Biological and biochemical properties of the Brazilian

Potamotrygon stingrays: Potamotrygon cf. scobina

and Potamotrygon gr. orbignyi

Kharita W. Magalhaes a, Carla Lima b, Ana Amelia Piran-Soares b, Elineide

E. Marques a, Clelia A. Hiruma-Lima c, Monica Lopes-Ferreira b,*

a Nucleus of Environmental Studies, Federal University of Tocantins, Tocantins, Brazilb Laboratory of Immunopathology, Butantan Institute Av. Vital Brazil, 1500, Butantan, 05503-009 Sao Paulo, SP, Brazil

c Physiology Department, Biosciences Institute, Sao Paulo State University, Sao 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

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).

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

K.W. Magalhaes et al. / Toxicon 47 (2006) 575–583576

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, 5 0-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 (Sao 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. Magalhaes et al. / Toxicon 47 (2006) 575–583 577

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 Cristalia; 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. Magalhaes et al. / Toxicon 47 (2006) 575–583578

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.

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)

Foo

tpad

thic

knes

s (m

m)

Potamotrygon cf. scobina venom

Potamotrygon gr. orbignyi venom

Foo

tpad

thic

knes

s (m

m)

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.

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

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

Noc

icep

tion

(s)

Noc

icep

tion

(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.

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.

K.W. Magalhaes et al. / Toxicon 47 (2006) 575–583 579

(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).

Potamotryg

Potamotryg

Venom + Mucus

Control Ps Po

A

B

Control 6.25 12

Veno

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

Potamotrygon g

Nec

rosi

s (p

rodu

ct o

fdi

amet

ers/

mm

2 )N

ecro

sis

(pro

duct

of

diam

eter

s/m

m 2 )

Fig. 3. Estimation of necrotizing activity. Necrosis was quantified after an i.d

of protein) contained in 50 ml of PBS into the shaved backs of the mice. The n

(50 mg) added with mucus (50 mg) (B). Two diameters were determined f

perpendicular to the longest. Results were expressed as the product of the d

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

oncf. scobina venom + Mucus

ongr. orbignyi venom + Mucus

.5 25 50 100

m ( µg of protein/paw)

. scobina venom

r. orbignyi venom

. injection the different doses of venom (6.25, 12.5, 25, 50, and 100 mg

ecrotic area was measured 72 h after injection of venom (A) or venom

or the necrotic spot by measuring the longest diameter and the one

iameters GSEM. *p!0.05 compared with control-group.

K.W. Magalhaes et al. / Toxicon 47 (2006) 575–583580

(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, 3.7G0.9 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

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.

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. 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.

1 2 3

66.2

45.0

35.0

25.0

18.4

A

B

Abs

orba

nce

(280

nm

)

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 63Potamotrygon 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. Magalhaes et al. / Toxicon 47 (2006) 575–583 581

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

K.W. Magalhaes et al. / Toxicon 47 (2006) 575–583582

slow rolling to firm adhesion, as postulated by Smith et al.

(2004).

Recently, it was reported that mice injected with venoms of

Thalassophryne nattereri or Thalassophryne maculosa fish

presented severe local tissue destruction accompanied by

thrombosis, without inducing haemorrhage (Lopes-Ferreira

et al., 2001; Sosa-Rosales et al., 2005). The data presented here

also demonstrate that injection of P. cf. scobina and P. gr.

orbignyi venoms in mice induced 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 Fundacao de Amparo a Pesquisa do Estado de Sao

Paulo (FAPESP).

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