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On-line version ISSN 1678-9199
J. Venom. Anim. Toxins incl. Trop. Dis vol.9 no.2 Botucatu 2003
M. F. D. FurtadoI; M. C. SantosII; A. S. KamigutiIII
ILaboratory of Herpetology/Venoms, Instituto Butantan, São Paulo, SP, Brazil
IIUniversity of Amazonas, Manaus, Brazil
IIIDepartment of Haematology, University of Liverpool, UK
An in vitro and in vivo comparative study was performed on the effects of Crotalus durissus terrificus venoms from a mother and its 15 newborns. The venoms were tested for protein content, lethality, proteolytic, myotoxic, hemorrhagic, and phospholipase A2 activity. The minimum coagulant dose in plasma and human fibrinogen, protrhombin, and Factor II activations were analyzed. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE). This showed that despite similar total protein content, the biological effects of the venoms were different. Venom from young snakes exhibited higher enzymatic and coagulant activities and higher myotoxicity compared to the mothers. In addition, the PLA2 content paralleled myotoxicity. However, no difference could be detected in their toxicity (LD50 0.08 mg/Kg). High incidence of blood coagulation disorders and elevated circulating myoglobin may characterize systemic envenoming by young C. d. terrificus.
Keywords: Age-related, biological activities, rattlesnake venom, Crotalus durissus terrificus.
The South American Crotalus durissus ("cascavel") is the most widely distributed rattlesnake species, with subspecies ranging discontinously from the Tropic of Cancer in Mexico to Northern Argentina (9). Although there are taxonomic problems relating to subspecies definition of different Crotalus, Crotalus durissus venom has been most extensively studied. Its neurotoxic components, particularly crotoxin, are of great interest to venom toxinologists (7). Others components have also been isolated and characterized from this venom, namely crotamine (11,31), giroxin (1,4), convulxin (35,41) and a thrombin-like enzyme (37). The genus Crotalus in Brazil is of special importance because of the high incidence of envenoming and mortality rates (3). Clinically, venom of this snake does not usually cause local effects at the bite site and is usually painless. However, the etiology progresses to systemic neurotoxic and myalgic symptoms, with frequent renal failure accompanied by acute tubular necrosis (3).
Previous investigations on Crotalus durissus venom have shown some regional differences in its activities (12,30,42,44). Variation in venom composition and activities in snakes of different ages has also been investigated in C. d. durissus (26). This study indicated that newborn snake venom was devoid of local hemorrhagic and edema-producing activity but had higher lethality compared to adult snake venom (26). Gutierrez et al. (19) demonstrated that newborn C. d. durissus venom is similar to adult C. d. terrificus venom. A drastic ontogenetic change appears to take place in venom composition of this subspecies (26). Very little is known about young C. d. terrificus venom.
In this study, we compared lethality, biological activity, and protein composition of venoms from adult and juvenile C. d. terrificus to investigate whether certain venom properties are age-dependent.
MATERIALS AND METHODS
Snakes and venom samples - A pregnant C. d. terrificus snake from Bauru, São Paulo State, Brazil, was brought to the Laboratory of Herpetology of the Instituto Butantan; a few weeks later it gave birth to 15 snakes. Seventy-two hours after birth, the mother and the young were weighed, measured, and venom was extracted. Venoms from newborn snakes were pooled. Venom samples were dried under vacuum and maintained at 4ºC. Venoms were reconstituted in sterile saline before use.
Protein content - Protein content was determined (27), using bovine albumin as a standard.
Toxicity - Lethal toxicity was assessed in out-bred Swiss mice (18-22 g) by IP venom injection in 0.5 ml physiological saline. Six mice were used at each venom dose. LD50 was calculated by probit analysis of deaths occurring within 48 hours of venom injection (15). Confidence limits (95%) were calculated.
Coagulation study - Plasma (MCD-P) and human fibrinogen (MCD-F) minimum coagulant doses were estimated (43). The minimum coagulant dose of a venom is defined as the minimum amount of venom resulting in clot formation within 60 sec at 37ºC.
Prothrombin activation was estimated using the two-stage prothrombin time test (6). Briefly, ten microliters (0.1 unit.) of Factor X (Sigma, U.K.), 100 µl of barbital buffer, pH 7.4, 100 µl of 25 mM calcium chloride, 25 µl of 0.5 mg/ml cephalin (Diagnostics Reagents, U.K.), and 50 µl of 125 µg/ml venom were incubated together at 37ºC for 10 min. Fifty microliters of this mixture were transferred to 0.2 ml of human fibrinogen (2 mg/ml, Kabi Diagnostics, Sweden) and clotting time recorded. The method used for estimating Factor II activation was similar except for the addition of 10 µl (0.1 unit.) of Factor II to the incubation mixture. Clotting times (CT) were transformed into coagulant activity (C.A.) using the formula: C.A.=1000/CT/mg venom.
Proteolytic activity - Proteolytic activity was estimated using fibrin, casein, and BAPNA (Bz-Arg-pNA; Sigma) as substrates. Fibrin plates were prepared (21). Thirty microliters of venom solutions were applied, and the system was incubated for 18 hours at 37ºC. Following incubation, two perpendicular diameters of the lysis area were measured. Caseinolytic activity was determined using casein-agar plates (34). Bovine pancreatic trypsin (Calbiochem, U.S.A.; 1-100 µg/ml protein) was used as standard. One unit of caseinolytic activity was defined as the venom amount that has the same activity as 1 µg/ml of trypsin. The system was incubated for 6 hours at 37ºC and the lysis area measured as described above.
Bapna - amidolytic activity of the venoms was determined (13). The results were analyzed by a linear curve obtained using venom dilutions. Activity was expressed as delta OD 405/min/mg venom protein.
Myotoxic activity - Crude venom was dissolved immediately before use in sterile 0.85% NaCl. Then, 0.1 ml of solution containing 2.0 µg venom was injected into the thigh muscle of the right hind leg of mice (18-22 g). Control mice were injected with 0.85% NaCl in the same manner. For each venom sample, six mice were used. All blood samples were collected by orbital plexus at various intervals after injection (1, 2, 3, 4, 6, 12, and 24 h). Blood was allowed to clot, centrifuged at 4ºC and 3000 r.p.m., and serum creatine kinase (CK) activity was determined by colorimetric method based on creatine formation through ADP/phosphocreatine reaction (CK activated with Nac; Merck-1-Test, Merck, Rio de Janeiro, Brazil). CK value was expressed in International units per liter (U/l) of serum.
Crotamine - For each assay, two mice (18-22 g) received IP venom injection of 0.25 mg in 0.5 ml saline solution (41). According to this method, the test is considered positive when spastic paralysis in the hind legs is observed within 30 minutes following venom injection. Crotamine detection was also investigated by SDS-PAGE (25).
Hemorrhagic assay - One microgram of each venom in 50 µl saline was injected ID into the shaven backs of Swiss mice (18-22 g, male). Four mice per venom sample were used. The mice were killed after 2 h, dorsal skins removed, and hemorrhagic lesions measured on skin inner surface (48).
Phospholipase A2 activity - This was calculated by estimating enzyme hemolytic activity. Briefly, agarose gels containing washed sheep erythrocytes, CaCl2, and egg yolk were prepared (18). Various venom concentrations were applied on these gels. Ten microliters of saline solution were used as control. After 20 h incubation at 37ºC, the diameters of hemolytic areas were measured and dose-response curves plotted. Data were estimated by comparing the curves obtained by phospholipase (standard curve) with venom dilutions. Activity was expressed in µg phospholipase/mg venom.
SDS-PAGE - Venoms (10 µl; 5 mg per ml 0.0lM tricine phosphate buffer, pH 8.7), crotamine fraction (10 µl, 5 mg per ml), and molecular weight calibration markers (PROMEGA) were subjected to 10% SDS-Tricine electrophoresis (25). Proteins were stained with Coomassie Blue R-250.
RESULTS AND DISCUSSION
Biometric data, venom yield, total protein content, and enzymatic activity of the venom from the adult C. d. terrificus and its 15 young are shown in Table 1. Total protein content did not show a substantial variation. Amidolytic, fibrinolytic, and phospholipase A activity was low in adult venom compared to pooled juvenile venom. Caseinolytic activity was absent in both groups. In another subspecies, C. d. durissus (26), a high hemolytic activity (PLA2 level) in newborn compared to of adult venom has been recorded. In contrast, there was an increase in proteolytic activity with age. In the timber rattlesnake (C. horridus horridus), an age-dependent increase in L-amino acid oxidase and a decrease in phosphodiesterase have been reported (8) . Also, venoms of young C. atrox (29), C. viridis helleri, and C. v. oreganus (23) have been described as possessing a lower protease activity than adults. There is a distinct difference in enzyme composition of Crotalus snake venoms depending on age, notably, the neurotoxic PLA2 component is higher in young snake venom (26).
|Table 1. Biometric data, venom yield, protein content, and enzymatic activity of venoms from C. d. terrificus mother and young.|
Table 2 shows lethality, hemorrhagic, and procoagulant activity of venom samples. There were no variations in LD50 of adult and juvenile venoms. However, newborn C. d. durissus venom was found to be seven times more lethal than adult (26); there is a similar relation between toxicity and snake age for C. atrox (29), C. v. viridis (14), C. h. horridus (8), and C. v. oreganus (23) in mice and for C. v. helleri and C. v. oreganus (28) in lizards. The higher LD50 of C. d. terrificus adult and juvenile venoms can be explained by the presence of crotoxin, which is a major component of C. d. terrificus venom - 60% (5). It is a very potent neurotoxin that possesses a PLA2 capable of blocking neuromuscular transmission (46,47). None of the venom samples exhibited hemorrhagic activity. C. d. terrificus newborn venom was ten times more potent both in clotting plasma (MCD-P) and fibrinogen (MCD-F) than the mothers. A similar result was observed in C. h. horridus (8) and C. atrox (38) venoms, and this also frequently applies to the Bothrops genus (17). Our results with C. d. terrificus showed that venom from newborn snakes was more active than that from adults. It is then possible that human envenoming by young C. d. terrificus snake may also result in a higher incidence of incoagulable blood symptoms, as it occurs with Bothrops jararaca (39). C. d. terrificus venom clotting activity has been investigated (32), and the enzyme responsible for this activity is a thrombin-like enzyme that has been isolated and characterized (37). In human envenoming by C. d. terrificus, blood incoagulability is due to fibrinogen consumption (2); approximately 40% of all cases have coagulation disturbances (22). Activation of factors II and X by both venoms is negligible compared to Bothrops venoms (33). As shown in Figure 1, venoms from both juvenile and adult snakes were qualitatively different as judged by their electrophoretic patterns, with a strong band around 18 kDa present in young venom but not in adult. Crotamine fraction is absent in both samples (compare rows 2 and 3 with rows 4). It has already been reported that this protein may or may not be present in C. d. terrificus venom (42,45) depending on the geographical distribution of this snake in São Paulo State. Schenberg (42) collected venom from 530 snakes, most of them in São Paulo State, analyzed their crotamine content, and found a transition from a region predominantly crotamine positive (Western São Paulo State) to a region mostly crotamine negative (Eastern and Central São Paulo State. Our snakes were captured in the central area of São Paulo State and are crotamine negative.
|Table 2. Comparative analysis of toxicity, hemorrhagic, edema-forming, and coagulant activity of venom from C. d. terrificus mother and young.|
|Figure 1. SDS-PAGE comparing C. d. terrificus adult and newborn venom demonstrating absence of crotamine toxin. Numbers on the left are molecular weight markers.|
Newborn snake venom is more myotoxic than adult (Figure 2). This could be due to the higher content of PLA2 found in young specimen venom (Table 1), since muscle necrosis is caused by phospholipase A2, a sub-unit of crotoxin (24). Newborn venom is approximately four times more active than the adult in myotoxic and phospholipase activity (19).
|Figure. 2. Myotoxic activity - Changes in creatine kinase (Units / liter) levels in plasma after IM. injection of 2.0 µg venom/mouse. Results presented as mean ± SEM (n= 6). Control ( ), C. d terrificus adult ( ), and C. d. terrificus newborn ( ).|
The South American rattlesnake (C. d. terrificus) venom exhibits age-dependency in its electrophoretic patterns and in biological properties, with variations in intensity of distinct activities. Inasmuch both the adult and juvenile venoms are very potent, as shown by LD50. The dietary requirements of juvenile rattlesnakes are similar to adults. It has been shown that Crotalus durissus from São Paulo State, Brazil, is almost entirely dependent on endothermic prey (40). Observations on captive specimens held in the Laboratory of Herpetology/Venoms at the Instituto Butantan, São Paulo (personal observation) have shown that from birth they readily feed on this type of prey. Studies on evolution of strike and release prey capture strategies have been done (23,36), demonstrating that envenoming degree and/or effects may vary with ontogeny (20); venom inducing rapid prey death may be an important feature in the evolution of their prey-capture strategies (10,16).
In conclusion, our investigation demonstrated that at least two major activities of the South American C. d. terrificus venom vary depending on snake age: both the coagulant and phospholipase A2 activities are higher in newborn snake venom.
We thank HANA SUZUKI for technical assistance. MFDF is fellowship of CNPq.
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M. F. D. Furtado
Laboratório de Herpetologia, Instituto Butantan
Av. Vital Brazil, 1500
05503-900, São Paulo, SP, Brasil
Received September 16, 2002
Accepted December 16, 2002