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Toxicon 52 (2008) 842–851

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Toxicon

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Interspecific variation in venom composition and toxicity of Braziliansnakes from Bothrops genus

Giselle Pidde Queiroz a, Lucas Alves Pessoa a, Fernanda C.V. Portaro a,Maria de Fatima D. Furtado b, Denise V. Tambourgi a,*

a Immunochemistry Laboratory, Butantan Institute, Av. Prof. Vital Brazil, 1500, CEP 05503-900, Sao Paulo, Brazilb Herpetology Laboratory, Butantan Institute, Av. Prof. Vital Brazil, 1500, CEP 05503-900, Sao Paulo, Brazil

a r t i c l e i n f o

Article history:Received 18 April 2008Received in revised form 5 September 2008Accepted 7 October 2008Available online 17 October 2008

Keywords:Bothrops spp.Snake venomsToxic activitiesAntivenomCross-reactivitySerum neutralization

* Corresponding author. Fax: þ55 11 3726 7222x2E-mail address: dvtambourgi@butantan.gov.br (D

0041-0101/$ – see front matter � 2008 Elsevier Ltddoi:10.1016/j.toxicon.2008.10.002

a b s t r a c t

The genus Bothrops spp. is responsible for 90% of envenomation by snakes in Brazil, andthe standard treatment for snakebites is the antivenom therapy. The anti-bothropic serumproduced by Butantan Institute is prepared by the hyperimmunization of horses witha pool of venoms from Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothropsmoojeni and Bothrops neuwiedi. In this study, the biochemical and biological characteristicsof the venoms from nineteen snakes of the genus Bothrops, responsible for human acci-dents in Brazil, were analysed. Venoms, particularly from Crotalidae and Viperidae snakes,are rich sources of serine proteases and metalloproteases and the ability of the Braziliananti-bothropic serum to neutralize the proteolytic activity of these venoms were alsotested. The results obtained here show the existence of a large range of variation in thecomposition and activities in Bothrops spp. toxins and demonstrate that the anti-bothropicserum is not able to fully neutralize the toxic activities of all analysed venoms. Thesesuggest that for the preparation of a fully effective therapeutic anti-bothropic serum, othervenoms should be included in the immunization mixture.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Snake venoms are a complex mixture of components,which have a diverse array of actions both on prey andhuman victims. Many of these components are biologicallyactive proteins that function to kill or immobilize the preyas well as assist in the digestion process (Kochva et al.,1983; Mackessy, 1988; Braud et al., 2000; Mackessy et al.,2003; Lu et al., 2005).

The genus Bothrops inflicts the vast majority of snake-bites in Central and South America, being responsible for90% of venomous ophidian accidents that occurs in Brazil(Ribeiro and Jorge, 1997; Ministerio da Saude, 1998; da Silvaet al., 2003). Envenomations are characterized by

106..V. Tambourgi).

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prominent local effects, including edema, hemorrhage andnecrosis, which can lead to permanent disability. Systemicmanifestations such as hemorrhage, coagulopathy, shockand acute renal failure may also occur (Braud et al., 2000;Gutierrez and Rucavado, 2000; White, 2005).

Snake venom composition may exhibit variations asso-ciated with the geographical origin, habitat, seasonal vari-ation, diet, age and gender (Mackessy, 1988; Chippaux et al.,1991; Mackessy et al., 2003; Furtado et al., 2006). Severalcomponents have been isolated from Bothrops venomsincluding proteases, such as serine and metalloproteases,phospholipase A2, L-amino acid oxidase, 50-nucleotidase,hyaluronidase and C-type lectins (Alam et al., 1996; Kini,2006), comprising more than 90% of their dry weight(Bieber, 1979). Proteomic and mass spectrometric analysisshowed that 51.5% and 14% of the total content of proteinspresent in the venom of B. jararaca are metallo and ser-ineproteases, respectively (Fox et al., 2006).

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851 843

The proteolytic activity in the Bothrops venoms has beenimplicated in the pathogenesis of the main local and thesystemic reactions. Snake venom serine proteases areenzymes affecting the hemostatic system. They act ona variety of components of the coagulation cascade, on thefibrinolytic and kallikrein–kinin systems and on cells tocause an imbalance of the hemostatic system of the prey(Pirkle,1998). On the other hand, the metalloproteases, alsocalled as SVMPs (Snake Venom Metalloproteases), actmainly as hemorrhagic factors, which exert their effects bydegradation of proteins such as laminin, nidogen, fibro-nectin, collagen type IV and proteoglycans from the endo-thelial basal membrane (Fox and Serrano, 2005). Bloodcoagulation proteins, such as fibrinogen (Garcıa et al., 2004)and von Willebrand factor (Kamiguti et al., 1996) are alsotargets of their proteolytic activity and enhancing thehemorrhagic state. Additionally, SVMPs can also inhibitplatelet aggregation (Laing and Moura-da-Silva, 2005) andtrigger the release of cytokines (Schattner et al., 2005).These two effects, associated with the proteolytic digestionof the basal membrane, are thought to be the majormechanism of SVMP-induced hemorrhage.

The usual treatment for snakebite is the serum therapy.The anti-bothropic serum, produced by Butantan Institute,is prepared by the hyperimmunization of horses witha pool of venoms from B. alternatus (12.5%), B. jararaca(50%), B. jararacussu (12.5%), B. moojeni (12.5%) andB. neuwiedi (12.5%) (Raw et al., 1991), and the neutralizingability is assessed by evaluating the capacity of this anti-venom to inhibit the lethal action of the reference venom,i.e., from B. jararaca, in a murine model (World HealthOrganization, 1981). The aim of the present study was toanalyse the biochemical and biological characteristics ofvenoms from 19 species of Bothrops snakes present in theBrazilian territory and the ability of the anti-bothropicserum to neutralize the proteolytic, phospholipase A2 andhyaluronidase activities of these venoms.

2. Materials and methods

2.1. Chemicals and reagents

Triton X-100, Tween 20, bovine serum albumin (BSA),ethylene diamine tetracetic acid (EDTA), 4-nitro-3(octanoyloxy) benzoic acid, cetyltrimethylammonium bromide(CTAB), ortho-phenylenediamine (OPD), hyaluronic acid andgoat anti-horse (GAH) IgG horseradish peroxidase (IgG-HRPO) were purchased from Sigma–Aldrich (St. Louis, MO,USA). Goat anti-horse (GAH) IgG labelled with alkalinephosphatase (IgG-AP), 5-bromo-4-chloro-3-indolyl-phos-phate (BCIP) and nitroblue tetrazolium (NBT) were fromPromega Corp. (Madison, Wisconsin, USA). FluorescentResonance Energy Transfer (FRET) substrate, Abz-F-R-S-S-R-Q-EDDnp (Abz-FRSSRQ-EDDnp), was synthesized andpurified according to Araujo et al. (2000).

2.2. Animals

Swiss strain male mice aged 2 months and weighing 18–22 g were obtained from Bioterio de Criaçao de Animaisfrom Butantan Institute, SP, Brazil. All the procedures

involving animals were in accordance with the ethicalprinciples in animal research adopted by the BrazilianCollege of Animal Experimentation.

2.3. Venoms

Venoms from Bothrops alternatus, Bothrops atrox, Bothropsbilineatus, Bothrops brazili, Bothrops castelnaudi, Bothropscotiara, Bothrops erythromelas, Bothrops fonsecai, Bothropshyoprorus, Bothrops insularis, Bothrops itapetiningae, Bothropsjararaca, Bothrops jararacussu, Bothrops leucurus, Bothropsmarajoensis, Bothrops moojeni, Bothrops neuwiedi, Bothropspirajai, Bothrops pradoi and Crotalus durissus terrificus weresupplied by Laboratorio de Herpetologia from ButantanInstitute, SP, Brazil. Stock solutions were prepared in PBS(10 mM Na Phosphate, 150 mM NaCl; pH 7.2) at 1.0 mg/mL.

2.4. Normal and hyperimmune sera

The anti-bothropic serum produced by the hyperim-munization of horses with a pool of venoms from B. alter-natus (12.5%), B. jararaca (50%), B. jararacussu (12.5%),B. moojeni (12.5%) and B. neuwiedi (12.5%) was obtainedfrom Seçao de Processamento de Plasmas Hiperimunes,Butantan Institute, SP, Brazil. The antivenom used (batchno. 0506110) had a protein concentration of 1.8 g/dL andeach milliliter was able to neutralize 6.61 mg of B. jararacavenom (lethality test in mice). Normal sera were obtainedfrom non-immunized animals.

2.5. Electrophoresis and western blotting

The Bothrops spp. venoms were solubilised in reducingsample buffer and separated in 12.5% SDS-PAGE gels(Laemmli, 1970). Gels were stained with Coomassie blue orblotted onto nitrocellulose (Towbin et al., 1979). Aftertransfer, the membranes were blocked with PBS containing5% BSA and incubated with anti-bothropic or normal horseserum (1:10000) for 1 h at room temperature. Immunore-active proteins were detected using GAH/IgG-AP (1:7500)in PBS/1% BSA for 1 h at room temperature. After washing 3times for 10 min with PBS/0.05% Tween 20, blots weredeveloped using NBT/BCIP according to the manufacturer’sinstructions (Molecular Probes, CA, USA).

2.6. ELISA

Microtitre plates were coated with 100 mL of Bothropsspp. venoms (10 mg/mL; overnight at 4 �C). Plates wereblocked with 5% BSA in PBS and dilutions of the anti-bothropic serum added. After 1 h of incubation at roomtemperature, plates were washed with PBS/0.05% Tween 20and incubated with the specific anti-IgG antibody conju-gated with HRPO for 1 h at room temperature. Plates werewashed and the reactions developed with OPD substrateaccording to the manufacturers conditions (Sigma). Theabsorbances were recorded in an ELISA reader (Multiskanspectrophotometer EX, Labsystems, Finland) at l 492 nm.The titre was established as the highest antiserum dilutionin which produced an absorbance eight times greater thanthat determined for the normal serum.

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851844

2.7. Determination of LD50

The lethal toxicity of Bothrops spp. venoms was assessedby intraperitoneal injection of different amounts of venomsin 500 mL of PBS into male Swiss strain mice. Six animalswere used for each venom dose (five doses). The LD50 wascalculated by probit analysis of death occurring within 48 hafter venom injection (Finney, 1971).

2.8. Phospholipase A2 activity

The phospholipase A2 activity of Bothrops spp. venomswas determined as described by Holzer and Mackessy(1996), with some modifications. Microtitre plates werecoated with samples of the venoms (8 mg) combined withbuffer (10 mM Tris–HCl, 10 mM CaCl2, 100 mM NaCl; pH8.0) to a final volume of 150 mL. The samples were mixedwith 15 mL of the substrate (0.3 mM 4-nitro-3-[octanoyloxy] benzoic acid diluted in acetonitrile) and incubated for20 min at 37 �C. The hydrolysis values were determined bymeasuring the OD at l 414 nm in a spectrophotometer(Multiskan EX, Labsystems, Finland). As positive andnegative controls, venom from C. d. terrificus (8 mg) and PBSwas used, respectively. The phospholipase action wasexpressed as specific activity in nanomoles of cleavedsubstrate per minute per mg of venom (U/mg) of threeindependent experiments.

2.9. Hyaluronidase activity

Hyaluronidase activity was measured following theprocedures of Pukrittayakamee et al. (1988), with slightmodifications. Microtitre plates were coated with samplesof Bothrops spp. venoms (8 mg), 20 mL of the hyaluronic acidsubstrate (0.5 mg/mL) and acetate buffer (0.2 M sodiumacetate-acetic acid, pH 6.0, containing 0.15 M NaCl) ina final volume of 100 mL. The mixtures were incubated for30 min at 37 �C. After incubation, 200 mL of CTAB 2.5% inNaOH 2% was added to the samples. The absorbances weremeasured at l 405 nm in a spectrophotometer (MultiskanEX, Labsystems, Finland) against a blank containing 100 mLof acetate buffer and 200 mL of CTAB. All assays were per-formed in duplicate. The turbidity reducing activity wasexpressed as percentage of the remaining hyaluronic acid,taking the absorbance of well in which no venom wasadded as 100%. Results were expressed in units of turbidityreduction (UTR) per mg of venom.

2.10. Proteolytic activity

The enzymatic activity of the Bothrops spp. venoms wasdetermined using the fluorescence resonance energytransfer (FRET) substrate and the relative inhibition witha metal chelating agent was determined in parallel. Theproteolytic activity of the venoms (1 mg) was measured inthe presence and in the absence of 100 mM EDTA. The bufferused was PBS pH 7.5 containing 2 mM of the FRET peptideAbz-FRSSRQ-EDDnp. The reactions were monitored bymeasuring hydrolysis in a fluorescence spectrophotometer(lem¼ 420 nm and lex¼ 320 nm), using 96-well microtitreplates (Victor 3�, Perkin–Elmer, MA, USA) at 37 �C, as

described by Araujo et al. (2000). All assays were made induplicate and the specific proteolytic activity was expressedas millimoles of cleaved substrate per minute per mg ofvenom (U/mg).

2.11. Serum neutralization of the venoms

The ability of the anti-bothropic serum to neutralize thevenoms phospholipase A2, hyaluronidase and proteolyticactivities was estimated by incubating Bothrops spp. venomswith the antivenom. Samples of Bothrops venoms wereincubated, at room temperature, in the presence andabsence of the anti-bothropic serum. The residual toxicactivities of the venoms were measured as described above(Sections 2.8–2.10). The volume of the anti-bothropic serumand the pre-incubation time for serum neutralization, ofeach of the toxic activities tested, were established by using,as positive control, the immunizing pool of Bothrops venoms(B. alternatus 12.5%, B. jararaca 50%, B. jararacussu 12.5%, B.moojeni 12.5% and B. neuwiedi 12.5%). For hyaluronidase andphospholipase A2 activities, samples of Bothrops spp.venoms (8 mg) were incubated for 10 min with 0.6 mL and2 mL of the anti-bothropic serum, respectively. For proteo-lytic activity, 250 ng of venoms were incubated with 2 mL ofthe anti-bothropic serum for 30 min.

3. Results

3.1. Immunochemical cross-reactivity ofBothrops spp. venoms

The protein profiles of Bothrops spp. venoms wereanalysed by SDS-PAGE followed by Coomassie blue stain-ing. Fig. 1A shows that the venoms of the 19 species differ incomposition, number and intensity of bands. However, themajority of these venoms contains components with Mr

between 64 and 25 kDa and of approximately 14 kDa.Exceptions to this are the venoms from B. castelnaudi and B.neuwiedi, which lack the 25 kDa and 60 kDa bands,respectively.

The ELISA results show that the anti-bothropic serumrecognized the Bothrops spp. venoms with different anti-body titres (Fig. 2). The higher cross-reactivities wereagainst the venoms of B. moojeni, B. jararaca and B. jarar-acussu, which are venoms used for serum production, and ofB. cotiara, B. pirajai, B. fonsecai, B. castelnaudi, B. itapetiningaeand B. pradoi. The serum presented intermediate antibodytitres for the venoms of B. alternatus, B. neuwiedi (alsopresent in the horse immunization mixture), B. atrox,B. hyoprorus, B. leucurus, B. brazili, B. erythromelas andB. marajoensis, and low titres for B. insularis and B. bilineatus.

By western blotting, it was demonstrated that the anti-bothropic serum could recognize several but not allcomponents present in the different Bothrops spp. venoms(Fig. 1B). The serum recognized two regions best, onearound 60 kDa and another around 15 kDa.

3.2. Toxic activities of Bothrops spp. venoms

In order to assess whether the venoms of Bothrops dis-played the same toxic activities, several functional assays

Fig. 1. SDS-polyacrylamide gel electrophoresis and Western blot analysis of Bothrops venoms. Samples (50 mg) of B. atrox, B. moojeni, B. cotiara, B. pirajai,B. fonsecai, B. castelnaudi, B. itapetiningae, B. pradoi, B. hyoprorus, B. insularis, B. alternatus, B. leucurus, B. bilineatus, B. brazili, B. erythromelas, B. jararaca,B. jararacussu, B. marajoensis and B. neuwiedi venoms were analysed by SDS-PAGE gel (12.5%) and stained by Coomassie blue [A] or electrotransfered tonitrocellulose membranes and incubated with anti-bothropic serum diluted 1:10000 followed by GAH/IgG-AP (1:7500) [B]. Recognized bands were visualizedwith NBT and BCIP.

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851 845

were carried out. Toxicity of Bothrops venoms was deter-mined after intraperitoneal injection of different doses ofthe venoms, and the death/survival ratio collected during48 h. The LD50 values, calculated by probit analysis at 95%confidence, were variable among the Bothrops venoms,with the more lethal the ones from B. castelnaudi, B. jar-araca and B. neuwiedi (Table 1).

Bothrops venoms also contain variable levels of PLA2

activity (Table 2). The venoms of B. hyoprorus, B. eryth-romelas and B. moojeni presented activities higher than thepositive control (C. d. terrificus). Lower activities wereobserved for B. cotiara and B. insularis.

Table 3 shows that all venoms presented hyaluronidaseactivity, being the values determined for B. jararaca,B. insularis, B. leucurus, B. fonsecae and B. erythromelashigher than the other Bothrops spp. venoms.

The proteolytic activity of the Bothrops venoms wastested using a FRET substrate. Table 4 shows that all venomswere able to hydrolyze the substrate, although withdifferent intensities. B. cotiara, B. insularis and B. jararacavenoms showed high activity (above 900 mM/min/mg) andB. moojeni, B. itapetiningae, B. erythromelas and B. mar-ajoensis lower activities (below 9 mM/min/mg). Theproteolytic activity of these venoms were also measured in

B. atrox

B. m

oojeni

B. cotiara

B. pirajai

B. fo

nsecai

B. caste

lnaudi

B. itapetiningae

B. pra

doi

B. hyopro

rus

B. insularis

B. altern

atu

s

B. leucuru

s

B. bilineatu

s

B. bra

zili

B. ery

thro

melas

B. jara

raca

B. jara

racussu

B. m

ara

joensis

B. neuwiedi

32768

An

tib

od

y T

iter [lo

g2]

16384

8192

4096

VENOMS

Fig. 2. Cross-reactivity of the anti-bothropic serum. ELISA plates were coated with 1 mg of Bothrops venoms and incubated with different dilutions of horse anti-bothropic serum, followed by GAH/IgG-HRPO (diluted 1:3000). The titres were established as the highest antiserum dilution which produces an absorbance eighttimes greater than that determined for the normal serum.

Table 2Phospholipase A2 activity and serum neutralization of venoms fromBothrops genus.

Specific activity (nmol/min/mg)

Venom Venomþ anti-botropic serum

Pool of venomsa 19.99� 0.64 6.45� 0C. d. terrificus 52.2� 0.6 n.d.

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851846

the presence of a metal chelating agent, EDTA, and Fig. 3shows a variation in the level of inhibition.

3.3. Serum neutralization

Fig. 4 shows the anti-bothropic serum neutralizationsupon the phospholipase A2 (Table 2), hyaluronidase(Table 3) and proteolytic (Table 4) activities of the venoms.The inhibitory potency was measured by pre-incubatingvenoms with the antivenom at room temperature (seeSections 2.8–2.11 for details). The best condition

Table 1Toxicity of Bothrops spp. venoms.

LD50 (mg/kg)

Median Confidence interval

B. atrox 130.2 115.2–147.9B. moojeni 90.8 79.6–97.4B. cotiara 53.8 65.3–40.1B. pirajai 93.0 75.5–111.3B. fonsecai 56.8 45.7–73.0B. castelnaudi 20.2 13.9–22.7B. itapetiningae 74.4 63.9–88.2B. pradoi 70.2 59.2–81.9B. hyoprorus 232.0 213.0–260.5B. insularis 62.3 55.9–71.2B. alternatus 63.7 51.3–73.1B. leucurus 63.7 58.0–71.4B. bilineatus 125.6 110.2–149.4B. brazili 151.6 135.1–179.4B. erythromelas 74.0 58.6–85.6B. jararaca 27.6 25.1–30.8B. jararacussu 70.4 62.4–81.4B. marajoensis 111.0 92.2–125.2B. neuwiedi 36.7 32.3–43.1

Various doses of Bothrops spp. venoms, diluted in PBS, were i.p. injected ingroups of mice. The LD50 values were calculated by probit analysis of deathoccurring within 48 h after of venom injection.

B. atrox 43.21� 0.6 38.05� 1.9B. moojenib 56.11� 1.9 30.9� 2B. cotiara 9.67� 0.6 3.87� 1.2B. pirajai 44.5� 0.6 42.57� 2.5B. fonsecai 21.28� 3.2 41.92� 0.6B. castelnaudi 20.64� 3.8 12.9� 1.2B. itapetiningae 24.51� 3.8 21.93� 1.2B. pradoi 24.51� 1.2 29.67� 1.2B. hyoprorus 76.11� 1.2 67.08� 2B. insularis 9.6� 0.6 10.32� 1.2B. alternatusb 14.8� 0.6 4.51� 0.6B. leucurus 21.9� 1.2 15.48� 1.9B. bilineatus 31.6� 1.9 30.3� 1.9B. brazili 29.67� 0 15.48� 1.2B. erythromelas 65.79� 0 69.7� 2.5B. jararacab 16.12� 0.6 12.9� 1.9B. jararacussub 35.6� 1.9 12.2� 0.6B. marajoensis 27.73� 1.9 25.8� 1.9B. neuwiedib 32.25� 1.2 31.6� 0.6

Samples of Bothrops spp. venoms (8 mg) were incubated with the substrate[4-nitro-3 (octanoyloxy) benzoic acid], at room temperature, in thepresence or absence of the 2 mL the anti-bothropic serum. The absorbancewas measured in a spectrophotometer at l 414 nm and the specific activityexpressed as nmol/min/mg venom. Buffer and venom from C. d. terrificus(8 mg) were used as negative and positive controls, respectively. Resultsare representative for three independent experiments� SD.n.d. not done.

a Pool of Bothrops spp. venoms used for serum production.b Venoms present in the immunization pool for the anti-bothropic

serum production.

Table 3Hyaluronidase activity and serum neutralization of venoms from Bothropsgenus.

Specific activity (UTR/mg)

Venom Venomþ anti-bothropic serum

Pool of venomsa 91.5� 1 0.0� 0.0B. atrox 76.7� 1 44.6� 2B. moojenib 58.5� 2.5 28.8� 1.5B. cotiara 112.6� 1 24.2� 0B. pirajai 59� 13 1.7� 0.5B. fonsecai 117.9� 1 37� 4B. castelnaudib 104.9� 5 8� 1B. itapetiningae 109.4� 1 49.1� 0.2B. pradoi 86.1� 0 50.2� 0.8B. hyoprorus 85.7� 3 32.8� 1B. insularis 142.9� 3 16� 2B. alternatusb 103� 4 13.5� 5B. leucurus 127.5� 2 20� 5B. bilineatus 84.5� 0.5 48.7� 0.7B. brazili 48.2� 1 30� 2B. erythromelas 113.7� 0 40.2� 0B. jararacab 190.7� 1.1 77.6� 1B. jararacussub 68.65� 1 48.9� 1B. marajoensis 96� 1 52� 0B. neuwiedib 73.6� 2 39� 0

Samples of Bothrops spp. venoms (8 mg) were incubated with the hyalur-onic acid, as substrate, in the presence or absence of 0.6 mL of the anti-bothropic serum. The absorbance was measured in a spectrophotometerat l 405 nm and the hyaluronidase activity expressed as reductionpercentage of turbidity (UTR) of the mixture. Results are representative forthree separated experiments and expressed as mean of duplicates� SD.

a Pool of Bothrops spp. venoms used for serum production.b Venoms present in the immunization pool for the anti-bothropic

serum production.

Table 4Proteolytic activity and serum neutralization of venoms from Bothropsgenus.

Specific activity (U/mg)

Venom Venomþ anti-bothropic serum

Pool of venomsa 184.5� 7.9 0.0� 0.0B. atrox 261.4� 16.8 186.5� 15.2B. moojenib 6.0� 0.6 2.5� 0.02B. cotiara 941.8� 74.3 566.2� 35.5B. pirajai 188.3� 7.5 193.0� 9.4B. fonsecai 351.3� 1.2 298.2� 2.0B. castelnaudi 330.2� 5.5 318.3� 36.6B. itapetiningae 9.27� 0.4 3.9� 0.3B. pradoi 13.9� 1.2 4.4� 0.1B. hyoprorus 37.3� 1.6 30.2� 1.8B. insularis 1467.4� 29.4 796.3� 49.1B. alternatusb 134.1� 0.7 53.3� 2.9B. leucurus 10.7� 1.8 12.4� 2.2B. bilineatus 63.9� 4.6 50.8� 27B. brazili 17.9� 0.08 12.2� 1.6B. erytromelas 8.4� 0.2 9.0� 0.3B. jararacab 1156.9� 72.8 206.0� 560B. jararacussub 56.1� 0.2 54.7� 2.6B. marajoensis 6.4� 0.2 5.1� 0.4B. neuwiedib 75.6� 6.5 25.4� 2.7

Samples of Bothrops spp. venoms (250 ng) were incubated for 30 min atroom temperature in the presence or absence of 2 mL of the anti-bothropicserum and the proteolytic activity measured using the FRET substrate. Allenzymatic assays were performed in duplicate and the results expressedas specific activity (U/mg)� SD.

a Pool of Bothrops spp. venoms used for serum production.b Venoms present in the immunization pool for the anti-bothropic

serum production.

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851 847

determined for maximum neutralization of the phospho-lipase A2 activity, present in the pool of Bothrops venomsused for the antivenom production, resulted in a serumneutralization of only 67% (Fig. 4A). However, the hyal-uronidase and proteolytic activities exhibited by theimmunization pool of Bothrops venoms were completelyneutralized by the anti-bothropic serum (Fig. 4B and C andTables 3 and 4).

The neutralization of the PLA2 activity of the venomsfrom B. moojeni, B. cotiara, B. alternatus, B. brazili and B. jar-aracussu, by the anti-bothropic serum, was above 40%; forthe venoms of B. pirajai, B. fonsecai, B. itapetiningae, B. pradoi,B. insularis, B. bilineatus, B. erythromelas, B. marajoensis andB. neuwiedi it was below 10% (Fig. 4A and Table 2). The anti-bothropic serum was able to neutralize the hyaluronidaseactivity of the Bothrops venoms with values of inhibitionhigher than 40%, with the exception of the B. jararacussuvenom, for which a serum neutralization of 29% wasobtained (Fig. 4B and Table 3). The serum neutralization ofthe proteolytic activity was high for B. jararaca (82%), fol-lowed by B. moojeni, B. itapetiningae, B. pradoi, B. alternatusand B. neuwiedi venoms, with neutralization values above50%. For B. atrox, B. cotiara, B. insularis and B. brazili venoms,neutralization values were between 29 and 50%. For thevenoms of B. pirajai, B. fonsecai, B. castelnaudi, B. hyoprorus,B. leucurus, B. bilineatus, B. erythromelas, B. jararacussu andB. marajoensis little or no neutralization of the proteolyticactivity was observed (Fig. 4C and Table 4).

4. Discussion

Better characterization of the biological activities ofBothrops snake venoms is very important, not only toelucidate the molecular mechanisms of the venom action,but also to seek new approaches for the patient treatment.Snakes of this genus are responsible for the vast majority ofsnakebites in Central and South America.

Many factors including phylogeny, sex, geographicorigin, season, age and prey preference may influence thevenoms composition (Chippaux et al., 1991). The Neotrop-ical pitviper genus Bothrops comprises about 20 species inBrazil, occurring in all main ecosystems. There is no strictconsensus in the systematics of this group. Morphology,habits and mitochondrial DNA sequence have been exten-sively used as characters to subdivide the genus Bothrops.Recently, it was proposed that B. bilineatus and B. cas-telnaudi should be included in the genus Bothriopsis andB. hyoprorus was placed in the genus Bothrocophias (Salo-mao et al., 1999; Campbell and Lamar, 2004; SBH, 2007).We included venoms from B. castelnaudi, B. hyoprorus andB. bilineatus in our study and here considered them asbelonging to the genus Bothrops. Humans envenomationinvolving these species are treated with the anti-bothropicserum.

Bothrops venoms, studied here, exhibit variations incomposition, number and intensity of protein bands, beingthe majority components with Mr between 64 and 14 kDa.The main components of Bothrops spp. venoms, describedup to now, are located in these regions and probably

B. atro

x

B. moo

jeni

B. coti

ara

B. pira

jai

B. fons

ecai

B. cas

telna

udi

B. itap

etinin

gae

B. prad

oi

B. hyo

prorus

B. insu

laris

B. alte

rnatus

B. leuc

urus

B. bilin

eatus

B. braz

ili

B. eryt

hromela

s

B. jarar

aca

B. jarar

acus

su

B. mara

joens

is

B. neu

wiedi

0

25

50

75

100

VENOMS

In

hib

itio

n [%

]

Fig. 3. Inhibition of the proteolytic activity. Samples of Bothrops venoms (1 mg) were incubated with the FRET substrate at 37 �C, in the presence or absence of100 mM EDTA. Results are representative of three separate experiments and were expressed as percentage of inhibition.

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851848

corresponding to metalloproteases with hemorrhagicactivities (64–40 kDa), serine proteases (40–20 kDa) andphospholipases A2 (16–13 kDa) (Moura-da-Silva et al.,1990; Kini, 2006).

The anti-bothropic serum used for human therapy andproduced by Butantan Institute is obtained through theimmunization of horses with a pool of venoms composedby B. alternatus, B. jararaca, B. jararacussu, B. moojeni andB. neuwiedi. ELISA and Western blot analysis showed thatthe antigens present in homologous and heterologousvenoms are recognized by the anti-bothropic serum,corroborating other studies already undertaken (Ferreiraet al., 1992; Lira et al., 2007). Components with molecularweight of 50 kDa and 14 kDa were more intensely recog-nized by the anti-bothropic serum; however, proteins withMr above 15 kDa, present in B. castelnaudi venom, wereweakly recognized by this antiserum.

The analysis of the lethal potential showed values varyingfrom 1.01 to 11.06 mg/kg, demonstrating a great toxicityvariation among venoms from snakes of the Bothrops genus.These results are in accordance with data obtained for Bogarınet al. (2000) studying the venoms of sixteen crotaline species.B. castelnaudi (1.01 mg/kg), B. jararaca (1.38 mg/kg) andB. neuwiedi (1.83 mg/kg) venoms were the most lethalvenoms, as tested in murine model; B. atrox (6.51 mg/kg), B.hyoprorus (11.6 mg/kg), B. brazili (7.58 mg/kg) and B. bilineatus(6.28 mg/kg) venoms were the lowest toxic among theBothrops spp. venoms here studied.

Phospholipase A2 (PLA2) molecules are considered animportant class of proteins found in Bothrops spp. snakevenoms, which exhibits diverse biological properties,including anticoagulant, cardiotoxic, myotoxic, neurotoxic,hemolytic, hemorrhagic and hypotensive activities (Moura-da-Silva et al., 1990; Gutierrez and Lomonte, 1995; Kini,2006). Our data not only show that the 19 Bothrops venomspresent PLA2 activity, but also that this is pronounced in thevenoms of B. hyoprorus, B. erythromelas and B. moojeni. It is

important to note that these venoms exhibited higher PLA2

activity when compared with the positive control, thevenom from C. d. terrificus. In the venom of C. d. terrificus,PLA2 is considered the main neurotoxic component, rep-resenting approximately 50% of the venom protein content(Rangel-Santos et al., 2004).

Hyaluronidase is present in many snake venoms andalso in the venoms of bees, spiders and scorpions. Itsactivity potentiates the toxicity of the venom by promotingloss of extracellular matrix integrity in soft connectivetissues surrounding the blood vessels increasing thesystemic influx of toxins (Girish et al., 2002; Kemparaju andGirish, 2006). For the majority of Bothrops spp. venomsanalysed here, hyaluronidase activity varied between 48and 190 UTR/mg, and the most active venoms found werefrom B. jararaca and B. insularis, with activities above140 UTR/mg. In contrast, low hyaluronidase activity wasdetected in the venoms of B. moojeni, B. brazili and B. pirajai,with activities below 60 UTR/mg. Similar results were notobtained by Lira et al. (2007), since these authors have notdetected hyaluronidase activity in B. jararaca and B. insu-laris venoms. These discrepancies may be due to thesensitivity of the methods used for the measurement of theactivity or to intraspecific variation in venom composition.

All venoms studied were able to hydrolyze the FRETsubstrate, though with variable intensities. B. moojeni,B. itapetiningae, B. erythromelas and B. marajoensis venomshad 250 times lower activity when compared with that ofB. insularis, which was the most active venom. In addition,it was demonstrated that EDTA could produce differentlevels of inhibition, also indicating variation in the contentof metalloproteases and other metal-dependent proteasesin the venoms from Bothrops genus, which is in accordancewith previously reported data (Braud et al., 2000; Matsuiet al., 2000; Lu et al., 2005).

The present study also aimed to evaluate the serumneutralization ability of the anti-bothropic serum, used in

20

0

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Venoms

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]

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f ven

oms

B. atro

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B. coti

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B. leuc

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2 activity

In

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]

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B. moo

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B. moo

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B. coti

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B. prad

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B. alte

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B. leuc

urus

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B. braz

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B. eryt

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s

B. jarar

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B. jarar

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B. mara

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B. neu

wiedi

A

B

C

Fig. 4. Serum neutralization of the proteolytic activity of venoms from Bothrops genus. Samples of Bothrops spp. venoms (8 mg) were incubated, at roomtemperature, for 10 min in the presence or absence of 2 mL or 0.6 mL of the anti-bothropic serum and the residual PLA2 [A] or hyaluronidase [B] activities,respectively, measured as described above (Sections 2.8 and 2.9). [C] Samples of Bothrops spp. venoms (250 ng) were incubated for 30 min, at room temperature,in the presence or the absence of 2 mL of the anti-bothropic serum and the proteolytic activity measured using the FRET substrate. Results are representative forthree separate experiments performed in duplicates. ‘‘Pool of venoms’’ corresponds to a mixture of Bothrops spp. venoms used for serum production.

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851 849

Brazil for human serum therapy, on the toxic effects ofBothrops spp. venoms. We have established and performedin vitro serum neutralization assays for the main toxiceffects of the Bothrops venoms, i.e., PLA2, hyaluronidase andproteolytic activities.

The anti-bothropic serum was capable of neutralizingmore than 40% of the PLA2 activity present in the venoms of B.moojeni, B. cotiara, B. alternatus, B. brazili and B. jararacussu; itwas less efficient or incapable of reducing this activity in theother venoms studied. All venoms had the hyaluronidase

G.P. Queiroz et al. / Toxicon 52 (2008) 842–851850

activity neutralized by the antivenom, with inhibitionpercentages above 40%. The substrate FRET, containinga universal sequence, recognized by proteases of differentcatalytic natures is used in the proteolytic studies. Using thismethod, it was possible to demonstrate that B. moojeni, B.itapetiningae, B. pradoi, B. alternatus, B. jararaca and B. neu-wiedi venoms were significantly neutralized by the anti-bothropic serum. Interestingly, B. itapetiningae and B. pradoivenoms are not used in the immunization pool.

On the other hand, the proteolytic activity exhibited bythe other venoms, including the homologous venom fromB. jararacussu, was poorly neutralized by the anti-bothropicserum. For B. jararacussu venom, the anti-bothropic serumwas able to block only 2.5% of the proteolytic activity;however, the PLA2 activity from this venom was betterneutralized (above 60%), which is in agreement withprevious reported observations (dos-Santos et al., 1992; deRoodt et al., 1999; Picolo et al., 2002; Beghini et al., 2005).The poor inhibition of the proteolytic components ofB. jararacussu venom indicates that the proteases in thisvenom are likely different from the ones present in theother venoms used in anti-sera production.

The neutralization of the PLA2 and proteolytic activitiesby anti-bothropic serum varied, in a general way, depend-ing on whether homologous or heterologous venoms wereused, indicating that these venoms contain non-homologous proteins, and this could be important for antigeniccross-reactivity and neutralization by the heterologousanti-sera. The inefficiency of the antivenom in neutralizingthe PLA2 and proteolytic activities of the heterologousvenoms may be due to primary sequence differences intheir enzymatic components.

Data presented here demonstrate the existence of a largerange of variation in the composition and activities in Bothropsspp. venoms, evidencing their diversity. The serum usedfor human therapy in Brazil is not fully able to neutralize thetoxic activities present in all venoms from snakes of thegenus Bothrops, indicating that for the preparation ofa universal bothropic serum, others venoms should beincluded in the immunization mixture. Moreover, the serumneutralization methods developed in this study may be a goodalternative to test the potency of the bothropic antivenom,since the in vivo methods are laborious and time-consumingand require large numbers of experimental animals.

Acknowledgements

This work was supported by FAPESP and CNPq. We aregrateful to Dr. Luiz Juliano Neto (Biophysics Department,UNIFESP, Sao Paulo, Brazil) for kindly providing the FRETsubstrate.

Conflict of interest

None declared.

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