1. 1. Toxicon 50 (2007) 676687 Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays Katia C. Barbaroa,, Marcela S. Liraa , Marlia B. Maltaa , Sabrina L. Soaresa , Domingos Garrone Netob , Joa o L.C. Cardosoc , Marcelo L. Santorod , Vidal Haddad Juniorc,e a Laboratory of Immunopathology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sao Paulo, SP, Brazil b Department of Zoology, UNESP, 18618-000 Botucatu, SP, Brazil c Hospital Vital Brazil, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sao Paulo, SP, Brazil d Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil 1500, 05503-900 Sao Paulo, SP, Brazil e Dermatology and Radiotherapy, UNESP, 18618-000 Botucatu, SP, Brazil Received 3 April 2007; received in revised form 1 June 2007; accepted 4 June 2007 Available online 22 June 2007 Abstract Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE), tissue extracts have similar bands above 80 kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was veried in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A2 and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specic sera reacted with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic, caseinolytic and brinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents. r 2007 Elsevier Ltd. All rights reserved. Keywords: Stingrays; Potamotrygon; Dasyatis; Venom; Toxin ARTICLE IN PRESS www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2007.06.002 Corresponding author. Tel.: +55 11 37267222x2278/2134; fax: +55 11 37261505. E-mail addresses: kbarbaro@butantan.gov.br, kbarbaro@usp.br (K.C. Barbaro).
2. 2. 1. Introduction In Brazil, freshwater stingrays are very common in northern, central-western and southeastern rivers, and marine stingrays are distributed throughout Atlantic Ocean coast. The family Potamotrygonidae comprises the only group of rays totally restricted to uvial systems (Rosa, 1985; Compagno and Cook, 1995), occurring only in the major river basins of South America; they are distributed in three genera: the monotypic genera Plesiotrygon (Plesiotrygon iwamae), Paratrygon (Paratrygon aiereba), and the Potamotrygon genus, which has 1618 valid species (Rosa, 1985; Charvet-Almeida et al., 2002; Carval- ho et al., 2003; Garrone Neto et al., 2007). Stingrays of the Dasyatidae family are marine, encompassing six genera and about 50 species that are widely distributed in tropical and temperate waters of Australia, Asia, Africa and America (Taniuchi, 1979; Roberts and Karnasuta, 1987; Taniuchi et al., 1991). Similar to other elasmobranchs, certain species of dasyatids are euryhaline, invade rivers and live in estuaries for extended periods of time (Thorson, 1983; Thorson et al., 1983). On account of that, some authors, based on physiological/ ecological ndings, suggest that potamotrygonids might be nested within dasyatids in a unique familyDasyatidae, with two subfamilies: Dasya- tinae and Potamotrygoninae (Nelson, 1994; Nishi- da, 1990). However, systematic studies with parasites suggested that potamotrygonids are most closely related to Pacic coast members of the genus Urolophus (Urolophidae) than dasiatids (Brooks et al., 1981; Lovejoy, 1996). Taxonomists consid- ered urolophids to be the sister group to the neotropical freshwater rays, and used them as the outgroup to assess intra-potamotrygonid relation- ships (Rosa, 1985; Rosa et al., 1987). Stingrays are commonly found in supercial waters, where they lay on the sandy or muddy bottom, or remain partially hidden beneath it (Halstead, 1966; Halstead, 1970). These animals have one or more retroserrated stingers on the tail, covered by integumentary and glandular tissues, where the venom is produced (Charvet-Almeida et al., 2002; Carvalho et al., 2003; Haddad et al., 2004). Such habits increase the possibility of accidents, which occur when humans accidentally step on the dorsal part of stingrays, and animals whip the tail to the stimulated site to defend themselves. Almost all patients present pain at the injury site, followed by edema and erythema. Skin necrosis and ulcers can occur, mainly if injuries are caused by freshwater stingrays, and they may take up to 3 months to heal (Haddad, 2000; Haddad et al., 2004). The most inicted anatomic region is the lower limbs (Haddad et al., 2004; Brisset et al., 2006; Lim and Kumarasinghe, 2007). Lethal injuries are very rare and usually occur when the stinger reaches vital organs (Isbister, 2001). In Brazil, injuries provoked by freshwater stingrays are common in the northern and middle- western regions, where they are considered as an important public health problem. In other regions, such as the southwestern one, due to the construc- tion of hydroelectric power plants, stingrays have gradually occupied larger areas in the last years, causing alterations in the epidemiological aspects of accidents caused by venomous animals. Thirty years ago, stingrays have been observed only in the Lower and Medium Parana River, in the boundaries with Paraguay and Argentina countries. Today, stingrays occur in areas located upstream, such as the Upper Parana River and tributaries, in Sa o Paulo and Mato Grosso do Sul States, Brazil. The risk of accidents increases when sand beaches are formed along the river, providing a touristic appeal to the region and increasing the possibility of an encounter between shermen or swimmers and stingrays (Garrone Neto et al., 2007). Several toxic activities have already been reported in extracts obtained from the tissue covering the stingers, and therefore many authors have named them as venom. Neurotoxic, cardiotoxic and lethal activities were observed by Vellard (1931, 1932). Nociception, dyspnea, agitation and convul- sion were also noted when experiments with the intact stinger of Dasyatis pastinaca were carried out in guinea pigs (Fleury, 1950). Cardiac and circula- tory disturbances observed in cats were likely due to the direct action of Urolophus halleri venom in myocardium cells (Russell and Van Harreveld, 1954; Russell et al., 1957). Recently, Magalha es et al. (2006) compared Potamotrygon cf. scobina and Potamotrygon gr. orbignyi venoms and showed that they could induce nociception, edema, necrosis and disturbances in murine microcirculation. These authors also showed that the mucus dispersed through the stingers could potentiate necrosis. Many enzymes were detected in marine and freshwater stingray venoms: phosphodiesterase and 50 -nucleotidase activities in U. halleri venom (Russell et al., 1958); 50 -nucleotidase, phospholi- pase, hyaluronidase and protease activities in ARTICLE IN PRESS K.C. Barbaro et al. / Toxicon 50 (2007) 676687 677
3. 3. Potamotrygon motoro (Magalha es, 2001), and hya- luronidase, caseinolytic, gelatinolytic and brino- genolytic activities in Potamotrygon falkneri venom (Haddad et al., 2004). A peptide with vasoconstric- tive activity was isolated from Potamotrygon orbignyi venom by Conceic-a o et al. (2006). Aiming to obtain detailed information about stingray tissue extracts, a comparative study of some biochemical, biological, enzymatic and im- munological features of freshwater (P. falkneri) and marine (Dasyatis guttata) stingray tissue extracts was carried out herein. 2. Materials and methods 2.1. Animals and tissue extracts Swiss male mice (1820 g) and adult rabbits (34 kg) were provided by Butantan Institute Animal House. Animals received food and water ad libitum. Specimens of P. falkneri and D. guttata were collected in Sa o Paulo and Mato Grosso do Sul States. Tissue extracts were obtained from the integumentary tissue covering the stinger as pre- viously described (Haddad et al., 2004). The protein content of tissue extract pools was determined by bicinchoninic acid method (Smith et al., 1985), using bovine serum albumin (BSA) as a standard. The procedures involving animals were conducted in conformity with national laws and policies controlled by Butantan Institute Animal Investiga- tion Ethical Committee (protocol no. 115/2002). 2.2. Production of sera against P. falkneri and D. guttata tissue extracts Sera against P. falkneri and D. guttata tissue extracts were obtained by immunization of two rabbits, one for each type of tissue extract. Tissue extracts (200mg) were diluted in 500ml of PBS and added to 500ml of complete Freunds adjuvant, and these mixtures were injected i.m. into rabbits. After 1 month, animals received ve additional boosters of antigen, suspended in incomplete Freunds adjuvant, at fortnight intervals. Blood was collected and sera was separated and stored at 201C until used. 2.3. ELISA Rabbit species-specic sera were titrated by ELISA, using P. falkneri and D. guttata tissue extracts (10 mg/ml) to coat the microplates (Nunc, USA), according to Theakston et al. (1977). The reaction was read using an ELISA reader (Multis- kan EX) and the titer determined as the reciprocal of the highest dilution that causes an absorbance greater than 0.050 at 492 nm, since non-specic reactions were observed below this value. 2.4. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) Proteins of P. falkneri and D. guttata tissue extracts (5mg) were analyzed by SDSPAGE (420%, Pierce, USA) under non-reducing conditions (Laemmli, 1970), and then proteins were silver stained (Blum et al., 1987). Myosin, b-galactosidase, BSA, carbonic anhy- drase, soybean trypsin inhibitor, lysozyme and aproti- nin were used as the molecular mass markers (Kaleidoscope prestained standards; BioRad, USA). 2.5. Western blotting Proteins of P. falkneri and D. guttata tissue extracts (20 mg) were rst fractionated by SDSPAGE as described above, and then electro- blotting was performed as described by Towbin et al. (1979). Nitrocellulose membranes were in- cubated with species-specic rabbit antisera, diluted at 1/250. Immunoreactive proteins were detected using peroxidase-labeled anti-rabbit IgG and the blot was developed with 0.05% (w/v) 4-chloro-1- naphtol in 15% (v/v) methanol, in the presence of 0.03% (v/v) H2O2. Non-immunized rabbit serum was used as a control. Pre-stained molecular mass markers (BioRad, USA) were used. 2.6. Proteolytic and hyaluronidase assays Zymography was used to assay protease and hyaluronidase activities. Casein (Merck, Darmstadt, Germany), gelatin and brinogen (Sigma, St. Louis, MO) were used as substrates to assay proteolytic activity (Heussen and Dowdle, 1980; Barbaro et al., 2005), and hyaluronic acid from rooster comb (Sigma, St. Louis, MO) to determine hyaluronidase activity (Miura et al., 1995; Barbaro et al., 2005). Samples of P. falkneri and D. guttata tissue extracts in non- reducing sample buffer were loaded into gels and run at 20mA/gel. Clear areas in the gel indicated regions of enzyme activity. When required, Na2-EDTA or 1,10-phenanthroline (Sigma, St. Louis, MO) was added in a nal concentration of 5mM to gel washing and incubation buffers, and then gel was stained as ARTICLE IN PRESS K.C. Barbaro et al. / Toxicon 50 (2007) 676687678
4. 4. usual. Pre-stained molecular mass markers (BioRad, Hercules, CA) were used. 2.7. Lethal activity Mice (n 4) were injected i.p. with 100, 200, 400 or 800 mg of P. falkneri and D. guttata tissue extracts diluted in 200 ml of PBS. Lethality was observed 24 and 48 h after i.p. tissue extracts injections. 2.8. Local reaction and dermonecrotic activity induced by P. falkneri and D. guttata tissue extracts Local reaction (edema/erythema and paleness/ ecchymosis areas) and dermonecrotic activity were determined by i.d. injection of 100, 200 or 400 mg of P. falkneri or D. guttata tissue extracts, dissolved in 0.1 ml of PBS, into the mouse dorsum skin (n 4). Animals were sacriced by CO2 inhalation and the inner dorsum skin was observed. Areas of edema/ erythema, paleness/ecchymosis and dermonecrosis were inspected 72 h after injection and reported as the mean of four different areas (mm2 ) for each parameter studied. Animals injected only with PBS were used as negative controls. 2.9. Nociceptive and edematogenic activities of P. falkneri and D. guttata tissue extracts To detect nociceptive activity, mice (n 8) were injected with different doses (1, 4, 16 and 64 mg) of P. falkneri and D. guttata tissue extracts dissolved in 30 ml of PBS into the right hind paw. Animals were individually placed under glass funnels on a mirror. Afterwards, the reactivity of animals to lick or bite the injected paw was measured, in seconds, during 30 min of experimental evaluation (Hunskaar et al., 1985). Animals injected only with PBS were used as negative controls. Edema-forming activity was evaluated at differ- ent time intervals (15 min, 1, 2, 24 and 48 h) as the difference of thickness (mm) measured with a caliper between the right hind pawinjected with different doses (1, 4, 16 and 64 mg) of P. falkneri and D. guttata tissue extracts diluted in PBS, or PBS alone (negative control)and the left hind paw of mice, which received no injection. 2.10. Estimation of myotoxic activity Mice (n 8) were injected i.m. into the right gastrocnemius muscle with 100, 200 or 300 mg of P. falkneri and D. guttata tissue extracts dissolved in 50 ml of PBS. The control group was injected with PBS alone. After 3 h, blood was collected from the ophthalmic plexus. Sera of mice were separated and was immediately assayed for creatine kinase (CK) activity (CK-NAC Liquiform, LABTEST, Brazil). One unit corresponds to the amount of enzyme that hydrolyzes 1 mmol of creatine per min at 25 1C. Myotoxic activity was expressed as U/mg of tissue extract of three independent experiments. Bothrops jararacussu snake venom (150 mg) was used as a positive control. 2.11. Coagulant activity Clotting time was performed according to Santoro and Sano-Martins (1993). P. falkneri and D. guttata tissue extracts (1, 4, 16 and 64 mg) diluted in 50 ml of PBS were added to 200 ml human plasma. Samples (duplicate) were observed for 5 min at 37 1C to determine the clotting time. Thereafter, to verify brinogen hydrolysis, 50 ml of bovine throm- bin (30 U/ml) (Sigma, USA) was added to the mixture. As a positive control, 50 ml samples of two- fold serially diluted B. jararaca snake venom (1.56200.0 mg) were used to determine the minimal coagulant dose (MCD). Experiments were carried out twice. 2.12. Direct hemolytic activity Human blood (type O, Rh+) was collected in the presence of 0.15 M sodium citrate (9:1) and cen- trifuged at 1900g for 15 min at 10 1C, as described by Boman and Ralleta (1957). Red blood cells were obtained and washed three times with PBS. Samples (50 ml) of PBS containing 3% of red blood cells were mixed with 100 ml of different doses (1, 4, 16 and 64 mg) of P. falkneri and D. guttata tissue extracts. Each sample (50 ml) was placed (duplicates) in microplates. As controls distilled water (100% hemolysis) and PBS (0% hemolysis) were used. Microplates were kept at room temperature for 3 h. The absorbance was read using an ELISA reader (Multiskan EX) at 595 nm. 2.13. Phospholipase A2 activity Phospholipase activity was determined as de- scribed elsewhere (Santoro et al., 1999). P. falkneri and D. guttata (15, 30, 60 and 120 mg) tissue extracts diluted in 15 ml of PBS, pH 7.4 were added to 1.5 ml ARTICLE IN PRESS K.C. Barbaro et al. / Toxicon 50 (2007) 676687 679
5. 5. of the reaction solution (100 mM NaCl, 10 mM CaCl2, 7 mM Triton X-100, 0.265% soybean lecithin, 98.8 mM phenol red, pH 7.6) in a spectro- photometer cuvette. The solution was immediately homogenized and read at 556 nm. The denition of 1 U of phospholipase A2 activity was taken as the amount of toxin (mg of protein/assay) producing a decrease of 0.001 absorbance units per min under the conditions described. Crotalus durissus terricus snake venom (6 mg) was used as a positive control. Phospholipase activity was expressed as U/mg of two independent experiments. 2.14. Statistical analysis Results were expressed as mean7SD. Two-way ANOVA followed by Bonferronis test was used to analyze data, using SigmaStat 3.0 software. Values with po0.05 were considered statistically signicant. 3. Results 3.1. Analyses of tissue extracts by SDS PAGE Fig. 1 shows the electrophoretic pattern of P. falkneri and D. guttata tissue extracts. After SDSPAGE, under non-reducing conditions, many components with similar molecular masses were noticed in both tissue extracts, mainly above 84 kDa, which were difcult to be separated. Some bands around 22 kDa were observed exclusively in P. falkneri tissue extract. A strong and diffuse band was observed between 43 and 65 kDa in P. falkneri tissue extract. Gels also showed major components below 18 kDa in both tissue extracts. At least 16 bands were distributed along the gel in both tissue extracts (Fig. 1). 3.2. Cross-reactivity determined by ELISA and Western blotting Table 1 shows the comparison of antibody titers obtained by ELISA between the two antisera assayed against homologous and heterologous antigens. Both tissue extracts were immunogenic and could induce high levels of antibodies in rabbits. Intense cross-reactivity between both tissue extracts was detected, and no signicant differences on titers were noticed (only variations higher than two-fold dilutions were considered signicant). Fig. 2 shows immunoblots of D. guttata and P. falkneri tissue extracts after incubation with species-specic sera produced in rabbits. Many components of both tissue extracts, mainly above 23 kDa, reacted with both antisera, likely due to the presence of many common proteins in the integu- mentary tissue. However, when compared with homologous antisera, heterologous antisera failed to react or faintly reacted with some components below 30 kDa, demonstrating the presence of unique components showing no immunological identity between each tissue extract. 3.3. Enzymatic activities of tissue extracts Casein, gelatin, brinogen or hyaluronic acid were incorporated as substrates in 12.5% acrylamide gels, ARTICLE IN PRESS Pf 83.91 133.08 205.73 41.56 31.35 17.25 7.01 Dg Fig. 1. Electrophoretic proles of P. falkneri (Pf) and D. guttata (Dg) tissue extracts (5 mg) after 420% SDSPAGE under non- reducing conditions. Gel was silver stained. Numbers on the right correspond to the position of molecular mass markers. Table 1 Antigenic cross-reactivity between D. guttata and P. falkneri tissue extracts using rabbit antisera by ELISA Antisera Tissue extracts D. guttata P. falkneri Anti-D. guttata 1,024,000a 1,024,000 Anti-P. falkneri 512,000 1,024,000 Normal o200b o200b a ELISA titers. Microplates were coated with each tissue extract, and then incubated with homologous or heterologous rabbit antisera. b Initial dilution 1/200. K.C. Barbaro et al. / Toxicon 50 (2007) 676687680
6. 6. and used to assay enzymatic activities of tissue extracts. Several components with molecular mass above 83kDa showed similar proles for caseinolytic, gelatinolytic and brinogenolytic activities (Fig. 3). However, some components below 60kDa showed these activities only in D. guttata tissue extract. Incubation with Na2- EDTA or 1,10-phenanthroline, inhibitors of metallo- proteinases, could not abolish the activity of most components (data not shown). No hyaluronidase activity was observed in D. guttata tissue extract (Fig. 3). A diffuse band between 41 and 65kDa was observed in P. falkneri tissue extract. Minimal phos- pholipase A2 activity was detected in both tissue extracts (2267U/mg to P. falkneri and 5200U/mg to D. guttata tissue extracts) even when high doses of tissue extract (120mg) were used. C. d. terricus snake venom (114,500U/mg) and PBS were used as a positive and negative control, respectively. 3.4. Toxic activities Only P. falkneri tissue extract could evoke a dose- dependent local reactionareas of edema/erythema and paleness/ecchymosis, and necrosisshowing an intense inammatory reaction at the site of injection (Table 2). No signs of inammatory reactions were observed in animals injected with D. guttata tissue extract, even when higher doses were administered ARTICLE IN PRESS Fig. 2. (A) Electrophoretic proles of P. falkneri (Pf) and D. guttata (Dg) tissue extracts (25 mg) after 12.5% SDSPAGE under non-reducing conditions. Gel was silver stained. (B) Antigenic cross-reactivity of P. falkneri (Pf) and D. guttata (Dg) tissue extracts was determined by Western blotting, using anti-D. guttata and anti-P. falkneri rabbit sera. Numbers on the left correspond to the position of molecular mass markers. Pf Dg 210- 131- 89- 41.3- 31.8- 18.1- Casein Pf Dg 210- 131- 89- 41.3- 31.8- 18.1- Gelatin Pf Dg 210- 131- 89- 41.3- 31.8- 18.1- Fibrinogen Hyaluronic Acid Pf Dg 17.26- 7.01- 31.35- 41.56- 83.91- 133.08- 205.73- Fig. 3. Caseinolytic, gelatinolytic, brinogenolytic (20 mg) and hyaluronidase (60 mg) activities of P. falkneri (Pf) and D. guttata (Dg) tissue extracts were determined using the technique of substrate SDSPAGE 12.5%. Numbers on the left correspond to the position of molecular mass markers. Clear areas in the gel indicate regions of enzymatic activity. K.C. Barbaro et al. / Toxicon 50 (2007) 676687 681
7. 7. (400 mg). Deaths could be observed only in animals injected with P. falkneri tissue extract. All animals injected with 800 mg of tissue extract died within 24 h after the tissue extract administration (Table 3). Both tissue extracts induced nociceptive activity (Fig. 4), which was dose dependent. However, only the highest dose of D. guttata tissue extract (64 mg) showed a statistically signicant difference with the control, whereas low doses of P. falkneri could induce pain. The edematogenic activity of both tissue extracts peaked at 15 min after the tissue extract injection. Edema decreased gradually until 24 h, when statistically signicant edema was no more observed (Fig. 5). No direct hemolytic activity was detected in both tissue extracts, even when 64 mg of each tissue extract was used. P. falkneri and D. guttata tissue extracts showed no coagulant activity. The MCD of B. jararaca snake venom (positive control) was 10.56 mg/ml. P. falkneri and D. guttata tissue extracts could not clot human plasma. When bovine thrombin (30 U/mg, nal concentration) was added to plasma previously incubated with all the tested doses of tissue extracts, clotting times were compar- able to those of plasma incubated with saline (1015 s). Fig. 6 shows that higher myotoxic activity was present in P. falkneri tissue extract than in D. guttata tissue extract. The latter showed CK levels similar to the control group (PBS). P. falkneri tissue extract, even in a low dose (100 mg), could induce a remarkable CK release, and this activity was dose dependent. Animals injected with PBS (7997367 U/l) or B. jararacussu (100 mg) snake venom (435071596 U/l) were used as negative and positive controls, respectively. 4. Discussion Stingrays have one or more retroserrated stingers adhered to their tail, which are covered by an integument composed of glandular tissues contain- ing toxic activities (Halstead, 1970). When intro- duced into the victim, the stinger causes a traumatic injury that may worsen if the integument is retained in the wound. Herein, we present a comparative study between tissue extracts of freshwater (P. falkneri) and marine (D. guttata) stingrays. By SDSPAGE, we observed several components of high molecular masses in P. falkneri and D. guttata tissue extracts. We suppose that many of those components belong to the tissue that recovered the stinger, once the stingrays do not have a venom-individualized gland. A great number of different components are observed below 84 kDa in both tissue extracts. Morphological studies of the stinger structure showed that more specialized cells with high protein content are observed in P. falkneri than in D. guttata venom-secreting tissue (Pedroso et al., submitted). This structural difference could explain the variation observed in the electrophoretic prole noticed between both tissue extracts. Both tissue extracts were immunogenic and could induce large amounts of antibodies. No signicant difference was observed between the titers of each species-specic antiserum, even when heterologous ARTICLE IN PRESS Table 2 Local reaction and necrosis induced by P. falkneri and D. guttata tissue extracts Tissue extracts Necrosis (mm2 ) Local reaction (mm2 ) D. guttata 100 mg 0 0 200 mg 0 0 400 mg 0 0 P. falkneri 100 mg 0 7.5711.4 200 mg 21.0718.5 31.5714.8 400 mg 13.3710.8 191.5716.5 Mice were injected i.d. with different doses of P. falkneri or D. guttata tissue extracts diluted in 0.1 ml of PBS. Local reaction (edema/erythema and paleness/ecchymosis) and necrosis were evaluated after 72 h. Data are expressed as mean7SD. Table 3 Lethal activity of P. falkneri and D. guttata tissue extracts Tissue extracts Lethality (h) 24 48 D. guttata 100 mg 0/4a 0/4 200 mg 0/4 0/4 400 mg 0/4 0/4 800 mg 0/4 0/4 P. falkneri 100 mg 0/4 0/4 200 mg 0/4 0/4 400 mg 2/4 3/4 800 mg 4/4 4/4 Mice (n 4) were injected i.p. with different doses of P. falkneri or D. guttata tissue extracts, diluted in 0.2 ml of PBS. Deaths were observed 24 and 48 h after tissue extract injection. a Dead/injected. K.C. Barbaro et al. / Toxicon 50 (2007) 676687682
8. 8. tissue extract was used to coat the microplates. Antigenic cross-reactivity was detected by ELISA and Western blotting, indicating the presence of many common epitopes among tissue extract components, especially above 41 kDa. This was expected since many common housekeeping pro- teins are shared by the constitutive tissue covering both stingers. Both antisera weakly reacted with components below 17 kDa, indicating that they are weakly immunogenic, since they are present in high quantities, as detected by electrophoresis. The intense inammation reaction observed in human injuries is suggestive of disturbances in the extracellular matrix. As previously reported ARTICLE IN PRESS Time (hours) Time (hours) Thickness(cm) 0.00 0.05 0.10 0.15 0.20 0.25 1 2 24 48 PBS 1 g 4 g 16 g 64 g # Thickness(cm) 0.00 0.05 0.10 0.15 0.20 0.25 0.25 1 2 24 PBS 1 g 4 g 16 g 64 g # # 48 Fig. 5. Edematogenic activity of P. falkneri (A) and D. guttata (B) tissue extract. To investigate edematogenic activity, different doses of tissue extract were diluted in 30 ml of PBS solution and injected intraplantarly, using vehicle as a negative control. Edema evaluation was carried out by measuring the difference in thickness (cm) between the injected and non-injected hind paws at different time intervals. *po0.001 and #po0.05statistically signicant difference between the experimental and control (PBS) groups. PBS 1 g 4 g 16 g 64 g Reactivity(seconds) 0 20 40 60 80 100 120 140 160 PBS P. falkneri D. guttata Fig. 4. Nociceptive activity of P. falkneri and D. guttata tissue extracts. To evaluate nociceptive activity, different doses of each tissue extract were diluted in 30 ml of PBS and injected intraplantarly. Vehicle was used a negative control. Reactivity was expressed as the time (in s) to animals to lick and/or bite the injected paw during a period of 30 min. *Statistically signicant (po0.05) difference between the experimental and control (PBS) groups. K.C. Barbaro et al. / Toxicon 50 (2007) 676687 683
9. 9. (Haddad et al., 2004), Zymographic analyses showed that P. falkneri tissue extract contains enzymes that can degrade distinct proteins such as casein, gelatin and brinogen. In fact, enzymatic activity was more intense in D. guttata than in P. falkneri tissue extract, and enzymes with mole- cular mass below 83 kDa could hydrolyze all substrates. In both tissue extracts, the prole of enzymatic degradation was similar using distinct substrates, indicating the presence of proteases with broad substrate specicity. These results suggest that such proteases could contribute to degradation of proteins and components present in the extra- cellular matrix, favoring the establishment of local injury. On the other hand, most of those enzymes are not metalloproteinases, once the incubation with Na2-EDTA or 1,10-ortho-phenantroline could not inhibit their enzymatic activity (data not shown). Under our experimental conditions, we only observed hyaluronidase activity in P. falkneri tissue extract, corroborating results described pre- viously (Magalha es, 2001; Haddad et al., 2004). The presence of hyaluronidases in P. falkneri tissue extract could amplify the local damage caused by toxins as well as the injury caused by the stinger. Several components with enzymatic activity have been described in venomous animals (Tan and Ponnudurai, 1992; Birkedal-Hansen et al., 1993; Veiga et al., 2000; Haddad et al., 2004; Lira et al., 2007). These enzymes could degrade components of extracellular matrix and function as diffusion factors, or they can act directly in the degradation of proteins, likely contributing to tissue injury. Biological activities were investigated according to the major symptoms described in human accidents. We observed that P. falkneri and D. guttata tissue extracts could induce a dose- dependent nociceptive activity. However, P. falkneri tissue extract was two times more active than D. guttata tissue extract. In human envenomation, pain is worsened by the mechanical damage caused by the stinger, which lacerates the local tissues, potentiating the action of toxins. Nociceptive activity might be associated with the direct action of toxic components, since it was observed imme- diately after tissue extract injection. Severe pain is also reported in human injuries (Isbister, 2001; Haddad et al., 2004). Besides, components with enzymatic activity could also promote a tissular injury, inducing an inammatory reaction with the release of mediators involved in nociception. Our results showed that both tissue extracts evoked a similar dose-dependent edema, which is more intense within 15 min after injection, returning to ARTICLE IN PRESS CreatinekinaseU/L 0 1000 2000 3000 4000 5000 6000 7000 PBS Bjssu Dg Pf Dg Pf Dg Pf 100 g 200 g 300 g # # # Fig. 6. Myotoxic activity of P. falkneri and D. guttata tissue extracts (100, 200 and 300 mg). *Statistically signicant difference between the experimental and negative control groups (PBS) (po0.05). # Statistically signicant difference between the experimental and the positive control group (Bjssu, Bothrops jararacussu snake venom, 150 mg) (po0.05). K.C. Barbaro et al. / Toxicon 50 (2007) 676687684
10. 10. basal levels within 24 h. Edema is also observed in the limbs of patients aficted by the stinger (Haddad et al., 2004). Under our experimental conditions, we veried that only the tissue extract of P. falkneri could induce necrosis and an intense inammatory reac- tion at the site of injection. These data are in agreement with reports of accidents in humans that demonstrate that necrosis and local inammation are much more prominent in injuries caused by freshwater stingrays (Haddad, 2000; Haddad et al., 2004). Our results agree with those reported by Castex et al. (1964), who stung or injected the mixed tegument, which covered the stinger of Potamotry- gonidae freshwater stingrays, i.m. or i.p. into guinea pigs. Magalha es et al. (2006) also observed edema, nociception and necrosis in Potamotrygon cf. scobina and P. gr. orbignyi venoms, and that the mucus covering the animal could augment this necrotic activity. The mechanism of the pain induction, edema and necrosis in the accidents for stingrays is still uncertain, but mucus certainly contributes to the injury caused by stingrays (Castex et al., 1964; Magalha es et al., 2006). Myotoxicity was found only in P. falkneri tissue extract. CK release could be due to the action of myotoxins, as it occurs in B. jararacussu envenoma- tion (Gutie rrez and Lomonte, 2003), or caused by the intense inammatory reaction, which can induce muscular damage, as observed in Loxosceles spider envenomation (Franc-a et al., 2002). In humans, the local damage caused by stingers could also con- tribute to expose muscular tissue to noxious enzymes and toxins. Lethal activity was detected only in P. falkneri tissue extract. High amounts of samples were necessary to cause the death of mice, probably because the sample used was a mixture of toxic components and integument constitutive tissue. Russell et al. (1957) reported that the LD50 of Urolophus stingray tissue extract was 28 mg/kg. That value is similar to that obtained in our experiments, since mortality started to be detected from 20 mg/kg. Other studies observed neurotoxic symptoms in animals experimentally injected with stingray tissue extracts, and i.v. injection could cause the death of mice (30 g) in 1020 min (Vellard 1931, 1932). We veried that D. guttata and P. falkneri tissue extracts did not induce direct hemolysis, conrming data published by Vellard (1931, 1932) using Potamotrygon sp. venom. Besides, tissue extracts could not prolong the clotting time of plasma nor consume plasma brinogen under our experimental conditions, indicating that these tissue extracts do not act directly in the coagulation cascade, as observed in many animal venoms, such as Bothrops snakes or Lonomia caterpillars (Sano-Martins and Santoro, 2003). The results presented herein enhance the informa- tion available about Brazilian stingray envenoma- tion. We veried that stingray tissue extract is a complex mixture of components, inducing different toxic activities depending on the species being studied. The morphological differences observed in the tegument that covers the stinger can also contribute to explain the different toxicity between tissue extracts (Pedroso et al., submitted). Our results suggest that P. falkneri and D. guttata tissue extracts have peculiar characteristics that can inuence their toxic activity, and consequently the clinical picture manifested by patients after stingray accidents. Since a specic treatment does not exist to stingray envenomation, and the therapeutical approach is symptomatic (use of anti-inammatory, analgesic drugs and antimicrobials to prevent infection), our data may contribute to understand the mechanisms of action of these tissue extracts. Acknowledgments This work was supported by FAPESP (03/06873-4). The authors thank Danieli M. Rangel, Guilherme C. Rocha, Thais A. Oliveira and Letcia M. P. Martins for technical assistance and Miss Ottilie Carolina Forster and Dr. Maria Jose Alencar Vilela, who provided some conditions to develop this work. The authors also thank the shermen in Ubatuba (Zeca, Bideco, Adalto, Quim, Elias, Major, Cebolinha, Paco, Nando, Santana, Amorim, Natalcio, Rafael, among others) and Tre s Lagoas (Marquinhos and Edmilson) cities for helping in the capture of stingrays. We also thank CNPq for the Grants of Katia C. Barbaro (306158/2004-3) and Domingos Garrone Neto (142985/2005-8). IBAMA provided animal collection permits (02027002992/ 2004-79) and CGEN provided the license for genetic patrimony access (041/05). References Barbaro, K.C., Knysak, I., Martins, R., Hogan, C., Winkel, K., 2005. Enzymatic characterization antigenic cross-reactivity and neutralization of dermonecrotic activity of ve Loxosceles ARTICLE IN PRESS K.C. Barbaro et al. / Toxicon 50 (2007) 676687 685
11. 11. spider venoms of medical importance in the Americas. Toxicon 45, 489499. Birkedal-Hansen, H., Moore, W.G., Bodden, M.K., Windsor, L.J., Birkedal-Hansen, B., DeCarlo, A., Engler, J.A., 1993. Matrix metalloproteinases: a review. Crit. Rev. Oral Biol. Med. 4, 197250. Blum, H., Beier, H., Gross, H.J., 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 9395. Boman, H.G., Ralleta, U., 1957. Chromatography of rattlesnake venoma separation of 3 phosphodiesterases. Biochim. Biophys. Acta 24, 619631. Brisset, I.B., Schaper, A., Pommier, P., Haro, L., 2006. Envenomation by amazoniam freshwater stingray Potamo- trygon motoro: 2 cases reported in Europe. Toxicon 47, 3234. Brooks, D.R., Thorson, T.B., Mayes, M.A., 1981. Fresh-water stingrays (Potamotrygonidae) and their helminth parasites: testing hypotheses of evolution and coevolution. In: Funk, V.A., Brooks, D.R. (Eds.), Advances in Cladistics. New York Botanical Garden, New York, pp. 147175. Carvalho, M.R., Lovejoy, N.R., Rosa, R.S., 2003. Family Potamotrygonidae (river stingray). In: Reis, R.E., Kullander, S.O., Ferraris, Jr., C.J. (Eds.), Check List f the Freshwater Fishes of South and Central Ame rica. Edipucrs, Porto Alegre, pp. 2228. Castex, M.N., Pedace, E., Maciel, I., Meyer, S.J., Murphy, M., Remonda, G., Alewaerts, F., 1964. La enfermedad para- trygo nica: cuadros clnicos experimentales y estudio anato- mopatolo gico. Prensa Med. Arg. 51, 10851095. Charvet-Almeida, P., Arau jo, M.L.G., Rosa, R.S., Rinco n, G., 2002. Neotropical freshwater stingrays: diversity and con- servation status. Shark News 14, 4751. Compagno, L.J.V., Cook, S.F., 1995. The exploitation and conservation of freshwater elasmobranchs: status of taxa and prospects for the future. J. Aquaric. Aquat. Sci. 7, 6290. Conceic-a o, K., Konno, K., Melo, R.L., Marques, E.E., Hiruma- Lima, C.A., Lima, C., Richardson, M., Pimenta, D.C., Lopes-Ferreira, M., 2006. Orpotrin: a novel vasoconstrictor peptide from this venom of the Brazilian stingray Potamo- trygon gr. orbignyi. Peptides 27, 30393046. Fleury, R., 1950. Lappareil venimeux des se laciens trygoni- formes. Mem. Soc. Zool. France 30, 137. Franc-a, F.O., Barbaro, K.C., Abdulkader, R.C., 2002. Rhabdo- myolysis in presumed viscero-cutaneous loxoscelism: report of two cases. Trans. R. Soc. Trop. Med. Hyg. 96, 287290. Garrone Neto, D., Haddad Jr., V., Vilela, M.J.A., Uieda, V.S., 2007. Registro de ocorre ncia de duas espe cies de Potamotrigondeos na regia o do Alto do Rio Parana e algumas considerac-o es sobre biologia. Biota Neotrop. Jan/ Abr 2007 7, 1. http://www.biotaneotropica.org.br/v7n1/pt/ abstract?shortcommunication+bn00707012007/http://www. biotaneotropica.org.br/v7n1/pt/abstract?short communication +bn00707012007S ISSN:1676-0603. Gutie rrez, J.M., Lomonte, B., 2003. Efectos locales en el envenenamiento ofdico en Ame rica Latina. In: Cardoso, J.L.C., Franc-a, F.O.S., Wen, F.H., Ma laque, C.M.S., Haddad, Jr., V. (Eds.), Animais Pec-onhentos no Brasil. Biologia, Clnica e terape utica dos Acidentes, Sarvier, pp. 310323. Haddad Jr., V., 2000. Atlas de animais aqua ticos perigosos do Brasil: guia me dico de identicac-a o e tratamento. Editora Rocca, Sa o Paulo, pp. 123128. Haddad Jr., V., Garrone, N.D., de Paula, N.J.B., Marques, F.P.L., Barbaro, K.C., 2004. Freshwater stingrays: study of epidemiologic, clinic and therapeutic aspects based on 84 envenomings in humans and some enzymatic activities of the venom. Toxicon 43, 287294. Halstead, B.W., 1966. Venomous marine animals of Brazil. Mem. Inst. Butantan 33, 126. Halstead, B.W., 1970. Poisonous and Venomous Marine Animals of the World. Gov Print Ofce: 3, Washington, DC, USA, pp. 2969. Heussen, C., Dowdle, E.B., 1980. Electrophoretic analysis of plasminogen in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102, 196202. Hunskaar, S., Fasmer, O.B., Hole, K., 1985. Formalin test in mice, a useful technique for evaluating mild analgesics. J. Neurosci. Methods 14, 6976. Isbister, G., 2001. Venomous sh stings in tropical northern Austra lia. Am. J. Emerg. Med. 19, 561565. Laemmli, U.K., 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680685. Lim, Y.L., Kumarasinghe, S.P.W., 2007. Cutaneous injuries from marine animals. Singapore Med. J. 48, 2528. Lira, M.S., Furtado, M.F., Martins, L.M.P., Lopes-Ferreira, M., Santoro, M.L., Barbaro, K.C., 2007. Enzymatic and im- munochemical characterization of bothrops insularis venom and its neutralization by polyspecic Bothrops antivenom. Toxicon 49, 982994. Lovejoy, N.R., 1996. Systematics of myliobatoid elasmobranchs: with emphasis on the phylogeny and historical biogeography of neotropical freshwater stingrays (Potamotrygonidae: Rajiformes). Zool. J. Linnean Soc. 117, 207257. Magalha es, K.W., Lima, C., Piran-Soares, A.A., Marques, E.E., Hiruma-Lima, C.A., Lopes-Ferreira, M., 2006. Biological and biochemical properties of the Brazilian Potamotrygon stin- grays: Potamotrygon cf. scobina and Potamotrygon gr. orbignyi. Toxicon 47, 575583. Magalha es, M.R., 2001. Estudos bioqumicos do veneno de raias Potamotrygon motoro (Chondrichthyes: Dasyatidae, Potamo- trygonidae)Puricac-a o e caracterizac-a o de uma hialuroni- dase. Dissertac-a o de Mestrado, Instituto de Cie ncias Biolo gicas, UFG. Miura, R.O., Yamagata, S., Miura, Y., Harada, T., Yamagata, T., 1995. Analysis of glycosaminoglycan-degrading enzymes by substrate gel electrophoresis (Zymography). Anal. Biochem. 225, 333340. Nelson, J., 1994. Fishes of the World, third ed. Wiley, New York, NY, 416pp. Nishida, K., 1990. Phylogeny of the suborder Myliobatidoidei. Mem. Fac. Fish. Hokkaido Univ. 37, 1108. Pedroso, C.M., Jared, C., Charvet-Almeida, P., Almeida, M.P., Garrone-Neto, D., Lira, M.S., Haddad Jr., V., Barbaro, K.C., Antoniazzi, M.M., 2007. Morphological characteriza- tion of the venom secretory epidermal cells in the stinger of marine and freshwater stingrays. Toxicon 50, 688697. Roberts, T.R., Karnasuta, J., 1987. Dasyatis laosensis, a new whiptailed stingray (family Dasyatidae), from the Mekong River of Laos and Thailand. Environ. Biol. Fish. 20, 161167. Rosa, R.S., 1985. A systematic revision of the South American freshwater stingrays (Chondrichthyes: Potamotrygonidae). ARTICLE IN PRESS K.C. Barbaro et al. / Toxicon 50 (2007) 676687686
12. 12. Unpublished D. Phil. Thesis, College of William and Mary, Williamsburg, Virginia. Rosa, R.S., Castello, H.P., Thorson, T.B., 1987. Plesiotrygon iwamae, a new genus and species of neotropical freshwater stingray (Chondrichthyes: Potamotrygonidae). Copeia 2, 447458. Russell, F.E., Van Harreveld, A., 1954. Cardiovascular effects of the venom of the round stingray, Urobatis halleri. Arch. Intern. Physiol. 62, 332333. Russell, F.E., Barrit, W.C., Fairchield, M.D., 1957. Electro- cardiographic patterns evoked by venom of the stingray. Proc. Soc. Exp. Biol. Med. 96, 634635. Russell, F.E., Fairchield, M.D., Michaelson, J., 1958. Some proper- ties of the venom of the stingray. Med. Arts Sci. 12, 7886. Sano-Martins, I.D., Santoro, M.L., 2003. In: Cardoso, J.L.C., Franc-a, F.O.S., Wen, F.H., Ma laque, C.M.S., Haddad, Jr., V. (Eds.), Animais Pec-onhentos no Brasil. Biologia, Clnica e terape utica dos Acidentes, Sarvier, pp. 289309. Santoro, M.L., Sano-Martins, I.S., 1993. Different clotting mechanisms of Bothrops jararaca snake venom on human and rabbit plasmas. Toxicon 31, 733742. Santoro, M.L., Sousa-e-Silva, M.C., Gonc-alves, L.R., Almeida- Santos, S.M., Cardoso, D.F., Laporta-Ferreira, I.L., Saiki, M., Peres, C.A., Sano-Martins, I.S., 1999. Comparison of the biological activities in venoms from three subspecies of the South American rattlesnake (C. durissus terricus, C. durissus cascavella and C. durissus collilineatus). Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 122, 6173. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 7685. Tan, N.H., Ponnudurai, G., 1992. Comparative study of the enzymatic, hemorrhagic, procoagulant and anticoagulant activities of some animal venoms. Comp. Biochem. Physiol. C 103, 299302. Taniuchi, T., 1979. Freshwater elasmobranchs from Lake Naujan, Perak River, and Indragiri River, Southeast. Asia Jpn. J. Ichthyol. 25, 273277. Taniuchi, T., Shimizu, M., Sano, M., Baba, O., Last, P.R., 1991. Descriptions of freshwater elasmobranchs collected from three rivers in northern Australia. Univ. Museum Univ. Tokyo Nat. Culture 3, 1126. Theakston, R.D.G., Lloyd-Jones, M.J., King, L.E., 1977. Micro- ELISA for detecting and assaying snake venom and anti- venom antibody. Lancet 2, 639641. Thorson, T.B., 1983. Observations on the morphology, ecology, and life history of the euryhaline stingray, Dasyatis guttata (Bloch and Schneider) 1801. Acta Biol. Venez. 11, 92125. Thorson, T.B., Brooks, D.R., Mayes, M.A., 1983. The evolution of freshwater adaptation in stingrays. Nat. Geol. Soc. Res. Rep. 15, 663694. Towbin, H., Staehelin, T., Gordon, J., 1979. Eletrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 43504354. Veiga, S.S., da Silveira, R.B., Dreyfuss, J.L., Haoach, J., Pereira, A.M., Mangili, O.C., Gremski, W., 2000. Identication of high molecular weight serine proteases in Loxosceles inter- media (brown spider) venom. Toxicon 38, 825839. Vellard, J., 1931. Venin des rais (Taeniura) du Rio Araguaya (Bre sil). C. R. Acad. Sci. 192, 12791281. Vellard, J., 1932. Mission scientique au Goyaz et au Rio Araguaya. Me m. Soc. Zool. France 29, 513539. ARTICLE IN PRESS K.C. Barbaro et al. / Toxicon 50 (2007) 676687 687