sign in sign up
<<
Home , Cape cobra, Chinese cobra, Javan spitting cobra, Naja nigricincta, Snouted cobra

Hindawi Journal of Toxicology Volume 2018, Article ID 6940798, 8 pages https://doi.org/10.1155/2018/6940798

Research Article Quantitative Characterization of the Hemorrhagic, Necrotic, Coagulation-Altering Properties and Edema-Forming Effects of Zebra ( nigricincta nigricincta)Venom

Erick Kandiwa,1 Borden Mushonga,1 Alaster Samkange ,1 and Ezequiel Fabiano2

1 School of Veterinary Medicine, Faculty of Agriculture and Natural Resources, Neudamm Campus, University of , P. Bag 13301, Pioneers Park, Windhoek, Namibia 2Department of Wildlife Management and Ecotourism, Katima Mulilo Campus, Faculty of Agriculture and Natural Resources, University of Namibia, P. Bag 1096, Ngweze, Katima Mulilo, Namibia

Correspondence should be addressed to Alaster Samkange; [email protected]

Received 30 May 2018; Revised 5 October 2018; Accepted 10 October 2018; Published 24 October 2018

Academic Editor: Anthony DeCaprio

Copyright © 2018 Erick Kandiwa et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Tis study was designed to investigate the cytotoxicity and haemotoxicity of the Western barred (zebra) spitting ( nigricincta) venom to help explain atypical and inconsistent reports on syndromes by Namibian physicians treating victims of human ophidian accidents. Freeze-dried venom milked from adult zebra was dissolved in phosphate bufered saline (PBS) for use in this study. Haemorrhagic and necrotic activity of venom were studied in New Zealand albino rabbits. Oedema-forming activity was investigated in 10-day-old Cobb500 broiler chicks. Procoagulant and thrombolytic activity was investigated in adult Kalahari red goat blood in vitro. Te rabbit skin minimum hemorrhagic dose (MHD) for N. n. nigricincta was 9.8�g. Te minimum necrotizing dose (MND) for N. n. nigricincta venom was 12.2�g. Te N. n. nigricincta venom showed linear dose-dependent procoagulant activity on goat blood (p<0.05). Likewise, N. n. nigricincta venom showed linear dose-dependent thrombolytic activity on goat blood (p<0.05, n = 6). Subplantar injection of N. n. nigricincta venom (25�g, 50�g, 75�g, and 100�g) into chick paw resulted in peak oedema of 35.5%, 38.5%, 42.9%, and 47.5%, respectively, two hours afer injection. Paw oedema subsided within fve hours to a mean volume ranging from 5% (25�gvenom)to17.6%(100�g venom). In conclusion, though N. n. nigricincta belongs to the , the current study has shown its venom to possess potent hemorrhagic, necrotic (cytotoxic), and paradoxically, both procoagulant and thrombolytic activity. Te authors propose further work to fractionate, isolate, and elucidate the structure of the various N. n. nigricincta venom as a prelude to the development of an antivenom.

1. Introduction and snake habitats, domiciliation of (main prey of most snakes), the nocturnal and heat seeking poikilothermic Since time immemorial, man has always sufered from nature of snakes, and accidents during snake handling. Some envenomation resulting from . Although accurate of these snakebites lead to fatalities and wound complications statistics have proved elusive, it is estimated that the global culminating in debilitating physical deformities in victims burden of snakebites stands at 1.2 to 5.5 million bites per year, [4, 5] and associated socioeconomic problems resulting from 25,000-125,000 deaths per year, and about 400,000 victims these disabilities [6, 7]. Te vast size of Namibia as a country lef with permanent disability [1]. In 2009, was also poses a potential problem of bringing emergency health declared a neglected tropical disease by the WHO [2]. Te care to such snakebite victims. World Health Organization declared snake envenomation as Venomous snakes belong to fve main families: Hydrophi- a signifcant Sub-Saharan disease problem [3]. inae, Elapidae, Viperidae, Crotalidae, and Colubridae [8, 9]. In Namibia, like in most other developing countries, the Tese snakes possess venom glands that can synthesize, majority of snake bites result from the overlap of human store, and secrete up to 50-60 proteins/peptides of varying 2 Journal of Toxicology structure but are capable of causing damage at the bite site of capture, and location of release were geo-referenced and and systemically [2, 10]. Te venom components are usually recorded on a database for surveillance of snakes in and fairly similar in snakes of the same family [1]. Venoms of around Windhoek. Venom was diluted with distilled water snakes belonging to the families Elapidae (mainly and and freeze-dried overnight using a Vitis Freeze Dryer (United mambas) and Hydrophiinae (mainly sea snakes) are highly Scientifc). Te resultant venom powder from each snake neurotoxic and produce faccid paralysis and respiratory was stored in a separate sealed and appropriately labelled ∘ paralysis in [2, 11–15]. Viperidae (vipers), Colubridae glass vial at -30 C until time of use. For this study, serial (back-fanged venomous snakes, e.g., Boomslang and the dilutions of venom (250 to 1000�g/ml) from a single snake Twig snake), and Crotalidae (pit-vipers) venoms produce were made by dissolving known quantities of venom powder in addition to systemic/lethal efects, striking local efects, in Phosphate Bufered Saline at pH 7.4 freshly prepared namely, hemorrhage, necrosis, and oedema [11, 16] as well from tablets (Sigma-Aldrich). Sterile 0.5ml needles were as alterations in coagulability of blood [17–19]. Te protein used to administer intradermal and subplantar injections of components of the of Naja sputatrix comprises solutions. the proteins, three-fnger toxins (3FTXs), phospholipase A2 (PLA2), nerve growth factors, and snake venom metallopro- 2.2. Animals. Male and female albino New Zealand rabbits teinaseinthatorder[15].TeZebrasnake(N. n. nigricincta) (about 2.5 kg weight) were obtained through City Pets, is a venomous spitting snake belonging to the Elapidae family Windhoek, and reared in the small stock section at Neudamm and found only in Namibia and Southern [20]. farm, University of Namibia. Day old broiler Cobb500 chicks Tough belonging to the family of Elapids, empirical were obtained from Poultry and reared in the Poultry evidence suggests that the Zebra snake has acquired highly section at Neudamm farm. Blood for thrombolytic studies potent cytotoxic, hemorrhagic, anticoagulant, and throm- was obtained from Kalahari red stud goats reared at the bolytic toxins whilst retaining their familial neurotoxins. Neudamm farm. Namibia has had a very high number of both human and victims of the Zebra snake (Buys, 2016; personal 2.3. Minimum Hemorrhagic Dose (MHD). Te MHD is communication). Snake antivenom immunoglobulins are defned as the least amount of venom (�gdryweight) the only specifc treatment for envenoming by snakebites which, when injected intradermally into rabbits, results in a [4, 21]. Clinically, administering antivenom to the afected hemorrhagic lesion of 10 mm diameter 24 hours later [25]. patient within a very limited time frame (<2 hours) efciently Aliquots of 0.1 ml PBS containing 7.5,10, 25, 50, 75, and 100�g reverses many of the detrimental systemic efects caused of venom were injected into the shaved dorsal skin of each of by snake venom [22]. South African Institute for Medical six adult rabbits marked with grids of 25mm squares (n=6). Research (SAIMR) polyvalent antivenom that is currently Tree replicates were performed for each dose on diferent used in Namibia at a cost of almost US$100.00 per vial is randomly chosen squares of the grid on each rabbit and obviously not afordable to the average rural dweller. Tis then mean values were determined for each concentration on polyvalent antivenom was developed against venoms from each animal. Te animals were sacrifced afer 24 hours, the puf adder, gaboon viper, rinkals, mambas, , forest dorsal skin was removed, and the diameter of the lesions was cobra, , and spitting cobra but not measured on the inner surface of the skin in two directions the zebra snake. at right angles using calipers and with the aid of background Te efcacy of SAIMR polyvalent antivenom against N. illumination. Te MHD was calculated using the regression n. nigricincta envenomation has reportedly not been satisfac- equations relating the doses of venom to the mean diameters tory as specifc treatment for this medically important and of the haemorrhagic lesions. almost exclusively Namibian snake (Buys 2016, personal com- munication). Tough fatalities are reportedly low, Namib- 2.4. Minimum Necrotic Dose (MND). Te MND is defned � ian physicians have resorted to fasciotomy/debridement of as the least amount of venom ( g dry weight) which, when necrotic lesions [23] followed by skin grafing due to the injected intradermally into rabbits, results in a necrotic lesion fact that in this vast country, victims ofen fail to receive of 5mm diameter 72 hours later [25]. Te method used was this polyvalent antivenom within the postulated 2-hr window thesameasthatfortheMHD,exceptthattheskinwas afer bite (Buys 2016, personal communication). Te aim of removed 72 hours afer injection. Te MND was calculated this study was to quantitatively characterize the cytotoxic using the regression equations relating the doses of venom to (necrotic), hemorrhagic, procoagulant, thrombolytic, and the mean diameters of the necrotic lesions. oedema-forming efects of the Namibian zebra snake venom using WHO approved protocols [24]. 2.5. Percentage Trombolysis. Venous blood was drawn from healthy adult male Kalahari red goats (n = 6) of which 2. Materials and Methods 500�L of blood was transferred to each of previously weighed microcentrifuge tubes to form clot. Phosphate bufered saline 2.1. Snake Venom. Venom was carefully and humanely (PBS) at pH 7.4 was added to lyophilized heparin vial (1000 milked by an expert snake handler from snakes that were I.U.) and mixed properly to create a stock solution from caught in suburban Windhoek and later relocated to the which serial dilutions of 0.05, 0.5, 5, and 50 I.U. heparin surrounding Savanna afer replenishment of venom were made for observation of thrombolytic activity of heparin gland stores. Approximate age, sex, length, girth, location using the in vitro method developed by Prasad et al., 2006. Journal of Toxicology 3

45 40 35 30 25 20 15 10 5 Diameter of lesion (mm) lesion of Diameter 0 0 100908070605040302010 Dose of venom (g) N. n. nigricincta Figure 1: Haemorrhagic lesions on internal aspect of rabbit skin 24 Log. (N. n. nigricincta) hours afer intradermal injection of varying doses of N. n. nigricincta venom. Figure 2: Te dose-dependent hemorrhagic activity of N. n. nigricincta venom on adult rabbit skin.

Tis protocol was adapted to measure the thrombolytic � � activity of 100 L solutions containing 25, 50, 75, or 100 g 2.8. Ethical Statement. All animals were used with the ethical � Zebra snake venom on 500 L goat blood. Tis experiment approval from the University of Namibia Ethical Clearance was repeated three times with blood from each animal and Committee (Certifcate: NCREC/01/2018/1). All procedures mean values were determined for each concentration for performed on animals and disposal of animals/animal tissues blood from each animal. followed a protocol approved by the University of Namibia Ethical Clearance Committee. In the course of this study, the 2.6. Coagulation-Altering Activity. Venous blood was drawn researchers strictly adhered to the WHO guidelines [24]. from healthy adult male Kalahari red goats (n = 6) of which 500�L of blood was transferred to each of previously weighed � 2.9. Statistical Analysis. Descriptive and inferential statistics 1.5ml Eppendorf tubes containing 100 L of 250, 500, 750, were performed in SPSS version 25 using one way ANOVA or 1000�g/ml of venom, 0.05, 0.5, 5, or 50 I.U. heparin ≤ � with Tukey’s post-hoc test. P values 0.05 were considered (positive control) or 100 L of PBS (negative control) and statistical signifcant. gently mixed to avoid haemolyzing the blood. Te mixtures ∘ were incubated at 37 C for 90 minutes to allow clots to form. Afer the incubation period, flter paper strips were used to 3. Results drain any liquid contents from the microcentrifuge tubes. Tis experiment was repeated three times and mean values As shown in Figure 1, intradermal injection of N. n. were determined for each concentration for blood from each nigricincta venom produced signifcant haemorrhagic lesions animal. Te clot weight was then determined and compared within 24hrs of injection. Maximum average diameter (40mm, n = 6) was recorded with the highest amount of N. n. with mean clot weight from tubes mixed with PBS. Percentage � coagulation was calculated using the equation below: nigricincta venom (100 g) injected. As shown in Figure 2, N. n. nigricincta venom showed a %Coagulation signifcant dose-dependent increase in the diameter of the hemorrhagic lesions with each increase in the amount of < = clot weight in tube with venom or heparin venom injected into each site (p 0.05, n = 6). Hemorrhagic (1) lesion diameter showed a very strong logarithmic depen- clot weight in tube with PBS 2 denceondoseofvenominjected(R = 0.90). Te MHD × 100 determined from this relationship for N. n. nigricincta was 9.8�g. 2.7. Edema-Forming Efects. Subplantar injection of known As shown in Figure 3, N. n. nigricincta venom showed quantities (25, 50, 75, and 100�g in 0.1ml PBS solution) of a signifcant dose-dependent increase in the diameter of the snake venom was performed into the right paws of 10-day- necrotic lesions with each increase in the amount of venom old chicks (about 250 - 300g weight). Eighteen chicks were injected into each site (p<0.05, n = 6). Necrotic lesion diame- ter showed a very strong logarithmic dependence on dose of used for this protocol. Te change in the paw volume was 2 quantifed using the chick paw edema method by Fereidoni venom injected (R = 0.93). Te MND was determined from and coworkers [26] and improved by Ainooson and others this relationship for N. n. nigricincta (12.4 �g). [27]. Formalin (2.5%) was used as a standard edema-forming Percentage thrombosis of goat blood showed an almost substance (positive control) and PBS was used as the negative perfect negative logarithmic dependence on the dose of 2 control. Te experiment was repeated three times using heparin (R = 0.9991) (Figure 4). Each increase in amount diferent chicks for each level of treatment and mean values of heparin (0.05 I.U., 0.5 I.U., 5 I.U., and 50 I.U.) incubated of proportional change in paw size per concentration were with goat blood showed a signifcant decrease in percentage determined. thrombosis (p<0.05, n = 6). Incubation of 500�L goat blood 4 Journal of Toxicology

18 100 16 90 14 80 12 70 10 60 8 50 6 40

4 % thrombosis 30

Diameter of lesion (mm) lesion of Diameter 2 20 0 10 0 20 40 60 80 100 120 0 0 20 40 60 80 100 120 Dose of venom (g) Amount of venom (ug) N. n. nigricincta Log. (N. n. nigricincta) Figure 5: Dose-dependent coagulative (thrombotic) activity of N. n. nigricincta venom on Kalahari red goat blood. Figure 3: Te dose-dependent necrotic activity of N. n. nigricincta venom on adult rabbit skin. 70 120 60 50 100 40 80 30 % clot lysis % clot 20 60 10 40 0 % thrombosis 025ug (0.05 I.U.) 50ug (0.5 I.U.) 75ug (5 I.U.) 100ug (50 I.U.) 20 Amount of venom/heparin 0 06010 20 30 40 50 N. n. nigricincta venom Linear (N. n. nigricincta venom) Amount of Heparin (I.U.) Heparin Figure 4: Te dose-dependent anticoagulative activity of heparin Linear (Heparin) on Kalahari red goat blood. Figure 6: Comparison of dose-dependent thrombolytic activity of heparin and N. n. nigricincta venom on Kalahari red goat blood clots. with 50 I.U. resulted in only 4.4% thrombosis whilst incuba- tion of same volume of blood with 0.05 I.U. (a 1000 times less venom showed 44.4% thrombolysis which was signifcantly heparin) resulted in 99.5% thrombosis. Tese results show higher than the 36.4% thrombolysis in the presence of 0.05 that heparin has signifcantly potent anticoagulant properties. I.U. heparin (p<0.05). As shown in Figure 5, percentage thrombosis of Kalahari Subplantar injection of N. n. nigricincta venom into red goat blood showed a very strong linear dependence on the 2 chick paw resulted in peak oedema 2-hrs afer injection and dose of N. n. nigricincta venom (R = 0.9892). An increase subsided within 5-hrs to a mean volume ranging from 5% in the amount of venom (25, 50, 75, and 100�g) resulted larger than the original volume (due to 25�gvenom)to in signifcantly higher levels of thrombosis (33.2%, 48.7%, 17.6% larger than the original volume (due to 100�gvenom) 75.9%, and 93.6%, respectively). Tese results show that N. (Figure 7). Te peak oedema was 35.5%, 38.5%, 42.9%, 47.5%, n. nigricincta venom has signifcantly potent procoagulant and 16.3% due to 25�g, 50�g, 75�g, and 100�gofvenomand properties (p<0.05). 2.5% formalin, respectively. Signifcant declines in oedema As shown in Figure 6, thrombolysis of Kalahari red goat were noticed with smaller quantities of venom (100ug > 75 blood clots showed very strong linear relationship with the ug > 50 ug, respectively) (p<0.05; n=18). 2 2 doses of heparin (R = 0.98) and N. n. nigricincta (R = 0.99). Oedema due to 50�g of venom was signifcantly greater At 100�g N. n. nigricincta venom showed 60% thrombolysis than that due to 25�gofvenomwhichinturnwasgreater which was signifcantly higher than the 45.6% thrombolysis that oedema caused by 2.5% formalin (p<0.05). Injection of inthepresenceof50I.U.heparin(p<0.05, n = 6). At PBS resulted in a peak increase in paw size of 4.6% within the 75�g N. n. nigricincta venom showed 55.9% thrombolysis frst 30mins. Paw size resolved back to normal (0% increase) which was signifcantly higher than the 43.9% thrombolysis within 2hrs of injection. Te change in paw size due to in the presence of 5 I.U. heparin (p<0.05, n = 6). At 50�g venom injection was signifcantly greater than that due to N. n. nigricincta venom showed 50.1% thrombolysis which PBS injection throughout the 5hrs of observation (p<0.05). was signifcantly higher than the 40% thrombolysis in the Te change in paw size due to 2.5% formalin was signifcantly presence of 0.5 I.U. heparin (n<0.05). At 25�g N. n. nigricincta greater than that due to PBS for the frst 3hrs afer injection Journal of Toxicology 5

50 Venom from spitting elapids contains 67-73% three fnger 45 40 toxins (3FTXs), 22-30% phospholipases A2 (PLA2), 2.1% 35 snake venom metalloproteinases (SVMPs), and minor quan- 30 25 tities of nucleotidases and cysteine-rich secretory proteins 20 (CRISPs) (Hus et al. [32]). Up to fve cytotoxins (cytotoxin 15 1, 2, 4, 5, and 11) have been isolated and strongly implicated 10 5 in the cytotoxicity of N. mossambica, N annulifera, and N.

% increase in paw oedema in paw % increase 0 pallida which are all close relatives of the N. n. nigricincta 0123456[32]. Similar fndings with another spitting cobra , Time afer injection (hrs) Naja sputatrix, revealed its venom to contain 3FTXs (64.2%),

25ug 50ug 75ug PLA2 (31.2%), nerve growth factor (1.82%), and SVMPs 100ug 2.5% formalin PBS (1.33%). About 48.08% of these 3FTXs were cytotoxins [15]. In addition, PLA2s (acidic PLA2 CM I, basic PLA2 I, and basic Figure 7: Dose-dependent oedema-forming efect of N. n. PLA2 CMIII) have also been implicated in the cytotoxicity nigricincta venom on 10-day-old chick paw. of N. mossambica. SVMPs (cobrin, atragin, and atrase) have been identifed and implicated as minor contributors to cytotoxic activity in N. atra and N. kaouthia venoms [33, 34]. Isolation and identifcation of specifc cytotoxic SVMPs (p<0.05). Tere was no signifcant diference in change in paw in Southern African spitting elapids is, however, not yet size between 2.5% formalin and PBS at 4hrs and 5hrs afer reported. It is therefore, logical to speculate that the Southern injection (p>0.05%). African spitting cobras also have a smaller contribution from SVMP’s towards their venoms’ cytotoxicity. CRISPs 4. Discussion (annuliferin, nawafarin, and natrin I) have been isolated and confrmed to contribute towards the cytotoxic activity of N. MHD and MND have been extensively used in the preclinical nigricollis and N. annulifera both of which are close relatives assessment of viperid and crotalid venoms as important of the N. n. nigricincta [32]. WHO approved protocols for haemorrhagic and necrotizing Te cytotoxic mechanisms of 3FTXs, PLA2s, and SVMPs venom toxins. Tough longstanding knowledge of cyto- mainly involve the disruption of microvascular basement toxins in spitting elapids exists, the presence of powerful membranes [35] as well as endothelial cell membranes to cytotoxins has also been well investigated and documented result in the observed haemorrhage, oedema, and myonecro- in nonspitting elapids. Tan and coworkers characterized a sis (resulting from disruption of plasma membranes of skele- signifcant cytotoxin contribution to the proteomic profle tal muscle cells) [32]. Tese mechanisms provide possible of the Malayan blue coral snake [28]. Te proteomic profle objectives in any further investigation of the cytotoxicity of of the Pakistan Naja naja was also recently documented N. n. nigricincta venom. [29], though there is no documentation of MHD and MND In vitro exposure of whole goat blood to N. n. nigricincta studies on the venoms from these elapids. Te assessment venom resulted in enhancement of coagulation but the of haemorrhagic activity in Micrurus pyrrhocryptus (a Latin exposure of pre-formed goat blood clots resulted in profound American elapid) venom using these protocols in mice and thrombolytic activity. It is not unusual to fnd one venom con- rats produced negative results [30]. Te MHD (rabbit) of N. taining both fbrinolytic (anticoagulant) and fbrinogenolytic n. nigricincta venom at 9.8�gwas,however,almostsimilar (coagulant) activities [36, 37]. Snake venom serine pro- to that of Bothrops atrox, a viper, determined using a similar teinases (SVSPs) have been found in elapid, viperid, and col- assay on mice by researchers in Colombia (Otero et al., ubrid venoms. Tese have been implicated in the interference 2000). At 12.4�g, the MND of N. n. nigricincta venom was with platelet aggregation, blood coagulation, fbrinolysis, less that the 39.3�gofEchis ocellatus (Nigeria), 47.15�gof complement system, and immune system [38]. Trombin- Echis leucogaster (Mali), the 24.9�gofEchis pyramidum like SVSPs (TLEs), however, have been implicated in procoag- leakeyi (Kenya), the 64.8�gofBitis arietans (Nigeria), and ulation through activation of factor V,VIII, XIII, possibly VII the 28.2�gofBitis gabonica (Nigeria) from a study by Segura and XI. TLEs have also been known to stimulate fbrinolysis et al. [31]. Tese fndings show that N. n. nigricincta venom and also activation of platelet aggregation [39]. Future studies has probably successfully acquired haemorrhagic activity with N. n. nigricincta venom can be guided towards proving equal to or even surpassing those of Viperidae.Inthis or disproving involvement of these mechanisms. study, an attempt to reduce the number of animals used by An L-amino acid oxidase with human platelet aggrega- using rabbits in place of rats and mice, however, resulted tion activity from Ophiophagus hannah (king cobra) venom in a major limitation when comparing the fndings from was isolated and characterized [40]. Cardiotoxin was iso- this study to those of other earlier studies. Te fndings lated from Naja naja atra () venom; this from this pioneering study with Zebra snake venom, how- was able to potentiate platelet aggregation induced by ever, provide a basis way for the use of WHO protocols ADP, thrombin, collagen, and venom phospholipase A2 [41]. involving large numbers of mice and rat for future work Cobra venom phospholipase A2 showed conficting efects to determine and compare the toxicity of this venom to on washed rabbit platelets, an initial reversible calcium- other dependent aggregation followed by an inhibition of platelet 6 Journal of Toxicology aggregation with longer incubation times [42]. Two three- References fnger toxins, hemextin A and hemextin B, were isolated and purifed from Hemachatus haemachatus () venom. [1]J.M.Guti´errez, T. Escalante, A. Rucavado, and C. Herrera, Individually, hemextin A prolongs blood coagulation, but “Hemorrhage caused by snake venom metalloproteinases: A journey of discovery and understanding,” Toxins,vol.8,no.4, hemextin B does not show any efect on blood clotting. article no. 93, 2016. However, hemextin AB complex inhibits coagulation by [2] S. K. Hirebail, H. Nagabushan, and G. M. Prakash, “A prospec- noncompetitively inhibiting the Tissue Factor–Factor VIIa tive study of efcacy and safety of olopatadine versus azelastine (extrinsic tenase) complex [43]. Studies to profle the pro- in allergic conjunctivitis at a tertiary care hospital,” International teomics of N. n. nigricincta venom would provide signifcant Journal of Basic & Clinical Pharmacology,vol.6,no.12,pp. and relevant information that can be applied in combating 2836–2842, 2017. envenomation from this species. [3] WHO, “WHO guidelines for the production, control and regu- Oedema-causing toxins in snake venom have not been lation of snake antivenom immunoglobulins,” Geneva, vol. 134, extensively studied. However, one study concluded that 2010. oedema induced by Bothrops snake venoms was multi- [4] D. Wood, C. Webb, and J. Demeyer, “Severe snakebites in factorial [44]. Other workers suggested that haemorrhagic northern KwaZulu-Natal: Treatment modalities and outcomes,” toxins, through disruption of the microvasculature, resulted South African Medical Journal,vol.99,no.11,pp.814–818,2009. in extravasation which characterizes observed oedema in [5] M. Wagener, M. Naidoo, and C. Aldous, “Wound infection some envenomations [45, 46]. Tese authors also suggested secondary to snakebite,” South African Medical Journal,vol.107, the involvement of other toxins which acted directly on the no. 4, pp. 315–319, 2017. endothelial cells of capillaries and venules thus increasing [6] M. M. Dalhat, “Socioeconomic aspects of snakebite in Africa their permeability. Histamine release from mast cells as a and the tropics,” in Toxinology: Clinical Toxinology in Asia result of phospholipases and cytotoxins was also a possible Pacifc and Africa, pp. 299–310, Springer, Dordrecht, Nether- mechanism [47]. Another suggested mechanism was the lands, 2015. release of prostaglandins resulting from phospholipase A2- [7] A. G. Habib and N. I. Brown, “Te snakebite problem and anti- induced liberation of arachidonic acid from plasma mem- venom crisis from a health-economic perspective,” Toxicon,vol. branes [44]. In one study it was illustrated that Bothrops 150, pp. 115–123, 2018. jararaca venom proteases activated plasma kininogens to [8]J.WhiteandJ.Meier,“Handbookofclinicaltoxicologyof bradykinin, an infammatory mediator responsible for vaso- animal venoms and poisons,” 2017. dilation (and thus oedema) at the site of infammation [48]. [9] I. Albaba, “Venomous snakes and envenomation in Palestine,” Journal of Entomology and Zoology Studies,vol.493,no.52,pp. Kallikrein released afer vascular damage was also suggestion 493–495, 2017. in the activation of kininogens to bradykinin [44]. Due to the [10] M. Choudhury, R. J. R. McCleary, M. Kesherwani, R. M. Kini, major diferences between viperid and elapids, suggestion of and D. Velmurugan, “Comparison of proteomic profles of the these mechanisms for elapid venom at best remains specula- venoms of two of the ‘Big Four’ snakes of India, the tive and needs to be investigated for N. n. nigricincta venom. (Naja naja) and the common krait (Bungarus caeruleus), and analyses of their toxins,” Toxicon,vol.135,pp.33–42,2017. 5. Conclusion [11] N. Sharma, S. Chauhan, S. Faruqi, P. Bhat, and S. Varma, “Snake envenomation in a north Indian hospital,” Emergency Medicine In conclusion, fractionation of N. n. nigricincta venom and Journal, vol. 22, no. 2, pp. 118–120, 2005. further investigations with cytotoxic, oedema-forming, pro- [12] U. Upadhyay, U. Upadhyay, N. K. Mohanty, and S. K. Behera, coagulant, and thrombolytic fractions separately may reveal “Neurotoxic snakebite cases with role of imaging,” Journal of the toxins responsible for observed activity of the venom Evidence Based Medicine and Healthcare,vol.4,no.90,pp.5418– in this study. Further work will then be required with N. 5423, 2017. n. nigricincta venom to unravel the mechanisms of action [13] C. H. Tan, K. Y. Tan, S. E. Lim, and N. H. Tan, “Venomics of of any discovered toxins. Te severity of the sequelae of the beaked sea snake, Hydrophis schistosus: A minimalist toxin the local cytotoxicity of this venom warrants a separate arsenal and its cross-neutralization by heterologous antiven- investigation into the formulation of efective intervention oms,” Journal of Proteomics,vol.126,pp.121–130,2015. measures (both antivenom and other emergency on-site [14]K.Y.Wong,C.H.Tan,andN.H.Tan,“Venomandpurifed measures including phytotherapy) to reduce the fatalities toxins of the spectacled cobra (Naja naja) from Pakistan: and bodily deformities resulting from envenomation by this Insights into toxicity and antivenom neutralization,” Te Amer- ican Journal of Tropical Medicine and Hygiene,vol.94,no.6,pp. snake. 1392–1399, 2016. [15]C.H.Tan,K.Y.Wong,K.Y.Tan,andN.H.Tan,“Venompro- Data Availability teome of the yellow-lipped sea krait, Laticauda colubrina from : Insights into subvenomic diversity, venom antigenicity and Te data used to support the fndings of this study are cross-neutralization by antivenom,” Journal of Proteomics,vol. available from the corresponding author upon request. 166,pp.48–58,2017. [16] P. K. Goswami, M. Samant, and R. S. Srivastava, “Snake venom, Conflicts of Interest anti-snake venom & potential of snake venom,” International Journal of Pharmacy and Pharmaceutical Sciences,vol.6,no.5, Te authors declare that they have no conficts of interest. pp.4–7,2014. Journal of Toxicology 7

[17]E.Alirol,S.K.Sharma,H.S.Bawaskar,U.Kuch,andF. [31] A.´ Segura, M. Villalta, M. Herrera et al., “Preclinical assessment Chappuis,“Snakebiteinsouthasia:areview,”PLOS Neglected of the efcacy of a new antivenom (EchiTAb-Plus-ICP5)forthe Tropical Diseases,vol.4,no.1,articlee603,2010. treatment of viper envenoming in sub-Saharan Africa,” Toxicon, [18] A. De Vries and I. Cohen, “Hemorrhagic and blood coagulation vol. 55, no. 2-3, pp. 369–374, 2010. disturbing action of snake venoms,” Recent Advances in Blood [32] K. Hus, J. Buczkowicz, V. Petrilla et al., “First look at the venom Coagulation,1969. of naja ashei,” Molecules,vol.23,no.3,pp.1–10,2018. [19] K. Y. Tan, N. H. Tan, and C. H. Tan, “Venom proteomics and [33] N. H. Tan, K. Y. Wong, and C. H. Tan, “Venomics of Naja antivenom neutralization for the Chinese eastern Russell’sviper, sputatrix, the : A short neurotoxin-driven Daboia siamensis from and ,” Scientifc Reports, venom needing improved antivenom neutralization,” Journal of vol. 8, no. 1, 2018. Proteomics,vol.157,pp.18–32,2017. [20] W. W¨uster, S. Crookes, I. Ineich et al., “Te phylogeny of [34] H.-W. Huang, B.-S. Liu, K.-Y. Chien et al., “Cobra venom cobras inferred from mitochondrial DNA sequences: Evolution proteome and glycome determined from individual snakes of venom spitting and the phylogeography of the African of Naja atra reveal medically important dynamic range and spitting cobras (Serpentes: Elapidae: Naja nigricollis complex),” systematic geographic variation,” Journal of Proteomics,vol.128, Molecular Phylogenetics and Evolution,vol.45,no.2,pp.437– pp. 92–104, 2015. 453, 2007. [35] A. G. Konshina, P. V. Dubovskii, and R. G. Efremov, “Structure [21] J. F´elix-Silva, A. A. Silva-Junior, S. M. Zucolotto, and M. de and dynamics of cardiotoxins,” Current Protein & Peptide Freitas Fernandes Pedrosa, “Medicinal plants for the treatment Science,vol.13,no.6,pp.570–584,2012. of local tissue damage induced by snake venoms: an overview [36] C. H. Oliveira, A. A. Sim˜ao,M.V.Trento,P.H.C´esar, and S. from traditional use to pharmacological evidence,” Evidence- Marcussi, “Inhibition of proteases and phospholipases A2 from Based Complementary and Alternative Medicine,vol.2017, Bothrops atrox and Crotalus durissus terrifcus snake venoms ArticleID5748256,52pages,2017. by ascorbic acid, vitamin E, and B-complex vitamins,” Anais da [22] S. E. Gasanov, R. K. Dagda, and E. D. Rael, “Snake Venom Academia Brasileira de Cienciasˆ ,vol.88,no.3,pp.2005–2016, Cytotoxins, Phospholipase A2s, and Zn2+-dependent Met- 2016. alloproteinases: Mechanisms of Action and Pharmacological [37] T. Matsui, Y. Fujimura, and K. Titani, “Snake venom proteases Relevance,” Journal of Clinical Toxicology,vol.4,no.1,2014. a´ı ecting hemostasis and thrombosis,” Biochimica et Biophysica [23]R.M.Stewart,C.P.Page,W.H.Schwesinger,R.McCarter,J. Acta (BBA),vol.1477,pp.146–156,2000. Martinez, and J. B. Aust, “Antivenin and fasciotomy/debride- [38] R. M. Kini, “Serine proteases afecting blood coagulation and ment in the treatment of the severe rattlesnake bite,” Te fbrinolysis from snake venoms,” Pathophysiology of Haemosta- American Journal of Surgery,vol.158,no.6,pp.543–547,1989. sis and Trombosis,vol.34,no.4-5,pp.200–204,2006. [24] WHO, “WHO Guidelines for the Production, Control and [39] T. Sajevic, A. Leonardi, and I. Krizaj,ˇ “Haemostatically active Regulation of Snake Antivenom Immunoglobulins,” Geneva, proteins in snake venoms,” Toxicon,vol.57,no.5,pp.627–645, 2016. 2011. [25] H. Kondo, S. Kondo, H. Ikezawa, and R. Murata, “Studies on the [40] Z.-Y. Li, T.-F. Yu, and E. C.-Y. Lian, “Purifcation and character- quantitative method for determination of hemorrhagic activity ization of l-amino acid oxidase from king cobra (Ophiophagus of habu snake venom,” Japanese Journal of Medical Science and hannah) venom and its efects on human platelet aggregation,” Biology,vol.13,pp.43–51,1960. Toxicon,vol.32,no.11,pp.1349–1358,1994. [26] M. Fereidoni, A. Ahmadiani, S. Semnanian, and M. Javan, “An accurate and simple method for measurement of paw edema,” [41] C.-M. Teng, W. Jy, and C. Ouyang, “Cardiotoxin from Naja Journal of Pharmacological and Toxicological Methods,vol.43, naja atra snake venom: A potentiator of platelet aggregation,” no.1,pp.11–14,2000. Toxicon,vol.22,no.3,pp.463–470,1984. [27] G. K. Ainooson, G. Owusu, E. Woode, C. Ansah, and K. Annan, [42] C.-P. Teng, Y.-P. Kuo, L.-G. Lee, and C. Ouyang, “Efect of “Trichilia monadelpha bark extracts inhibit carrageenan- cobra venom phospholipase A2 on platelet aggregation in induced foot-oedema in the 7-day old chick and the oedema comparison with those produced by arachidonrc acid and associated with adjuvant-induced arthritis in rats,” African Jour- lysophophatidylcholine,” Trombosis Research,vol.44,no.6,pp. nal of Traditional, Complementary, and Alternative Medicines : 875–886, 1986. AJTCAM/African Networks on Ethnomedicines,vol.9,no.1,pp. [43] Y. Banerjee, R. Lakshminarayanan, S. Vivekanandan, G. S. 8–16, 2012. Anand, S. Valiyaveettil, and R. M. Kini, “Biophysical character- [28]C.H.Tan,S.Y.Fung,M.K.K.Yap,P.K.Leong,J.L.Liew, ization of anticoagulant hemextin AB complex from the venom and N. H. Tan, “Unveiling the elusive and exotic: Venomics of of snake Hemachatus haemachatus,” Biophysical Journal,vol.93, the Malayan blue coral snake (Calliophis bivirgata faviceps),” no. 11, pp. 3963–3976, 2007. Journal of Proteomics,vol.132,pp.1–12,2016. [44] J. M. Guti´errez, B. Lomonte, and J. M. Guti´errez, “Local [29]K.Y.Wong,C.H.Tan,K.Y.Tan,N.H.Quraishi,andN.H. tissue damage induced by Bothrops snake venoms - A review,” Tan, “Elucidating the biogeographical variation of the venom Memorias´ do Inst. Butantan,vol.51,no.4,1989. ofNajanaja(spectacledcobra)fromPakistanthroughavenom- [45] C. L. Ownby, “Pathology of rattlesnake envenomation,” in Rat- decomplexing proteomic study,” Journal of Proteomics,vol.175, tlesnake Venoms, pp. 163–209, Marcel Dekker, New York, NY, pp. 156–173, 2018. USA, 1982. [30] J. C. Dokmetjian, S. del Canto, S. Vinz´on, and M. B. de [46] A. Ohsaka, “Hemorrhagic, Necrotizing and Edema-Forming Jim´enez Bonino, “Biochemical characterization of the Micrurus Efects of Snake Venoms,” in Snake Venoms, C.-Y. Lee, Ed., vol. pyrrhocryptus venom,” Toxicon,vol.53,no.3,pp.375–382, 52 of Handbook of Experimental Pharmacology,pp.480–546, 2009. Springer, Heidelberg, Berlin, 1979. 8 Journal of Toxicology

[47] L. Preciado, J. A. Perea˜nez, V. Nu˜nez, and T. Lobo-Echeverri, “Characterization of the most promising fraction of Swietenia macrophylla active against myotoxic phospholipases A2: Iden- tifcation of catechin as one of the active compounds,” Vitae,vol. 23,no.2,pp.124–133,2016. [48] M. Rocha e silva, W.T. Beraldo, and G. Rosenfeld, “Bradykinin, a hypotensive and smooth muscle stimulating factor released from plasma globulin by snake venoms and by trypsin,” Amer- ican Journal of Physiology-Endocrinology and Metabolism,vol. 156, no. 2, pp. 261–273, 1949. M EDIATORSof INFLAMMATION

Canadian Journal of The Scientifc Autoimmune Infectious Diseases and Scientica Diseases Medical Microbiology Hindawi World Journal www.hindawi.com Volume 2018 Hindawi Hindawi Publishing Corporation Hindawi Hindawi www.hindawi.com Volume 2018 http://www.hindawi.comwww.hindawi.com Volume 20182013 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018

International Journal of Emergency Medicine Medicinal Chemistry International

Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018

Submit your manuscripts at www.hindawi.com

Pain Journal of Research and Treatment Addiction Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018

Stroke Neurology Journal of Anesthesiology Journal of Research and Treatment Research International Pharmaceutics Research and Practice Tropical Medicine Hindawi Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018

Advances in BioMed Journal of Pharmacological Journal of Research International Toxicology Arthritis Sciences Drug Delivery Hindawi Hindawi Hindawi Hindawi Hindawi www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018 www.hindawi.com Volume 2018