrillová M, Łyskowski A, Legáth J, et al. First Look at the Venom of Naja ashei. Molecules. 2018;23:609.

Andrejcakova Z, Vlckova R. Comparisons of the African Cobras using electrophoretical analysis of venom and their morphology. International Research Journal of Natural and Applied Sciences. 2015;4 May:58–68.
Wüster W, Broadley DG. Get an eyeful of this: A new species of giant spitting cobra from eastern and north-eastern Africa (Squamata: Serpentes: Elapidae: Naja). Zootaxa. 2007;68:51–68.
Antolikova NR, Kello M, Zigova M, Tischlerova V, Petrilla V, Pirnik Z, et al. Naja ashei venom induces mitochondria-mediated apoptosis in human colorectal cancer cells. Acta Biochimica Polonica. 2019;66:207–13.
Aitken A, Learmonth MP. The protein protocols handbook. Edited by. 2002;:3–7.
Habermann E, Hardt KL. A sensitive and specific plate test for the quantitation of phospholipases. Analytical Biochemistry. 1972;50:163–73.
Felix-Silva J, Souza T, Menezes YAS, Cabral B, Camara RBG, Silva-Junior AA, et al. Aqueous leaf extract of Jatropha gossypiifolia L. (Euphorbiaceae) inhibits enzymatic and biological actions of Bothrops jararaca snake venom. PLoS ONE. 2014;9.
Iwanaga S, Suzuki T. Enzymes in snake venom. In: Snake venoms. Springer; 1979. p. 61–158.
Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE j, McLaughlin JL. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Medica. 1982;45:31–4.
Parra AL, Yhebra RS, Sardiñas IG, Buela LI. Comparative study of the assay of Artemia salina L. and the estimate of the median lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts. Phytomedicine. 2001;8:395–400.

Bliss CI. the Calculation of the Dosage‐Mortality Curve. Annals of Applied Biology. 1935;22:134–67.
Finney DJ. Probit analysis: statistical treatment of the sigmoid response curve. Cambridge university press, Cambridge; 1952.
Clarkson C, Maharaj VJ, Crouch NR, Grace OM, Pillay P, Matsabisa MG, et al. In vitro antiplasmodial activity of medicinal plants native to or naturalized in South Africa. Journal of ethnopharmacology. 2004;92:177–91.
About Us | Kenya Snakebite Research and Intervention Centre. https://ksric-asrg.org/about-us/. Accessed 1 Jun 2020.
Harrison RA, Oluoch GO, Ainsworth S, Alsolaiss J, Bolton F, Arias A-S, et al. Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa. PLoS neglected tropical diseases. 2017;11:e0005969.
Pucca MB, Ahmadi S, Cerni FA, Ledsgaard L, Sørensen C V., McGeoghan FTS, et al. Unity Makes Strength: Exploring Intraspecies and Interspecies Toxin Synergism between Phospholipases A2 and Cytotoxins. Frontiers in Pharmacology. 2020;11 May.
Phospholipases A, Sørensen C, Pucca MB, Ahmadi S, Cerni FA, Ledsgaard L, et al. Unity Makes Strength:Exploring Intraspecies and Interspecies Toxin Synergism between Phospholipases A 2 and Cytotoxins. 2020; May.
Damotharan P, Veeruraj A, Arumugam M, Balasubramanian T. Isolation and characterization of biologically active venom protein from sea snake Enhydrina schistosa. Journal of biochemical and molecular toxicology. 2015;29:140–7.
Veeruraj A, Pugazhvendan SR, Ajithkumar TT, Arumugam M. Isolation and identification of cytotoxic and biological active toxin from the pufferfish Arothron stellatus. Toxicological Research. 2016;32:215–23.
Hahn ST, O’Connor JM. An investigation of the biological activity of bullrout (Notesthes robusta) venom. Toxicon. 2000;38:79–89.
Abirami P, Arumugam M, Giji S, Nagarajan S. Bio-prospecting of catfish sting venom Arius maculatus available along South East coast of India. Int J Pharm Pharm Sci. 2014;6:110–5.
Nair MSR, Cheung P, Leong I, Ruggieri GD. A non-proteinaceous toxin from the venomous spines of the lionfish Pterois volitans (Linnaeus). Toxicon. 1985;23:525–7.
Bajo L, Tan R, Lagas KF, Perocho RCJ, Gomez E. Biological characterization of Conus striatus venom for medical applications. Science International-Lahore. 2017;29 November 2017:1277–81.
Wu X, Shao X, Guo Z-Y, Chi C-W. Identification of neuropeptide Y-like conopeptides from the venom of Conus betulinus. Acta Biochim Biophys Sin. 2010;42:502–5.
Kumari A, Ameri S, Ravikrishna P, Dhayalan A, Kamala-Kannan S, Selvankumar T, et al. Isolation and Characterization of Conotoxin Protein from Conus inscriptus and Its Potential Anticancer Activity Against Cervical Cancer (HeLa-HPV 16 Associated) Cell Lines. International Journal of Peptide Research and Therapeutics. 2019;:1–9.
Xiao J, Zhao X, Zhong W-T, Jiao F-R, Wang X-L, Ma L, et al. Bufadienolides from the Venom of Bufo Bufo gargarizans and Their Enzyme Inhibition Activities and Brine Shrimp Lethality. Natural Product Communications. 2018;13:1934578X1801300710.
Abrantes AF, Rocha TC da, Lima ABS de, Cavalcanti MT. Honeybee venom: influence of collection on quality and cytotoxicity. Ciência Rural. 2017;47.
Bickler PE. Amplification of Snake Venom Toxicity by Endogenous Signaling Pathways. Toxins. 2020;12:1–26.
Rigoni M, Paoli M, Milanesi E, Caccin P, Rasola A, Bernardi P, et al. Snake phospholipase A2 neurotoxins enter neurons, bind specifically to mitochondria, and open their transition pores. Journal of Biological Chemistry. 2008;283:34013–20.
Paoli M, Rigoni M, Koster G, Rossetto O, Montecucco C, Postle AD. Mass spectrometry analysis of the phospholipase A2 activity of snake pre‐synaptic neurotoxins in cultured neurons. Journal of neurochemistry. 2009;111:737–44.
de Silva HA, Ryan NM, de Silva HJ. Adverse reactions to snake antivenom, and their prevention and treatment. British journal of clinical pharmacology. 2016;81:446–52.
Sapan C V, Lundblad RL, Price NC, Boulevarde C, Raton B, Healthcare B, et al. Colorimetric protein assay techniques. 1999;108:99–108.

Table 1: The dose-response relationship of svPLA₂ in Naja ashei venom

Concentration of venom (µg/mL)	Log₁₀ concentration	Mean (SD) (n=6)	%svPLA₂ activity
0	-	0.0 (0.0)	0.0 (0.0)
0.5	-0.3010	9.5 (0.5) ^a	49.3 (3.3) ^a
1.0	0.0000	10.0 (0.6) ^a	52.0 (5.6) ^a
2.0	0.3010	11.8 (1.6) ^ab	61.5 (8.9) ^ab
4.0	0.6021	12.2 (1.0) ^ab	63.3 (8.1) ^ab
8.0	0.9031	12.0 (0.9) ^ab	62.5 (8.3) ^ab
10.0	1.0000	13.8 (1.6) ^b	72.0 (10.6) ^bc
12.5	1.0970	14.4 (2.4) ^bc	75.4(17.2) ^bcd
15.0	1.1760	17.1 (2.1) ^cd	88.6(10.4) ^cde
17.5	1.2430	18.8 (1.5) ^d	97.0 (3.4) ^e
20.0	1.3010	16.7 (1.2) ^cd	86.3 (3.7) ^cde
22.5	1.3522	17.7 (0.4) ^d	91.8 (7.6) ^de

svPLA₂: Snake venom phospholipase A₂. Means with different superscripts along the columns are significantly different from each other at p<0.05(ANOVA and Bonferroni post hoc test)

Table 2: The brine shrimp cytotoxicity profile of Naja ashei venom, two commercial antivenoms, and vincristine sulphate (standard cytotoxic agent)

Mortality per test dose

Toxicity

Sample

Duration of exposure

µg/ml

100

µg/ml

1000

µg/ml

LC₅₀ (µg/ml)

Meyer’s toxicity

index [16]

Clarkson’s toxicity

index [20]

Vincristine sulphate (positive control)

171.83

2.10

All dead

Toxic

Highly toxic

Antivenom

No mortality

2346.23

5268.05

Non toxic

Antivenom

No mortality

599,484,250.30

1622.89

Non toxic

Naja

ashei

venom

63.02

4.73

0.15

Toxic

Highly toxic

Dr.MitchelSupplementarymaterial.docx

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published 06 Jul, 2020

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Submission checks completed at journal
29 Jun, 2020
Editorial decision: Accept
29 Jun, 2020

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Enzymatic activity and brine shrimp lethality of venom from the large brown spitting cobra (Naja ashei) and its neutralization by antivenom

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Abstract

Figures

Introduction

Materials And Methods

Results

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 4