Saldarriaga (2003) Ontogenetic variability of Bothrops atrox and Bothrops asper by Cesar Alexander Ortiz Rojas

Toxicon 42 (2003) 405–411 www.elsevier.com/locate/toxicon

Ontogenetic variability of Bothrops atrox and Bothrops asper snake venoms from Colombia Mońica Marıá Saldarriagaa, Rafael Oteroa,*, Vitelbina Nuń˜eza, Maria Fabiola Toroa, Abel Dıáza, Jose´ Marıá Gutie´rrezb a

Programa de Ofidismo/Escorpionismo, Facultad de Medicina, Universidad de Antioquia, Cra. 50A No 63-65/A.A. 1226, Medellı´n 1226, Colombia b Facultad de Microbiologı´a, 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, edemaforming 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, carbohydrates, 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 (Gutie´rrez et al., * Corresponding author. Tel.: þ57-4-263-1914; fax: þ 57-4-2638282. E-mail addresses: rotero@catios.udea.edu.co, rotero@epm.net. co (R. Otero).

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 nephrotoxicity (Rosenfeld, 1971; Otero et al., 1992, 2002; Gutie´rrez, 1995). In Colombia, Bothrops asper and Bothrops atrox inflict at least 70 – 90% of the 3000 bites

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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 electrophoretic, chromatographic and pharmacological characteristics 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, Medellı´n. 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 2 20 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 computer 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 Gutie´rrez 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 Gutie´rrez 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 Gutie´rrez 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-polyacrylamide gel electrophoresis (SDS-PAGE) using 15% acrylamide 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, anticrotalic, 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 chromogenic 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.

Values in parenthesis are 95% confidence limits. Results are expressed as mean ^ SD of three experiments. Values with different superscripts (a, b, c, d) are significantly different (Newman– Keuls test, p , 0:05). A, B. asper; B, B. atrox; n, number of snakes in each group. MHD, minimum hemorrhagic dose; MED, minimum edema-forming dose; MCD, minimum coagulant dose; MIHD, minimum indirect hemolytic dose (see the text).

17.3a 1.8 ^ 0.1a 18.1 ^ 0.21b 3.3 ^ 0.4b 19.9 ^ 0.15d 5.4 ^ 0.2c

Lethality (LD50, mg/mouse) Hemorrhage (MHD, mg/mouse) Edema-forming (MED, mg/mouse) Coagulant (MCD, mg) Indirect hemolytic (MIHD, mg)

5.3 ^ 0.3c

1.0 ^ 0.02a 0.3a

19.3 ^ 0.1c

2.5 ^ 0.10d 3.4 ^ 0.01d 1.5 ^ 0.04c 1.0 ^ 0.04c 1.2 ^ 0.02b

0.7 ^ 0.04a 0.9 ^ 0.03a

0.8 ^ 0.03b

2.0 ^ 0.3b 2.2 ^ 0.2b 0.8 ^ 0.06a 1.3 ^ 0.1a 0.8 ^ 0.06a

0.3 ^ 0.10a 0.3 ^ 0.04a

1.0 ^ 0.15a

1.8 ^ 0.20c 1.5 ^ 0.1d 0.7 ^ 0.10b 0.8 ^ 0.2c 0.3 ^ 0.06a

51.8 (37.9–65.7)a 42.9 (35.5–50.3)a

0.5 ^ 0.06b

67.1 (60.1–74.1)b 65.5 (54.1–76.9)a 49.4 (40.7–58.1)a

A ðn ¼ 15Þ A ðn ¼ 20Þ B ðn ¼ 33Þ A ðn ¼ 45Þ

B ðn ¼ 23Þ

2 1

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).

,0.5

3.2. Immunochemical characterization

Age of specimen (years)

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:

Activity

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:

Table 1 Ontogenetic variability in the pharmacological and enzymatic effects of B. asper and B. atrox venoms from Colombia

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:

B ðn ¼ 22Þ

3.1. Pharmacological and enzymatic activities

59.7 (54.0–65.4)a

3. Results

A ðn ¼ 15Þ

B ðn ¼ 21Þ

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.

48.1 (43.1–53.1)a

2.4. Statistical analysis

407 81.4 (80.2–83.6)b

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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 3years old) were separated (150 mA, 60 min). MWM, molecular mass markers.

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 (Gutie´rrez 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; Gutie´rrez 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 (Gutie´rrez 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 (Gutie´rrez 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). Lo´pez-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

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409

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.

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 metalloproteinases (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 coagulopathies. 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 (GutieÂ´rrez et al., 1980; Chaves et al.,

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1992) and immunochemical observations (Gutie´rrez 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, edemaforming and coagulant than venoms from adult specimens.

Acknowledgements This work was supported by the Instituto Colombiano para el Desarrollo de la Ciencia y la Tecnologıá Francisco Jose´ de Caldas (COLCIENCIAS), Banco Interamericano de Desarrollo (BID), the Universidad de Antioquia and Vicerrectorıá de Investigacioń, Universidad de Costa Rica (project 741-A1-027).

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