Contribution of mast cells and snake venom metalloproteinases to
the hyperalgesia induced by Bothrops jararaca venom in rats
Andre ´ Gustavo C. Bonavitaa, Aline S. da Costaa, Ana Lucia A. Piresa,
Ana G.C. Neves-Ferreirab, Jonas Peralesb, Renato S.B. Cordeiroa,
Marco A. Martinsa, Patrı ´cia M.R. e Silvaa,*
aLaboratory of Inflammation, Department of Physiology and Pharmacodynamics, Oswaldo Cruz Institute, Oswaldo Cruz Foundation,
Av. Brasil, CEP 21040-900, 4365 Rio de Janeiro, Brazil
bLaboratory of Toxinology, Department of Physiology and Pharmacodynamics, Oswaldo Cruz Institute, Oswaldo Cruz Foundation,
Av. Brasil, CEP 21040-900, 4365 Rio de Janeiro, Brazil
Received 27 December 2005; revised 24 February 2006; accepted 24 February 2006
Available online 24 March 2006
Bothrops jararaca venom (Bjv) is known to induce local inflammation and severe pain. Since, mast cells are able to secrete
mediators involved in algesic processes, in this study we examined the putative role of these cells in the hyperalgesia triggered
by Bjv in the rat paw. We noted that treatment with mast cell stabilizer sodium cromoglicate as well as with histamine and 5-
hydroxytriptamine receptor antagonists meclizine and methysergide, respectively, inhibited the Bjv-induced hyperalgesia. In
addition, we showed that stimulation of isolated rat peritoneal mast cells with Bjv in vitro resulted in the release of stored and
neo-generated inflammatory mediators such as histamine and leukotriene C4, respectively. Bjv-induced histamine secretion was
clearly sensitive to treatment with sodium cromoglicate and sodium nedocromil. We further observed that metalloproteinase
inhibitors 1,10-phenantroline and DM43 inhibited mast cell degranulation in vitro, under conditions where inhibitors of
phospholipase A2as well as of serine- and cysteine-proteinases were inactive. Altogether, our findings indicate that mast cells
seem to contribute to the hyperalgesia caused by Bjv in the rat paw, and also provide evidence that this response might be
dependent on the ability of the Bjv to activate directly mast cells.
q 2006 Elsevier Ltd. All rights reserved.
Keywords: Bothrops jararaca venom; Hyperalgesia; Mast cells; Metalloproteinase
The large majority of ophidic accidents in latin America
are caused by species from Bothrops genus and, in Brazil,
Bothrops jararaca (Bj) is responsible for most of the
envenomation cases (Ministe ´rio do Brasil, 2001). Viper
snake venoms are known to produce local effects in humans
and animals characterized by haemorrhage, necrosis,
edema, leucocyte infiltration and intense pain (Gutie ´rrez
and Lomonte, 1989; Trebien and Calixto, 1989). The
pathogenesis of Bothrops envenomation is complex,
involving the combined action of serine-proteinases,
metalloproteinases and phospholipases from the venom
(Rothschild and Rothschild, 1979; Gutie ´rrez and Lomonte,
1989), as well as the release of various chemical mediators
originated either from plasma or from inflammatory cells
(Chacur et al., 2002; Moura-da-Silva et al., 1996; Teixeira
et al., 1994; Trebien and Calixto, 1989). Some pieces of
evidence indicate that macrophages can participate in the
Toxicon 47 (2006) 885–893
0041-0101/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
*Corresponding author. Tel.: C55 21 2598 4392x227; fax: C55
21 2590 9490.
E-mail address: firstname.lastname@example.org (P.M.R. e Silva).
inflammatory response caused by Bj venom (Bjv), by a
mechanism at least partially dependent on the activity of the
snake venom metalloproteinase (SVMP) jararhagin (Clissa
et al., 2001; Costa et al., 2002). Despite these observations,
since the profile of cells involved in the triggering of Bj
envenomation is not entirely understood, additional studies
are still necessary in order to clarify this point.
Mast cells are considered as important effector cells in
acquired immunity, but they can also represent a central
component of innate host defense against several injuring
stimuli (Mekori and Metcalfe, 2000). The location of mast
cells in large numbers at sites that are exposed to the
external environment and their close association with blood
vessels, allows them to have a crucial sentinel role as host
defenders. Upon activation, they often rapidly secrete a
wide range of preformed and neo-synthetised inflammatory
mediators (Metcalfe et al., 1997). Thus, the present study
was undertaken to investigate the potential role of mast cells
in the nociceptive response caused by Bjv in the rat paw.
The effect of the venom on mast cells in vitro was also
assessed. Our results support the idea that mast cells seems
to contribute to the Bjv-induced plantar hyperalgesia in rats
and demonstrate, for the first time, the ability of Bjv to
stimulate mast cells directly. The mechanism responsible
for Bjv-induced mast cell activation seems to be, at least
partially, associated with the metalloproteinase component
of the venom.
2. Material and methods
Wistar rats (200–250 g) of both sexes were obtained
from the Oswaldo Cruz Foundation Breeding (Rio de
Janeiro, Brazil) and used in accordance with the guidelines
of the Committee on Use of Laboratory Animals of the
Oswaldo Cruz Foundation (license 0085-02).
2.2. Venoms and reagents
Lyophilized crude B. jararaca venom (Bjv) was
obtained from Instituto Butantan (Sa ˜o Paulo, Brazil).
Jararhagin, jararhagin C and the SVMP inhibitor
DM43 were purified as described by Neves-Ferreira et al.
(2002). Sodium heparin was purchased from Roche (Sa ˜o
Paulo, Brazil). Sodium cromoglycate, meclizine, methyser-
gide, bovine serum albumin, o-phtaldialdehyde (OPT),
Hank’s balanced salt solution (HBSS), substance P,
compound 48/80, (4-amidino-phenyl)-methane-sulfonyl
fluoride (APMSF), L-trans-epoxysuccinyl-leucylamide-(4-
guanido)-butane (E-64), 1,10-phenantroline, p-bromophe-
nacyl bromide (p-BPB) and Percoll were obtained from
Sigma Chemical Co (St Louis, MO). All solutions were
freshly immediately prepared before use.
2.3. Thermal hyperalgesia
Thermal hyperalgesia was evaluated as previously
described (Lavich et al., 2005). After intraplantar injection
of Bjv (2.5–30 mg/paw; 100 mL), and isotonic saline
(100 mL) into left and right hind paws, respectively, the
animals were placed individually on a hot plate with the
temperature adjusted to 51 8C (Ugo Basile, Varese, Italy).
The withdrawal response latency of each hind paw was
determined at 30, 60, 120, 180 and 240 min after Bjv
challenge. The heat source was maintained at constant
intensity, which produced a stable withdrawal latency of
approximately 8–10 s in vehicle-challenged paws. Hyper-
algesia to heat was defined as a decrease in withdrawal
latency and calculated as follows: D paw withdrawal latency
(s)Zright paw withdrawal latencyKleft paw withdrawal
2.4. Mast cell purification and stimulation
Mast cells were recovered from the peritoneal cavity of
naive rats and purified by means of isoosmotic Percoll
gradient as previously described (de Oliveira Barreto et al.,
2003). After peritoneal wash with heparinized (10 U/mL)
HBSS calcium and magnesium free (HBSSK), the cells
were centrifuged at 150!g for 10 min, the supernatant was
discarded and the pellet ressuspended in HBSSKcontaining
0.1% bovine serum albumin (BSA). Cell suspension was
mixed with Percoll (90%) and overlaid with 1 mL of
HBSSKto be further centrifuged at 150!g for 25 min.
Pelleted mast cells were washed twice with HBSSKand
purity was over 96% as attested by toluidine blue staining.
Viability was higher than 95% as attested by trypan blue
exclusion. Isolated mast cells (1!105/mL) were added to
24-well plates and incubated with Bjv (2.5–0 mg/mL) or
preheated Bjv (60 and 100 8C) for 30 min at 37 8C in 5%
CO2atmosphere. Mast cells were also stimulated with the
P-III metalloproteinase jararhagin (EC 184.108.40.206) (5 mg/mL)
(Paine et al., 1992) and the proteolitically processed form
jararhagin C (5 mg/mL) (Usami et al., 1994). The
degranulating agent compound 48/80 (50 mg/mL) were
used for comparison. Co-stimulatory effects were evaluated
by incubating mast cells with jararhagin (5 mg/mL),
jararhagin C (5 mg/mL) or substance P (1.4 mg/mL) plus a
low dose of Bjv (2 mg/mL). After incubation, the plates were
centrifuged at 150!g, the supernatant was collected and
stored at K20 8C. In another set of experiments, mast cells
were pretreated with sodium cromoglicate and sodium
nedocromil (1 mM) at 37 8C, 30 min before Bjv challenge.
The divalent metal chelating 1,10-phenantroline (1 mM)
and inhibitors of phospholipase A2(p-BPB; 1.8 mM), snake
venom metalloproteinase (DM43; 0.5–5 mg/mL) and of
serine- (APMSF; 50 and 100 mM) and cysteine-proteinases
(E64; 1 and 10 mM) were also tested. In this set of
experiments, cells were incubated with Bjv in the presence
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893886
or the absence of the drugs, and the analysis was made
30 min after stimulation.
2.5. Mast cell morphological changes after Bjv stimulation
Mast cell morphological alterations caused by Bjv (2.5–
10 mg/mL) were evaluated 30 min after stimulation by
means of an inverted-type light microscope coupled on a
video-camera (Optronics Engineering, DEI-750). The
camera output was processed and analyzed by an image
analyzer software (Image-Pro Plus 4).
2.6. Quantification of histamine and LTC4from mast cells
Histamine release (%) was assayed fluorimetrically
according to Shore et al. (1959). Samples were collected,
added to 0.8 N perchloric acid (1:2 dilution) and centrifuged
at 170!g for 10 min. The supernatant was diluted with
0.1 N HCl followed by 0.8 N of NaOH and further addition
of the substrate o-phtaldialdehyde (OPT) was performed.
After 4 min incubation, the reaction was stopped with 3 N
HCl and the fluorescence measured in a Shimadzu RF1501
spectrofluorophotometer (Japan) (excitation at 360 nm;
emission at 450 nm). LTC4was measured by means of an
enzymatic immune assay (EIA) kit following the manu-
facturer’s instructions (Cayman Chemical Company, USA).
Sodium cromoglicate (30 mg/kg), meclizine (30 mg/kg)
and methysergide (4 mg/kg) were intraperitoneally admi-
nistered 1 h before the venom. All drugs were dissolved in
sterile saline, except meclizine that was dissolved in Tween
80 and further diluted with sterile isotonic saline.
2.8. Statistical analysis
The data were reported as meanGstandard error of the
mean (SEM) and statistically analyzed by Student’s t-test or
ANOVA followed by the Newman–Keuls–Student’s t-test.
Probability values (p) of 0.05 or less were considered
3.1. Involvement of mast cells in hyperalgesia induced by
Bjv in the rats
Injection of Bjv (2.5–30 mg/paw) into the rat hind paw
led to a marked hyperalgesic response, which set in very
rapidly, peaking from 30 to 60 min and decreasing there-
after. Maximal hyperalgesia was evoked by 10 mg/paw of
the venom and this dose was chosen for further experiments
(Fig. 1). Intraperitoneal treatment of rats with mast cell
stabilizer sodium cromoglicate (30 mg/kg), 1 h before
stimulation, abolished Bjv-induced hyperalgesia (Fig. 2A).
Likewise, the hyperalgesic response was entirely blocked by
treatment with selective histamine H1receptor antagonist,
meclizine (30 mg/kg, i.p.) (Fig. 2B) as well as by selective
5-HT1, 5HT2and 5-HT7receptor antagonist methysergide
(4 mg/kg, i.p.) (Fig. 2C).
3.2. Degranulation by Bjv of isolated mast cells in vitro
Fig. 3 shows, by means of inverted light microscopy, that
exposure of mast cells to Bjv resulted in morphological
alterations (Fig. 3B–D) compared with non-stimulated cells
(Fig. 3A). This phenomenon was shown to be dose-
dependent (2.5–10 mg/mL) and characterized by loss of
plasma membrane integrity and secretion of cytoplasmic
granules (Fig. 3B–D). In parallel, we noted that stimulation
of mast cells with increasing concentrations of Bjv caused
release of stored histamine as well as of neogenerated LTC4
(Fig. 4A and B, respectively). The latter was noted only at
the highest concentration of Bjv. Venom-evoked histamine
release was clearly sensitive to treatment with membrane
stabilizers sodium cromoglicate (1 mM) and sodium
nedocromil (1 mM). Values were 60.6G4.25% for
untreated cells and 33.3G4.4% (nZ4, P!0.01) and
32.1G2.2% (nZ4, P!0.01) for cells treated with sodium
cromoglicate and sodium nedocromil, respectively. We
showed that preheated Bjv had no stimulatory activity on
mast cells, suggesting the involvement of heat labile
enzymes in such process. Values of histamine dropped
from 50G4.3% (nZ4) in control cells to 2.0G1.7% (nZ4,
P!0.01) and to 8.5G3.0% (nZ4, P!0.01) in cells
stimulated with venom heated at 100 or 60 8C, respectively.
Basal histamine levels were 4.2G1.5%.
060 120 180240
∆ Latency (s)
Fig. 1. Time course of thermal hyperalgesia caused by intraplantar
injectionof Bjv (2.5–30 mg/paw). Injection of saline (opensymbols)
was used as negative control. Values are the meanGSEM for at
least six animals.CP!0.05 significantly different from negative
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893887
3.3. Effect of proteinase and PLA2inhibitors on mast cell
degranulation induced by Bjv
Bjv comprises a complex mixture of pharmacologically
active substances (Gutie ´rrez and Lomonte, 1989; Markland,
1998). In order to elucidate the components potentially
involved in the activation of mast cells by Bjv, several
selective inhibitors were tested. We found that incubation of
Bjv with the divalent metal chelator 1,10-phenantroline
(10 mM) abolished the venom ability to activate mast cells,
under conditions where the response triggered by compound
48/80 (50 mg/mL) was unaltered (Fig. 5). As illustrated in
Fig. 6, the SVMP inhibitor DM43 (0.5–5 mg/mL) (Neves-
Ferreira et al., 2000) inhibited, dose-dependently, histamine
secretion from mast cells. In contrast, inhibitors of serine-
proteinase APMSF (50 and 100 mM) and of cysteine-
proteinase E 64 (1 and 10 mM), did not interfere with Bjv
stimulatory activity (Table 1). PLA2inhibitor p-bromophe-
nacyl bromide (1.8 mM) also failed to impair mast cell
degranulation by Bjv (Table 1).
3.4. Effect of jararhagin on mast cells in vitro
Jararhagin is a P-III metalloproteinase isolated from Bjv
and was shown to produce several biological effects (for
review see Laing and Moura-da-Silva, 2005). Since this
toxin constitutes the major component of the metallopro-
teinases present in Bjv, we investigated its potential effect
on mast cells directly. As illustrated in Fig. 7A, no histamine
secretion was detected after stimulation with jararhagin
(5 mg/mL). In order to eliminate the possibility that the lack
of jararhagin effect on mast cells was due to previous
inactivation, we tested its fibrinogenolytic activity by means
SDS-PAGE gel and observed a typical hydrolysis of
fibrinogen Aa chain (data not shown). In addition, we
evaluated the stimulation of mast cells with jararhagin
(5 mg/mL) plus low concentration of Bjv (2 mg/mL) and
found that, under this particular condition, histamine
secretion was detected (Fig. 7A). Mast cells appeared
unaffected after co-stimulation with jararhagin and the
neuropeptide substance P (Fig. 7B). Likewise, no histamine
release was noted after mast cell stimulation with jararhagin
C (5 mg/mL), the proteolytically processed form of
jararhagin lacking the metalloproteinase domain, plus a
low dose of Bjv (2 mg/mL) (Fig. 7C).
Bothrops snake venoms are known for their ability to
produce intense local tissue damage through a mechanism
which is not fully understood. Usually, commercial
antivenoms are quite inefficient to neutralize venom-
induced local effects, even if administered before or
immediately after venom inoculation in different animal
species (Borkow et al., 1997; Theakston et al., 2003).
Venom toxins act very rapidly in the affected tissues and
trigger inflammatory responses associated with the release
of several endogenous mediators before the antivenom can
appropriately neutralize the activity of venom components.
Thus, to understand the mechanisms underlying snake
venom local effects is of great importance to achieve
successfully treatment for patients bitten by venomous
snakes. Although ophidic accidents are usually very painful
experiences, few studies on local effects induced by snake
venoms deal with the problem (Battellino et al., 2003;
∆ Latency (s)
0 60120180 24030
∆ Latency (s)
Fig. 2. Effect of sodium cromoglicate (30 mg/kg, i.p.) (A) meclizine
(30 mg/kg, i.p.) (B) and methysergide (4 mg/kg, i.p.) (C) on thermal
hyperalgesia triggered by intraplantar injection with Bjv
(10 mg/paw). Saline (C) and Bjv (&) were injected into the right
and left hid paws, respectively. Treated groups are indicated by
open circles. Values are the meanGSEM for at least six animals.
CP!0.05 significantly different from the negative control group;
*P!0.05 significantly different from positive control group.
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893888
Gutierrez et al., 1998; Picolo et al., 2002). In the current
study we investigated the involvement of mast cells in the
nociceptive response caused by Bjv in the rat paw. We
found that treatment with mast cell stabilizer sodium
cromoglicate inhibited Bjv-induced thermal hyperalgesia.
Also, the incubation of isolated rat mast cells with Bjv
resulted in marked degranulating response, a phenomenon
selectively suppressed by SVMP inhibitor DM43.
Altogether, our data indicate that mast cells are implicated
in the hyperalgesia caused by Bjv in rats, and suggest that
this response may be at least partially dependent on the
ability of Bjv to activate mast cells.
Sensitization of primary nociceptors often depends on
production and/or release of inflammatory mediators at the
site of the injury and mast cells are reported by their ability
to secrete several algesic stimuli (Dines and Powell, 1997;
Parada et al., 2001; Zuo et al., 2003). Most data about
hyperalgesic mechanisms derive from experimental models
in which overt behavioral responses can be indirectly
triggered by an exogenous secondary stimulus, as in the rat
paw withdrawal test evoked by either pressure (Randall and
Selitto, 1957) or heating (Hargreaves et al., 1988). In this
study, the paw withdrawal reflex threshold following plantar
thermal stimulus (Lavich et al., 2005) was used to measure
the noniceptive response stimulated with Bjv in rats. The
threshold in the area of local tissue damaged created by Bjv
was dose-dependent and about 2–6 s lower than the
threshold of the contralateral paw injected with saline. We
found that the thermal hyperalgesia was markedly
suppressed after treatment with mast cell membrane
stabilizer sodium cromoglicate, strongly suggesting the
involvement of mast cells in the nociceptive response
caused by Bjv. In addition, treatment with histamine H1
receptor antagonist meclizine as well as 5-HT1, 5HT2and 5-
HT7receptor antagonist methysergide also blocked Bjv-
induced hyperalgesia. Similar results were obtained when
the classical method of Hargreave’s (Hargreaves et al.,
1988) was used for measuring thermal hyperalgesia induced
by Bjv (data not shown). Our data are in agreement with
those obtained by Rocha et al. (2000) indicating that
vasoactive amines do contribute to thermal hyperalgesia
triggered by Bjv in the mice paw. Nevertheless, these
findings contrast with previous observations by Chacur et al.
(2002), demonstrating that bradykinin but not vasoactive
amines play a role in Bjv-induced mechanical hyperalgesia.
It is likely that this discrepancy may be accounted for by the
distinct hyperalgesia applied. Of note, mechanically
insensitive C-fibre nociceptors able to respond to thermal
Fig. 3. Effect of Bjv on isolated rat mast cells. Cells were incubated with medium (A) or various concentrations of Bjv: 2.5 mg/mL (B) 5 mg/mL
(C) and 10 mg/mL (D). The analysis was performed under inverted light microscope, 30 min after stimulation. Arrowhead indicates intact mast
cell membrane and arrow indicates granule released (!600).
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893 889
stimuli have been identified (Julius and Basbaum, 2001),
suggesting that distinct chemical modulation may be
implicated in thermal and mechanical hyperalgesia systems.
Venom toxins were reported for their property of
activating some constitutive as well as inflammatory cells
(Cardoso et al., 2001; Farsky et al., 2000; Landucci et al.,
1998; Martins et al., 2003; Schweitz et al., 1989). We
demonstrated herein that Bjv induced a dose- and time-
dependent degranulation of mast cells, a phenomenon
shown to be sensitive to treatment with stabilizers sodium
cromoglicate and sodium nedocromil. These findings
Bjv C 48/80
Histamine release (%)
Fig. 5. Effect of 1,10 phenantroline on histamine release (%) from
mast cells stimulated with Bjv (5 mg/mL) or 48/80 (50 mg/mL).
Cells were incubated with 1,10-phenantroline (10 mM) (hatched
bars) plus Bjv or medium (open bars) and the analysis was
performed 30 min post-stimulation. Dotted line indicates basal
levels of released histamine. Each column represents the meanG
SEM from at least four independent experiments.
significantly different from non-stimulated cells; *P!0.05 signifi-
cantly different from stimulated cells.
DM43 (µg/mL):- 0.51.02.5 5.0
Histamine release (%)
Fig. 6. Effect of the metalloproteinase inhibitor DM43 (0.5–
5 mg/mL) on histamine release (%) from mast cells stimulated with
Bjv (5 mg/mL). Cells were incubated with DM 43 (0.5–5 mg/mL) or
medium (open bars) and the analysis was performed 30 min post-
stimulation. Dotted line indicates basal levels of histamine release.
Each column represents the meanGSEM from at least four
non-stimulated cells; *P!0.05 significantly different from Bjv-
CP!0.05 significantly different from
Histamine release (%)
Bjv (µg/mL):5 2.510-
Fig. 4. Release of histamine (A) and LTC4(B) from isolated mast
cells after stimulation with Bjv (2.5–10 mg/mL) in vitro. The
analysis was performed 30 min post-stimulation. Dotted line
indicates basal levels of histamine release (A). The data are
expressed as meanGSEM. from at least four independent
experiments.CP!0.05 significantly different from non-stimulated
Effect of protease and phospholipase A2inhibitors on Bothrops
jararaca venom (Bjv)-induced mast cell degranulation in vitro
Stimuli Treatment (mM) Histamine
Isolated mast cells were incubated with phospholipase A2pBPB as
well as with serine- and cysteine-protease inhibitors APMSF and E
64, respectively, 30 min before stimulation with B. jararaca venom
(Bjv)(5 mg/mL).Cellsincubatedwithmediumwere usedascontrol.
Values are the meanGSEM of cells from at least four gradients.
*P!0.01 as compared to medium-stimulated cells.
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893890
provided, for the first time, evidence that Bjv shows the
ability to stimulate mast cells directly.
Previous heating or treatment of Bjv with divalent metal
chelator 1,10-phenantroline clearly abolished the venom
effect on mast cells, indicating that metalloproteinases
and/or PLA2might play role in this system. We further
demonstrated that the selective metalloproteinase inhibitor
DM43 neutralized mast cell degranulation induced by the
venom. In contrast, inhibitors of phospholipase A2as well
as of serine- and of cysteine-proteinases had no effect at all.
Taken together, our findings support the idea that
metalloproteinases play a relevant role in Bjv-triggered
mast cell activation. Consistently, DM43 was reported to
block local and systemic effects produced by Bjv including
hyperalgesia, paw edema and lethality, and also to
neutralize the metalloproteinase activity of Bjv or
jararhagin (Neves-Ferreira et al., 2000; Rocha et al.,
2000; Dale et al., 2004). Nevertheless, different from
what observed by our group, Landucci et al. (1998),
demonstrated that phospholipase A2 enzymes were
involved in mast cell activation caused by Bothrops
jararacussu venom. Diversity and complexicity of venom
molecular composition—a common feature among all
snake venoms—as well as genetic variation, environmental
factors and venom processing are possible explanations for
this discrepancy (Daltry et al., 1996; Moura-da-Silva et al.,
Jararhagin is a representative example of P-III
metalloproteinases and constitutes the major part of the
Bothrops venom (Moura-da-Silva et al., 2003). Dale et al.
(2004) recently showed that jararhagin induces hyperalge-
sia and oedema in rats, phenomena inhibited by the
chelator Na2-EDTA and to the synthetic peptide
mS100A9p—a catalytic site blocker. We found that
jararhagin itself showed no ability to stimulate mast cells
in vitro. However, the combination of jararhagin with Bjv,
at a dose, which alone produced a small effect, led to the
release of significant amount of histamine from mast cells.
Jararhagin C, the proteolitically processed form of
jararhagin, did not show the same ability as the full-length
protein did. These data strongly suggest the existence of an
interaction between jararhagin and other proteinases
present in Bjv, in a way dependent on the presence of its
catalytic site (Borkow et al., 1993). In addition, the
potential inespecific effect of jararhagin facilitating mast
cell response was ruled out as its combination with
substance P failed to activate the cells. Further experiments
are now underway in order to clarify the mechanism
involved in the interaction between jararhagin and other
metalloproteinases from Bjv to produce mast cell
In conclusion, our findings indicate that mast cells seem
to contribute to the hyperalgesic process caused by Bjv in
the rat paw. They also provide evidence that Bjv can
stimulate mast cells directly, by a mechanism which appears
Histamine release (%)
Histamine release (%)
Histamine release (%)
Fig. 7. Histamine release (%) after co-stimulation of mast cells with
jararhagin (5 mg/mL) plus Bjv (5 mg/mL) (A) or substance P (SP)
(1.4 mg/mL) (B) and jararhagin-C (5 mg/mL) plus Bjv (5 mg/mL)
(C). The analysis was made after 30 min after stimulation. Dotted
line indicates basal levels of histamine release. Each column
represents the meanGSEM from at least four independent
experiments.CP!0.05 significantly different from jararhagin- or
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893 891
to be dependent on the metalloproteinase component of the
This study was supported by grants from Conselho
Nacional de Desenvolvimento Cientı ´fico and Tecnolo ´gico
(CNPq) and Fundac ¸a ˜o de Amparo a ` Pesquisa do Estado do
Rio de Janeiro (FAPERJ), Brazil. AGCB thanks CNPq for
his PhD scholarship.
Battellino, C., Piazza, R., da Silva, A.M., Cury, Y., Farsky, S.H.,
2003. Assessment of efficacy of bothropic antivenom therapy on
microcirculatory effects induced by Bothrops jararaca snake
venom. Toxicon 41, 583–593.
Borkow, G., Gutierrez, J.M., Ovadia, M., 1993. Isolation and
characterization of synergistic hemorrhagins from the venom of
the snake Bothrops asper. Toxicon 31, 1137–1150.
Borkow, G., Gutierrez, J.M., Ovadia, M., 1997. Inhibition of toxic
activities of Bothrops asper venom and other crotalid snake
venoms by a novel neutralizing mixture. Toxicol. Appl.
Pharmacol. 147, 442–447.
Brasil, M.d., 2001. Manual de diagno ´stico e tratamento de acidentes
por animais pec ¸onhentos, vol. 112. Fundac ¸a ˜o Nacional da
Sau ´de, Brası ´lia.
Macedo, M.S., Farsky, S.H., 2001. Role of crotoxin, a
phospholipase A2 isolated from Crotalus durissus terrificus
snake venom, on inflammatory and immune reactions.
Mediators Inflamm. 10, 125–133.
Chacur, M., Picolo,G.,Teixeira,C.F., Cury,Y.,2002.Bradykininis
involved in hyperalgesia induced by Bothrops jararaca venom.
Toxicon 40, 1047–1051.
Clissa, P.B., Laing, G.D., Theakston, R.D., Mota, I., Taylor, M.J.,
Moura-da-Silva, A.M., 2001. The effect of jararhagin, a
metalloproteinase from Bothrops jararaca venom, on pro-
inflammatory cytokines released by murine peritoneal adherent
cells. Toxicon 39, 1567–1573.
Costa, E.P., Clissa, P.B., Teixeira, C.F., Moura-da-Silva, A.M.,
2002. Importance of metalloproteinases and macrophages in
viper snake envenomation-induced local inflammation. Inflam-
mation 26, 13–17.
Giorgi, R., 2004. The C-terminus of murine S100A9 inhibits
Daltry, J.C., Wuster, W., Thorpe, R.S., 1996. Diet and snake venom
evolution. Nature 379, 537–540.
de Oliveira Barreto, E., de Frias Carvalho, V., Diaz, B.L.,
Balduino, A., Cordeiro, R.S., Martins, M.A., Rodrigues e
Silva, P.M., 2003. Adoptive transfer of mast cells abolishes the
inflammatory refractoriness to allergen in diabetic rats. Int.
Arch. Allergy Immunol. 131, 212–220.
Dines, K.C., Powell, H.C., 1997. Mast cell interactions with the
nervous system: relationship to mechanisms of disease.
J. Neuropathol. Exp. Neurol. 56, 627–640.
Farsky, S.H., Goncalves, L.R., Gutierrez, J.M., Correa, A.P.,
Rucavado, A., Gasque, P., Tambourgi, D.V., 2000. Bothrops
asper snake venom and its metalloproteinase BaP-1 activate the
complement system. Role in leucocyte recruitment. Mediators
Inflamm. 9, 213–221.
Gutie ´rrez, J.M.,Lomonte,B.,1989.Local tissuedamageinducedby
Bothrops snake venoms. A review. Mem. Inst. Butantan 51,
Gutierrez, J.M., Leon, G., Rojas, G., Lomonte, B., Rucavado, A.,
Chaves, F., 1998. Neutralization of local tissue damage
induced by Bothrops asper (terciopelo) snake venom. Toxicon
Hargreaves, K., Dubner, R., Brown, F., Flores, C., Joris, J., 1988. A
new and sensitive method for measuring thermal nociception in
cutaneous hyperalgesia. Pain 32, 77–88.
Julius, D., Basbaum, A.I., 2001. Molecular mechanisms of
nociception. Nature 413, 203–210.
Laing, G.D., Moura-da-Silva, A.M., 2005. Jararhagin and its
multiple effects on hemostasis. Toxicon 45, 987–996.
Landucci, E.C., Castro, R.C., Pereira, M.F., Cintra, A.C.,
Antunes, E., De Nucci, G., 1998. Mast cell degranulation
induced by two phospholipase A2 homologues: dissociation
J. Pharmacol. 343, 257–263.
Lavich, T.R., Cordeiro, R.S., Silva, P.M., Martins, M.A., 2005.
A novel hot-plate test sensitive to hyperalgesic stimuli and
non-opioid analgesics. Braz. J. Med. Biol. Res. 38, 445–
Markland, F.S., 1998. Snake venoms and the hemostatic system.
Toxicon 36, 1749–1800.
Martins, A.M., Lima, A.A., Toyama, M.H., Marangoni, S.,
Fonteles, M.C., Monteiro, H.S., 2003. Renal effects of
supernatant from macrophages activated by Crotalus durissus
cascavella venom: the role of phospholipase A2 and cyclo-
oxygenase. Pharmacol. Toxicol. 92, 14–20.
Mekori, Y.A., Metcalfe, D.D., 2000. Mast cells in innate immunity.
Immunol. Rev. 173, 131–140.
Metcalfe, D.D., Baram, D., Mekori, Y.A., 1997. Mast cells. Physiol.
Rev. 77, 1033–1079.
Moura-da-Silva, A.M., Laing, G.D., Paine, M.J., Dennison, J.M.,
Politi, V., Crampton,J.M., Theakston, R.D.,1996. Processingof
pro-tumor necrosis factor-alpha by venom metalloproteinases: a
hypothesis explaining local tissue damage following snake bite.
Eur. J. Immunol. 26, 2000–2005.
Assakura, M.T., Butera, D., Lebrun, I., Shannon, J.D.,
Serrano, S.M., Fox, J.W., 2003. Evidence for heterogeneous
forms of the snake venom metalloproteinase jararhagin: a factor
contributing to snake venom variability. Arch. Biochem.
Biophys. 409, 395–401.
Neves-Ferreira, A.G., Cardinale, N., Rocha, S.L., Perales, J.,
Domont, G.B., 2000. Isolation and characterization of DM40
and DM43, two snake venom metalloproteinase inhibitors from
Didelphis marsupialis serum. Biochim. Biophys. Acta 1474,
Neves-Ferreira, A.G., Perales, J., Fox, J.W., Shannon, J.D.,
Makino, D.L., Garratt, R.C., Domont, G.B., 2002. Structural
and functional analyses of DM43, a snake venom metallopro-
teinase inhibitor from Didelphis marsupialis serum. J. Biol.
Chem. 277, 13129–13137.
S.,Oliveira, B., Cirino,G.,
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893892
Paine, M.J., Desmond, H.P., Theakston, R.D., Crampton, J.M., Download full-text
1992. Purification, cloning, and molecular characterization
of a high molecular weight hemorrhagic metalloprotease,
jararhagin, from Bothrops jararaca venom. Insights into the
disintegrin gene family. J. Biol. Chem. 267, 22869–22876.
Parada, C.A., Tambeli, C.H., Cunha, F.Q., Ferreira, S.H., 2001. The
major role of peripheral release of histamine and 5-hydroxy-
tryptamine in formalin-induced nociception. Neuroscience 102,
edema induced by Bothrops jararaca and Bothrops asper snake
venoms. Braz. J. Med. Biol. Res. 35, 1221–1228.
Randall, L.O., Selitto, J.J., 1957. A method for measurement of
analgesic activity on inflamed tissue. Arch. Int. Pharmacodyn.
Ther. 111, 409–419.
Rocha, S.L., Frutuoso, V.S., Domont, G.B., Martins, M.A.,
Moussatche, H., Perales, J., 2000. Inhibition of the hyperalgesic
activity of Bothrops jararaca venom by an antibothropic
fraction isolated from opossum (Didelphis marsupialis) serum.
Toxicon 38, 875–880.
Rothschild, A.M., Rothschild, Z., 1979. Liberation of pharmaco-
logically active substances by snake venoms. In: Lee, C.Y.
Snake Venoms—Handbook of Experimental Pharmacology,
vol. 52. Springer, Berlin(Ed.), pp. 591–628.
Schweitz, H., Bidard, J.N., Maes, P., Lazdunski, M., 1989.
Charybdotoxin is a new member of the KC channel toxin
family that includes dendrotoxin I and mast cell degranulating
peptide. Biochemistry 28, 9708–9714.
Shore, P.A., Burkhalter, A., Cohn Jr., V.H., 1959. A method for the
fluorometric assay of histamine in tissues. J. Pharmacol. Exp.
Ther. 127, 182–186.
Teixeira, C.F., Cury, Y., Oga, S., Jancar, S., 1994. Hyperalgesia
induced by Bothrops jararaca venom in rats: role of eicosanoids
and platelet activating factor (PAF). Toxicon 32, 419–426.
Theakston, R.D., Warrell, D.A., Griffiths, E., 2003. Report of a
WHO workshop on the standardization and control of
antivenoms. Toxicon 41, 541–557.
Trebien, H.A., Calixto, J.B., 1989. Pharmacological evaluation of
rat paw oedema induced by Bothrops jararaca venom. Agents
Actions 26, 292–300.
Usami, Y., Fujimura, Y., Miura, S., Shima, H., Yoshida, E.,
Yoshioka, A., Hirano, K., Suzuki, M., Titani, K., 1994. A 28
kDa-protein with disintegrin-like structure (jararhagin-C) pur-
induced platelet aggregation.Biochem.Biophys. Res. Commun.
Zuo, Y., Perkins, N.M., Tracey, D.J., Geczy, C.L., 2003.
Inflammation and hyperalgesia induced by nerve injury in the
rat: a key role of mast cells. Pain 105, 467–479.
A.G.C. Bonavita et al. / Toxicon 47 (2006) 885–893893