Cytotoxiciy of Naja nubiae ( Serpentes : Elapidae ) and Echis ocellatus ( Serpentes : Viperidae ) Venoms from Sudan

In Sudan, as in many African countries, no local specific antivenom is manufactured resulting in snake bite victims being treated by antivenoms imported from abroad. In the present work we measured the cytotoxic effect of the recently described spitting cobra (Naja nubiae) and the carpet viper (Echis ocellatus) snake venoms using a cell based assay. We also investigated the efficacy of four antivenoms CSL (Australia), SAIMR (South Africa), snake venom antiserum (India), and EchiTAb-Plus-ICP (Cost Rica) to neutralize the cytotoxic effect of the two venoms. The venoms resulted in a remarkable inhibition of cell viability with N. nubiae being more cytotoxic than E. ocellatus. The four antivenoms studied were effective in neutralizing N. nubiae cytotoxicity. However, only partial efficacy in neutralizing the cytotoxic effect of E. ocellatuswas achieved using CSL (Australia) and SVA (India) antivenoms. Based on the cross neutralization by the four antivenoms, the Sudanese N. nubiae venommost likely has homologous epitopes with similar snakes from Australia, South Africa, India, and Cost Rica, while E. ocellatus venom from Sudan shares little homology with similar snakes from other countries.


Introduction
Snakes belonging to the genus Echis (saw-scaled viper) and Naja (cobras) are widely distributed in Africa and are of great medical importance [1][2][3].Some species in the genus Naja (nonspitting cobras) are predominantly neurotoxic and produce progressive paralysis without necrosis [3].The other species (spitting cobras) are characterized by cytotoxic pattern of envenomation which includes swelling at the bite site with blistering and bruising that may lead to necrosis [3,4].The abundance of cytotoxins and cytotoxic PLA 2 s in the venom of spitting cobras is suggested to be the main factor responsible for these clinical features [5,6].The venom of the recently described spitting cobra, Naja nubiae [7], is unique as it displays both cytotoxic and neurotoxic properties [5].The preclinical testing of cytotoxicity in the case of cytotoxic Naja deserves attention since local tissue damage is a major consequence [3,8].
The saw-scaled or carpet viper (genus Echis) is the most important cause of snakebite mortality and morbidity in the sub-Saharan savannah region [3,9].Bites by these snakes produce moderate to severe local swelling, blistering, and necrosis with sever systematic haemostatic disorders [3,10,11].
Recently, a MTS based cell cytotoxicity assay is being widely used as an alternative model for testing the cytotoxic effect of venoms as well as the efficacy of antivenoms with the advantage of avoiding the use of experimental animals [6,12,13].In this study, a rat skeletal muscle cell line, L6, was used to examine the cytotoxic activity of Naja nubiae and Echis ocellatus venoms.Four antivenoms, CSL (Australia), SAIMR (South Africa), snake venom antiserum (India), and EchiTAb-Plus-ICP (Cost Rica), were also used to determine their ability to neutralize the cytotoxic effect of the two venoms.venom (1.647-8 g/mL) and E. ocellatus venom (0.668-50 g/mL) resulted in a concentration-dependent inhibition of cell viability with an IC 50 value of 4.27 g/mL for N. nubiae and 10.33 g/mL for E. ocellatus ( = 4; Figure 1).Previously the same cell-based assay examined the cytotoxic effect of the Naja nigricollis and Naja mossambica venoms on L6 skeletal muscle cell line [13] and found IC 50 = 7.2 ± 0.6 g/mL for N. nigricollis and IC 50 = 3.1 ± 0.4 g/mL for N. mossambica.This indicates that N. nubiae possesses venom comparable with the highly cytotoxic spitting cobras.It was observable that N. nubiae venom was markedly more cytotoxic than E. ocellatus since the concentration used for N. nubiae venom was sixfold less than E. ocellatus venom.The high cytotoxicity of N. nubiae venom is attributed to the presence of high concentrations of cytotoxic components such as cytotoxins and cytotoxic PLA 2 .Cytotoxins account for 58%-73% of the venom proteins of spitting cobras [5,6].The results of this study agreed with Chiam-Matyas and Ovadia [14] who postulated that the cytotoxic activity of elapids is higher than vipers.It is also supported by a cell-based study which examined myonecrotic PLA 2 of Naja nigricollis and Vipera russelli snakes [15].In agreement with the current finding, Naja nigricollis PLA 2 was more potent at eliciting the myotoxic effect than Vipera russelli.These findings are consistent with the general consideration that spitting cobras are mainly cytotoxic with little, if any, neurotoxicity [8,16].It also agrees with clinical features that characterize envenoming by spitting cobras [3,4].

The Effect of Antivenoms on N. nubiae Venom Cytotoxicity.
L6 cells were incubated with serially diluted N. nubiae venom (1.647-8 g/mL) in a media supplemented with CSL, SAIMR, SVA, or EchiTAb-Plus-ICP antivenoms.The effects of the four antivenoms on the venom concentrations that caused 100% (IC 100 ) and 50% (IC 50 ) cell death are shown in Figure 2. All the antivenoms significantly inhibited the cytotoxic effect of N. nubiae venom at a venom concentration that caused 100% and 50% cell death (one-way ANOVA,  < 0.05).The four antivenoms were effective in neutralizing the cytotoxic effect of N. nubiae venom despite the fact that none of the antivenoms contain N. nubiae in its immunization mixture.This cross neutralization indicates the presence of antibodies capable of neutralizing N. nubiae cytotoxins in the four tested antivenoms.SAIMR polyvalent antivenom was raised against the venom of 10 species of viperid and elapid snakes including N. mossambica venom whereas EchiTAb-Plus-ICP antivenom contains N. nigricollis venom in its immunization mixture.The venoms of both N. mossambica and N. nigricollis are highly cytotoxic [3].Since all spitting cobras have similar cytotoxic PLA 2 s [5], it is not surprising that antibodies derived from N. nigricollis and N. mossambica cytotoxins are capable of neutralizing N. nubiae cytotoxicity.This finding is supported by a recent cell-based assay which found SAIMR antivenom effective against the cytotoxic effect of both N. nigricollis and N. mossambica venoms [13].Another cellbased assay by Méndez et al. reported effective neutralization of N. nubiae venom cytotoxicity by EchiTAb-Plus-ICP antivenom [6].Interestingly, they found the antivenom efficiency higher in the case of N. nubiae venom than N. nigricollis, the venom used in the manufacture of EchiTAb-Plus-ICP which explains the complex factors participating in the neutralization of snake venom toxins.Moreover, recent investigation of the efficacy of EchiTAb-Plus-ICP antivenom against PLA 2 activity of N. nubiae venom revealed high antivenomic neutralization potency [5].The CSL polyvalent antivenom which is produced against group of Australian elapids is also capable of neutralizing the cytotoxicity of N. nubiae venom.This indicates the presence of antibodies capable of neutralizing N. nubiae cytotoxins in the CSL antivenom.The polyvalent SVA antivenom contains the cytotoxic Indian cobra Naja naja venom in its immunization mixture which may contribute to its efficacy.

The Effect of Antivenoms on E. ocellatus Venom Cytotoxicity.
L6 cells were incubated with serially diluted E. ocellatus venom (0.668-50 g/mL) in a media supplemented with CSL, SAIMR-Echis, SVA, or EchiTAb-Plus-ICP antivenoms (5 units/mL).The effects of the four antivenoms on the venom concentrations that caused 100% (IC 100 ) and 50% (IC 50 ) cell death are shown in (Figure 3).All the antivenoms failed to  significantly inhibit the cytotoxic effect of E. ocellatus at IC 100 .However, CSL and SVA were able to significantly inhibit the effect of E. ocellatus venom at IC 50 (one-way ANOVA,  < 0.05).It is surprising that SAIMR-Echis and EchiTAb-Plus-ICP antivenoms were unable to inhibit the cytotoxic effect of E. ocellatus venom at IC 100 and IC 50 .The former is monospecific antivenom against E. ocellatus while the latter is polyspecific antivenom against E. ocellatus, B. arietans, and N. nigricollis.This deficit is more likely to be due to lack of neutralizing antibodies against the cytotoxic PLA 2 and cytotoxins in the two antivenoms in spite of the fact that E. ocellatus has been used in the immunization mixture for the production of the two antivenoms.Furthermore it is surprising that the effect in the case of EchiTAb-Plus-ICP antivenom led to more cell death than the venom alone.A possible explanation for this might be the large volume of antivenom used coupled with its low efficacy which results in media dilution and more cell death.Our results are constant with previous studies which observed impairment in the neutralization of some snake cytotoxins.A cell-based assay found death adder antivenom (CSL Ltd.) unable to prevent the cell death caused by the death adder (Acanthophis spp.) even  at high antivenom concentration [13], although death adder antivenom was able to inhibit the myotoxic and the neurotoxic effects of Acanthophis spp.[17].Moreover Gowda and Middlebrook reported impaired neutralization of N. nigricollis PLA 2 by rabbit antiserum produced against the same toxin [15].However, rabbit antiserum prepared the same way easily neutralized the lethal effects of the snake venom PLA 2 neurotoxins [18].On the other hand, the findings of the current study do not support previous reports which showed that EchiTAb-Plus-ICP antivenom was effective in eliminating the myotoxic effect of mice injected intramuscularly with E. ocellatus venom [19].This difference in the antivenom potency may be explained by the difference in the assay used for assessment (in vivo and in vitro) or it may be due to the geographical difference between the two venoms examined (E.ocellatus from East and West Africa).CSL, which is polyspecific antivenom produced against group of Australian snakes, was found effective in neutralizing the cytotoxic effect of E. ocellatus venom at IC 50 while not effective at IC 100 .Since all medically important Australian snakes are elapid, this partial neutralization may be attributed to the presence of neutralizing antibodies against elapid cytotoxins and cytotoxic PLA 2 in CSL polyvalent antivenom.Partial neutralization was also reported with the Indian SVA which is polyvalent antivenom raised against two elapids (Naja naja and Bungarus caeruleus) and two vipers (Echis carinatus and Daboia russelii).Although E. carinatus is included in the immunization mixture, it seems that the presence of the elapid cytotoxins has a crucial role in the antivenom potency.
The results of this study indicate a low immunogenicity of E. ocellatus cytotoxins and suggest that their neutralization depends on the presence of antibodies against cytotoxic components belonging to other venoms such as elapids.However, previous reports showed that elapids and vipers appeared to have different mode of cytotoxicity.Elapids venom causes irreversible depolarization of the cell membrane which results in cell death within the first hour of incubation, displaying a cellular necrosis effect.While in the case of vipers, the cells become rounded, lose the attachment with the substrate, and finally die, displaying an apoptotic effect [14].

Cells.
The rat skeletal muscles myoblast cells line, L6, was purchased from the American Tissue Type Collection (ATTC Virginia, USA).

Preparation of the Venoms.
The freeze-dried venoms were suspended in milliQ water and filtered through 0.22 m millipore membrane (Millipore; Bedford, MA, USA).The protein concentration was determined using the bicinchoninic acid reagent (BCA) protein assay kit (Pierce; Illinois, USA).BSA was used as standard solution at dilutions (1-0.025mg/mL) and milliQ water as blank.The venom samples were diluted to 1 : 2 and 1 : 4. Twenty-five microliters (25 L) of the standard solutions and the venom samples were added to 96-well microtiter plate and the absorbance was measured at 562 nm in a fusion  microplate reader according to manufacturer's instructions (Packard bioscience; Connecticut, USA).
Venom samples were divided into aliquots and stored at −20 ∘ C till used.
3.5.Cell Culture.L6 cells were cultured in 175 cm 2 flask (Greiner Bio-One; Frickenhausen, Germany) using DMEM culture media.Media were supplemented with 5% FCS and 1% penicillin/streptomycin (5% DMEM).Cells were incubated at 37 ∘ C and media was changed every second day in an atmosphere of 5% CO 2 until 50% confluence assessed under light microscope.Cells were lifted using trypsin and pelleted.The cell pellet was resuspended in 30 mL culture media and seeded at 100 L/well in 96-well cell culture plates.Plates were incubated at 37 ∘ C in an atmosphere of 5% CO 2 and the media were changed every second day until cells were 90% confluent (assessed by eye using light microscope).Media were discarded from wells and replaced with DMEM media supplemented with 2% FCS and 1% penicillin/streptomycin 2% DMEM enabled the differentiation of myoblasts into myocytes.Plates were incubated at 37 ∘ C at atmosphere of 5% CO 2 and the media changed every second day until the differentiation was observed under light microscope.

MTS Assay
3.6.1.The Effect of the Venom on Cell Proliferation.Media were removed from wells and the venom stock solutions were diluted in culture media (2% DMEM) to a final concentration of 8 g/mL for N. nubiae venom and subsequently serially diluted 1.1-fold 15 times (1.647-8 g/mL).For E. ocellatus media were diluted to a final concentration of 50 g/mL and then serially diluted 1.3-fold 15 times (0.668-50 g/mL).Samples (100 L/well) were added to the 96-well cell culture plate in quadruplicate.Control samples (cells + media, without venom) and media blanks (no cells) were also run in parallel and the plates were incubated at 37 ∘ C in an atmosphere of 5% CO 2 for 24 h.The cell culture plates were then removed from the incubator and washed three times with prewarmed PBS.DMEM (50 L) and MTS solution (10 L) were added to each well and the plates were incubated for 3 h at 37 ∘ C at atmosphere of 5% CO 2 .Absorbance was measured at 492 nm utilizing a fusion  plate reader (Packared Bioscience; Connecticut, USA).

The Effect of Antivenoms on Cell Proliferation.
The protein concentrations of the antivenoms were as follows: CSL polyvalent snake antivenom 814.2 mg/mL, SAIMR 481.6 mg/mL, SAIMR-Echis 548.5 mg/mL, EchiTAb-Plus-ICP 230 mg/mL, and snake venom antiserum (SVA) 173.9 mg/mL.The cell culture was carried out as described above; antivenoms (5 U/mL) were added to the cell media before serially diluting the venom 1.1-fold for N. nubiae and 1.3-fold for E. ocellatus.Control samples (cells + media + antivenoms) and media blanks (media + antivenom) were also run in parallel.When the number of units is not indicated in the vial, the antivenom was used in a mass to mass ratio as antivenom with units/mL indicated.The plates were incubated 24 h at 37 ∘ C with 5% CO 2 .The plates were washed, MTS was added, and the absorbance was measured as described above.
3.6.3.Data Analysis.Data were analyzed using the Graphpad Prism 5 software (Graphpad software Inc., California, USA, 2007).The sigmoidal growth curve displayed as a percentage of cell viability versus log venom concentration and the IC 50 (the half maximal inhibitory concentration) was calculated for each venom.The results were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison test with statistical significance indicated when  < 0.05.

Conclusions
Using the cell-based assay, higher cytotoxicity was reported to N. nubiae venom compared to E. ocellatus venom.
The antivenoms CSL (Australia), SAIMR (South Africa), SVA (India), and EchiTAb-Plus-ICP (Cost Rica) showed high potency against N. nubiae cytotoxicity suggesting presence of homologous epitopes.E. ocellatus venom mostly likely has diverse immunological epitopes and was only partially neutralized with CSL (Australia) and SVA (India).

Figure 2 :
Figure 2: L6 cells incubated with N. nubiae venom and (a) CSL antivenom, (b) SAIMR antivenom, (c) SVA antivenom, or (d) EchiTAb-Plus-ICP antivenom using two venom concentrations: the first concentration caused 100% cell death (IC 100 ) and the second caused 50% cell death (IC 50 ) ( = 4).Cell viability is expressed as a percentage of the control.Statistical analysis was made by comparing venom with venom + antivenom using a one-way ANOVA,  < 0.05 followed by Bonferroni multiple comparison test.

Figure 3 :
Figure 3: L6 cells incubated with E. ocellatus venom and (a) CSL antivenom, (b) SAIMR-Echisantivenom, (c) SVA antivenom, or (d) EchiTAb-Plus-ICP antivenom using two venom concentrations.The first concentration caused 100% cell death (IC 100 ) and the second concentration caused 50% cell death (IC 50 ) ( = 4).Cell viability is expressed as a percentage of the control.Statistical analysis was made by comparing venom with venom + antivenom using a one-way ANOVA,  < 0.05 followed by Bonferroni multiple comparison test.