Ontogenetic variability of Bothrops atrox and Bothrops asper

snake venoms from Colombia

Monica Marıa Saldarriagaa, Rafael Oteroa,*, Vitelbina Nuneza, Maria Fabiola Toroa,Abel Dıaza, Jose Marıa Gutierrezb

aPrograma de Ofidismo/Escorpionismo, Facultad de Medicina, Universidad de Antioquia, Cra. 50A No 63-65/A.A. 1226,

Medellın 1226, ColombiabFacultad de Microbiologıa, Instituto Clodomiro Picado, Universidad de Costa Rica, San Jose, Costa Rica

Received 26 March 2003; accepted 19 June 2003

Abstract

The lancehead snakes Bothrops asper and Bothrops atrox inflict 70–90% of the 3000 bites reported every year in Colombia.

In this work, the venoms of B. atrox from Meta (Villavicencio, 33 specimens) and B. asper from Antioquia (San Carlos, 45

specimens), all of them born in captivity, were obtained at different ages (0–6 months; 1, 2 and 3-years old) and compared in

terms of their pharmacological and immunochemical characteristics. A conspicuous ontogenetic variability was observed in

venom samples from both species. Venoms from newborn and juvenile specimens showed higher lethal, hemorrhagic, edema-

forming and coagulant activities, whereas venoms from 3-year old specimens showed higher indirect hemolytic, i.e.

phospholipase A2 activity, being more significant in the case of B. asper. SDS-polyacrylamide gel electrophoresis of whole

venom for both species evidenced a predominance of high mol. mass bands in the venoms from specimens of ,1 year of age,

with a change towards bands having lower mol. mass as snakes aged. Gel filtration chromatography showed five peaks in the

venoms of B. asper of ,6 months and in those from 3-year old specimens. Venom of adult specimens showed a higher number

of peaks with indirect hemolytic activity than venom of newborn specimens. Polyvalent antivenom produced in Costa Rica

recognized all the bands of both venoms from specimens at all ages tested, when assayed by Western blotting.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: Bothrops atrox; Bothrops asper; Colombia; Venom variability

1. Introduction

Snake venoms constitute complex mixtures of proteins,

which contain a variety of enzymes, peptides, carbo-

hydrates, salts and water (Meier and Stocker, 1995).

Variation in venom composition has been described not

only at the interspecies level, but also within a single

species. In addition, there is a prominent ontogenetic

variation in the biochemical characteristics and in the

pharmacological profile of snake venoms (Gutierrez et al.,

1980; Mebs and Kornalik, 1984; Meier, 1986; Chippaux

et al., 1991; Furtado et al., 1991; Tun-Pe et al., 1995; Daltry

et al., 1996; Rael et al., 1997). Such venom variability has

implications for the biological role of venoms, for the

clinical characteristics of envenomations and for the

selection of venoms to be used in the immunization of

animals for antivenom production.

Ninety percent of snakebites in Latin America are

inflicted by pit vipers of the genus Bothrops. These venoms

induce a complex pathophysiological picture characterized

by local and systemic effects such as edema, myonecrosis,

blistering, hemorrhage, defibrination, shock and nephro-

toxicity (Rosenfeld, 1971; Otero et al., 1992, 2002;

Gutierrez, 1995). In Colombia, Bothrops asper and

Bothrops atrox inflict at least 70–90% of the 3000 bites

0041-0101/03/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0041-0101(03)00171-5

Toxicon 42 (2003) 405–411

www.elsevier.com/locate/toxicon

* Corresponding author. Tel.: þ57-4-263-1914; fax: þ57-4-263-

8282.

E-mail addresses: rotero@catios.udea.edu.co, rotero@epm.net.

co (R. Otero).

reported every year (Otero, 1994; Silva, 1989). These

species are widely distributed in tropical rainforest up to

1200 m, their range including east (both species) and west

(B. asper) of the Colombian Andes (Campbell and Lamar,

1989). Owing to the medical relevance of these species, and

to the fact that bites are inflicted by specimens of different

ages, a comparative study was performed on the electro-

phoretic, chromatographic and pharmacological character-

istics of B. atrox and B. asper venoms from specimens at

different ages and two localities in Colombia.

2. Materials and methods

2.1. Animals and venoms

For the in vivo experiments, Swiss Webster mice (18–

20 g body weight) were used. One litter of B. atrox from

Meta (Villavicencio) (33 specimens) and 1 l of B. asper

from Antioquia (San Carlos) (45 specimens), both born in

captivity, were used throughout this study. They were

maintained in the same environment and fed with mice at

the Serpentarium of the Universidad de Antioquia, Medel-

lın. The venoms were obtained by manual extraction at

different ages (,0.5, 1, 2 and 3-years old). Venoms were

centrifuged at 3000g for 15 min, and supernatants

were lyophilized and stored at 220 8C until used. Pools

were prepared for each age and species.

2.2. Characterization of pharmacological and enzymatic

activities

2.2.1. Lethality

Groups of four mice were injected i.p. with various doses

of venom dissolved in 0.5 ml phosphate-buffered saline

(PBS) solution, pH 7.2; controls received 0.5 ml PBS in

identical conditions. Deaths were recorded during 48 h and

the median lethal dose (LD50) was calculated by the

Spearman–Karber method (WHO, 1981), using the com-

puter program ‘Toxicalc’ (Robles and Gene, 1990).

2.2.2. Edema

Groups of four mice were injected s.c. in the right

footpad, with various doses of venom dissolved in 50 ml

PBS. The left footpad received 50 ml of PBS alone. After

6 h, mice were sacrificed by ether inhalation and both feet

weighed, following the method described by Yamakawa

et al. (1976), modified by Gutierrez et al. (1986). The

minimum edema-forming dose (MED) was the venom dose

that induced 30% edema in 6 h.

2.2.3. Hemorrhage

Groups of four mice were injected i.d. in the abdomen,

with various doses of venom dissolved in 0.1 ml PBS.

Controls received 0.1 ml PBS under identical conditions.

Two hours later, mice were sacrificed by ether inhalation

and the hemorrhagic areas were measured. The minimum

hemorrhagic dose (MHD) was the venom dose that induced

a hemorrhagic area of 10 mm diameter, according to the

method of Gutierrez et al. (1985, 1988a).

2.2.4. Coagulant effect

Coagulant activity was determined in human citrated

plasma (200 ml), adding various amounts of venom

dissolved in 100 ml PBS. The minimum coagulant dose

(MCD) was defined as the amount of venom which induced

coagulation of plasma in 60 s (Theakston and Reid, 1983;

Gene et al., 1989).

2.2.5. Indirect hemolysis

The method described by Habermann and Hardt (1972),

modified by Gutierrez et al. (1988b), was followed using

agarose–erythrocyte–egg yolk gels. The minimum indirect

hemolytic dose (MIHD) was the venom dose that produced

a hemolytic halo of 20 mm diameter in 20 h.

2.3. Immunochemical characterization

2.3.1. Electrophoresis

The whole venoms or the fractions obtained by gel

filtration chromatography were analyzed by SDS-polyacryl-

amide gel electrophoresis (SDS-PAGE) using 15% acryl-

amide gels (Laemmli, 1970). Samples of 20 mg of venom or

fractions were separated under non-reducing conditions and

gels were stained with Coomassie Brilliant Blue G-250.

Molecular weight markers were run in parallel.

2.3.2. Western blot

Western blots were performed by electrotransferring

proteins (150 mA, 2 h) from SDS-PAGE gels (15%) onto

nitrocellulose membranes, followed by incubation with a

horse-derived antivenom (Polyvalent antibothropic, antic-

rotalic, antilachesic antivenom from Instituto Clodomiro

Picado—ICP—Costa Rica, batch 2790996 LQ). After a

washing step, goat peroxidase-labeled anti-horse IgG was

added and the reaction was evidenced using the chromo-

genic substrate 4-chloro-1-naphtol.

2.3.3. Gel filtration chromatography

B. asper venoms obtained at the ages of 0–6 months and

3-years were subjected to gel filtration as follows: 70 mg

venom, dissolved in PBS, were applied to a Sephacryl S-200

column (60 £ 2.6 cm2), equilibrated previously with the

same buffer. Fractions of 7 ml were collected at a flow rate

of 1 ml/min, dialyzed against distilled water during 48 h at

4 8C, and then lyophilized. All of them were tested for

hemorrhagic and indirect hemolytic activities as described

earlier.

M.M. Saldarriaga et al. / Toxicon 42 (2003) 405–411406

2.4. Statistical analysis

The significance of the differences observed between

mean values was determined by a one way analysis of

variance (ANOVA). When the values were significantly

different ðp , 0:05Þ; the significance of the differences

between pairs of means was determined by the Newman–

Keuls test.

3. Results

3.1. Pharmacological and enzymatic activities

3.1.1. Lethality

The venoms from specimens of both species of ,0.5, 1

and 2 years of age did not differ in their LD50 values, all of

them having higher toxicity than the venoms obtained from

3-year old specimens (Table 1). B. asper venom from adult

specimens (3-years old) was more lethal than that from B.

atrox ðp , 0:05Þ:

3.1.2. Hemorrhagic, coagulant and edema-forming

activities

For both species, the highest hemorrhagic and coagulant

activities were observed in the venoms from newborn and

juvenile specimens, when compared with the venoms

obtained from snakes of 3 years of age (Table 1). A similar

pattern of ontogenetic variability was observed regarding

edema-forming activity. Coagulant activity was higher for

the venoms from newborn and juvenile specimens of

B. asper when compared with B. atrox venom at similar

ages ðp , 0:05Þ:

3.1.3. Indirect hemolytic activity

In the case of B. asper venom, indirect hemolytic activity

highly increased as the snakes aged (Table 1). In contrast,

very minor differences were observed in the venoms of

B. atrox from specimens at different ages. B. asper venom

had higher indirect hemolytic activity than B. atrox venom,

at all ages tested ðp , 0:05Þ:

3.2. Immunochemical characterization

Conspicuous ontogenetic differences were observed in

the SDS-PAGE pattern of the venoms from both species.

Venoms from young specimens (0.5–1.0 year) showed high

mol. mass bands, whereas a band of approximately 14 kDa

was evident in the venoms of 1, 2 and 3-years old

specimens, especially in those from B. atrox (Fig. 1). A

band of 24 kDa was present in all venom samples analyzed

(Fig. 1). Polyvalent antivenom recognized protein bands of

various mol. masses for both venoms, as observed in

Western blots. The highest reactivity was observed against

bands of high mol. mass (data not shown). Tab

le1

On

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49

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5.5

(54

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76

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67

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(80

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83

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.

M.M. Saldarriaga et al. / Toxicon 42 (2003) 405–411 407

3.3. Gel filtration chromatography

The chromatographic analysis of B. asper venom from

specimens ,0.5 and 3-years old showed the same number

of peaks (five), although the elution volume and the

eletrophoretic profile of some peaks differed (Fig. 2).

Venom from newborn specimens (Fig. 2(A)) showed two

hemorrhagic fractions (peaks one and two) associated with

high mol. mass bands (60–100 kDa), and two peaks

exerting indirect hemolytic activity (peaks three and four).

In contrast, the venom from adult specimens (Fig. 2(B))

showed one hemorrhagic fraction (peak one) with the

presence of bands of 53 and 31 kDa, and four peaks with

indirect hemolytic activity (peaks two to five).

4. Discussion

Snake venoms vary in their biochemical composition

and pharmacological profile, not only between different

species, but also within a single species and in snakes of

different ages (Gutierrez et al., 1980; Mebs and Kornalik,

1984; Minton and Weinstein, 1986; Meier, 1986; Chippaux

et al., 1991; Tan et al., 1993; Tun-Pe et al., 1995; Daltry

et al., 1996; Rael et al., 1997; Cavinato et al., 1998).

Ontogenetic variation in venom composition has been

demonstrated in a number of species. In some cases,

venoms from newborn and juvenile specimens have higher

toxicity than those from adult specimens, as demonstrated

for Crotalus durissus in Central America (Lomonte et al.,

1983; Gutierrez et al., 1991). In contrast, venoms from adult

specimens are of higher toxicity in other species of Bothrops

from Brazil (Furtado et al., 1991), and the venom from

newborn specimens of Lachesis muta from Costa Rica is

almost devoid of toxicity (Gutierrez et al., 1990).

Ontogenetic variability of B. atrox and B. asper venoms

from Colombia has similarities with results described for B.

asper venom from Costa Rica (Gutierrez et al., 1980;

Chaves et al., 1992). Venoms from newborn and juvenile

specimens have higher lethal, hemorrhagic, edema-forming

and coagulant activities than those from adult specimens,

whereas the latter have higher indirect hemolytic, i.e.

phospholipase A2 activity. Our observations also indicate

that venoms from newborn and juvenile specimens present a

higher number of high mol. mass electrophoretic bands than

venoms from adult specimens. These results are in

agreement with previous observations carried out with the

venom of B. atrox from Brazil (Meier, 1986). Lopez-Lozano

et al. (2002) demonstrated by SDS-PAGE that the most

intense bands in the venom from adult specimens of B. atrox

corresponded to proteins of 23 and 50 kDa, which are

probably metalloproteinases.

Besides their intrinsic biological relevance, studies on

the ontogeny of snake venoms have practical clinical and

immunological implications (Warrell, 1997). Results

obtained with B. atrox and B. asper venoms suggest that,

despite the low amount of venom that young specimens may

inject in a bite, envenomations by newborns and juveniles

may cause prominent vasculotoxic effects, i.e. hemorrhage

and edema. The high hemorrhagic activity of these venoms

Fig. 1. SDS-PAGE of B. asper venom from Antioquia (A) and B. atrox venom from Meta (B), run under non-reducing conditions in 15%

acrylamide gels and stained with Coomassie Brilliant Blue. Samples of venom (20 (mg) from both species at different ages (,0.5, 1, 2 and 3-

years old) were separated (150 mA, 60 min). MWM, molecular mass markers.

M.M. Saldarriaga et al. / Toxicon 42 (2003) 405–411408

correlates with the abundance of high mol. mass bands in

electrophoresis, since it has been demonstrated that the most

potent hemorrhagic toxins are high mol. mass metallopro-

teinases (Kini and Evans, 1992; Paine et al., 1992;

Bjarnason and Fox, 1994).

Moreover, owing to the strong coagulant activity of

venoms from newborn and young specimens, these

envenomations are probably associated with coagulopa-

thies. Observations with a number of species of Bothrops

from Brazil demonstrated that venoms from newborn

specimens have high procoagulant activity (Furtado et al.,

1991). Clinical observations on Bothrops jararaca and

B. asper (from B. atrox complex in a previous classification)

envenomations in Brazil and Colombia, respectively, agree

with these experimental findings (Ribeiro and Jorge, 1989;

Otero et al., 1996).

Since a number of snakebites are inflicted by newborn

and juvenile specimens, it is important to test if antivenoms,

which are produced by immunizing horses predominantly

with venoms from adult specimens, are effective against

toxic activities of venoms from specimens of all ages.

Neutralization studies (Gutierrez et al., 1980; Chaves et al.,

Fig. 2. Chromatographic and electrophoretic profiles of B. asper venom from Antioquia (Colombia) at different ages. (A) Newborn specimens

venom (,0.5 years). (B) Adult specimens venom (3-years old). Whole venom (70 mg) was re-suspended in PBS pH 7.2 and applied to a

Sephacryl S-200 column previously equilibrated with the same buffer. All the fractions were tested for hemorrhagic and indirect hemolytic

activities, and their electrophoretic patterns under reducing conditions were also determined.

M.M. Saldarriaga et al. / Toxicon 42 (2003) 405–411 409

1992) and immunochemical observations (Gutierrez et al.,

1980; this work) suggest that the polyvalent antivenom

produced in Costa Rica is effective in the neutralization of

venoms from newborn and juvenile specimens of B. atrox

and B. asper. However, as a general rule, it is recommended

that venoms from specimens of different ages should be

used in the preparation of pools for immunizing animals in

antivenom production centers (WHO, 1981).

The adaptive role of the observed ontogenetic variations

in venom composition and actions is not clear at present, and

conflicting points of view have been presented on this

subject (Daltry et al., 1996; Sasa, 1999). Despite the fact that

all specimens used in this work were fed with mice since

they were born, in natural conditions B. atrox and B. asper

specimens of ,1 year of age feed mainly of amphibians and

reptiles, whereas at later stages their diet shifts mainly to

rodents (Silva, 1998). It has been demonstrated that snake

venom composition is inherited rather than environmentally

induced, being under a strict genetic control (Daltry et al.,

1996). Thus, it is likely that the characteristics of B. atrox

and B. asper venoms from different ages herein described

are similar for wild specimens. In conclusion, prominent

ontogenetic changes occur in the venoms of B. atrox and

B. asper from Colombia, as venoms from newborn and

juvenile specimens are more lethal, hemorrhagic, edema-

forming and coagulant than venoms from adult specimens.

Acknowledgements

This work was supported by the Instituto Colombiano

para el Desarrollo de la Ciencia y la Tecnologıa Francisco

Jose de Caldas (COLCIENCIAS), Banco Interamericano de

Desarrollo (BID), the Universidad de Antioquia and Vice-

rrectorıa de Investigacion, Universidad de Costa Rica

(project 741-A1-027).

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