Preliminary report

Studies on pharmacological properties of mucus and sting venom ofPotamotrygon cf. henlei

Juliane Monteiro-dos-Santos a, Katia Conceição b, Carla Simone Seibert a, Elineide Eugênio Marques a,Pedro Ismael Silva Jr.b, Anderson Brito Soares a, Carla Lima b, Mônica Lopes-Ferreira b,⁎

a Universidade Federal de Tocantins, Brazilb LETA (Laboratório Especial de Toxinologia Aplicada) Center for Applied Toxinology (CAT/CEPID), Butantan Institute, São Paulo, SP, Brazil

a b s t r a c ta r t i c l e i n f o

Article history:

Received 9 February 2011Received in revised form 21 March 2011Accepted 22 March 2011Available online 8 April 2011

Keywords:

StingrayPotamotrygon

VenomMucusPharmacological and biochemical activities

Stingrays from the Potamotrygon cf. henlei species are widely distributed in high numbers throughout therivers of central-west Brazil, being the source of numerous envenomations occurring in the dry season, posinga serious public health problem even if not properly reported. The accidents usually involve fishermen andbathers, and to date there is no effective treatment for the injured. Considering these facts and limitations ofstudies aiming at understanding the effects induced by P. cf. henlei envenoming, this study aimed to describethe principal pharmacological and certain biochemical properties of the mucus and sting venom. We foundthat mucus and sting venom is toxic to mice having nociceptive, edematogenic and proteolysis activities. Ourresults also indicate that the inflammatory cellular influx observed could be triggered by the venom andmucus. Furthermore the venom and mucus were partially purified by solid-phase extraction tested forantimicrobial activity in which only the mucus presented activity. It could be inferred from the present studythat P. cf. henlei venom possesses a diverse mixture of peptides, enzymes and pharmacologically activecomponents.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Venomous fish are diverse with representatives spread across fourorders, distributed worldwide but being largely concentrated intropical areas [1–4]. Their envenomation cause numerous reportedinjuries with symptoms as intense pain, skin necrosis, blisters, ulcers,fever, and rarely death probably associated with bacterial infections[2,4–6]. Although accidents caused by these animals are consideredsevere, it is surprising that only a few studies have ever examined thisrelationship [7–9], given all the pharmaceutical benefits and healththreats posed by venomous aquatic animals [10].

Stingrays are found in shallow tropical coasts worldwide totemperate waters (including marine and freshwater species) [11–14]. In South America, freshwater stingrays are included in thePotamotrygonidae family, which comprise three valid genera: Plesio-trygon, Paratrygon and Potamotrygon, the last being more diversified,with 19 described species [15,16]. Freshwater stingrays in Brazil arevery common in northern, central-western and southeastern rivers,and marine stingrays are distributed throughout the Atlantic Oceancoast [16].

Stingraysmayhaveone to seven spines on theirwhip-like tail,whichare responsible for numerous injuries worldwide [17–19]. Their spinesare hard, sharp, bilaterally retroserrated and covered by an integumen-tary sheath with a ventrolateral glandular groove containing venomglands along either edge [5]. The spine, called a venom apparatus, isoften covered with a film of venom and mucus also covering the entirebody of these animals. Envenomation by stingrays is relatively commonamongfisherman, bathers and swimmers inwhich .injuriesmay be verypainful, causing complications such as nausea, vomiting, salivation,sweating, respiratory depression, muscle fasciculation, convulsions,edema, and ischemic necrosis [2,20–24].

In addition tovenom, the skin offishplays apassive role inprotectiveimmunity, serving as ananatomical andphysiological barrier against theexternal environment. Skinmucus, secretedbymucous cells localized inthe epidermis, is considered thefirst lineof defense, as observed inotherfish species [25]. The mucus, such as that produced by the skin of thestingrays, may include amino acids, peptides, complex carbohydrates,glycopeptides, glycolipids and other chemicals [26]. The mucus isknown to comprise a number of immunecomponents suchas lysozyme,immunoglobulin, complement, carbonic anhydrase, lectins, crinotoxins,calmodulin, C-reactive protein, proteolytic enzymes and antimicrobialpeptides [27,28]. Therefore, the presence of one or several of thesecompounds could increase the severity of injuries caused by freshwaterstingrays as presented by Magalhães et al. [14].

Despite that a wide variety of fish species have been described,there is little information in the literature on stingray mucus. Most

International Immunopharmacology 11 (2011) 1368–1377

⁎ Corresponding author at: Laboratório Especial de Toxinologia Aplicada Center forApplied Toxinology (CAT/CEPID), Butantan Institute, Avenida Vital Brazil 1500, SãoPaulo-SP, 05503-900, Brazil. Tel.: +55 11 3726 1024.

E-mail address: mlferrei@usp.br (M. Lopes-Ferreira).

1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.intimp.2011.03.019

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research thus far has been on sting venom, which causes severalbiological effects. In one study, our group [14] demonstrated that thevenom of Potamotrygon gr. orbignyi can induce edematogenic andnociceptive responses, necrosis in mice as well as the presence ofenzymatic activity. This work leads us to a better and more detailedstudy of this venom and its compounds, purifying toxins to allowpharmacological investigations. By this approach, we isolated twobioactive peptides from the venom of the P. gr orbignyi freshwaterstingray [29,30]. These peptides, called orpotrin and porflan, shown tobe effective in the microcirculatory environment, induce strongvasoconstriction and inflammation, respectively. Caseinolytic, gelati-nolytic and hyaluronidase activity was identified in Potamotrygon

falkneri venom [22]. Barbaro et al. [18], comparing extracts from tissueof marine and freshwater stingrays Dasyatis guttata and Potamorygon

falkneri, observed edematogenic, gelatinolytic, caseinolytic andfibrinogenolytic activity in both extracts. Nociceptive activity wasverified in both tissue extracts; however, P. falkneri presented two-fold greater activity than D. guttata. Lethal, dermonecrotic, myotoxicand hyaluronidase activities were observed only in the tissue extractof P. falkneri.

In central-west rivers of Brazil, there is a wide distribution ofstingrays from Potamotrygon cf. henlei stingray species, with thehighest incidence of envenoming occurring in the dry season,resulting in a serious public health problem. The accidents usuallyinvolve fishermen and bathers, and presently, there is no effectivetreatment for this injury. Considering these facts and limitations instudies aiming at understanding the effects induced by P. cf. henlei

envenoming, this study aimed to describe the principal pharmaco-logical and certain biochemical properties of the mucus and stingvenom from P. cf. henlei. Furthermorewe investigated the action of themucus collected near the sting and back of the animals.

2. Materials and methods

2.1. Animals

Groups of 5 Swiss mice weighing 18–22 g were used throughoutthe study. The animals provided by the Instituto Butantan animalhouse were kept in temperature and humidity-controlled rooms, andreceived food and water ad libitum. All the procedures involving micewere in accordance with the guidelines provided by the BrazilianCollege of Animal Experimentation (number 730/10).

2.2. Venom and mucus

Specimens from adult female and male (n=15) P. cf. henlei fishwere collected from the Manoel Alves River in the state of Tocantins,Brazil. Venom produced by venom glands (Vn) and mucus dispersedthroughout the spine were collected after scraping both theepithelium and mucus, respectively. The mucus venom dispersed onthe dorsal area of the animal was collected and named ‘far’ (Mf) fromthe sting and the mucus ‘near’ (Mn) was the mucus collected in theregion of the tail next to the sting. Both mucus and venom wereobtained by scraping the skin and sting with a glass slide, beingimmediately stored on ice, and then diluted in sterile saline,homogenized, and centrifuged for collection of the supernatant. Thesting venom extraction was done by scraping the sting. Thesupernatant was collected and stored at −20 °C. Protein contentwas determined by the method of Bradford [31] using bovine serumalbumin (Sigma Chemical Co., St Louis, MO) as standard protein.

2.3. Estimation of nociceptive activity

Nociceptive activity of the venom and mucus was assayedaccording to the Hunskaar et al. [32]. Samples (Mucus near, Mucusfar and Venom) of 30 μl containing different doses of proteins (25, 50

and 100 μg), were injected (i.pl.) in the right footpad of mice. Thecontrol group was injected only with sterile PBS. Then, each mousewas kept in an adapted chamber mounted on a mirror for 10 min.Each animal was then returned to the observation chamber, and theamount of time spent licking or biting each hind paw was recorded.Each bar represents the mean±SEM.

2.4. Estimation of edema-inducing activity

Edematogenic activity was assayed according to the Lima et al.[33]. Samples (Mucus near, Mucus far and Venom) of 30 μl containingdifferent doses of protein (25, 50 and 100 μg) were injected in theright footpad of mice (i.pl.) and local edema was quantified 2 h afterinjection. Additionally, mice were injected with 100 μg of venom ordifferent mucus, and edema was measured at 0.5, 1, 2, 3, 4 and 5 hafter injection. Mice injected with 30 μl of sterile PBS were consideredthe control group. Local edema was quantified by measuring thethickness of injected paws with a paquimeter (Mytutoyo SulAmericana, SP, Brazil). Results were expressed by the differencebetween experimental and control footpad thickness. Each barrepresents mean±SEM.

2.5. Evaluation of the vascular permeability

For permeability analysis, Evans blue dye, 20 mg/kg in 200 μl ofPBS was i.v. (intravenously) administered 20 min before i.p. (intra-peritoneal) administration of the mucus near, mucus far and venom(25, 50 and 100 μg) or PBS (control group). After 2 h, mice weresacrificed, and their peritoneal cavities were washed with 2 ml of ice-cold PBS and 0.1% bovine serum albumin (BSA). The cells were spundown, and the optical density (OD) of the supernatant was measuredat 620 nm as an indicator of Evans blue leakage into the peritonealcavity [34]. The results were expressed in μg of Evans blue/ml, and theconcentration of Evans blue was calculated from a standard curve of aknown concentration.

2.6. Intravital microscopy

The dynamic of alterations in the microcirculatory network weredetermined using intravital microscopy by transillumination of micecremaster muscle after subcutaneous application 100 μg of proteinvenom or mucus of Potamotrygon cf. henlei dissolved in 20 μl of sterilesaline. Administration of the same amount of sterile salinewas used ascontrol. In three independent experiments (n=4), mice wereinjected with 0.4% Xilazine (Coopazine®, Schering-Plough) and thenanaesthetizedwith 0.2 g/kg chloral hydrate, and the cremastermusclewas exposed for microscopic examination in situ as described by Baez[35] and Lomonte et al. [36]. The animals were maintained on a boardthermostatically controlled at 37 °C, which included a transparentplatform on which the tissue to be transilluminated was placed. Afterthe stabilization of the microcirculator, the number of roller cells andadherent leukocytes in the postcapillary venules were counted 10 minafter venom injection. The study of the microvascular system of thetransilluminated tissue was accomplished with an optical microscope(Axio Imager A.1, Carl-Zeiss, Germany) coupled to a camera (IcC 1,Carl-Zeiss, Germany) using a 10/025 longitudinal distance objective/numeric aperture and 1.6 optovar.

2.7. Induction of a local inflammatory reaction

The induction of an inflammatory reaction by the sting venom ormucus of P. cf. henleiwas assayed according to Lima et al. [33]. Samples(Mucus near, Mucus far and Venom) of 100 μg of protein in 30 μl ofPBS were injected in the intraplantar region of the right hind footpad.Animals injected with 30 μl PBS were considered a control group. Twohours after injection, animals were sacrificed, and the right pawswere

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amputated, the tissue was disrupted with scissors and homogenisedwith a glass piston in 200 μl of PBS to reach a 1 ml of cell suspension.

2.8. Cell harvesting and counting

Leukocyte migration was assessed 2 h after Potamotrygon cf. henlei

sting venom, mucus near, mucus far or PBS administration in thefootpad. The samples were immediately centrifuged at 3000 rpm, at4 °C, for 20 min. The supernatants were stored at −20 °C for futuredeterminations. The cell pellets were resuspended in 1 ml of PBS+0.1% BSA for cell counts. Total cell counts were performed in ahemocytometer and differential leukocyte counts in cytocentrifugepreparations stained with Wright–Giemsa. Cells were differentiallycounted by microscopy, evaluating 300 cells per slide. The resultsrepresent the mean±SEM per millilitre of cell suspension of threeindependent experiments.

2.9. Histological assessment of leukocyte influx

Mice injected with 100 μg of P. cf. henlei sting venom, mucus near,mucus far or PBS were killed 2 h later and footpads were removed,immediately fixed in 10% buffered formalin. The tissue was processedand embedded in paraffin. Five-micrometer tissue sections wereprepared and stained by the Hematoxilin and Eosin method. All slideswere examined with light microscopy at a magnification of ×40 (AxioImager A1, Carl Zeiss, Germany) calibrated with a referencemicrometer slide. For each group of four mice, four stained footpadsections from eachmouse were analysed. Positive control of leukocyteinflux was induced in mice by injecting 50 μl of a 0.5% solution ofcarrageenan (Type IV Lambda; Sigma-Aldrich) in PBS.

2.10. Quantification of cytokines and chemokines

Cytokines and chemokines were measured in the supernatant ofthe peritoneal exudate lavage fluid or from macrophage cultures by aspecific two-site sandwich ELISA, using the BDTM OpteIA ELISA Setsfor Interleukin-1 beta (IL-1β), and Interleukin-6 (IL-6), KC (Chemo-kine family with homology to human IL-8), and Monocyte chemoat-tractant protein-1 (MCP-1) according to the manufacturer'sinstructions (B&D Pharmingen, Oxford, UK). Binding of biotinylatedmonoclonal antibodies was detected using streptavidin–biotinylatedhorseradish peroxidase complex and 3,3′,5,5′-tetramethylbenzidine(B&D Pharmingen, Oxford, UK). Samples were quantified by compar-ing standard curves of recombinant mice cytokines and chemokines.The results were expressed as the arithmetic mean±SEM fortriplicate samples. Detection limits were 7.8 pg/ml for each cytokineand chemokine.

2.11. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE)

SDS-PAGE was carried out according to Laemmli [37]. Protein(10 μg) of the mucus near, mucus far and sting venoms were analysedby SDS-PAGE 4–20% acrylamide gradient under reducing conditions.Prior to electrophoresis, the samples were mixed 1:1 (v/v) withsample buffer. The gel was stained with the silver stain method.

2.12. Zymography

Zymography with gelatin gels was performed to visualizegelatinolytic enzymatic activity in the venoms. After electrophoresis(SDS-PAGE), gels containing 10% polyacrilamyde and 1 mg/mL gelatinwere washed for 30 min in a buffer containing 50 mM Tris–HCl(pH 7.5), 5 mM CaCl2 and 2.5% Triton X-100, and incubated overnight(16 h) in incubation buffer at 37 °C [50 mM Tris–HCl, 5 mM CaCl20.02% NaN3, pH 7.6 (all reagents from Sigma-Aldrich)] and 1% Triton

X-100. Gels were stainedwith Coomassie blue and destained by aceticacid in methanol and H2O (1:3:6), both for 60 min. In this method, theproteolytic activity in gelatin is detected as colourless bands on theotherwise blue gel after staining with Coomassie blue [38].

2.13. Chromatographic profile of the mucus and venoms

The venoms or mucus were processed in the same way aspreviously described. Aliquots of 1 mg of the venoms were dissolvedin 1 mL of deionized water in 0.1% TFA and centrifuged at 5000×g for20 min (10 °C). The supernatants were applied to a system ofreversed-phase binary HPLC (Äkta basic, Amersham Biosciences —

Sweden) for the sample separation. The venoms were loaded in a ACEC18 column (5 μ, 4.6×250 mm, ACT-Aberdeen, Scotland) in a two-solvent system: (A) trifluoroacetic acid (TFA)/H2O (1:1000) and (B)TFA/Acetonitrile (ACN)/H2O (1:900:100). The column was eluted at aflow rate of 1.0 mL/min with a 10 to 80% gradient of solvent B over40 min. The HPLC column eluates weremonitored by their absorbanceat 214 nm.

2.14. Antibacterial assay

Pooled venom and mucus (male and female) were dissolved indeionized water and centrifuged at 5000×g for 20 min (roomtemperature). The supernatants were loaded onto solid phaseextraction cartridges Sep-Pak, C18 (Waters Corporation, Taunton,MA, USA) equilibrated in acidified water (0.1% trifluoroacetic acid). Asingle aliquot of 3 mg diluted in 3 mL of 0.1% TFA was loaded and theelution was performed with 40 and 80% acetonitrile, and was furtherconcentrated by a vacuum centrifuge. Antimicrobial activity wasmonitored by a liquid growth inhibition assay against Micrococcus

luteus A270, Escherichia coli SBS 363 and Candida albicans MDM8, asdescribed by Bulet et al. [39] and Ehret-Sabatier et al. [40]. Pre inoculaof the strains were prepared in Poor Broth (1.0 g peptone in 100 mL ofH2O containing 86 mM NaCl at pH 7.4; 217 mOsM for M. luteus and E.

coli and 1.2 g potato dextrose in 100 mL of H2O at pH 5.0; 79mOsM forC. albicans) and incubated at 37 °C with shaking. The absorbance at595 nm was determined and one aliquot of this solution was taken toobtain cells in logarithmic growth (A595nm~0.6), and diluted 600times (A595 nm=0.0001). The venom, mucus and fractions weredissolved in sterile Milli-Q water, at a final volume of 100 μl (10 μl ofthe peptide and 90 μl of the inoculum in PB broth). After incubationfor 18 h at 30 °C the inhibition of bacterial growth was determined bymeasuring absorbance at 595 nm.

2.15. Statistical analysis

All results were presented as means±SEM of at least four animalsin each group. Differences among data were determined by ne wayanalysis of variance (ANOVA) followed by Dunnett's test. Differencesbetween twomeans were determined using unpaired Student's t-test.Data were considered significant at pb0.05.

3. Results

3.1. Pharmacological activities induced by venom and mucus

Mice were first injected with different doses (25, 50 and 100 μg) ofvenom ormucus (near and far) from P. cf. henlei andwere analysed forthe presence of nociception, edema induction and increase of vascularpermeability.

The nociceptive response observed after injection of venom andmucus into the mouse right hind-paw are quite similar in all samples,with a dose-dependent manner of paw licking during 30 min thatreached its maximum at 100 μg of protein (Fig. 1A). The neurogenic(0–5 min after venom injection) and inflammatory (15–40 min) phases

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of the nociception test were also induced for all samples, with theinflammatory phase of nociception being more intense in all samplegroups tested (Fig. 1B). Groups tested with PBS (control group)presented no significant activity.

The results depicted in Fig. 2A demonstrate that the subplantarinjection of tested doses of the venom and mucus caused substantialpaw edema with significant response and similar patterns. Therefore,comparing groups revealed that the sting venom group presented ahigher increase in footpad thickness persisting up to 5 h after venominjection (Fig. 2B).

To evaluate the induction of vascular permeability, mice receivedintraperitoneal injection of the entire venom, mucus or vehicle (PBS)as the control. After 120 min, the mice were killed and the dyeexudates were measured. The venom and mucus induced an increase,dose dependent in vascular permeability (Fig. 3) compared to themice that received only the vehicle (PBS). The major differencebetween the tested groups was observed in the mucus far group.

To assess the effects of the sting venom and mucus in themicrocirculation, intravital microscopy was employed. This method-ology allows the direct observation of changes in themicrocirculation,and it is also a plain and noninvasive approach used for understandingbiological effects [41]. The topical application of 100 μg of mucus andsting venom induced an increase of cellular recruitment characterizedby the number of leukocyte rolling (Fig. 4). For mucus (near and far)the cellular recruitment started at 10 min after topical application.Interestingly for the sting venom, an earlier recruitment wasobserved, in the first 5 min, after the venom application (Fig. 4inset), and this profile of recruitment was elevated at least two-fold inanimals compared to the tested groups. Fig. 5A shows the increase in

the number of adherent cells at the vascular endothelium inducedafter topical application of sting venom only. This activity was morepronounced after 20 to 30 min of experiment (Fig. 5B). No change innumbers of adhered cells was observed in the other groups or controlanimals receiving PBS.

Leukocyte recruitment to the site of injury after mucus and stingvenom injections were evaluated in mice. Fig. 6 shows the incrementin cell recruitment into footpad tissue of animals receiving all testedsamples (venom and mucus) at 2 h. The cellular recruitment inducedby the sting venom (Fig. 6 inset) was characterized by highrecruitment of neutrophils followed by macrophages, whereas themucus (near and far) induced significant cellular influx characterizedonly by the recruitment of neutrophils in this period.

Knowing that the IL-1β and IL-6 cytokines are importantinflammatory mediators recognized for their role in the modulationof cell recruitment, it was important to evaluate their production afterP. cf. henlei mucus and sting venom injection. Thus, we evaluated thelevels of cytokines in the footpad homogenate after injecting 100 μg ofthe samples at 2 h. Compared with the control-group, we observed asignificant increase in IL-1β and IL-6 levels for all experimental groups(Fig. 7A and B).

Additionally, MCP-1 and KC chemokines are critical for theregulation of monocyte and neutrophil trafficking, respectively.These molecules were also measured 2 h after sample injection inthe footpad homogenates. As shown in Fig. 7 C, the injection of mucusand sting venom provoked a higher release of KC, andMCP-1was onlydetected in higher levels only for the sting venom group (Fig. 7D).

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Fig. 1. Estimation of nociception-inducing activity. Samples of 30 μl containing differentdoses of venom and mucus (25, 50, and 100 μg of protein) were injected (i.pl.) in theright footpad of mice. The control group was injected only with sterile PBS. Each animalwas then returned to the observation chamber and the amount of time spent (s) lickingor biting each hind paw was recorded for 30 min (A) or 0–5 and 15–40 min (B) andtaken as the index of nociception. Each bar represents mean±SEM. *pb0.05 comparedwith control-group.

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Fig. 2. Determination of edema-inducing activity. (A) Samples of 30 μl containingdifferent doses of venom andmucus (25, 50, and 100 μg of protein) were injected (i.pl.)in the right footpad of mice. Local edema was quantified 2 h after injection. (B) Localedemawas quantified in 1/2, 1, 2, 3, 4, and 5 h after injection of 30 μl containing 25 μg ofprotein/animal. Mice injected with sterile PBS were considered the control-group. Theresults were expressed by the difference between experimental and control footpadthickness. Each point represents mean±SEM. *pb0.05 compared with control-group.

1371J. Monteiro-dos-Santos et al. / International Immunopharmacology 11 (2011) 1368–1377

3.2. Antimicrobial activity and biochemical characterization of the

venoms

The venom and mucus (Mn and Mf) were partially purified bysolid-phase extraction and divided into 3 fraction eluates with 0, 40and 80% acetonitrile. A total of 12 samples (including the crudevenom and mucus) were then tested for antimicrobial activityagainst Micrococcus luteus (Gram-positive), Escherichia coli (Gram-negative) and Candida albicans. Of the samples tested, only themucus far and two eluates (Mucus far 40 and 80%) inhibited thegrowth of C. albicans. The most potent antibacterial activity wasdetected in the Mf 80% ACN eluate.

To determine the eletrophoretical profile, the samples weresubmitted to an 8–12% SDS-Page (10 μg of protein/well). SDS-PAGEanalysis of the venoms showed a very similar eletrophoretic profile asshown in Fig. 8A. The P. cf. henlei venoms showed intense bands withapproximately 70 kDa, around 40 and 50 kDa, and one last bandpresented approximately 15 kDa. Analysis of gelatinolytic activityusing 5, 10 and 20 μg of the venoms is presented in Fig. 8B. Thevenoms showed a similar gelatin hydrolysis profile with several bandswith enzymatic activity, except for one band at 35 kDa observed onlyin the sting venom in all tested concentrations.

The tested samples showed significant amounts of enzymaticactivities against gelatin, in which the venom presented two weakbands at 60 and 18 kDa, and two bands around 30 and 35 kDa. Bothmucus tested presented similar gelatin hydrolysis profiles with bandsaround 30, 20, 18 and 16 kDa (Fig. 8B).

Despite similar identity of venoms identified by SDS-PAGE, byreverse phase chromatography we observed the opposite. Typical UVchromatograms are shown in Fig. 8C, D and E. Although somesimilarities of retention times and relative concentration of certaincomponents seem to occur, the overall profile is quite distinct. Theheterogeneity of venoms demonstrates and confirms several compo-nents evenly distributed throughout the profile. We observed that thegeneral shape and peak distribution between the venoms are unlike,with at least two peaks being different in each chromatogram. In thefirst 20 min of HPLC separation, some components apparently are notshared between the samples as the two higher peaks in the mucusnear sample. However it is worth mentioning that importantcomponents are separated at around 30–40 min retention time.

4. Discussion

Although P. cf. henlei is a Brazilian stingray causing serious humaninjuries, little has been reported on its venom. For this reason, in thepresent communication a comparative analysis regarding venom andmucus composition of P. cf. henlei collected in Tocantins, together witha general biochemical and biological characterization of the samplesof venom and mucus studied are reported. To the best of ourknowledge, there is only a couple of reports dealing with theexperimental toxicity of Potamotrygon stingray venoms [14] and[17], the characterization of two peptides [29,30] and the presence ofa protein with hyaluronidase activity [42]. The venom of P. cf. henlei,compared with P. cf. scobina and P. gr. orbignyi is similar regardingactivities such as induction of nociception, edema and leukocyterecruitment.

Local intense pain and edema in stung areas are common signs inpatients suffering stingray attacks, being especially evident in the legsand foot region [43]. The edematogenic activity by P. cf. henlei mucusand sting venom in the footpad of mice reproduced a localinflammatory lesion similar to that described in humans. Studies onstonefish, toadfish and stingray fish venom are known to induceintense and sustained edematogenic responses in mice [14,33,44,45].Edematogenic activity is dependent on a synergism betweenmediators that increase vascular permeability and those that increaseblood flow [46]. The plasma extravasation and edema formation inresponse to venom and mucus strongly suggested the involvement ofvasoactive mediators derived from mast cell granules. Serotonin andhistamine are mediators found in large amounts in mast cells [47].These biogenic amines released from inflammatory cells participate inthe genesis of acute inflammatory events and in various stages of theimmune response [48].

Leukocytes are key components in the inflammatory response dueto their activities. An increase in leukocyte adhesion is a marker of aninflamed and dysfunctional endothelium [49]. The current studyshowed that venom andmucus of P. cf. henlei in mice induce increasedleukocyte rolling, with the venom group followed by a gradualincrease in firmly adherent cells to the endothelium in intravitalexperiments. Meanwhile the ability of the venom and mucus todevelop a nociceptive response was also demonstrated, and thenociception induced during the inflammatory period (15–40 min)could be associated with the augmented rolling and adhesion ofleukocytes to the endothelium of cremaster mice induced by venom.The first phase of this experiment is characterized by neurogenic paincaused by direct chemical stimulation of nociceptors and reflectscentrally mediated pain. The second phase is characterized byinflammatory pain triggered by a combination of stimuli, includinginflammation of the peripheral tissues and mechanisms of centralsensitization [50] and results from the action of inflammatorymediators in peripheral tissues such as prostaglandins, serotonin,histamine and bradykinin.

The selectin family of adhesion molecules is associated with theinitial phase of leukocyte recruitment characterized by leukocyterolling [51]. P-selectin is more critical in the initial rolling and slowingof recruited leukocytes [52], whereas E-selectin is more important inleukocyte arrest or the transition from slow rolling to firm adhesion,as postulated by Smith et al. [53]. In addition, -1 and -2 integrinadhesion molecules (VCAM-1, ICAM-1) are critical for firm attach-ment of activated leukocytes in the recruitment cascade [51]. Ourresults support the hypothesis that the venom of P. cf. henlei wouldlead to local release of vasoactive mediators, cytokines, and chemoat-tractants, up-regulating the expression of important adhesion mole-cules for leukocyte recruitment.

Neutrophils are recruited rapidly into sites of acute infection anddominate the initial influx of leukocytes [54]. Later in inflammation,leukocytes of the monocyte/macrophage lineage replace neutrophilsas the predominant leukocyte, suggesting a bimodal recruitment

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Fig. 3. Evaluation of vascular permeability. Evans blue dye, 20 mg/Kg in 200 μl of PBSwas i.v. administered 20 min before different doses of venom and mucus (25, 50, and100 μg of protein) or PBS i.p. administration. After 2 h, mice were sacrificed, and theirperitoneal cavity was washed with PBS, 0.1% BSA. The cells were spun down and the ODof the supernatant at 620 nm was measured as an indicator of Evans blue leakage intothe peritoneal cavity. Five mice were used for each group per experiment, and theexperiments were conducted three times. The results were expressed in μg of EvansBlue/ml.

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Fig. 5. Adhered leukocytes in postcapillary venules of cremaster muscle. Adhered leukocytes were observed after application of 100 μg of venom, mucus and sterile saline (20 μl,control). (A) Representative graph showing leukocyte adhering to endothelium after topical application of samples. Determinations were performed for 30 min after venominjection and values averaged from three independent experiments. (B) Intravital micrograph of cremaster venule showing adherent leukocytes after samples application.Photographs were obtained from digitized images on the computer monitor. Each point represents mean±SEM. *pb0.05 compared with the control group of three independentexperiments.

Fig. 4. Number of leukocytes rolling. Representative graph showing leukocyte rolling in postcapillary venules of the mice cremaster muscle after subcutaneous application of 100 μgof venom, mucus and sterile saline (20 μl, control) at different times. Determinations were performed for 30 min after application and values averaged from three independentexperiments. Inset: Leukocyte rolling after 15 min of sting venom administration (arrow); Photographs were obtained from digitized images on the computer monitor. Each pointrepresents mean±SEM. *pb0.05 compared with control group of three independent experiments.

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pattern involving a switch from neutrophils to monocytes. Recruitedneutrophils are thought to mediate this switch by releasing solublefactors into the early inflammatory site that initiate monocyterecruitment [55,56]. Macrophages play a central role in the inflam-matory response, releasing cytokines that control key events in theinitiation, resolution, and repair processes of inflammation (reviewed

in [57]). In general, the major chemokine responsible for recruitingmonocytes to inflammatory sites is the chemokine MCP-1, andneutrophils are instrumental in mediating a chemokine switchpromoting monocyte chemoattraction [58]. Interestingly, we showedhere a profile of leukocyte recruitment unique for the venom with apredominance of neutrophils followed by macrophage influx intomice footpads, and elevated levels of MCP-1 and KC protein at 2 h.

Proteolytic activity detected in venom compares well withproteolytic activity in Thallassophryne maculosa, stingrays, Potamo-

trygon cf. scobina and P. gr. orbignyi, and Scatophagus. argus venom[14,59,60]. Mild proteolytic activity has been observed in bullrout,Notesthes robusta, venom [61].

In addition, antimicrobial activity was found in the mucus farsample eluted with 40% and 80% acetonitrile. This indicates that thehydrophilic and/or high hydrophobic substances are not the potentantimicrobial compounds in the mucus. A report on the antibacterialactivity of crude mucus extracts from farmed cod pointed out that theresponse to the tested bacterial strains varied with individual fish[62,63]. Histone H2B and two ribosomal proteins with antibacterialactivity have been isolated from epidermal mucus of Atlantic cod [64].AMPs were found to be important innate defence components in theepidermal mucosal layer of Moses sole fish (Pardachirus marmoratus),winter flounder (Pleuronectes americanus), catfish (Parasilurus aso-

tus), Atlantic halibut (Hippoglossus hippoglossus), rainbow trout(Oncorhynchus mykiss), Atlantic cod (Gadus morhua) and Hagfish(Myxine glutinosa L.) [28,65–67]. The bactericidal activity of fishepidermalmucus AMPs has been shown to be retained in the presenceof sodium salt and divalent cations [68,69]. These observationssuggest that fish epidermal mucus is a good source of novel AMPsfor fish and human health-related applications.

As presented in the HPLC profiles, another conclusion drawn fromthis study is that there must be venom variations among thespecimens collected in certain areas. For example, in the study

*A B

0

100

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IL-1

(p

g/m

L) *

Control Vn Mn Mf

*

0

500

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(p

g/m

L)

*

Control Vn Mn Mf

**

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1000

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MC

P-1

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L)

*

0

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500

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*

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Control Vn Mn Mf

**

C D

Fig. 7. Quantification of cytokines and chemokines in homogenates of footpad from mice injected with P. cf. henlei sting venom and mucus. Samples (100 μg in 30 μl of PBS) wereinjected in the intraplantar region of the right hind footpad. Mice only injected with PBS were considered the control group. After 2 and 24 h, animals were sacrificed and the rightpaws were amputated, and homogenised for ELISA cytokine determinations. Each bar represents mean±SEM. pb0.05 for triplicate samples compared with the control-group.

Fig. 6. Effect of P. cf. henlei sting venom and mucus on leukocyte recruitment. Samples(100 μg in 30 μl of PBS) were injected in the intraplantar region of the right hind footpad.Animals injected with 30 μl PBS were considered the control group. Six hours afterinjection, animals were sacrificed and the right paws were amputated, the tissue wasprocessed for cell count. Inset: Fotomicrograph from the footpadofmice injectedwith P. cf.

henlei sting venom (400X, HE). The results represent the mean±SEM. * pb0.05 of threeindependent experiments compared with control-group.

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realized with P gr. orbignyi collected in the same region, we can noticethe presence of two hydrophilic peaks in the venom chromatogramnot seen in the P. cf. henlei venom profile. These two peaks contain thebioactive peptides orpotrin and porflan [29,30]. Although thesepopulations of stingrays seem to be very close, the composition andpotency of their venoms is slightly different.

Discoveries of toxins from venoms, especially from aquaticresources, are racing ahead because of their extremely complex andunique action on various physiological systems. After all, venoms arepart of an organism's defence and/or predatory mechanisms, whosespecificity has beenhoned over amillion years of evolution. Venomousfish represent a vast source of novel molecules that may prove usefuleither as research tools or therapeutic agents, and have been objects of

study for several research groups [45,70–72]. The production of toxinsby fish is an important strategy that guarantees survival in a highlycompetitive ecosystem. These animals produce an enormous numberof molecules such as alkaloids, steroids, peptides and proteins withchemical and pharmacological proprieties differing from thosepresented by venoms of terrestrial animals [73].

In conclusion, the present study contributes to the biological studyof venom in mice correlated with local manifestations described inP. cf. henlei envenomation in humans as well as proteins in the venomthat contribute to potentiating local tissue destruction. The spectrum ofactivity in experimental animals resembles those of other fish venomspreviously studied. This study found that P. cf. henlei mucus and stingvenom is toxic tomice, havingnociceptive, edematogenic andproteolytic

Fig. 8. (A) Analysis by SDS-PAGE of venoms using a polyacrylamide gradient gel 4–20% and stained with Silver (A). Lanes: 1) Sting venom; 2) Mucus near; 3) Mucus far. Numbers atleft corresponded to position of Mwmarkers; (B) Gelatin zymography 12% P. cf. henleimucus and sting venoms. Lanes: 1) Sting venom 5 μg; 2) Mucus near 5 μg; 3)Mucus far 5 μg; 4)Sting venom 10 μg; 5) Mucus near 10 μg; 6) Mucus far 10 μg; 7) Sting venom 20 μg; 8) Mucus near 20 μg; 9) Mucus far 10 μg. Numbers at left correspond to position of Mwmarkers;Comparison of the preparative reverse phase HPLC profiles obtained for the sting venom (C), mucus near (D) and mucus far (E) from P. cf. henlei venom. The amount of proteininjected into the column was 1 mg. The presence of peptides or proteins in the eluate was detected by measuring the UV absorption at 214 nm. Selected peaks (arrows) show themajor differences between the samples. All other experimental details are given in materials and methods section.

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activity. Our results also indicate that the inflammatory cellular influxobserved could be triggered venom and mucus. It could be inferredfrom the present study that P. cf. henlei venom possesses a diversemixture of peptides, enzymes and pharmacologically active compo-nents. Though exact relationships of these components in envenom-ation have not been previously traced, the involvement of proteinsand peptides in envenomation cannot be ruled out. Further studiesare required to isolate and find the mechanism of action of thesemolecules in the venom and mucus as well as the elucidation of themechanisms involved in the inflammatory reaction induced by thevenom.

Acknowledgement

This work has been supported by a FAPESP (2007/nn-9) and CNPq.We also wish to thank Jim Hesson for editing of the English language.

References

[1] Halstead BW. Venomous marine animals of Brazil. Mem Instituto Butantan1966;33:1–26.

[2] Halstead BW. Poisonous and Venomous Marine Animals of the World. Princeton,New Jersey, NJ: The Darwin Press; 1988. p. 1168.

[3] Church JE, Hodgson WC. The pharmacological activity of fish venoms. Toxicon2002;40:1083–93.

[4] Haddad Jr V, Martins IA, Makyama HM. Injuries caused by scorpionfishes(Scorpaena plumieri Bloch, 1789 and Scorpaena brasiliensis Cuvier, 1829) in theSouthwestern Atlantic Ocean (Brazilian coast): epidemiologic, clinic and thera-peutic aspects of 23 stings in humans. Toxicon 2003;42:79–83.

[5] Halstead BW. Poisonous and Venomous Marine Animals of the World, 3.Washington, DC: US Government Printing Office; 1970.

[6] Vetrano SJ, Lebowitz JB, Marcus S. Lionfish envenomation. J Emerg Med 2002;23:379–82.

[7] Lopes-Ferreira M, Barbaro KC, Cardoso DF, Moura-Da-Silva AM, Mota I. Thalasso-phryne nattereri fish venom: biological and biochemical characterization andserum neutralization of its toxic activities. Toxicon 1998;36:405–10.

[8] Lopes-Ferreira M, Moura-da-Silva AM, Mota I, Takehara HA. Neutralization ofThalassophryne nattereri (niquim) fish venom by an experimental antivenom.Toxicon 2000;38:1149–56.

[9] Sivan G, Venketasvaran K, Radhakrishnan CK. Characterization of biologicalactivity of Scatophagus argus venom. Toxicon 2010;56:914–25.

[10] Smith WL, Wheeler WC. Venom evolution widespread in fishes: a phylogeneticroad map for the bioprospecting of piscine venoms. J Hered 2006;97:206–17.

[11] Fenner JP. Dangers in the ocean: the traveler and marine envenomation, II. Marinevertebrate. J Travel Med 1998;5:213–6.

[12] Scharf JM. Cutaneous injuries and envenomation from fish, shark and ray.Dermatol Ther 2002;15:47–57.

[13] Uzel PA, Massicot M, Jean M. Stingray injury to the ankle. Eur J Orthop SurgTraumatol 2002;12:115–6.

[14] Magalhães KW, Lima C, Piran-Soares AA, Marques EE, Hiruma-Lima CA, Lopes-Ferreira M. Biological and biochemical properties of the Brazilian Potamotrygonstingrays: Potamotrygon cf. scobina and Potamotrygon gr. orbignyi. Toxicon2006;47:575–83.

[15] Charvet-Almeida P, Araújo MLG, Rosa RS, Rincón C. Neotropical freshwaterstingrays: diversity and conservation status. Shark News 2002;14:1–2.

[16] Carvalho MR, Lovejoy NR, Rosa RS. Family potamotrygonidae. In: Reis RE,Kullander SO, Ferraris. CJ, editors. Checklist of the Freshwater Fishes of Southand Central America (CLOFFSCA). Porto Alegre: Edipucrs; 2003. p. 22–9.

[17] Thorson TB, Langhammer KJ, Oetinger IM. Periodic shedding and replacement ofvenomous caudal spines, with special reference to South American freshwaterstingrays, Potamotrygon spp. Env Biol Fish 1988;23:299–314.

[18] Barbaro KC, Lira MS, Malta MB, Soares SL, Garrone Neto D, Cardoso JL, Santoro ML,Haddad Junior V. Comparative study on extracts from the tissue covering thestingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata)stingrays. Toxicon 2007;50:676–87.

[19] Dehghani H, Sajjadi MM, Rajaian H, Sajedianfard J, Parto P. Study of patient'sinjuries by stingrays, lethal activity determination and cardiac effects induced byHimantura gerrardi venom. Toxicon 2009;54:881–6.

[20] Lalwani K. Animal toxins: scorpaenidae and stingrays. Br J Anaesth 1995;75:247.[21] Fenner JP, Williamson JA, Burnett JW. Clinical aspects of envenomation by marine

animals. Toxicon 1996;34:145.[22] Haddad Jr V, Neto DG, de Paula Neto JB, de Luna Marques FP, Barbaro KC.

Freshwater stingrays: study of epidemiologic, clinic and therapeutic aspects basedon 84 envenomings in humans and some enzymatic activities of the venom.Toxicon 2004;43:287–94.

[23] Perkins AR, Morgan SS. Poisoning, envenomation, and trauma from marinecreatures. Am Fam Physician 2004;69:885–90.

[24] Forrester BM. Pattern of stingray injuries reported to Texas poison centers from1998 to 2004. Hum Exp Toxicol 2005;24:639–42.

[25] Zhao X, Findly RC, Dickerson HW. Cutaneous antibody-secreting cells and B cells ina teleost fish. Dev Comp Immunol 2008;32:500–8.

[26] Klesius PH, Shoemaker CA, Evans JJ. Flavobacterium columnare chemotaxis tochannel catfish mucus. FEMS Microbiol Lett 2008;288:216–20.

[27] Alexander JB, Ingram GA. Noncellular nonspecific defence mechanism of fish.Annu Rev Fish Dis 1992;2:249–79.

[28] Birkemo GA, Lüders T, Andersen Ø, Nes IF, Nissen-Meyer J. Hipposin, a histone-derived antimicrobial peptide in Atlantic halibut (Hippoglossus hippoglossus L.).Biochim Biophys Acta 2003;1646:207–15.

[29] Conceição K, Konno K, Melo RL, Marques EE, Hiruma-Lima CA, Lima C, RichardsonM, Pimenta DC, Lopes-Ferreira M. Orpotrin: a novel vasoconstrictor peptide fromthe venom of the Brazilian stingray Potamotrygon gr. orbignyi. Peptides 2006;27:3039–46.

[30] Conceição K, Santos JM, Bruni FM, Klitzke CF, Marques EE, Borges MH, Melo RL,Fernandez JH, Lopes-Ferreira M. Characterization of a new bioactive peptidefrom Potamotrygon gr. orbignyi freshwater stingray venom. Peptides 2009;30:2191–9.

[31] Bradford MM. A rapid and sensitive method for quantitation of microgramquantities of protein utilizing the principle of protein dye binding. Anal Biochem1976;72:248–54.

[32] Hunskaar S, Fasmer OB, Hole K. Formalin test in mice, a useful technique forevaluating mild analgesics. J Neurosci Methods 1985;14:69–76.

[33] Lima C, Bianca Clissa P, Piran-Soares AA, Tanjoni I, Moura-da —Silva AM, Lopes —Ferreira M. Characterisation of local inflammatory response induced by Thalasso-phryne nattereri fish venom in a mice model of tissue injury. Toxicon 2003;42:499–507.

[34] Sirois MG, Jancar S, Braquet P, Plante GE, Sirois P. PAF increases vascularpermeability in selected tissues: effect of BN —52021 and L —655,240.Prostaglandins 1988;36:631–44.

[35] Baez S. An open cremaster muscle preparation for the study of blood vessels by invivo microscopy. Microvasc Res 1973;5:384–96.

[36] Lomonte B, Lundgren J, Johansson B, Bagge U. The dynamics of local tissue damageinduced by Bothrops asper snake venom and myotoxin II on the mouse cremastermuscle. An intravital and electron microscopic study. Toxicon 1994;32:41–55.

[37] Laemmli UK. Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature 1970;227:680–5.

[38] Woessner JF. Quantification of matrix metalloproteinases in tissue samples.Methods Enzymol 1995;248:510–28.

[39] Bulet P, Dimarcq JL, Hetru C, Lagueux M, Charlet M, Hegy G, Van Dorsselaer A,Hoffmann JA. A novel inducible antibacterial peptide of Drosophila carries an O —

glycosylated substitution. J Biol Chem 1993;268:14893–7.[40] Ehret-Sabatier L, Loew D, Goyffon M, Fehlbaum P, Hoffmann JA, Van Dorsselaer

AV, Bulet P. Characterization of novel cysteine-rich antimicrobial peptides fromscorpion blood. J Biol Chem 1996;271:29537–44.

[41] Lubbers DW. Optical sensors for clinical monitoring. Acta Anaesthesiol ScandSuppl 1995;104:37–54.

[42] Magalhães MR, da Silva NJ, Ulhoa CJ. A hyaluronidase from Potamotrygon motoro(freshwater stingrays) venom: isolation and characterization. Toxicon 2008;51:1060–7.

[43] Fenner JP, Williamson JA, Skinner RA. Fatal and non-fatal stingray envenomation.Med J Aust 1989;151:621–5.

[44] Poh CH, Yuen R, Khoo HE, Chung M, Gwee M, Gopalakrishnakone P. Purificationand partial characterization of stonustoxin (lethal factor) from Synanceja horridavenom. Comp Biochem Physiol B 1991;99:793–8.

[45] Khoo HE, Yuen R, Poh CH, Tan CH. Biological activities of Synanceja horrida(stonefish) venom. Nat Toxins 1992;1:54–60.

[46] Brain SD, Williams TJ. Inflammatory mechanims of inflamed-tissue factor. AgentsActions 1985;3:348–56.

[47] Nagata K, Fugimiva M, Sugiura H, Uehara M. Intracellular localization of serotoninin mast cells of the colon in normal and colitis rats. Histochem J 2001;33:559–68.

[48] Mossner R, Lesch KP. Role of serotonin in the immune system and inneuroimmune interactions. Brain Behav Immun 1998;12:249–71.

[49] Ulbrich H, Eriksson EE, Lindbom L. Leukocyte and endothelial cell adhesionmolecules as targets for therapeutic interventions in inflammatory disease. TrendsPharmacol Sci 2003;24:640–7.

[50] Tjølsen A, Berge DG, Hunskaar S, Rosland JH, Hole K. The formalin test: anevaluation of the method. Pain 1992;51:5–17.

[51] Ley K. Molecular mechanisms of leukocyte recruitment in the inflammatoryprocess. Cardiovasc Res 1996;32:733–42.

[52] Robinson SD, Frenette PS, RayburnH, CummiskeyM, Ullman-CullereM,Wagner DD,HynesRO.Multiple, targeteddeficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment. Proc Natl Acad Sci USA 1999;96:11452–7.

[53] Smith ML, Sperandio M, Galkina EV, Ley K. Autoperfused mice flow chamberreveals synergistic neutrophil accumulation through P-selectin and E-selectin. JLeukoc Biol 2004;76:985–93.

[54] Issekutz AC, Movat HZ. The in vivo quantitation and kinetics of rabbit neutrophilleukocyte accumulation in the skin in response to chemotactic agents andEscherichia coli. Lab Invest 1980;42:310–7.

[55] Ryan GB, Majno G. Acute inflammation. Am J Pathol 1977;86:185–274.[56] Doherty DE, Downey GP, Worthen GS, Haslett C, Henson PM. Monocyte retention

and migration in pulmonary inflammation: requirement for neutrophils. LabInvest 1988;59:200–13.

[57] Adams DO, Hamilton TA. Macrophages as destructive cells in host defence. In:Gallin JI, Goldstein IM, Snyderman R, editors. Inflammation: Basic Principles andClinical Correlates. 2nd ed. New York, NY: Raven Press; 1992. p. 637–62.

1376 J. Monteiro-dos-Santos et al. / International Immunopharmacology 11 (2011) 1368–1377

[58] Kaplanski G, Marin V, Montero-Julian F, Mantovani A, Farnarier C. IL-6: a regulatorof the transition from neutrophil to monocyte recruitment during inflammation.Trends Immunol 2003;24:25–9.

[59] Sosa-Rosales JI, Piran-Soares AA, Farsky SH, Takehara HA, Lima C. Lopes-FerreiraM. Important biological activities induced by Thalassophryne maculosa fish venom.Toxicon 2005;45:155–61.

[60] Sivan G, Venketesvaran K, Radhakrishnan CK. Biological and biochemicalproperties of Scatophagus argus venom. Toxicon 2007;50:563–71.

[61] Hahn ST, O'Connor JM. An investigation of the biological activity of bullrout(Notesthes robusta) venom. Toxicon 2000;38:79–89.

[62] Mozumder MMH. Antibacterial activity in fish mucus from farmed fish. Universityof Tromsø, Norway: Norwegian College of Fishery Science; 2005.

[63] Ruangsri J, Fernandes JM, Brinchmann M, Kiron V. Antimicrobial activity in thetissues of Atlantic cod (Gadus morhua L.). Fish Shellfish Immunol 2010;28:879–86.

[64] Bergsson G, Agerberth B, Jornvall H, Gudmundsson GH. Isolation and identificationof antimicrobial components from the epidermal mucus of Atlantic cod (Gadusmorhua). FEBS J 2005;272:4960–9.

[65] Shai Y, Fox J, Caratsch C, Shih YL, Edwards C, Lazarovici P. Sequencing andsynthesis of pardaxin, a polypeptide from the Red Sea Moses sole with ionophoreactivity. FEBS Lett 1988;242:161–6.

[66] Cole AM, Darouiche RO, Legarda D, Connell N, Diamond G. Characterization of afish antimicrobial peptide: gene expression, subcellular localization, and spectrumof activity. Antimicrob Agents Chemother 2000;44:2039–45.

[67] Subramanian S, Ross NW, MacKinnon SL. Myxinidin, a novel antimicrobial peptidefrom the epidermal mucus of hagfish, Myxine glutinosa L. Mar Biotechnol NY2009;11:748–57.

[68] Cole AM, Weis P, Diamond G. Isolation and characterization of pleurocidin, anantimicrobial peptides in the skin secretions of winter flounder. J Biol Chem1997;272:12008–13.

[69] Noga EJ, Silphaduang U. Piscidins: a novel family of peptide antibiotics from fish.Drug News Perspect 2003;16:87–92.

[70] Khoo HE. Bioactive proteins from stonefish venom. Clin Exp Pharmacol Physiol2002;29:802–6.

[71] Lopes-Ferreira M, Moura-da-Silva AM, Piran-Soares AA, Ângulo Y, Lomonte B,Gutiérrez JM, Farsky SH. Hemostatic effects induced by Thalassophryne nattererifish venom: a model of endothelium-mediated blood flow impairment. Toxicon2002;40:1141–7.

[72] Carrijo LC, Andrich F, de Lima ME, Cordeiro MN, Richardson M, Figueiredo SG.Biological properties of the venom from the scorpionfish (Scorpaena plumieri) andpurification of a gelatinolytic protease. Toxicon 2005;45:843–50.

[73] Russell FE. Poisonous marine animals. New York: TFH Publications; 1971.

1377J. Monteiro-dos-Santos et al. / International Immunopharmacology 11 (2011) 1368–1377