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Toxicon 49 (2007) 982–994

www.elsevier.com/locate/toxicon

Enzymatic and immunochemical characterization ofBothrops insularis venom and its neutralization by

polyspecific Bothrops antivenom

M.S. Liraa, M.F. Furtadob, L.M.P. Martinsa, M. Lopes-Ferreiraa,M.L. Santoroc, K.C. Barbaroa,�

aLaboratory of Immunopathology, Butantan Institute, Av Vital Brazil 1500, 05503-900, Sao Paulo, SP, BrazilbLaboratory of Herpetology, Butantan Institute, Av Vital Brazil 1500, 05503-900, Sao Paulo, SP, Brazil

cLaboratory of Pathophysiology, Butantan Institute, Av Vital Brazil 1500, 05503-900, Sao Paulo, SP, Brazil

Received 14 August 2006; received in revised form 16 January 2007; accepted 17 January 2007

Available online 1 February 2007

Abstract

Herein we compared the biological activities of Bothrops insularis and Bothrops jararaca venoms as well as their

neutralization by polyspecific Bothrops antivenom (PBA). On account of that, we investigated their antigenic cross-

reactivity and the neutralization of lethal, myotoxic and defibrinating activities by polyspecific and species-specific

antivenoms. Silver-stained SDS-PAGE gels evidenced many common bands particularly above 47 kDa between B. jararaca

and B. insularis venoms. However, some protein bands between 46 and 28 kDa were observed exclusively in B. jararaca

venom. Both venoms presented gelatinolytic, caseinolytic, fibrinogenolytic and phospholipase A2 activities. No

hyaluronidase activity was detected in both venoms by zymography. Polyspecific and species-specific antivenoms showed

similar titers to B. jararaca and B. insularis venoms by ELISA, and recognized similar components by immunoblotting.

The PBA was effective in neutralizing the lethal, myotoxic and defibrinating activities of both venoms as well as to

abrogate microcirculatory disturbances induced by B. insularis venom. No statistically significant difference was observed

for minimal hemorrhagic doses between both venoms. Antigenic cross-reactivity was evident between both venoms. Since

toxic and enzymatic activities were similar, we speculate that B. insularis venoms can induce a local damage in humans

comparable to that observed in other Bothrops venoms. Besides, the PBA was effective in neutralizing the toxic activities of

B. insularis venom.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Bothrops; Snake venom; Polyspecific Bothrops antivenom; Bothrops insularis; Bothrops jararaca; Microcirculation

e front matter r 2007 Elsevier Ltd. All rights reserved

xicon.2007.01.009

ng author. Tel.: +55 11 37267222x2278/2134;

61505.

sses: kbarbaro@usp.br,

ntan.gov.br (K.C. Barbaro).

1. Introduction

Around 20,000 snakebites are reported annuallyin Brazil, and 90% of them are inflicted by the genusBothrops (Araujo et al., 2003), which comprisesmore than 20 species distributed throughout Brazil(Melgarejo, 2003). Envenomation by Bothrops

.

ARTICLE IN PRESSM.S. Lira et al. / Toxicon 49 (2007) 982–994 983

snakes is characterized by acute inflammation at thesite of bite, hemorrhage and blood coagulationdisturbances, and, according to the severity of theclinical picture of patients at hospital admission, itcan be classified as mild, moderate or severe. Acuteinflammatory activity is induced by several venomcomponents, which induce release of inflammatorymediators and tissue damage (Franc-a and Malaque,2003). Hemorrhagic activity is caused by severalvenom components that disturb hemostasis (Sano-Martins and Santoro, 2003). Patients are treatedwith polyspecific Bothrops antivenom (PBA) and thenumber of antivenom ampoules will depend on theseverity of envenomation. In severe cases, besidesantivenom administration, intense hemorrhage,shock and renal failure may occur, which promptspecial therapeutic approaches (Franc-a andMalaque, 2003).

Bothrops insularis (common name, jararacailhoa) is an endemic species found only in QueimadaGrande Island, coast of Sao Paulo State(Brazil). Differentiation between B. insularis and

B. jararaca (the continental species) snakes isexplained by the model of allopatric speciation,i.e., two populations are separated by a geographi-cal barrier and undergo speciation over time(Marques et al., 2002). B. insularis is a closedsister taxa of B. jararaca (Wuster et al., 2005) andpresents some biological and behavior peculiarities.They live on trees, are active during the day(Campos and Mello-Filho, 1966) and have lightcoloration, mimicking the environment aroundthem (Marques et al., 2002). Due to the absenceof small terrestrial mammals in the island, jararaca-ilhoa has a diet based mainly on birds. For thatreason, B. insularis venom is considered much moretoxic to birds than B. jararaca venom (Amaral,1920).

Few investigations have dealt with B. insularis

venom. Hitherto, B. insularis is known to presentcoagulant (thrombin-like, prothrombin and factorX activating enzymes) (Nahas et al., 1979; Selistreand Giglio, 1987; Modesto et al., 2005) andhemolytic activities (Campos and Mello-Filho,1966), to induce edema formation due to the actionof proteases and phospholipases (Selistre et al.,1990) and to present a possibly neuromuscularaction mediated by phospholipase A2 (PLA2) (Cogoet al., 1998). Barbosa et al. (2003) demonstrated thatB. insularis venom induced edema mediated by therelease of histamine, and generation of nitric oxideand cyclooxygenase products.

Our purpose was to contribute further to thecharacterization of B. insularis venom, investigatingsome of its immunochemical and enzymatic aspects.Furthermore, this work also investigated the effi-cacy of PBA to neutralize some toxic activities ofB. insularis venom.

2. Material and methods

2.1. Animals

Adult male Swiss mice (18–22 g) were obtainedfrom Butantan Institute Animal House. Animalsreceived food and water ad libitum. All proceduresinvolving mice were carried out in conformity withnational and international laws and policies, con-trolled by Butantan Institute Animal InvestigationEthical Committee, protocol no. 142/2003.

2.2. Venom and commercial antivenom

Eight adult B. insularis specimens were collectedin Queimada Grande Island and maintained incaptivity in individual cages at room temperature(2672 1C) in the Laboratory of Herpetology,Butantan Institute (Sao Paulo, Brazil). Food supply(mice) was provided to snakes monthly. Severalvenom samples of each specimen were collected bymanual extraction and then were frozen, pooled,lyophilized and stored at �20 1C. At the moment ofuse, venom was dissolved in 0.15M NaClor phosphate-buffered saline (PBS). A sample ofB. jararaca venom was withdrawn from a large poolof frozen-dried venom, which was extracted from2500 specimens of snakes from all regions where thisspecies inhabits. The protein content of venompools was determined using bicinchoninic acidassay, according to Smith et al. (1985), using bovineserum albumin as standard.

PBA (batch 970783) was produced by ButantanInstitute by immunization of horses with a mixtureof B. jararaca (50%), B. jararacussu (12.5%),B. moojeni (12.5%), B. neuwiedi (12.5%) andB. alternatus (12.5%) venoms. It is composed ofthe F(ab0)2 portion of antibodies (Raw et al., 1991).

2.3. Production of specific B. insularis and

B. jararaca antivenoms

Specific antivenoms against B. insularis or B.

jararaca venoms were obtained by immunization ofrabbits. Venom (200 mg) was diluted in 500 ml of

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PBS and added to 500 ml of complete Freund’sadjuvant, and these mixtures were injected i.m.After one month, animals received five additionalboosters in incomplete Freund’s adjuvant at fort-night intervals. Blood was collected, and sera wasseparated and stored at �20 1C until use.

2.4. Sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE)

Proteins (10 or 15 mg) of B. insularis andB. jararaca venoms were separated in 12.5% SDS-PAGE gels (Laemmli, 1970) under non-reducingconditions, and then stained by Coomassie blue orsilver (Blum et al., 1987). The following pre-stainedmolecular mass markers (BioRad, Hercules, CA,USA) were used: myosin, b-galactosidase, bovineserum albumin, carbonic anhydrase, soybean tryp-sin inhibitor, lysozyme and aprotinin.

2.5. ELISA

Titration of PBA and rabbit species-specificantivenoms was accomplished by ELISA, usingB. insularis and B. jararaca venoms (10 mg/ml) tocoat plates (Nunc, USA). The reaction was readusing an ELISA plate reader (Titertek Multiskanplus) and the titer determined as the reciprocal ofthe highest dilution that shows an absorbancegreater than 0.050 at 492 nm, as non-specificreactions were observed below this value.

2.6. Western blotting

B. insularis and B. jararaca venoms (20 mg) werefirst fractionated by SDS-PAGE as described above,and transferred to nitrocellulose membranes(Towbin et al., 1979) at 385mA for 2 h. Membraneswere then incubated with either PBA or rabbitspecies-specific antivenoms, diluted at 1:1000 and1:10,000. Immunoreactive proteins were detectedusing peroxidase-labeled anti-horse IgG and anti-rabbit IgG, and then blots were developed with0.05% 4-chloro-1-napthol in 15% methanol (v/v),in the presence of 0.03% H2O2 (v/v). Non-immu-nized horse and rabbit sera were used as controls.

2.7. Protease and hyaluronidase assays

Zymography was employed to assay protease andhyaluronidase activities, using casein, gelatin orfibrinogen (Heussen and Dowdle, 1980; Barbaro

et al., 2005), and hyaluronic acid from rooster comb(Sigma, USA) (Miura et al., 1995; Barbaro et al.,2005), respectively, as substrates. Samples ofB. insularis or B. jararaca venom (10 mg), dissolvedin non-reducing sample buffer, were loaded, andgels were run at 20mA/gel and 4 1C. Clear areas inthe gel indicated regions of enzyme activity.Loxosceles gaucho (10 mg) spider venom was usedas a positive control in the hyaluronidase assay.When required, the metal chelating agent 1,10-phenanthroline (Sigma Chemicals, St Louis, MO)was added in a final concentration of 3mM to everygel washing and incubation buffer, and then gelswere stained as usual.

2.8. PLA2 activity

PLA2 activity was determined colorimetrically(Lobo de Araujo and Radvanyi, 1987), as modi-fied by Santoro et al. (1999). Briefly, an aliquot(15 ml) of B. insularis or B. jararaca venoms(0.33mg/ml in PBS, pH 7.4) was added to 1.5mlof reaction solution (100mM NaCl, 10mM CaCl2,7mM Triton X-100, 0.265% soybean lecithin,98.8 mM phenol red, pH 7.6) in a spectrophotometercuvette and read at 558 nm. The definition of 1 unitof PLA2 activity was taken as the amount ofvenom in assay producing a decrease of 0.001absorbance units per min under the conditionsdescribed. Crotalus durissus terrificus (5 mg)snake venom was used as a positive control. PLA2

activity was expressed as U/mg of two independentexperiments.

2.9. Microcirculatory disturbances

Mice (n ¼ 4) were anesthetized with 200 ml ofxylazine (50mg/kg) and after 10min with 200 ml of2.5% chloral hydrate (i.p.). Surgical procedures forintravital studies were performed as previouslydescribed by Lomonte et al. (1994). Animals weremaintained on a special board thermostaticallycontrolled at 37 1C, which included a transparentplatform on which the tissue to be transilluminatedwas placed. The dynamics of disturbances in themicrocirculatory network was determined usingintravital microscopy of cremaster muscle, aftertopical application of 5 mg of each venom diluted in20 ml of PBS. Administration of the same volume ofsterile saline was used as control. Experiments werealso carried out to evaluate the efficacy of PBA, byincubating a constant amount of venom (5 mg) with

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15 ml of antivenom for 1 h at 37 1C, and thereafterapplying 20 ml of the resulting solution topicallyto the internal spermatic fascia of animals. Thefollowing parameters were observed: hemo-rrhagic lesion formation, assessed by the latencytime for the beginning of lesions, number of lesions/tissue area and area of hemorrhagic lesions after40min.

2.10. Evaluation of myotoxic activity and

neutralization by PBA

Mice (n ¼ 8–12) were injected intramuscularly(i.m.) in the right gastrocnemius muscle with 50 mgof B. insularis or B. jararaca venom in 50 ml of sterilePBS. The ability of PBA to neutralize the myotoxicactivity was estimated by incubating 50 mg of B.

insularis and B. jararaca venom with 50 ml ofantivenom (dilution 1:2) for 30min at 37 1C.Following incubation, the mixture was injectedi.m. into mice. Control groups were injected withvenom or PBA alone. After 3 h, blood was collectedfrom the ophthalmic plexus. Sera of mice wereseparated and immediately assayed for creatinekinase activity (CK 520, Sigma, USA). One unitcorresponds to the amount of enzyme that hydro-lyzes 1 mmol of creatine per min at 25 1C. Enzymeactivity was expressed in U/L.

2.11. Neutralization of defibrinating activity

Groups of four mice were injected i.v. withvarious amounts of venoms (dissolved in 0.1ml of0.15M NaCl solution). One hour later, animalswere bled through the ophthalmic plexus, and bloodfrom each mouse was placed in a separate test tubeand left at 37 1C for 30min. The minimal defibrinat-ing dose (MDD) was defined as the lowest amountof venom that elicited blood incoagulability in mice(Gene et al., 1989). Neutralization experiments wereperformed by incubating two MDD of each venomwith 0.1ml of several dilutions of antivenom, inorder to obtain several antivenom/venom ratios.Incubations were carried out at 37 1C for 30min,and then the mixture was injected i.v. in mice. After1 h, a blood sample was taken as described above,and coagulation was assessed. The neutralizationability of antivenom was expressed as the effectivedose, defined as the lowest antivenom/venomratio in which blood coagulation was observed infour mice.

2.12. Lethal dose 50% (LD50) and neutralization of

lethality by antivenoms

Groups of three Swiss mice were injected i.p. with0.5ml of 0.15M NaCl containing different concen-trations (0.5–3.0mg/kg body weight) of B. insularis

or B. jararaca venoms. The number of dead micewithin 48 h was used to calculate LD50 by probitanalysis (Litchfield and Wilcoxon, 1949). Theexperiment was carried out in duplicate. In orderto determine the ability of PBA and rabbit species-specific antivenoms to neutralize the lethal activityof venoms, 3 LD50 and 5 LD50 (recommended edict174, Ministry of Health/SUS, Brazil, 1996) ofB. insularis or B. jararaca venoms were mixed with0.2ml of each sera. Non-immunized horse or rabbitsera incubated with both venoms were used ascontrols. The neutralizing ability of PBA was alsoexpressed as effective dose 50% (ED50), defined asmicroliters of antivenom per milligram of venom atwhich half of the injected animals survived(Gutierrez et al., 1990). The challenge dose usedfor B. insularis venom and B. jararaca venoms was 5LD50. The mixture was incubated for 1 h at 37 1C,centrifuged and the supernatant injected i.p. intomice. The number of surviving animals was assessedafter 48 h.

2.13. Hemorrhagic activity

The hemorrhagic activity was evaluated by amodification of Kondo’s method (Kondo et al.,1960). Groups of five mice were depilated on theback and then intradermically (i.d.) injected withdifferent doses of B. jararaca or B. insularis venoms(0.38–4.5 mg) in 100 ml of PBS. The skins wereexcised 2 h later and the diameters of hemorrhagicspots were measured on the internal surfaces. Theminimum hemorrhagic dose (MHD) was defined asthe amount (mg) of venom needed to produce ahemorrhagic spot of 10mm in diameter.

2.14. Statistical analyses of data

Depending on the activity being comparedbetween B. insularis and B. jararaca venoms,statistical analyses of data were carried out usingeither one-way analysis of variance (ANOVA),followed by Tukey’s test, or Student’s t-test.Statistical analyses were performed using the soft-ware SigmaStatTM, version 3.1. Estimates and 95%confidence intervals of MHD were calculated by

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inverse regression analyses (Santoro et al.,1999). Differences with po 0.05 were consideredstatistically significant. Data were expressed asmean7SEM.

3. Results

3.1. Comparison of venom profiles by SDS-PAGE

Fig. 1 shows the banding patterns of B. insularis

and B. jararaca venoms by SDS-PAGE in absenceor presence of 2-mercaptoethanol. At least 16 bandswere detected by SDS-PAGE in both venoms.Many components of similar molecular masses werenoticed in both venoms, but some specific bandsbetween 17 and 46 kDa were observed exclusively inB. jararaca venom. In the presence of 2-mercap-toethanol, several bands were noticed between 31and 63 kDa, especially in B. jararaca venom, andmost bands above 132 kDa and below 25 kDa couldnot be visualized anymore.

Fig. 1. SDS-PAGE (12.5% resolution gels) of B. insularis (Bi)

and B. jararaca (Bj) venoms (10mg/lane) in absence (1) or

presence (2) of 2-mercaptoethanol. Gels were silver stained.

3.2. Cross-reactivity determined by Western blotting

Fig. 2 shows that PBA and rabbit species-specificantivenoms reacted with B. insularis and B. jararaca

venoms. Separated components of both venomswere recognized similarly by all antivenoms. Com-ponents above 24 kDa of both venoms were moreimmunogenic than those of lower molecular masses.Bands below 21 kDa were strongly stained onlywhen antivenoms were diluted at 1:1000. Using anti-B. jararaca rabbit antivenom and PBA, bandsbetween 20 and 13 kDa were recognized only in B.

jararaca venom. Both rabbit antivenoms and PBAfailed to recognize B. insularis components in thisregion. However, components with approximately7 kDa of both venoms reacted with differentantisera.

3.3. Cross-reactivity determined by ELISA

Intense cross-reactivity between B. insularis andB. jararaca venoms was detected by ELISAusing PBA and rabbit species-specific antivenoms(Table 1). Differences in titers of either PBA orspecies-specific rabbit antivenoms against eachvenom could not be demonstrated, since valuesonly above two-fold dilutions are consideredsignificant.

3.4. Enzymatic activities of venoms

Zymography was used to detect proteolyticactivity in B. insularis and B. jararaca venoms, byusing casein, gelatin and fibrinogen as substrates(Fig. 3). Both venoms showed activity against thesesubstrates. B. jararaca and B. insularis venomspresented caseinolytic spots between 25 and 57 kDa;however, the use of 1,10-phenanthroline, an inhi-bitor of metalloproteases, decreased the intensity ofsome bands in both venoms. Gelatinolytic activity

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Fig. 2. (A) SDS-PAGE (12.5% resolution gels) of B. insularis (Bi) and B. jararaca (Bj) venoms (10mg/lane). Gels were stained by

Coomassie blue. (B) B. insularis (Bi) and B. jararaca (Bj) venoms were fractionated by SDS-PAGE, transferred to nitrocellulose

membranes and developed after incubation with polyspecific Bothrops antivenom (PBA), anti-B. jararaca and anti-B. insularis rabbit

antivenoms diluted 1:1000 and 1:10,000. Numbers on the right correspond to position of molecular mass markers.

Table 1

Antigenic cross-reactivity between B. insularis and B. jararaca

venoms by ELISA

Antivenoms Antibody titers

B. insularis venom B. jararaca venom

Anti-B. insularis (rabbit) 640,000 640,000

Anti-B. jararaca (rabbit) 1,280,000 1,280,000

Polyspecific (horse) 1,280,000 2,560,000

Titers represent the reciprocal of the highest dilution that causes

an absorbance greater than 0.050 at 492 nm. ELISA plates were

coated with either venom (10mg/ml), and then developed with

anti-B. insularis (rabbit), anti-B. jararaca (rabbit) and polyspecific

Bothrops (horse) antivenoms.

M.S. Lira et al. / Toxicon 49 (2007) 982–994 987

was similarly observed in both venoms, with manybands distributed between 29 and 127 kDa. Bandingintensity of some proteins decreased after incuba-tion with 1,10-phenanthroline, especially inB. jararaca venom. Fibrinogenolytic activity wasnoticed between 51 and 26 kDa. For all threesubstrates, differences and similarities were

observed in intensity, quantity and position ofvenom components. Hyaluronidase activity wasnot detected in B. insularis and B. jararaca venomsusing the protocol described herein (Fig. 3). SimilarPLA2 activities were noticed for B. insularis and B.

jararaca venoms (Fig. 4), which were nonethelesslower than that of Crotalus durissus terrificus

venom, used as a positive control.

3.5. Myotoxic activity and neutralization by PBA

Myonecrosis occurred when both venoms wereadministered to mice, since high levels of creatinephosphokinase enzyme (CK) were present in serum.Fig. 5 shows the myotoxicity induced by theinjection of 50 mg of B. insularis and B. jararaca

venoms in mice. Myotoxicity was higher inB. jararaca venom (po 0.001), and could not becompletely neutralized by PBA. On the other hand,the CK release induced by B. insularis venom couldbe abrogated by incubation with PBA (1:2 dilution),reaching values similar to controls (PBS).

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Fig. 3. Zymography of enzymatic activities of venoms. Casein, gelatin, fibrinogen and hyaluronic acid were used as substrates in SDS-

PAGE for analyzing B. jararaca (Bj) and B. insularis (Bi) venoms enzymes in absence (A) or presence (B) of 1,10 phenanthroline

(metalloprotease inhibitor). Clear areas in the gel indicate regions of enzymatic activity. Numbers on the right correspond to the position

of molecular mass markers. Loxosceles gaucho (Lg) spider venom (10 mg) was used as a positive control for the hyaluronidase activity

assay.

M.S. Lira et al. / Toxicon 49 (2007) 982–994988

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3.6. Neutralization of microcirculatory disturbances

and MHD by PBA

B. jararaca and B. insularis venoms inducedhemorrhage following topical application on thecremaster muscle of mice. Hemorrhagic activityinduced by B. jararaca and B. insularis venomsstarted 5min after application and intensified after10min. Nonetheless, B. jararaca venom provoked a

Phospholip

ase a

ctivity (

U/m

g)

0

10000

20000

30000

40000

*

*

C. d. terrificus B. jararacaB. insularis

Fig. 4. Phospholipase A2 activity (U/mg) of B. insularis and B.

jararaca venoms (5 mg). Crotalus durissus terrificus venom (5mg)was used as positive control. The absorbance was determined at

558 nm using a spectrophotometer. *Statistically significant

difference with the positive control.

U/L

0

50

100

150

200

PBS Bi

∗

∗ α

Bj

Fig. 5. Myotoxic activities of B. jararaca (Bj) and B. insularis (Bi) ven

(dilution 1:2) to neutralize them. Animals injected only with PBS or

different from the control (PBS), po 0.001; #—statistically significan

significant different from the Bj group, po 0.001; b—statistically signi

faster appearance of great hemorrhagic areas (3–5spots) (Fig. 6B, C), while B. insularis venomprovoked the appearance of several and smallhemorrhagic spots (10–15 spots) (Fig. 6F, G). Theneutralizing ability of PBA was evaluated by pre-incubating B. insularis and B. jararaca venoms withantivenoms at 37 1C. Both venoms were neutralizedby PBA (Fig. 6D, H). The MHD of both venomswas also determined in mice; it was 1.42 mg toB. insularis venom (95% confidence interval,1.24–2.42 mg) and 0.65 mg to B. jararaca venom(95% confidence interval, 0.61–2.28 ml). These va-lues were not statistically different.

3.7. Defibrinating activity and neutralization by PBA

The MDD determined for B. insularis and B.

jararaca venom was 0.2 and 2.0 mg, respectively.Two MDD of B. jararaca and B. insularis venomscould be neutralized by 2 and 10 ml of PBA,respectively.

3.8. Neutralization of lethality induced by B.

insularis and B. jararaca venoms by antivenoms

No statistically significant difference was shownbetween the LD50 of B. jararaca (1.66mg/kg, con-fidence interval: 1.32–2.17mg/kg) and B. insularis

PBA

#β∗

#

Bj + PBA Bi + PBA

oms and effectiveness of polyspecific Bothrops antivenom (PBA)

PBA were used as controls. (n ¼ 8–12). *Statistically significant

t different from the Bj or Bi groups, po 0.001; a—statistically

ficant different from the control (PBA), po 0.001.

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Fig. 6. Disturbances of murine microcirculatory network after the topical application of B. jararaca or B. insularis venoms (5mg) oncremaster muscle (A and E–before venom administration; B and F—after 5min; C and G—after 10min). Neutralization of disturbances

by previous incubation of B. jararaca (D) and B. insularis (H) venoms (5mg) in PBS with 15 ml of polyspecific Bothrops antivenom for 1

hour at 37 1C; the pictures show the panorama 10min after topical application. Arrows—hemorrhagic spots.

M.S. Lira et al. / Toxicon 49 (2007) 982–994990

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Table 2

Neutralization of lethal activity of B. insularis and B. jararaca venoms by antivenoms

Sera Survival

3 DL50 5 DL50

B. insularis B. jararaca B. insularis B. jararaca

Non-immunized horse 2/6a 1/6 0/6 1/6

Non-immunized rabbit 1/6 2/6 0/6 1/6

Anti-B. insularis (rabbit) 4/6 5/6 2/6 4/6

Anti-B. jararaca (rabbit) 6/6 6/6 2/6 6/6

Polyspecific (horse) 6/6 6/6 6/6 6/6

Groups of six mice were injected i.p. with the venom of each species (3 or 5 LD50), previously incubated for 1 h at 37 1C with 0.2ml of

polyspecific Bothrops antivenom or species-specific rabbit antivenoms. The number of survival animals was recorded 48 h after venom

injection. Non-immunized horse and rabbit sera incubated with both venoms were used as controls.aSurvival animals/injected animals.

M.S. Lira et al. / Toxicon 49 (2007) 982–994 991

(2.00mg/kg, confidence interval: 1.59–2.53mg/kg)venoms in mice. On account of the neutralizingactivity of antivenoms (Table 2), PBA was effectivein neutralizing 100% of the lethal activity inducedby 3 and 5 LD50 of B. insularis and B. jararaca

venoms. The ED50 was 26.6 ml/mg to B. insularis

venom (95% confidence limit, 15.9–50.4 ml) and23.5 ml/mg to B. jararaca venom (95% confidencelimit, 12.4–39.3 ml), no statistically significant dif-ference was noticed between ED50. Rabbit species-specific antivenoms were less effective; anti-B.

jararaca antibodies were more effective than thoseof B. insularis, and only failed to protect animalsinjected with 5 LD50 of B. insularis venom.However, anti-B. insularis antibodies could notcompletely neutralize the lethal activity of bothvenoms, even if 3 LD50 were used.

4. Discussion

B. jararaca is an abundant continental speciesthat occurs in southeastern Brazil, and it is the mostimportant snake in this area if the number ofsnakebite victims is taken into account (Araujo etal., 2003). On the other hand, B. insularis is aninsular snake, and it is a relatively recent isolatedderivative of B. jararaca (Wuster et al., 2005).

The results presented in this study showed thatthere are more similarities than differences betweenB. insularis and B. jararaca venoms. By SDS-PAGE,at least 16 bands were detected in B. insularis

venom, similar to the results reported by Cogo et al.(1998). The electrophoretic pattern of B. insularis

was very similar to B. jararaca venom, mainly inregions above 47 kDa, but different components

under 46 kDa were detected exclusively inB. jararaca venom. After reducing samples with2-mercaptoethanol, no bands of high and lowmolecular masses could be visualized, especially inB. jararaca venom. Important components ofBothrops sp. venoms have been shown to be locatedin this region, such as proteins with coagulantactivity, metalloproteases with fibrinolytic andhemorrhagic activities, phospholipase A2 (Theak-ston and Kamiguti, 2002) and myotoxins (Gutierrezand Lomonte, 2003).

Antigenic cross-reactivity between B. insularis

and B. jararaca venoms was observed using equinepolyspecific and rabbit species-specific antivenoms.By Western blotting, a great number of immuno-genic components were detected above 24 kDa inboth venoms. Our results corroborate studiescarried out by Ferreira et al. (1992), showingantigenic cross-reactivity among nine species ofBothrops genus by ELISA and Western blotting.Furthermore, it was interesting to observe thatantibodies present in PBA could also recognizeB. insularis venom components, which is notincluded in the immunization pool of horses.

Both venoms presented caseinolytic, gelatinolyticand fibrinogenolytic activities. In our study, theenzymatic activity of many bands decreased afterincubation with 1,10-phenanthrolin in both venoms,demonstrating the presence of metalloproteases inB. insularis venom. However, this effect was morevisible in B. jararaca venom. On the other hand, nohyaluronidase activity (spreading factor) was foundin these venoms using zymography, confirmingprevious reports showing that Bothrops venomsshow a lower hyaluronidase activity compared to

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other animal venoms (Tan and Ponnudurai, 1991,1992). In fact, metalloproteases have been reportedto degrade extracellular matrix components (lami-nin, fibronectin and collagen type IV) in vitro, toinduce hemorrhage (Gutierrez and Rucavado,2000), and to contribute to hemostatic disturbances(Kamiguti et al., 1996). Thus, the activity ofmetalloproteases may be correlated with the severityof local symptoms that occur in Bothrops sp.envenomation.

PLA2 activity was also present in B. insularis

venom, confirming it is ubiquitous in Bothrops sp.venoms (Moura-da-Silva et al., 1991, 1997; Valienteet al., 1992). PLA2 is an enzyme capable ofcatalyzing the hydrolysis of phospholipids, whichwill produce important inflammatory mediators (Sixand Dennis, 2000) and will damage muscular cells(Gutierrez and Lomonte, 2003). Since both venomspresented similar PLA2 activities, but B. jararaca

venom showed an increased myotoxic activity, itcan be stated that other components in B. jararaca

venom, other than PLA2, induce myonecrosis.Using our protocol, PBA could neutralize themyotoxic activity induced by both venoms. How-ever, it was more effective to neutralize B. insularis

myotoxic activity, which was lower than B. jararaca

venom. This data agree with Zamuner et al. (2004),who observed that PBA, after incubation for 2 hwith venoms, neutralized differently the myotoxicactivity of B. jararaca (87.5%), B. neuwiedi (84.2%),B. moojeni (79.7%), B. erythromelas (41.5%) andB. jararacussu (30.4%) venoms.

Both venoms presented defibrinating activity, butB. insularis venom was 10 times more active than B.

jararaca venom. PBA was effective to neutralize 2MDD of both the venoms, demonstrating that itmay be used to treat hemostatic disturbances ofpatients bitten by B. insularis.

The MHD was also determined in mice, and nostatistically significant difference was observed forboth venoms, although a dose 2 times higher ofB. insularis venom was necessary to cause ahemorrhagic spot of 10mm. By intravital micro-scopy, B. insularis induced the appearance ofhemorrhagic spots, similarly to B. jararaca venom.Nonetheless, the appearance of hemorrhagic spotsinduced by B. insularis was smaller and occurredlater than those induced by B. jararaca venom. Oncemore, PBA was effective in neutralizing the hemor-rhagic activity induced by both venoms. These dataagree with those shown by Battellino et al. (2003),who verified that PBA pre-incubated with B.

jararaca before topical application in rats neutra-lized fibrin clot formation, vessel stasis, hemorrha-gic lesions, increased vascular permeability anddisturbances of leukocyte adhesion to endothelium.

Although Bogarın et al. (2000) demonstrateddifferences in venom toxicities among 11 species ofBothrops snakes (range of 37–114 mg/g), we did notfind statistically significant differences in lethalityinduced by B. jararaca and B. insularis venomsusing mice as experimental model. However, Amar-al (1921) and Campos and Mello-Filho (1966)reported that B. insularis was more toxic thanB. jararaca venom for pigeons, probably becauseB. insularis feed birds. However, this may be alsodue to the high coagulant activity of B. insularis

venom; once these authors used i.v. administrationof venoms to assess toxicity. PBA was effective toneutralize the lethality induced by 5 LD50 of bothvenoms. Besides, no differences were observed inED50 to PBA to both venoms. Nevertheless, species-specific antivenoms produced in rabbit could notcompletely protect animals against the lethalactivity of 5 LD50 of B. jararaca and B. insularis

venoms, probably due to the high toxicity ofvenoms. If a dose of 3 LD50 was used instead,rabbit anti-B. jararaca serum could neutralize 100%of the lethal activity of both venoms, whereas anti-B. insularis serum could protect 66.7% and 83.3%of animals injected with B. insularis and B. jararaca

venoms, respectively. The low efficiency of hetero-logous rabbit antivenoms may be ascribed todifferences found between venoms, mainly in theregion below 46 kDa. On the other hand, our resultsconfirm a previous report (Bogarın et al., 2000) thatshows the neutralization of 4 LD50 of severalBothrops sp. venoms by PBA produced by ButantanInstitute, demonstrating its effectiveness to neutra-lize the lethal effects of these venoms. The highcapacity of neutralization of PBA may result fromthe hyperimmunization of horses, which raisesantivenom titers uppermost. Moreover, the poolused to produce PBA is composed of a mixture ofvenoms from five Bothrops species, increasingantigenic diversity. As a result, a high neutralizationcapacity of this antivenom is obtained.

The great similarity found between B. insularis

and B. jararaca venoms might be caused by splittingof a common ancestral snake during the increase inocean level 9000 years ago (Marques et al., 2002).Since B. insularis and B. jararaca venoms are alike,B. insularis could cause a clinical picture similar toB. jararaca envenomation. Thus, we suggest that in

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case of snakebites by B. insularis, PBA can beadministered to patients, since no antivenomspecific to B. insularis is available.

Acknowledgements

This investigation was supported by FAPESP(Fundac- ao de Amparo a Pesquisa do Estado de SaoPaulo). L.M.P. Martins was a fellow from FUN-DAP, and K.C. Barbaro from CNPq. The authorsthank Vivian Lopes Silva for technical assistance.

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