Toskwi, Vol . 30, No . 9, pp. 1099-1109, 1992. Printed in Otat Htinln.

0041-0101/92 55 .00 + .00 ® 1992 moo Pew Ltd

PATHOLOGICAL AND BIOCHEMICAL CHANGES INDUCED IN MICE AFTER INTRAMUSCULAR INJECTION OF VENOM FROM NEWBORN SPECIMENS OF THE SNAKE BOTHROPS ASPER (TERCIOPELO) FSRNwNDO CHwvFS .l Josi~ MwRlw GUT~RREZI

and

FERNwNDO BRENx~

IInstituto Clodomiro i?icado, Facultad de Microbiologfa, Univcrsidad de Costa Rica, San José, Costa Rica; and ~Lahoratorio de i°atologla Experimental, Hospital México, Caja Coatarricense del Seguro Social, Costa Rica (Received 9 October 1991 ; accepted 20 February 1992)

F . CHwvas, J. M . GuTiIéRREZ and F. BRENFS. Pathological and biochemical changes induced in mice after intramuscular injection of venom from newborn specimens of the snake Bothrops riper (Terciopelo) . Toxicon 30, 1099-1109, 1992.-Venom from newborn Bothrops riper snakes has higher lethal, hemorrhagic, edema-forming, proteolytic and defibrinating activities than venom from adult B. riper specimens. Electrophoretic analysis confirmed the variation between these venoms . Intramuscular injection of 100 ug of venom from newborn specimens in mice induced defibrination, together with moderate increments of serum levels of lactate dehydrogenase, creatine kinase, hemoglobin and total proteins . A conspicuous hemorrhage developed in injected muscle rapidly after envenomation, probably due to a drastic alteration in capillaries and larger blood vessels. Other histological alterations included moderate myonecrosis, lung collapse and prominent renal damage, characterized by tubular necrosis and hyalinization. Polyvalent antivenom effectively neutralized lethal, hemorrhagc and indirect hemolytic activities of newborn B. riper venom, although requiring higher antivenom doses than neutralization of venom from adult B. riper.

INTRODUCTION

Bothrops riper is responsible for the majority of snakebites in Central America (BoL.wI~os, 1982). The effects induced by venoms obtained from adult B. riper specimens have been studied clinically and experimentally, and include a complex series of local and systemic alterations such as local myonecrosis, hemorrhage and edema, as well as defibrination, systemic hemorrhage, cardiovascular shock and acute renal failure (MEKBEL and C~s>~FS, 1963 ; PEfirw-CHwvwRRfw et al., 1970; Gtrrt~eREZ et al., 1980a, 1982; Bot.wfiros, 1982; BARRANTES et al., 1985 ; CHwvgs et al., 1989). It has been reported that many bites in Central America are caused by newborn and juvenile specimens of B. riper (Swss, 1979 ; our unpublished observations). A previous study demonstrated that newborn B. riper venom differs markedly from adult venom in several biochemical and pharmacological parameters (GvTt~eREZ et al., 19806) . 1099

1100

F. CHAVES et al.

Accordingly, it is important to characterize the pathological alterations induced by the venom of newborn B. riper snakes . In the present work we have used an experimental model based on the i.m. injection of the venom from newborn B. riper specimens, in order to study the development of local and systemic pathological effects, as well as the changes in several biochemical parameters in blood and muscle tissue . In addition, the ability of the polyvalent antivenom produced in Costa Rica to neutralize some of these activities has been investigated . MATERIALS AND METHODS Venom and antivenom Venom was a pool obtained from specimens of five different litters, that were born at the aerpentarium of the Instituto Clodomiro Picado . The mothers had been collected in the Atlantic region of Costa Rica. Venom was obtained once during the first 30 days after birth. It was then pooled, lyophilized and stored at -20°C. For neutralization tests the polyvalent antivenom (batch 159), produced at the Instituto Clodomiro Picado according to the method of Bot.nAos and Ceenes (1980), was used . In the production of this antivenom, horses were immunized with a mixture of venoms of adult spatimens of Bothrops riper, Crotahcr durtxnv durirsus and Lachesis mots stenophrys (Ho~f~os and Ceanes, 1980). Electrophoresis of the vtnoms Venoms from newborn and adult B. riper specimens were analyzed by SDS-polyacryLunide ®el electrophoresis (SDS-PAGE), as described by L~tu (1970) and using 12% polyacryhunide gels . Fnzynatic activities Proteolytic activity on casein was determined as described by Loe~orrrE and GvriéanFZ (1983) and Gtrrd~nx$z et al. (198 . Phospholipase Az activity was estimated by the indirect hemolytic assay on agarose gels (Gvxtt;axsz et al., 198ßa) . Neutralization of hemolytic activity by polyvalent antivenom was studied according to the method of Gv~t~téaxez et al. (19ß8a) . lethality assays Groups of four mice (16-18 g) were injected i.p. with various doses of venom, diluted with phosphate-buffered saline solution, pH 7.2 (PBS), in a final injection volume of 0.5 ml . Deaths were recorded for 48 hr and lethal dose 50 (~~ was estimated by the Spearman-Karber method (Grtvé and ROBLPB, 1987). For neutralization of lethality, mixtures containing a constant amount of venom and various dilutions of antivenom were prepared, in order to have several ratios of pl antivenom/mg venom. Mixtures were incubated at 37°C for 30 min, and then groups of four mice (16-18 g) were injected i.p. with 0.5 ml of the mixtures, containing an amount of venom corresponding to a challenge dose of four so- Control mice received venom only. Deaths were recorded for 48 hr and neutralization was expressed as effective dose 50 (tm~, defined as the ratio pl antivenom/mg venom that protects 50% of the population injected (Gu'r~axez et al., 19886) . Hemorrhr~gic activity The technique of Koxno et al. (1960), as modified by Gvr~aaFZ et al. (1985), was used. Briefly, groups of four mice (20-22 g) were injected inr*A~~++, atty with various doeev of venom dissolved in 100 pl of PBS. Two hours after injection animals were sacrificed (ether inhalation), their skins removed and the hemoahagic areas measured. The minimum hemorrhaic dose (MHD) was defined as the amount of venom that induces a hemorrhaic lesion of 10 mm diameter. For neutralization studies, mixtures having a constant amount of venom and various dilutions of antivenom were prepared and incubated at 37°C for 30 min. Then, groups of four mice (20-22 g) were injected as described with 100pl of each mixture, containing a challenge dose of venom corresponding to 10 MHD. Hemorrhage was evaluated as described and neutralization was expressed as effective dose 50 (ens~, defined as the ratio of pl antivenom/mg venom that reduces hemorrhagic activity by 50%. In order to study the development of hemorrhage in muscle tissue, the technique of Owivsx et al. (1984) was used, with the modiflcations introduced by GvrdfleYR 7 et al. (1987) . Groups of four mice (20-22 g) were injected i.m . in the right thigh with 100 pg of venom, diluted in 100pl of PBS. Control mice were injected with PBS. At diffet+ent time intervals (15 and 30 min, and 1, 3, 6, 24 and 48 hr) animals were sacrificed by cervical dislocation. A sample of muscle tissue was obtained from the thigh and immediately weighed and homogenized in 3.0 ml of Drabkin solution, using a Brinkmann homogenizer PT 10/35 (Polyfron, Switzerland). Then, samples were

Newborn B. riper Venom-induced Pathology

1101

centrifuged 10 min at 9000 g and 1 .0 ml of the supernatant was added to 2.5 ml of a Drabkin solution containing l'/e Triton X-100. The absorbana at 540 nm was recorded and the amount of hemoglobin was estimated by using a cyanometahemoglobin standard (Sdwz et al., 1987). Hemorrhage in muscle was expressed as mg hemoglobin per gram of muscle wet weight . Edemo-Jorming acttvtty The method of Y~xew~ et al. (1976) wa~ used. Groups of four mice (20-22 g) were injected s.c ., in the right foot pad, with various amounts of venom dissolved in 501e1 of PBS. The left foot pad received 5014 of PBS only . Mice were sacriffced bY ceNical dislocation after 6 hr and both feet were cut and weighed. The minimum edemaforming dose (MED) was defined as the amount of venom inducing an edema of 30%. Effects of venom ore coagulation The coagulant activity of venom on citrated human plasma was studied trr vitro, according to the procedure of T~xsrox and Rstn (1983), as modified by Gwé et al. (1989) . Different doses of venom, in a volume of 1001e1 of PHS, were added to 2001x1 of plasma. Clotting times were recorded with a Fibrometer (BBL, Maryland, U.S .A .) . The minimum coagulant concentration (MCC) was defined as the amount of venom inducing coagulation of plasma in 60 sec. Defibrinating activity was studied in mice in two different ways : (a) i.v. injection of various doses of venom ~~roN and Rte, 1983; Gtwé et al., 1989); controls received PBS. Then, after 1 hr, animals were anesthetized with ether and bled by cardiac puncture . Blood was placed in tubes and incubated at 22-25°C for 2 hr . The minimum defibrinating dose (MDD) was defined as the lowest amount of venom causing inhibition of blood coagulation in all animals tested . (b) i.m, injection of 1001îg of venomin the thighof mice (20-22 g) . Then, at 30 min, and 1, 3, 6 and 24 hr, blood was collected as described, placed in tubes, and coagulation times recorded. Bloclremical clrarrges to blood Groups of four to five mice (20-22 g) were injected i.m . in the thigh with 100 pg of venom, whereas control mice received 1001x1 of PBS. At 1, 3, 6, 24 and 48 hr mice were sacrificed by ether inhalation and their thoracic c8vitiea were immediately opened . Blood samples were obtained by cutting the aorta. Hematocrit was determined in heparinized capillary tubes. Blood ATP was determined according to the Sigma Kit 366 UV . Blood was allowed to clot and serum was obtained by centrifugation . The following determinations were carried out in serum: total protein (Srecrase, 1978); creative kinase, CK (Sigma Kit 520 ; one unit results in the phosphorylation of l nmole of croatine per min at 25°C); lactate dehydrogenase, LDH (Sigma Kit 228 UV ; one unit results in the formation of l lanole of NADH per min); aspartate aminotransferase, AST (Sigma kit 58 UV ; one unit results in the formation of l bole of NAD per min); alanine aminotransferase, ALT (Sigma kit 59 UV ; one unit results in the formation of l lanok of NAD per min). In addition, serum hemoglobin wncentration was determined by the benzidine reaction (Sr(errz et al., 1987). Histological studies Groups of four mice (20-22 g) were injected i .m . in the thigh with 100 pg of venom. Controls were injected with PBS. Animals were sacrificed by ether inhalation at 15 aced 30 min, and 1, 3, 6, 24 and 48 hr, and samples of skeletal muscle, kidneys, heart, lungs and liver were obtained, fixed in a solution of Duboscq-Brasil which contains formalin, ethanol, acetic acid and picric acid (Mt~rnr, 1980), processed routinely and embedded in paraffin . Sections were stained with hematoxilin-eosin and examined under a light microscope . Statistical analysis The Student's t-test was used to determine the significance of the differences observed between the means of the two experimental groups. RESULTS

Toxic and enzymatic activities

Table 1 depicts the lethal, hemorrhagic, edema-forming, coagulant, defibrinating, indirect hemolytic and proteolytic activities of venom from newborn B. riper specimens. For comparison, the activities reported for venom from adult specimens are also included .

F. CHAVES et al .

1102

TABLE 1 . Lgrfui., EE~oRRxAatc, ®nu-t+oR~ta, coAauLANT D~RUVA~tuva, nvDmEC.-r ~oLrnc AND I7t0180LYTIC ACrIV1TIBS OF NEWBORN AND ADULT B . riper v~to~s Activities Lethal (~so Pg/g)t Edema-forming (MED)$ Coagulant (MCC~ Defibrinating (MDD)II Hemonhagic (MHD)~j Indirect hemolytic (MHD)" ' Proteolytic (MPD)tt

Newborn 1 .97 (1 .81-2.13) 0 .32 t 0 .05 S .S f 0.3 1 .0 0.95 f 0 .06 1 .40 f 0.27 0.6ßt0.06

Adult' 3.94 (3 .77 3.5 1 .63 5.0 1 .5 1 .8 1 .2

.11)

Results in newborn venom concerning edema-forming, coagulant, hemorhagic, indirect-hemolytic and proteolytic effects are presented as mean f S.E . (n = 4) . " Data for adult venom were obtained from Gur~eREZ et d. (1985, 1986) and GENE: et a1 . (1989) . j~LD (f+g/g) for mice i .p route. Values represent mean (95% fiducial limits). $MED: Minimum edema-forting dose. Amount of venom (in Rg) that induced edema of 30% . ¢MCC : Minimum coagulant concentration . Venom concentration (final ~gJml) that induced coagulation of plasma in 60 sec . IIMDD : Minimum defibrinating done . Amount of venom (in Rg) that induced incoagulability 1 hr after injection . ~jMHD: Minimum hemorhagic dose. Amount of venom (in Rg) which induced a hemorhagic spot of 10 mm in diameter . °"MHD : Minimum hemolytic dose. Amount of venom (in Wg) which induced a hemolytic halo of 15 mm diameter. ttMPD : Minimum proteolytic dose . Amount of venom (mg) that induces a change in absorbance at 280 nm of 0 .5 .

FYa. 1 . $DS-POLYACRILAI~E sEL ELECTROP}~ORFSIS (12%) OF VENOèlS OBTAINFD FROlS NEWBORN AND ADULT srECnQNS oa~ Botlvops arper . (1), (4) and (6) represent 3 .75, 2 .5 and 1 .25 Pg, respectively, of venom from newborn specimens . (2), (~ and (7) represent 3.75, 2 .5 and 1 .25 pg, respectively, of venom from adult specimens . (3) and (8) are mol . wt standards: a, 94,000 ; b, 67,000; c, 43,000; d, 30,000; e, 20,100; and f, 14,400. All samples were reduced with 2-mercaptoethanol.

Newborn B. riper Venom-induced Pathology

1103

A

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LEVELS of TIC ENZVtIrns ucrATE DEHVnROCIENASE, LDH (A); cREATINE ruvASE, CK AMINOTRA~A~, ALT (C) ; AND ASPARTATE AMINO'raANSFERASe, AST (D), AT DQrFERENT T~ INTERVALS AFTER 1 .m. INJECTION OF 100 Pg OF NEWBORN B. asptr VENOM.

FYc . 2 . SERtJId (B) ; ALANINE

Results are expressed as mean f S.E . (n = 4) . Values with asterisk ( " ) are significantly different from control values obtained from mice injected with PBS .

Venoms from newborn specimens induce higher lethal, hemorrhagic, edema-forming, defibrinating and proteolytic activities. Electrophoresis

Figure 1 shows the presence of conspicuous differences in the electrophoretic patterns of venoms from newborn and adult B. riper . Venom from newborn specimens has predominantly bands of high mol. wt with only two faint bands of mol. wt below 20,000 . In contrast, adult venom has a strong band of 16,000, having fewer bands in the high mol. wt region . Biochemical alterations in blood

A moderate increment in serum levels of creatine kinase and lactate dehydrogenase was observed, these reaching their maximum values 6 hr after venom injection (Fig . 2). On the other hand, there was only a slight increase in aspartate aminotransferase and alanine aminotransferase, these reaching their highest levels at 24 hr (Fig . 2). Table 2 shows that there was a significant reduction in hematocrit as a consequence of envenomation, with values of 27.3 t 2.0% (n = 5) at 48 hr (control hematocrit = 44.4 f 1 .6%) . Serum mx so=sue

1104

F. CHAVES et al. TABLE 2. CHANOH4 nv rorAt, seteu~t FROr~N coxcwTRAnoN, ~rwTOCRtr, seacrx HElIOOLOBIN AND HLOOD ATP AT D~ENT TI103 INTERVAIS IN lI1CH II30CIJLATED W1TH VENOlI OF 2~WHORN SFBCIIISEN3 OF B. Q.4Per

Tmu (~)

Hematocrit (%)

C

Serum hemoglobin (me/~)

$Crunl proteins (g/~)

5 .0t0 .7 27f6 " 30t7 " 30t8 " 30t3 " 46f I "

7.7f1 .1 S.8f0.5 7.3f0.2 5.7t0 .4 10.1t0.1 " 9.6t0.5

45t1 44f2 41t2

1 3 6 24 48

39t2' 32f3 " 27t2 "

ATP (ltmol/dl) 47t4 52f2 Slt2

n.d

39f2 49f3

Results are expressed as mean t S .E. (n = 4-5) . Control value (C) were obtained from groups of four mice that were not envenomated. " P < 0 .05; n .d. = not determined .

hemoglobin increased significantly, reaching its highest level by 48 hr (Table 2). On the other hand, an increment in total serum protein was observed at 24 hr (Table 2). Changes in muscle hemoglobin levels Newborn B. riper venom induced a rapid and drastic increment in muscle hemoglobin

levels (Fig. 3). Highest values were observed at 3 hr, but a significant accumulation of hemoglobin, as a consequence of hemorrhage, was observed in samples collected 15 min after injection (Fig. 3). Alterations of blood coagulation in vivo

When injected i.v., venom from newborn B. riper specimens induced a dose-dependent defibrination, rendering blood unclottable (Table 1). Mice injected i.m . with PBS had clotting times of 1 .30 f 0.24 min (n = 3). When 100 Fig of venom was injected i.m., eo. so . O

20

to 0

d ~é

24 TIARE (M)

48

Ftc . 3. INCREASE 1N HElIOOIABIN IN THIGH MUSCLE AT DII+PERSNT TIl~ INIERVAL4 AFTER 1NIEG'I70N OF I00 /!g OF NEWBORN B. riper VENOM. Results are presented as mean t S .E. (n = 4) .

i.m .

Newborn B. riper Venom-induced Pathology

1105

coagulation time was 1.34 f 0.45 min (n = 4) in samples collected after 1 hr. On the othér hand, coagulation did not occur in samples collected at 3 and 6 hr. By 24 hr, coagulation time was 1 .00 t 0.02 min (n = 4). Lethality after i.m. injection

When mice were not sacrificed during the first 24 hr after injection of 100 ~g of venom, between 15 and 25% of them died in the following 24 hr. Before death, animals were prostrated and lethargic, with no evidence of paralytic or respiratory problems . Histological changes

The most important alterations were observed in skeletal muscles, kidneys and lungs. Prominent hemorrhage was present in thigh skeletal muscle at all time periods, together with drastic alterations in the walls of arteries and veins (Fig. 4). A moderate myonecrosis developed after the first hour of envenomation, with the majority of necrotic muscle cells having a hyaline morphology . In kidneys, tubular cells were vacuolated as early as 15 min after envenomation. Many tubules contained erythrocytes and hematic casts by 1 hr. Renal lesions progressed and, by 24 hr, there were foci of cortical necrosis, as well as hyalinization and degeneration of proximal tubular cells (Fig . 4). On the other hand, lung collapse startod to develop as early as 1 hr after venom injection, being more prominent at 3 and 6 hr, when degenerative changes in alveolar cells were evident, Histological examination of liver and heart showed no degenerative, necrotic or hemorrhagc changes at any time . There was a moderate congestion of liver sinusoids during the first 24 hr after envenomation . Neutralization by antivenom

Polyvalent antivenom was effective in neutralizing lethal, hemorrhagc and indirect hemolytic activities of newborn B. riper venom (Table 3). For comparison, effective doses 50% obtained with the same antivenom against the venom of adult B. riper specimens are also shown in Table 3. It is evident that, although antivenom neutralizes venom from newborn specimens, it is more effective in the neutralization of venom from adult specimens. DISCUSSION

Venom obtained from newborn (less than 30 days old) B. riper specimens has striking differences when compared to that of adult specimens. These differences are evident not only in their electrophoretic pattern, but also in their enzymatic and toxic activities. Our results agree with previous observations of Gu~rI~xxEZ et al. (1980) in that newborn B. riper venom has higher lethal, hemorrhagc, edema-forming and proteolytic activities . Moreover, our data also indicate that venom of newborn B. riper showed higher defibrinating activity . Interestingly, venom from newborn specimens has lower myotoxic effect than that of adult specimens, as evidenced by the histological observations in muscle tissue . This finding agrees with the immunochemical observations of LoMOx~tE et al. (198 . Intramuscular injections of 100 pg of venom from newborn B. riper in mice induced a rather complex and severe pathophysiological picture, characterized not only by local

1106

F. CHAVES et al.

FYa. 4. L~axr eacaooaerx o~ (A) xouss ~nax ~~ 6 hr etrrm i.m . Ix1SCI10N OF 100 Pg o~ xt=.waosav B. riper v~wu exn o~ (B) sanxsr n~vs 48 hr eNtEa i .m . nvJecnox as+ 100 Kg of ro waoRx B. toper vawoM. Notice abundant hemorrhage in the muscle . Some muscle fibers show evidence of degeneration and necrosis . Notice in the kidney degeneration and hyalinization of proximal tubular cells . Bar represents 200 um in kidney and 100 Ean in muscle .

Newborn B. after Venom-induced Pathology

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TABLE 3. Coe~ARATIVE r~rRALIZATION of LETHAL, FIHèIORBHAGIC AND INDIRECT HEMOLYTIC ACTIVITIES OF NEWBORN AND ADULT B, wiper VENOMS BY A POLYVALENT AN7iVENOM

Activities Lethal Hemorrhagic Indirect hemolytic

Neutrali7stion (EDSO)" Newborn Adult"' 746 1500 5000

333 135 770

'NeutraliTation is expressed as effective dose 50 (©~, defined as the ratio of Wl of antivenom/ mg venom that reduced the activity of the venom by 50%. "'Data of adult venom were obtained from BoLAI~os and CbenAS (1980) and Gur~RRPa et at. (1988a, b) .

effects, but also by prominent systemic alterations leading to death in some mice. This contrasts with the effects previously described resulting from the i.m . injection of 100 ~g of adult B. riper venom (CI-IAVES et al., 1989). In the latter case there was a moderate envenomation, characterized by prominent local and mild systemic effects. Thus, i.m . injection of 100 pg of newborn B. riper venom in mice might be a useful experimental model of a severe envenomation with systemic alterations including hemorrhagc, hemolytic, coagulant and nephrotoxic effects. Local tissue damage induced by newborn B. riper venom differs from that induced by adult venom in several ways : (1) hemorrhage is more extensive after injection of newborn venom, in agreement with its lower minimum hemorrhagc dose; (2) newborn venom induces more drastic effects on arteries and veins, as well as a more prominent edema; and (3) adult venom has a stronger myotoxic action than newborn venoms . This was observed histologically, as well as by the higher increments in muscle-derived enzymes in serum (Gt1TIf~1tEZ et al., 1980; CHAVFS et al., 1989), and is probably due to the higher concentration of myotozins in adult venom (LOMONTE et al., 1987). These myotoxins are molecules with phospholipase Az structure that induce acute muscle damage by first affecting the integrity of muscle plasma membrane (Gu~rl>~xez et al., 1984a,b, 1986 : GuTdRxEZ and LOMONTE, 1989). In contrast, myonecrosis observed after injection of newborn venom is probably a consequence of the ischemia which ensues in skeletal muscle after the drastic vasculotoxic effect exerted by the venom. This is supported by the morphology of muscle fibers and by the fact that increments in muscle hemoglobin preceded muscle necrosis . Renal alterations were prominent and could be due to the combination of the following elements : (1) renal ischemia due to diminished renal perfusion, as a consequence of hemorrhage and hypovolemia; (2) intravascular coagulation, a factor that has been known to contribute to the pathogenesis of acute renal failure after snakebites ($ITPRUA and BOONPUCKNAVIG, 1979); and (3) direct action of venom components on tubular cells, as has been shown in the case of Vipers russelli venom (Sol: er al., 1990). The drop in hematocrit may be a consequence of extensive blood loss . In addition, intravascular hemolysis might contribute to changes in hematocrit . The cause of this hemolysis is not clear, since the venom does not have a direct hemolytic effect (results not shown) . It is likely that intravascular coagulation, in combination with vessel wall

1108

F . CHAVES et d.

alterations induced by hemorrhagic toxins, constitute mechanical factors that affect erythrocyte membrane stability (CONDREA, 1979). More studies are required to test this hypothesis . Blood incoagulability occurs in mice injected with newborn and adult B. riper venoms (CHAVES et al., 1989 ; this work). This effect is related to a defibrination secondary to the coagulant activity of the venoms (BARRANTES et al., 1985 ; GEIV~ et al., 1989). Clinically, prolongation of prothrombin time has been described in envenomations by adult specimens (PENA-CHwvARRfA et al., 1970; BARRANTES et al., 1985). Clinical observations also indicate that bites by newborn specimens induce a similar effect (Ko1tNALfrc and Volu.ovx, 1990; our unpublished observations). Thus, routine determinations of prothrombin or coagulation times might also be useful in order to monitor the evolution of envenomations induced by newborn B. riper bites. In this regard, observations carried out in B.jararaca bites indicate that human envenomations induced by newborn specimens present incoagulability more frequently than similar envenomations caused by bites of adult specimens (RIBSIRO and JORGE, 1989). Polyvalent antivenom is used in Central America for the treatment of envenomations induced by Crotaline snakes (BoLAfitas and CERDAS, 1980; Boi.Afiios, 1982). Since venoms used to produce this antivenom are from adult specimens, it was important to test the antivenom for its ability to neutralize newborn B. riper venom. Our results indicate that antivenom was effective, although higher antivenom/venom ratios were required to achieve neutralization . Acknowledgements - The authors thank Joxc~ SexweAte, Jev~t NuAez, Arm, ROat~s, Metuo S~(rtct~z and

MwwA nn. Cea~r Osermo for their valuable support in the laboratory work, as well as Dr BRUxo Lo~oxm for critically reading the manuscxipt . This study was supported by Viocrroctoria de Investigaci6n, Universidad de Costa Rica, project 741-85-008 . F. Cxevt~ and J . M . Gvrtl axFZ are recipients of a research career award from the Costa Rican National Scientific and Technological Research Council (CONICIT). Thin work was done in partial fulfilment of the requirements for the M .Sc . Degree for FBttvermo Cnev~ at the Univerridad de Costa Rica .

REFERENCES BexaeN~rfs, A ., Sor3s, V. and HotwRtaa, R. (1985) Alteraci6n de loa mecanismos de coagulacibn en et envenenamieato par Botkropa riper (terciopelo) . Toxicon 23, 39907. HoLeAos, R. (1982) Las aerpientea venenosas de Centroamérica y et problems del ofidismo . Primera parte: aspectoe zool6gicos, epidemiol6gicos y biomédicos. Rev. Coat . Gentian. AfEd. 3, 165-184. Hor.eAos, R . and Ones, L. (1980) Produoci8n y control de sueras andolidicoa en Conta Rica . BolerLr. Of.

Sanft. Parrain. g8, 189-196.

Gtwv~, F., Gtrr~ereEZ, J. M ., LoMOrrre, B . and C©enes, L . (1989) Histopatological and biochemical alterations induced by intramuacular injection of BotJrropa saper (terciopelo) venom in mice. Toxicon 27, 1085-1093. CorvnRrw, E. (1979) Hemolytic effects of snake venoms. In : Snake Yenonrr, pp. 448-479 (L .~, C. Y., Ed .) . Berlin : Springer . Gm~, J. A . and ROa[PS, A . (1987) Det~+++++nA~ 6n de la doris étal 50'/o par et método de Speatmsn Karber .

Reviata MEdica Hospitd National de Nirioa Catta Rica 1, 35-40.

Gt?r~, J . A ., Rov, A., Roes, G., Gvr>i~raz, J . M . and genes, L . (1989) Comparative study on coagulant, defibrinating, fibrinolytic and fibrinogtnolytic activities of Costa Rican crotaline snake venoms and their neutraliTation by a polyvalent antivenom . Toxlrnn 27, 841-848. Gvrréaresz, J . M. and LoatoxtE, B. (1989) Local tissue damage induced by Bothropa snake venoms . A review .

Mem. Inat. Brrtantan Sl, 211-223.

Gvr>~et?z, J . M ., ARROYO, O. and HOLei~os, R. (1980a) Mionecrosis, hemorragia y edema inducidos par et veneno de Bothropa saper en rat6n blanoo. Toxicon 218, 60310. Grm~teRaz, J . M ., C~uv>$, F. and Bor efios, R . (19808) Eatudio comparativo de venenos de ejemplarea tecién nacidos y adultos de Botlrropa saper. Revtata de Biologic Tropitd 28, 341-351.

Newborn B. riper Venom-induced Pathôlogy

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Pathological and biochemical changes induced in mice after intramuscular injection of venom from newborn specimens of the snake Bothrops asper (Terciopelo).

Venom from newborn Bothrops asper snakes has higher lethal, hemorrhagic, edema-forming, proteolytic and defibrinating activities than venom from adult...
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