Toxicon Vol. 36, No. 11, pp.

1529±1538, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0041-0101(98)00145-7 0041-0101/98 $19.00 + 0.00

NEUTRALIZATION OF LOCAL TISSUE DAMAGE

INDUCED BY BOTHROPS ASPER (TERCIOPELO)
SNAKE VENOM

JOSEÂ MARIÂA GUTIEÂRREZ,* GUILLERMO LEOÂN,

GUSTAVO ROJAS, BRUNO LOMONTE,
ALEXANDRA RUCAVADO and FERNANDO CHAVES
Instituto Clodomiro Picado, Facultad de MicrobiologõÂ a, Universidad de Costa Rica, San JoseÂ,
Costa Rica

(Received 9 December 1997; accepted 19 January 1998)

J. M. GutieÂrrez, G. LeoÂn, G. Rojas, B. Lomonte, A. Rucavado and F.

Chaves. Neutralization of local tissue damage induced by Bothrops asper
(terciopelo) snake venom. Toxicon 36, 1529±1538, 1998.ÐLocal tissue
damage represents a serious consequence of Bothrops asper envenomations.
It encompasses a complex series of alterations, including myonecrosis, der-
monecrosis, hemorrhage and edema. Due to its rapid development it is di-
cult to neutralize by antivenoms, especially if there is a delay in serotherapy.
Experimental studies with this venom and the polyvalent (Crotalinae) antive-
nom produced in Costa Rica indicate that antivenom is e€ective in neutraliz-
ing these toxic activities when incubated with the venom prior to injection.
However, if venom and antivenom are injected independently in mice, neu-
tralization of these e€ects is only partial. Moreover, neutralization is not
complete even if homologous or heterologous antibodies are present in the
circulation before venom is injected. Despite di€erences in their pharmacoki-
netic pro®les, equine whole IgG and F(ab')2 antivenoms show similar ecacy
in the neutralization of edema, hemorrhage and myonecrosis induced by B.
asper venom, suggesting that the use of antivenoms made of antibody frag-
ments may not improve neutralization of these e€ects. This is due, at least in
part, to the fact that microvessel disruption by venom components favors a
similar antibody concentration in the a€ected tissues. Recent advances in the
development of neutralizing substances of rapid di€usion, that could be
injected locally in the ®eld, may contribute to the neutralization of metallo-
proteinases and phospholipases A2. In addition, the rapid administration of
antivenoms with high antibody titers against locally-acting toxins is very im-
portant in the treatment of these e€ects. # 1998 Elsevier Science Ltd. All
rights reserved

* Author to whom correspondence should be addressed.

1529
1530 J. M. GUTIERREZ et al.

INTRODUCTION

Local tissue damage, i.e. hemorrhage, myonecrosis, dermonecrosis and edema, constitu-
tes one of the most common and serious consequences of crotaline snake envenomation
(Fan and Cardoso, 1995; GutieÂrrez, 1995; Warrell, 1996). The pathogenesis of these
alterations is rather complex, as they are induced by di€erent toxins present in the
venoms, such as myotoxins (Mebs and Ownby, 1990; GutieÂrrez and Lomonte, 1997),
hemorrhagic metalloproteinases (Ownby, 1982; Bjarnason and Fox, 1994), and various
edema-inducing components (Bjarnason et al., 1983; Teng et al., 1989; Lomonte and
GutieÂrrez, 1989). In addition, local tissue damage can be complicated by bacterial infec-
tion and the development of compartmental syndrome (Gar®n et al., 1985; Cardoso et
al., 1993; Warrell, 1996).
Neutralization of these e€ects by antivenoms is dicult to achieve, not only due to
the variety of components inducing these alterations, but also to the rapid onset of
these pathological changes. Thus, the delay in antivenom administration that occurs in
many rural areas of tropical countries hampers the success of serotherapy. Moreover,
some aspects of local tissue damage are caused by venom-induced release of endogenous
mediators, such as eicosanoids, vasoactive amines, kinins and cytokines (Rothschild and
Rothschild, 1979; Moura da Silva et al., 1996). It has been stated that the evaluation of
antivenoms should include not only the neutralization of lethality, but also of other rel-
evant pathophysiological e€ects, such as those characteristic of local tissue damage
(WHO, 1981; GutieÂrrez et al., 1990; Theakston et al., 1995). Several studies have been
performed in our laboratory in order to investigate the ability of the polyvalent
(Crotalinae) antivenom produced in Costa Rica to neutralize local e€ects induced by
the venom of Bothrops asper, the most important poisonous snake in Central America.
These results and their implications are discussed in this contribution.

ASSAYS WITH PREINCUBATION AND ASSAYS WITH INDEPENDENT INJECTION OF VENOM

AND ANTIVENOM

A basic methodology to approach the study of neutralization of pharmacological ac-

tivity of venoms by antivenoms was adapted in our laboratory (see GutieÂrrez et al.,
1990 and references therein). It includes two basic types of test: (a) assays with preincu-
bation of venom and antivenom, and (b) assays with independent injection of venom
and antivenom. In the ®rst type of assay, a ``challenge dose'' of venom is selected for
each e€ect to be evaluated, on the basis of dose±response studies. Then, a constant
amount of venom is mixed with various dilutions of antivenom, in a constant ®nal
volume, and incubated for 30 min at 378C. Controls include venom alone, physiologic
saline solution alone and antivenom alone. After incubation, aliquots of the mixtures,
containing the ``challenge dose'' of venom, are tested in the corresponding assay system.
The neutralizing ability of antivenom is expressed as e€ective dose 50%, de®ned as the
ratio of ml antivenom/mg venom in which the e€ect of venom alone is neutralized by
50%. On the other hand, assays with independent injection of venom and antivenom
attempt to reproduce the natural course of envenomation. In these assays, the ``chal-
lenge dose'' of venom is injected ®rst and then, at various time intervals, antivenom is
administered intravenously.
Several test systems are used for the study of local e€ects. Hemorrhagic activity is
quanti®ed by the skin test (Kondo et al., 1960; GutieÂrrez et al., 1985) and by the spec-
trophotometric determination of hemoglobin in muscle (Ownby et al., 1984; GutieÂrrez
 Neutralization of Tissue Damage Induced by Bothrops Venom 1531

et al., 1987). Edema-forming activity is evaluated in the rodent foot pad model either
plethysmographically (Trebien and Calixto, 1989; Chaves et al., 1995) or by determining
the increments in weight (Yamakawa et al., 1976; GutieÂrrez et al., 1986) or in thickness
(Lomonte et al., 1993a; Rucavado and Lomonte, 1996). Myonecrosis is quanti®ed either
histologically (Ownby et al., 1982; Preston et al., 1990; Lomonte et al., 1993b) or by
measuring the creatine kinase activity in plasma (GutieÂrrez et al., 1981) or in tissue
homogenates (GutieÂrrez et al., 1987). Local necrotic activity is studied by a skin test in
which necrotic lesions are observed 72 h after venom injection (Theakston and Reid,
1983).
When these tests were applied to study the ability of polyvalent antivenom to neutral-
ize local e€ects induced by B. asper venom, a consistent picture emerged: antivenom
was e€ective in experiments where venom and antivenom were incubated before injec-
tion, but neutralization was only partial when antivenom was administered after enve-
nomation (Fig. 1 and 2) (GutieÂrrez et al., 1981, 1985). Similar results were obtained
when testing the neutralization of other Central American crotaline snake venoms
(GutieÂrrez et al., 1985, 1986, 1987; Rojas et al., 1987). Thus, the polyvalent antivenom
produced in Costa Rica contains antibodies capable of neutralizing locally-acting toxins
if incubated prior to injection. This conclusion has been strengthened by the determi-
nation of antibody titres, by ELISA, against myotoxins present in Bothrops venoms
(Lomonte et al., 1991). However, neutralization is not achieved if antivenom is injected
after envenomation, probably due to the extremely rapid development of local tissue
damage. This hypothesis is supported by the studies on the pathogenesis of hemorrhage
(GutieÂrrez et al., 1984; Moreira et al., 1992), myonecrosis (GutieÂrrez et al., 1984) and
edema (GutieÂrrez et al., 1980; Chaves et al., 1995) induced by B. asper venom in mice.
Moreover, this has been corroborated in intravital microscopy studies after application

Fig. 1. Neutralization of hemorrhagic (Q), myotoxic (R) and edema-forming (.) activities of
Bothrops asper venom by polyvalent (Crotalinae) antivenom in assays in which venom and anti-
venom are incubated prior to injection. For each e€ect, a constant amount of venom was incu-
bated with various dilutions of antivenom for 30 min at 378C. Then, aliquots of the mixtures
were injected in mice and the e€ects evaluated by standard laboratory assays. Results are
expressed as percentage of activity, taking as 100% the activity of samples in which venom was
incubated with saline solution instead of antivenom.
1532 J. M. GUTIERREZ et al.

Fig. 2. Neutralization of hemorrhagic (q), myotoxic (diagonally lined) and edema-forming (hori-
zontally lined) activities of Bothrops asper venom by the polyvalent (Crotalinae) antivenom in
assays in which venom and antivenom are injected independently in mice. In these experiments,
venom was injected intradermally, intramuscularly or subcutaneously, depending on the e€ect to
be studied, and then antivenom was administered intravenously, at di€erent time intervals (im-
mediately, 15 and 30 min after envenomation). Results are expressed as percentage of activity,
taking as 100% the e€ect induced by venom alone, i.e. in mice injected with venom and not
receiving antivenom.

of venom on the mouse cremaster muscle (Lomonte et al., 1994a). In the three cases the
e€ects were prominent very soon after venom injection.
These methods have been also utilized in the evaluation of other antivenoms distribu-
ted in Central America. In general, antivenoms have high antibody titers against hemor-
rhagic toxins, as shown by the ecient neutralization in preincubation-type assays
(Bogarõ n et al., 1995), although they are partially ine€ective in neutralizing hemorrhage
induced by B. asper venom in assays with independent injection of venom and antive-
nom. On the other hand, drastic di€erences in antibody titers against myotoxins were
observed in various commercial antivenoms, suggesting that some of them might be
ine€ective in the neutralization of B. asper-induced local myotoxicity (Lomonte et al.,
1991; Bogarõ n et al., 1995). Such disparity in antivenom ecacy is probably based on
the antigenic variability of venom components between species and, sometimes, between
populations of the same species (Warrell, 1997). Thus, since antivenoms are produced
using di€erent immunizing mixtures of venoms, an antivenom e€ective against a venom
in a particular region might not be e€ective against a di€erent venom in another
country or region, as has been demonstrated in many instances (Theakston et al., 1995;
Theakston, 1996). These observations stress the relevance of evaluating the neutralizing
ecacy of antivenoms against the most important venoms of each country or region.

NEUTRALIZATION OF LOCALLY-ACTING TOXINS WHEN ANTIBODIES ARE INJECTED

BEFORE ENVENOMATION

Since experiments in which antivenom was administered after envenomation demon-

strated only partial neutralization of local e€ects, a study was performed to determine
the success of neutralization if antibodies are present in experimental animals before
 Neutralization of Tissue Damage Induced by Bothrops Venom 1533

venom injection (Rucavado and Lomonte, 1996). This was investigated in two exper-
imental ways: (a) mice immunized with B. asper venom and then challenged with
venom, and (b) mice injected intravenously with antivenom and then receiving venom.
In both cases it was demonstrated that the antibodies present in blood would neutralize
the venom injected in preincubation-type experiments.
When actively-immunized mice were challenged with B. asper venom, local tissue
damage was signi®cantly, but not totally, reduced. Acute edema, i.e. edema at 1 h, was
not inhibited, but at subsequent time intervals it was signi®cantly neutralized when com-
pared with mice injected with venom alone. Hemorrhage was reduced by about 70%
and myonecrosis by 80% (Rucavado and Lomonte, 1996). On the other hand, hemor-
rhage was almost completely neutralized in animals passively immunized with heter-
ologous antibodies (equine polyvalent antivenom) either 5 or 120 min before
envenomation. Edema was not neutralized at the ®rst time intervals evaluated, but
decreased signi®cantly when compared to mice injected with venom and not pretreated
with antivenom, at later time intervals. Myotoxicity was neutralized by about 65% in
passively immunized mice, with no signi®cant di€erence if antivenom was administered
either 5 or 120 min before envenomation.
These results clearly show that, although a signi®cant reduction in local tissue
damage is achieved when antibodies are present in the bloodstream before venom injec-
tion, such neutralization is not complete, and hemorrhage, edema and myonecrosis
develop to some extent. It is likely that the antibody concentration in the tissues by the
time venom is injected is insucient to achieve neutralization of locally-acting toxins.
These results are important not only to understand the basis of antibody neutralization
of locally-acting toxins, but also to simulate the e€ect of active immunization with
venoms as a future strategy to confront the problem of local tissue damage.

ARE F(ab')2 FRAGMENTS MORE EFFECTIVE THAN WHOLE IgE MOLECULES IN THE
NEUTRALIZATION OF LOCAL EFFECTS?

Neutralization of locally-acting toxins may depend on the concentration of antibodies

in the tissues at the time venom is injected. Based on this assumption, a study was per-
formed to test the hypothesis that an antivenom made of F(ab')2 fragments would be
more e€ective than a whole IgG antivenom in the neutralization of myonecrosis, hemor-
rhage and edema induced by B. asper venom. This hypothesis is supported by pharma-
cokinetic studies which demonstrate that, due to their lower molecular weight, F(ab')2
fragments have a larger volume of distribution and reach the tissues more rapidly than
whole IgG antibodies (Ismail and Abd-Elsalam, 1996).
Both types of antivenoms were prepared from the same batch of hyperimmune equine
plasma, and it was assured that they had the same neutralizing potency in experiments
with preincubation of venom and antivenom (LeoÂn et al., 1997). Therefore, any di€er-
ence in neutralization would depend on the di€erent pharmacokinetic pro®le of the pro-
ducts, and not on variations in antibody titers against locally-acting toxins. This is
particularly relevant since many studies have compared the neutralizing ability of anti-
venoms having di€erent potencies. In these cases it is not possible to conclude with cer-
tainty if the di€erences observed are due to the varying potencies or to the
pharmacokinetic characteristics of the antivenoms. In the study performed by LeoÂn et
al. (1997), B. asper venom was injected locally and a high dose of antivenom was admi-
nistered intravenously at 0, 15 and 30 min after envenomation. In some experiments,
antivenom was injected by other routes (intramuscular and intraperitoneal).
1534 J. M. GUTIERREZ et al.

No signi®cant di€erences were observed in neutralization between these two types of

antivenom (Fig. 3), thus indicating that administration of antibody fragments of lower
molecular weight than whole IgG molecules does not improve neutralization of B.
asper-induced local e€ects. In agreement with previous works cited above, neutralization
of local tissue damage was only partial even if antivenom was administered immediately
after envenomation. Myonecrosis was reduced by 80%, whereas hemorrhage and edema
were neutralized only by 25% and 30%, respectively (LeoÂn et al., 1997). Antivenoms
were even less e€ective if the time lapse between envenomation and serotherapy
increased. Although these results cannot be simplistically extrapolated to other venom
and antivenom systems, Lomonte et al. (1996) obtained similar results when comparing
neutralization by F(ab')2 and Fab fragment antivenoms of hemorrhage induced by
Vipera berus venom. The ecacy of an Fab antivenom to neutralize local e€ects
induced by B. asper venom is currently under investigation.
Several hypotheses can be proposed to explain these ®ndings:
(1) It might be that, despite a higher concentration of F(ab')2 fragments in the tissues,
their amount is still too low to e€ectively neutralize edema-forming, hemorrhagic and
myotoxic venom components. Moreover, since these e€ects develop so fast after venom
injection, this would further hamper the possibility of neutralization.
(2) Alternatively, it might be that B. asper venom-induced microvessel damage, i.e.
edema and hemorrhage, induces a similar extravasation of antibodies, independently of
their molecular weight and of their normal pharmacokinetic behavior. This hypothesis
has been recently demonstrated in our laboratory (unpublished results), since antibody
concentration increased signi®cantly in envenomated muscle tissue, as compared with
muscle tissue of non-envenomated mice injected with antivenoms. Moreover, a similar
concentration of IgG and F(ab')2 was observed in the tissue. Thus, by disrupting the
integrity of the microvasculature, B. asper venom promotes a prominent, and similar,

Fig. 3. Neutralization of hemorrhagic and myotoxic activities of Bothrops asper venom by poly-
valent antivenoms made of either whole IgG molecules or F(ab')2 fragments. Mice were injected
with venom and then, at various time intervals (immediately, 15 and 30 min after envenomation),
a constant volume of antivenom was administered intravenously. Results are expressed as per-
centage of activity, taking as 100% the e€ect induced by venom alone, i.e. in mice injected with
venom and not receiving antivenom. Neutralization of hemorrhage: IgG antivenom (.); F(ab')2
antivenom (T). Neutralization of myotoxicity: IgG antivenom (Q); F(ab')2 antivenom (R).
 Neutralization of Tissue Damage Induced by Bothrops Venom 1535

extravasation of both IgG and F(ab')2 antibodies. These observations may be also rel-
evant for other hemorrhagic venoms and corroborate that antibody pharmacokinetics is
di€erent in normal and in envenomated animals, thus stressing the need of comparative
pharmacokinetic studies in both situations.
(3) Despite the demonstration of increased extravasation of antibodies in venom-
damaged tissues, another possibility to interpret these results would be that neutraliz-
ation of toxin occurs in the vascular compartment, and that formation of toxin±anti-
body complexes causes a redistribution of toxins from tissues to blood, where they are
neutralized. Such hypothesis is supported by pharmacokinetic studies showing an
increase in total venom concentration in blood after intravenous administration of anti-
venom (Choumet et al., 1996; Riviere et al., 1997). Thus, it is important to maintain
high antibody concentrations in blood during prolonged periods of time in order to pro-
mote a complete redistribution of venom components to the circulation. Such neutraliz-
ation mechanism would not be completely e€ective in neutralizing locally-acting toxins,
since they would have already caused tissue damage by the time of redistribution.

IS THERE A SOLUTION? FUTURE TRENDS IN THE NEUTRALIZATION OF VENOM-INDUCED

LOCAL EFFECTS

Results obtained so far concerning neutralization of local e€ects induced by B. asper

venom are discouraging. However, it is expected that the knowledge being gained on
the biochemical nature, immunochemistry and mechanism of action of hemorrhagic,
myotoxic and edema-forming toxins will be of value in the search for novel avenues to
confront this problem. It is necessary to search for novel neutralizing agents. For
instance, several monoclonal antibodies were produced against B. asper myotoxin I
(Lomonte and Kahan, 1988). In preincubation-type experiments, two of these antibodies
completely neutralized local myotoxicity induced by crude venom and myotoxin I, and
one of them neutralized myotoxin II, a lys-49 phospholipase A2 present in this venom
(Lomonte et al., 1992). However, preliminary studies using independent injection of
venom and antibodies revealed the same partial neutralization described for polyclonal
equine antibodies (Lomonte, unpublished results).
A variety of natural and synthetic molecules that inhibit metalloproteinases and phos-
pholipases A2 have been characterized (see for example Xue et al. (1996) and Mihelich
et al. (1997)) and some of them are being used in the treatment of in¯ammatory diseases
and other pathologies associated with these endogenous enzymes. Based on the simi-
larities described between some venom enzymes and these endogenous metalloprotei-
nases and phospholipases A2, it is likely that some of these inhibitors may be e€ective
in the neutralization of venom components. For instance, in the case of B. asper venom,
a recent study demonstrated that a novel neutralizing mixture, composed by CaNa2
EDTA, polyvalent antivenom and an inhibitor of hemorrhagic toxins isolated from the
blood of B. asper was quite e€ective in the neutralization of hemorrhage induced by
this venom, even when the mixture was administered after envenomation (Borkow et
al., 1997). CaNa2 EDTA is being used successfully in reducing the extent of local tissue
damage in horses immunized with venoms for the production of polyvalent antivenom
in Costa Rica (LeoÂn et al., 1998).
Moreover, heparin binds to B. asper myotoxin II in a region located near the C-ter-
minus of the molecule, neutralizing its myotoxicity (Lomonte et al., 1994b,c). Similar
observations have been performed by Melo et al. (1993) with heparin and B. jararacussu
venom. Nevertheless, when these encouraging results obtained in preincubation-type
1536 J. M. GUTIERREZ et al.

experiments were performed in experiments with independent injection of venom and

heparin, neutralization was poor (Lomonte, unpublished results). More recently, Lizano
et al. (1997) characterized a plasma protein from B. asper which is highly e€ective, in
preincubation-type experiments, in neutralizing myotoxicity induced by the four myo-
toxins isolated from B. asper venom. This natural antidote is currently being evaluated
in independent injection tests. Plant extracts constitute another rich source of substances
of potential use in venom neutralization (Mors et al., 1989; Melo et al., 1994). Thus, the
search for natural and synthetic inhibitors of high di€usibility and devoid of toxicity
that could be applied locally at the site of venom injection is a relevant area of
research.
Finally, it is necessary to remark that, despite the partial ine€ectiveness of antivenoms
to neutralize B. asper venom-induced local e€ects, it is very important to test the ability
of commercial antivenoms to neutralize myonecrosis, hemorrhage, dermonecrosis and
edema in experiments with preincubation of venom and antivenom. This would assure
that antivenoms with high antibody titers are being used therapeutically. If these pro-
ducts are administered in the clinics rapidly after envenomation, some degree of neutral-
ization would be achieved. Antivenom producers should continue their e€orts to
improve the antibody titers against locally-acting toxins, and quality control labora-
tories must guarantee that antivenoms being used in the clinics have high titers against
these toxins. The combination of rapid administration of antivenoms having high anti-
body titers, together with the local injection of e€ective toxin inhibitors, may greatly
improve our ability to cope with this relevant medical problem.

Acknowledgements ÐThe authors thank the sta€ of Instituto Clodomiro Picado for their permanent support in
this research throughout these years. These studies have been supported by VicerrectorõÂ a de InvestigacioÂn,
Universidad de Costa Rica (project 741-89-057) and by the International Foundation for Science (projects F/
0883-4 and F/1388-3F).

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