raia

1. Biological and biochemical properties of the Brazilian
Potamotrygon stingrays: Potamotrygon cf. scobina
and Potamotrygon gr. orbignyi
Kharita W. Magalha˜es a
, Carla Lima b
, Ana Ame´lia Piran-Soares b
, Elineide
E. Marques a
, Cle´lia A. Hiruma-Lima c
, Moˆnica Lopes-Ferreira b,
*
a
Nucleus of Environmental Studies, Federal University of Tocantins, Tocantins, Brazil
b
Laboratory of Immunopathology, Butantan Institute Av. Vital Brazil, 1500, Butantan, 05503-009 Sa˜o Paulo, SP, Brazil
c
Physiology Department, Biosciences Institute, Sa˜o Paulo State University, Sa˜o Paulo, SP, Brazil
Received 9 November 2005; revised 25 January 2006; accepted 26 January 2006
Available online 24 March 2006
Abstract
Stingrays of the family Potamotrygonidae are widespread throughout river systems of South America that drain into the
Atlantic Ocean. Some species are endemic to the most extreme freshwater environment of the Brazil and cause frequent
accidents to humans. The envenomation causes immediate, local, and intense pain, soft tissue edema, and a variable extent of
bleeding. The present study was carried out in order to describe the principal biological and some biochemical properties of the
Brazilian Potamotrygon fish venoms (Potamotrygon cf. scobina and P. gr. orbignyi). Both stingray venoms induced significant
edematogenic and nociceptive responses in mice. Edematogenic and nociceptive responses were reduced when the venom was
incubated at 37 or 56 8C. The results showed striking augments of leukocytes rolling and adherent cells to the endothelium of
cremaster mice induced by both venoms. The data also presented that injection of both venoms induced necrosis, low level of
proteolytic activity, without inducing haemorrhage. But when the venoms of both stingray species were injected together with
their mucus secretion, the necrotizing activity was more vigorous. The present study provided in vivo evidence of toxic effects
for P. cf. scobina and P. gr. orbignyi venoms.
q 2006 Elsevier Ltd. All rights reserved.
Keywords: Potamotrygon venoms; Stingrays; Biological and biochemical activities
1. Introduction
The production of toxins from aquatic animals is an
important strategy that guarantees their survival in a highly
competitive ecosystem. These animals produce an enor-
mous number of metabolics, whose combinations result in a
great variety of chemical structures and complex molecules,
as alkaloids, steroids, peptides and proteins with chemical
and pharmacological properties, different from that pre-
sented by the poisons of terrestrial animals (Russell, 1971).
Despite the fact that studies of the terrestrial animal venoms
have been advanced, there are few reports related to
Brazilian fish venoms. In this country, a special attention
has been given to the Thalassophryne nattereri fish venom,
which presented several biological, biochemical, and
pharmacological activities characterized (Lopes-Ferreira
et al., 2004).
Stingrays of the family Potamotrygonidae are wide-
spread throughout river systems of South America that
drain into the Atlantic Ocean. While some members of the
Toxicon 47 (2006) 575–583
www.elsevier.com/locate/toxicon
0041-0101/$ - see front matter q 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.toxicon.2006.01.028
* Corresponding author. Tel.: C55 113 726 7222x2134/2087;
fax: C55 113 676 1392.
E-mail address: mlferrei@usp.br (M. Lopes-Ferreira).

2. other family of rays may complete their entire life cycle in
freshwater (Compagno and Roberts, 1982; Johnson and
Snelson, 1996), the potamotrygonid stingrays are
considered to be the only group of elasmobranchs to
have speciated exclusively within freshwaters (Lovejoy,
1996). Some species of the Potamotrygonidae are
endemic to the most extreme freshwater environment
of the Brazil, of the Parana´ River, Tocantins River and its
tributaries, and cause frequent accidents to humans.
Stingrays have one to four venomous stingers on the
dorsum of an elongated, whip-like caudal appendage. The
tapered, vasodentine spines are bilaterally retroserrated
(saw-edged, with the cutting cartilage pointing away from
the apex of the spine). Each spine is enveloped by
an integumentary sheath with a ventrolateral glandular
groove containing venom glands along either edge
(Halstead, 1970). The spine is often covered with a film
of venom and mucus.
Stingrays do not attack people, however if they are
stepped on, stingrays utilize its spine as a form of defense.
Although being pierced by the stingray’s spine is painful,
it is rarely life threatening to humans. When the wings of
the ray are touched, its tail whips in response, thrusting
the spine into the victim and causing a puncture wound or
jagged laceration. The integumentary sheath overlying the
spine ruptures, and venom is released into the wound-
along with mucus, pieces of the sting sheath, and
fragments of spines. The entire tip of the spine may
remain embedded in the wound (Fenner et al., 1989;
Evans and Davies, 1996).
The envenomation causes immediate, local, and
intense pain, soft tissue edema, and a variable extent of
bleeding. Pain peaks after 30–60 min, may radiate
centrally, and can last for 48 h. Most minor punctures
resemble cellulites and do not lead to serious infection or
tissue loss. A severe wound initially appears dusky
or cyanotic and progresses to assume an erythematous or
mottled appearance, with rapid hemorrhage and necrosis
of fat and muscle. Stingrays venom may cause extensive
necrosis. Systemic symptoms include vomiting, seizures,
generalized edema (with a truncal wound), limb
paralysis, hypotension, and bradycardia (Fenner et al.,
1989).
There appear to be several different chemicals in the
venom, but not all of these have been well characterized to
date. Some authors describe neurotoxicity (Vellard, 1931;
1932), cardiotoxicity (Fleury, 1950) and circulatory dis-
turbances (Russell and Van Harreveld, 1954; Russell et al.,
1957; Rodrigues, 1963; 1972). Some studies demonstrated
that venoms of rays contain serotonin, 50
-nucleotidase and
phosphodiesterases (Fenner et al., 1989).
In view of these facts, the present study was carried out
in order to describe the principal biological and some
biochemical properties of the Brazilian Potamotrygon fish
venoms (Potamotrygon cf. scobina and P. gr. orbignyi).
2. Materials and methods
2.1. Animals
Seven to eight weeks-old male Swiss mice (nZ6)
obtained from a colony at Butantan Institute (Sa˜o Paulo,
Brazil) were maintained at the animal house facilities of the
Laboratory of Immunopathology, under specific pathogen-
free conditions. All the procedures involving mice were in
accordance with the guidelines provided by the Brazilian
College of Animal Experimentation.
2.2. Venom and mucus
Specimens (nZ10) of both sexes of Potamotrygon cf.
scobina and Potamotrygon gr. orbighnyi were collected on
Parana´ River and Tocantins River both in the state of
Tocantins, Brazil, and transferred immediately to laboratory
to extract of the venom. Venom produced by venom glands
and mucus dispersed through the spines were collected after
scratching both the epithelium and mucus, respectively.
Total mucus and venom dissolved in PBS pH 7.4 were
immediately centrifuged at 6000!g for 15 min. Venom and
mucus were stored at K208 until use. Protein content was
determined by the method of Bradford (1976) using bovine
serum albumin (Sigma Chemical Co., St Louis, MO) as
standard protein.
2.3. Estimation of edema-inducing activity
Edematogenic activity of the venom was assayed
according to the Lima et al. (2003). Samples of 30 ml
containing different doses of venom (6.25, 12.5, 25, 50, and
100 mg of protein) were injected (i.pl.) in the right footpad of
mice. Local edema was quantified by measuring the
thickness of injected paws with a paquimeter (Mytutoyo
Sul Americana, SP, Brazil) in 2, 4, 6, 8, 10 and 12 h after
injection. Mice injected with 30 ml of sterile PBS were
considered as control-group. The results were expressed by
the difference between experimental and control footpad
thickness. Each point represents meanGSEM.
2.4. Estimation of nociceptive activity
Nociceptive activity of the venom was assayed accord-
ing to the Hunskaar et al. (1985). Samples of 30 ml
containing different doses of venom (6.25, 12.5, 25, 50,
and 100 mg of protein) were injected (i.pl.) in the right
footpad of mice. Then, each mouse was kept in an adapted
chamber mounted on a mirror for 10 min. The control-group
was injected only with sterile PBS. Each animal was then
returned to the observation chamber and the amount of time
spent licking or biting each hind paw was recorded. Each
point represents meanGSEM.
K.W. Magalha˜es et al. / Toxicon 47 (2006) 575–583576

3. 2.5. Determination of thermolability of the venom
To determine the effect of the high temperature on
venom lability, the venom was heated at 37 or 56 8C for
30 min. or remained at room temperature for 24 h. Then,
50 mg of venom protein were used for induction of edema
and nociception such as described previoulsly.
2.6. Estimation of haemorrhagic activity
Haemorrhagic activity was assayed according to the
Ferreira et al. (1992). Briefly, mice were shaved in the backs
and injected (i.d.) with 50 ml of the solutions containing
different doses of venom (6.25, 12.5, 25, 50, and 100 mg of
protein). After 2 h, the mice were killed; the skin stripped off
from the dorsum and placed on a plank. Two diameters were
determined for the haemorrhagic spot by measuring the
longest diameter and the one perpendicular to the longest.
Results were expressed as the product of the diameters
GSEM.
2.7. Estimation of necrotizing activity
Necrotizing activity of the venom was assayed according
to the Ferreira et al. (1992). Necrosis was quantified after an
i.d. injection of different doses of venom (6.25, 12.5, 25, 50,
and 100 mg of protein) contained in 50 ml of PBS into the
shaved backs of the mice. Another group of mice were
injected with 50 mg of venom protein added with 50 mg of
mucus. After 72 h, the animals were killed with ether, and
the skin removed. The necrotic area was measured. Two
diameters were determined for the necrotic spot by
measuring the longest diameter and the one perpendicular
to the longest. Results were expressed as the product of the
diameters G SEM.
2.8. Estimation of proteolytic activity
Proteolytic activity was estimated using casein as
substrate, as described by Mandelbaum et al. (1990). One
milliliter of 1% casein was incubated for 2 h at 37 8C with
400 ml of test solutions containing different doses of venom
(3, 10, 30, and 100 mg of protein), in the presence of
0.008 M CaCl2 at pH 8.8. Reaction was stopped with 5%
trichloracetic acid and the hydrolyzed peptides contained in
the supernatants quantified according to Lowry et al. (1951).
One unit was defined as the amount of enzyme yielding an
increase in absorbance of 1.0 per min at 750 nm. Results
were expressed in U/mg of venom.
2.9. Microcirculatory alterations
The dynamic of alterations in the microcirculatory
network were determined using intravital microscopy by
transillumination of mice cremaster muscle after subcu-
taneous application of venom (25 mg of protein dissolved in
50 ml of sterile saline). Administration of the same amount
of sterile saline was used as control. In three independent
experiments (nZ4) mice were anaesthetized with pento-
barbital sodium (Hypnolw
Crista´lia; 50 mg/kg, intraperito-
neal route) and the cremaster muscle was exposed for
microscopic examination in situ as described by Baez
(1973) and Lomonte et al. (1994). The animals were
maintained on a special board thermostatically controlled at
37 8 C, which included a transparent platform on which the
tissue to be transilluminated was placed. After the
stabilization of the microcirculator, the number of roller
cells and adherent leukocytes in the postcapillary venules
were counted 10 min after venom injection. The study of the
microvascular system of the tissue transilluminated was
accomplished with optical microscope (Axiolab, Carl-Zeiss,
Oberkochen, DE) coupled to a photographic camera
(Coolpix 990-Nikon) using an !10/025 longitudinal
distance objective/numeric aperture and 1.6 optovar.
2.10. Sodium dodecyl sulphate-polyacrylamide
gel electrophoresis (SDS-PAGE)
SDS-PAGE was carried out according to the method of
Laemmli (1970). The proteins (10 mg) of venom were
analyzed by SDS-PAGE a 4–20% acrylamide gradient
under non-reducing conditions. Prior to electrophoresis, the
samples were mixed 1:1 (v/v) with sample buffer.
Phosphorylase B (97,000), Albumin (68,000), Ovalbumin
(43,000), Anidrase carbonic (29,000) and b-lactoglobulin
(18,400) (Sigma Chemical Company, St Louis, MO, USA)
were used as molecular mass markers. The gel was stained
with the Comassie Blue staining method.
2.11. Fractionation of the venoms
Venoms samples were isolated using a FPLC system
(Pharmacia, Uppsala, Sweden) by gel filtration chromatog-
raphy. Five milligrams of each venom were dissolved in
500 ml of Milli Q water (Millipore, UK) and centrifuged
immediately before fractionation. Samples were applied to
the Superdex 12-HR equilibrated with a 50 mM sodium
phosphate buffer, pH 7.2 containing 0.15 M NaCl (PBS).
The optical density of the eluant was monitored at 280 nm.
The flow rate was 0.5 ml/min and 1 ml fractions were
collected.
2.12. Statistical analysis
One Way Analysis of Variance (ANOVA) followed by
Dunnett’s test was used to determine the levels of difference
between all groups. Differences were considered statisti-
cally significant at p!0.05. The SPSS statistical package
(Release 8.0, Standard Version, 1997) was employed.
K.W. Magalha˜es et al. / Toxicon 47 (2006) 575–583 577

4. 3. Results
3.1. P. cf. scobina and P. gr. orbignyi venoms induced
edematogenic activity in mice
Swiss mice were injected with P. cf. scobina or P. gr.
orbignyi venoms and the thickness of the right footpad was
measured 2 h after injection. As shown in Fig. 1A both
stingray venoms induced significant edematogenic activity
in all doses analysed (6.25–100 mg), as compared with
control-group of mice. The dose of 25 mg of both venoms
induced a sustained edematogenic response until 10 h after
injection (Fig. 1B). The edematogenic response was absent
in the last time-point analysed for both venoms.
3.2. Nociceptive effect of P. cf. scobina and P. gr.
orbignyi venoms
Injection of stingray venoms into the mouse right hind-
paw induced a dose-related increase of the paw licking
duration during 30 min that reached its maximum at 25 mg
of venom protein, and stabilized thereafter (Fig. 2A). The
neurogenic (0–5 min after venom injection) and inflamma-
tory (15–40 min) phases of the nociception test were also
induced for P. cf. scobina and P. gr. orbignyi venoms
(Fig. 2B).
3.3. Thermolability of the venoms
The increase in the temperature of storage of the venom
promoted a crescent decrease of the edematogenic activity
induced by P. cf. scobina and P. gr. orbignyi venoms
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Control 6.25 12.5 25 50 100
Venom( µg of protein/paw)
Footpadthickness(mm)
Potamotrygon cf. scobina venom
Potamotrygon gr. orbignyi venom
Footpadthickness(mm)
0
0.5
1
1.5
2
2 4 6 8 10 12
Potamotrygon cf. scobina venom
Potamotrygon gr. orbignyi venom
PBS
Hours after injection
A
B
Fig. 1. Estimation of edema-inducing activity. (A) Samples of 30 ml
containing different doses of venom (6.25, 12.5, 25, 50, and 100 mg
of protein) were injected (i.pl.) in the right footpad of mice. Local
edema was quantified 2 h after injection. (B) Local edema was
quantified in 2, 4, 6, 8, 10, and 12 h after injection of 30 ml
containing 25 mg of protein/animal. Mice injected with sterile PBS
were considered as control-group. The results were expressed by the
difference between experimental and control footpad thickness.
Each point represents meanGSEM. *p!0.05 compared with
control-group.
Potamotrygon cf.scobina venom(PsV)
Potamotrygon gr.orbignyi venom(PoV)
0
50
100
150
200
250
300
Control 6.25 12.5 25 50 100
Venom( µg of protein/paw)
0
20
40
60
80
100
Control PsV PoV Control PsV PoV
0-5 min 15 - 40 min
Nociception(s)Nociception(s)
A
B
Fig. 2. Estimation of nociception-inducing activity. Samples of
30 ml containing different doses of venom (6.25, 12.5, 25, 50, and
100 mg of protein) were injected (i.pl.) in the right footpad of mice.
The control group was injected only with sterile PBS. Each animal
was then returned to the observation chamber and the amount of
time spent licking or biting each hind paw was recorded for 30 min
(A) or 0–5 and 15–40 min (B) and taken as the index of nociception.
Each point represents meanGSEM. *p! 0.05 compared with
control-group.
K.W. Magalha˜es et al. / Toxicon 47 (2006) 575–583578

5. (approximately 15 and 9%, respectively, for room tempera-
ture; 44 and 42%, respectively, for 37 8C; and 63 and 61%,
respectively, for 56 8C). Both venoms also induced
diminished response of nociception after incubation at
56 8C (12.5 and 12%, respectively, for room temperature; 33
and 29%, respectively, for 37 8C; and 44 and 47%,
respectively, for 56 8C) (Table 1).
3.4. Estimation of necrotizing, proteolytic,
and hemorrhagic activities
The ability of the both venoms induced necrosis in mice
is presented in Fig. 3. The two lower doses (6.25 and
12.5 mg) were not capable to induce necrosis, but only the
doses of 25, 50 and 100 mg of both venoms induced necrosis
Table 1
Effect of heating on biological activities induced by Potamotrygon venoms
K70 8C RT 37 8C 56 8C
P. cf. scobina Nociception (s) 130G25 111G19 73G12 48G0.9
Edema (mm) 1.8G0.4 1.6G0.3 1.2G0.3 1.0G0.3
P. gr. orbignyi Nociception (s) 110G31 100G21 64G13 43G8.0
Edema (mm) 1.7G0.3 1.5G0.4 1.2G0.2 0.9G0.1
Venom activities were assayed as described in Section 2 and are expressed as the mean of three independent experiments GSD. *p!0.05
compared with control-group; #p!0.05 compared with venom stored at K70 8C.
Potamotrygoncf. scobina venom + Mucus
Potamotrygongr. orbignyi venom + Mucus
Venom + Mucus
Control Ps Po
A
B
Control 6.25 12.5 25 50 100
Venom ( µg of protein/paw)
3.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
0.0
0.5
1.0
1.5
2.0
2.5
Potamotrygon cf. scobina venom
Potamotrygon gr. orbignyi venom
Necrosis(productof
diameters/mm2
)
Necrosis(productof
diameters/mm2
)
Fig. 3. Estimationof necrotizing activity. Necrosiswas quantifiedafter an i.d.injectionthe differentdoses ofvenom (6.25,12.5,25, 50, and 100 mg
of protein) contained in 50 ml of PBS into the shaved backs of the mice. The necroticarea wasmeasured 72 h after injection of venom (A) or venom
(50 mg) added with mucus (50 mg) (B). Two diameters were determined for the necrotic spot by measuring the longest diameter and the one
perpendicular to the longest. Results were expressed as the product of the diameters GSEM. *p!0.05 compared with control-group.
K.W. Magalha˜es et al. / Toxicon 47 (2006) 575–583 579

6. (Fig. 3A). Interestingly, when the venoms of both stingray
species were injected added with their respective mucus the
necrotizing activity was more vigorous (two-fold) than that
observed in mice injected only with the venoms (Fig. 3B).
Neither P. cf. scobina nor P. gr. orbignyi venoms
injected-mice differed from the control-group in regard to
hemorrhagic activity (data not shown), but low level of
proteolytic activity was detectable for both Potamotrygon
venoms using 50 or 100 mg of protein (P. cf. scobina, 3.7G
0.9 U/mg and 5.0G1.3 U/mg, respectively; P. gr. orbynyi,
3.3G0.9 U/mg and 4.8G1.1 U/mg, respectively).
3.5. Leukocyte-endothelial interactions
The contribution of venom to leukocyte interactions was
examined by determining the number of rolling and adhered
cells in postcapillary venules of the cremaster muscle of mice
injected with P. cf. scobina and P. gr. orbignyi venoms.
Under basal conditions, the rolling and adherence of
leukocyte were not different between venom- or control-
groups (data not shown). As shown in Fig. 4A, both venoms
increased the number of rolling leukocytes after subcu-
taneous injection (56 and 75%, respectively). In both group
of mice injected with P. cf. scobina or P. gr. orbignyi venoms
it was observed a large increase in the number of adherent
leukocytes, as demonstrated by arrows (Fig. 5A and B). No
change in rolling leukocyte velocity and adhered cells was
seen over time in control-animals (Figs. 4B and 5B).
3.6. Chromatographic and eletrophoretical profile
of Potamotrygon stingray venoms
SDS-PAGE analysis of the P. cf. scobina or P. gr.
orbignyi venoms showed a similar eletrophoretic profile,
Fig. 4. Number of leukocytes rolling. Rolling leukocytes in
postcapillary venules of the mice cremaster muscle after subcu-
taneous application of venoms (25 mg of protein dissolved in 50 ml
of sterile saline) or sterile saline (50 ml, control). Determinations
were performed 10 min after venom injection and values averaged.
Results were obtained in recorded images on optical microscope
(Axiolab, Carl-Zeiss) coupled to a photographic camera (Coolpix
990-Nikon) using an !10/025 longitudinal distance objective/nu-
meric aperture and 1.6 optovar.
Fig. 5. Adhered leukocytes in postcapillary venules of cremaster
muscle after injection with venom. Adhered leukocytes were
observed after application of venoms (25 mg of protein dissolved in
50 ml of sterile saline) or sterile saline (50 ml, control). Determi-
nations were performed 10 min after venom injection and values
averaged. Venom results were determined as described in Fig. 4.
K.W. Magalha˜es et al. / Toxicon 47 (2006) 575–583580

7. and a broad band starting at 15 kDa was observed under
non-reducing conditions (Fig. 6A). In contrast to P. cf.
scobina the venom of the P. gr. orbignyi venom presented
two exclusive bands at 66.2 kDa and one close of 25 kDa.
Chromatographic separation on Superdex 12-HR at pH 7.2
resulted in five peaks in both stingray venoms (Fig. 6B).
4. Discussion
Some species of the Potamotrygonidae are endemic to
the most extreme freshwater environment of Brazil, of the
Parana´ River, Tocantins River and its tributaries, and cause
frequent accidents to humans, mainly the species P. cf.
scobina and P. gr. orbignyi. The envenomation causes
immediate, local, and intense pain, edema, and in a variable
number of victims skin necrosis was demonstrated (Fenner
et al., 1989; Haddad et al., 2004).
In this study we shown that the Brazilian venoms of
P. cf. scobina and P. gr. orbignyi can induce edematogenic
and nociceptive responses, and necrosis in mice. Our results
showed a striking augment in leukocytes rolling and
adherent cells to the endothelium of cremaster mice.
These toxic effects were similar in P. cf. scobina and
P. gr. orbignyi venoms. Neither P. cf. scobina nor P. gr.
orbignyi venoms showed hemorrhagic activity. The proteo-
lytic activity detected for both Potamotrygon venoms was
similar to that observed for Thalassophryne nattereri and
Thalassophryne maculosa venoms (Lopes-Ferreira et al.,
1998; Sosa-Rosales et al., 2005).
Edema formation is a common feature of the cutaneous
inflammatory processes and is dependent on a synergism
between mediators that increase vascular permeability and
those that increase blood flow (Brain and Williams, 1985).
Injection of P. cf. scobina and P. gr. orbignyi venoms in
footpad of mice induced a similar dose-related increase of
edematogenic response that reached the maximum at 25 mg
for both venoms. As expected from previous reports (Lima
et al., 2003; Sosa-Rosales et al., 2005) fish venoms are
known to induce intense and sustained edematogenic
response in mice. In contrast, we demonstrated here that
the edematogenic response induced by Potamotrygon
venoms was less intense and fast (remained until 10 h
after injection).
Numerous inflammatory mediators are produced and
released in the course of inflammation (prostaglandins,
bradykinin, histamine, ATP and acetylcholine, and others),
and they cause the classical signs of inflammation, i.e.
swelling, redness, hyperthermia, and pain. Nociceptors
express receptors for the transduction of mechanical,
thermal or chemical stimuli into electrical potentials. During
inflammation the swelling may more effectively open the
cation channels than under normal conditions leading to a
depolarisation of the sensory ending (Clatworthy et al.,
1995; Dray, 1995; Wood and Docherty, 1997; Wagner et al.,
1998; Cui et al., 2000). The ability of both venoms to
develop a nociceptive response was also demonstrated, and
the nociception induced during inflammatory period
(15–40 min) could be associated with the augmented rolling
and adhesiveness of leukocytes to the endothelium of
cremaster mice induced by both venoms.
Our findings show that the deleterious local processes
induced by P. cf. scobina and P. gr. orbignyi venoms in
mice could be initiated at the microcirculatory level, as
reveled by intravital microscopy. The topical application of
both venoms at microcirculatory net of cremaster mice
would lead to local release of the vasoactive mediators,
cytokines, and chemoattractants. Finally, these mediators
can up-regulate the expression of adhesion molecules
favoring leukocyte mobilization. The selectin family of
adhesion molecules is associated with the initial phase of
leukocyte recruitment characterized by leukocyte rolling
(Ley, 1996). This is in accordance with the notion that
P-selectin is more critical in the initial rolling and slowing of
recruited leukocytes (Robinson et al., 1999) while E-selectin
is more important in leukocyte arrest, or the transition from
1 2 3
66.2
45.0
35.0
25.0
18.4
A
B
Absorbance(280nm)
Fractions
0
0.2
0.4
0.6
0.8
1.0
0
3
6
9
12
15
18
21
24
27
30
33
36
39
42
45
48
51
54
57
60
63
Potamotrygon gr. scobina venom
Potamotrygon cf. orbignyi venom
Fig. 6. Fractionation of Potamotrygon cf. scobina and Potamo-
trygon gr. orbygnyi venoms. (A) Venoms were analyzed by SDS-
PAGE using polyacrylamide gradient gel 4–20%. And stained with
Comanssie Blue. Numbers at left corresponded to position of Mw
markers. (B) Three milligrams of venoms were fractionated on a
FPLC Mono S HR 5/5 column equilibrated with 20 mM Tris/HCl at
pH 8.3 containing 0–2 M NaCl. The flow was 1.0 ml/min and 1 ml
fractions were collected. Protein elution was monitored at 280 nm.
1. Molecular markers. 2. Potamotrygon cf. orbignyi. 3. Potamo-
trygon gr. scobina
K.W. Magalha˜es et al. / Toxicon 47 (2006) 575–583 581

8. slow rolling to firm adhesion, as postulated by Smith et al.
(2004).
Recently, itwas reported thatmiceinjectedwithvenomsof
Thalassophryne nattereri or Thalassophryne maculosa fish
presented severe local tissue destruction accompanied by
thrombosis, without inducing haemorrhage (Lopes-Ferreira
etal.,2001; Sosa-Rosales etal.,2005).Thedata presentedhere
also demonstrate that injection of P. cf. scobina and P. gr.
orbignyi venoms inmiceinduced moderate necrosis, low level
of proteolytic activity, without induncing haemorrhage.
Interestingly, when the venoms of both stingray species
were injected added with their respective mucus the
necrotizing activity was more vigorous (two-fold), indicating
that proteins presented in epithelial mucus secretion can
potentiate the toxic effect of the venoms.
Mucus layer of aquatic organisms are under constant
attack from microorganisms. When threatened or injured,
catfish secretes a thick gel-like layer of proteinaceous
materials, which includes antibodies and proteases, to its
skin surface mainly from the unicellular glands of the
epidermis (Ourth, 1980; Lobb, 1987; Al-Hassan et al.,
1987). Several reports have established antimicrobial
peptides as the host-defense effector molecules that protect
the mucus epithelia from invading microbes (Bevins, 1994;
Park et al., 1998). This may underscore the importance of
other factors as peptides in the mucus in the induction of
toxic activities induced by the Potamotrygon venoms.
Generally, the extraction of the venom of stingrays is
difficult due to the dangerous form of capture of the animals.
Thus, the quantity of the extracted venom is usually very
low and this situation is worsened by its termolability
(Russell and Van Harreveld, 1954; Russell et al., 1957;
Russell et al., 1958). Consistent with these studies, our
results show that heat almost abrogated the edema or the
nociception induced by the P. cf. scobina and P. gr. orbignyi
venoms. Finally, we suggest that the similar biological
activities induced by Potamotrygon venoms are probably
the result of the combined action of several components
present in venoms.
The present study provided in vivo evidence of toxic
effects for P. cf. scobina and P. gr. orbignyi venoms on
target cells in microcirculatory environment. This study also
demonstrated for the first time that toxic effects provoked by
injection of both venoms in mice show moderate levels of
intensity, as well as the presence of proteins within the
mucus that contribute potentiating the local tissue destruc-
tion. Given the critical importance of stingrays accidents in
several river systems of Brazil, this study clarifies the
principal toxic activities of the P. cf. scobina and P. gr.
orbignyi venoms.
Acknowledgements
We are grateful to NEAMB (Nucleus of Environmental
Studies) for support the fieldwork. This work was supported
by the Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o
Paulo (FAPESP).
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