e. Soelo5503-900, Sa03-90
Article history:Received 7 May 2008Accepted 23 May 2008Available online 4 June 2008
nish in venom claws. Such structures are connected tothe short and cylindrical venom gland through a venomduct (Jangi, 1984; Pedroso et al., 2007). Centipedes are
are distributed in all continents except in Antarctica(Barnes et al., 1995). The major biological diversity ofcentipedes is found in subtropical and temperate regions(Knysak and Martins, 1999). The order Scolopendromorphacontains the largest centipedes in the world, and severalspecies are medically important. This order is divided intothree families: Scolopendridae (16 genera), Cryptopidae(7 genera) and Scolopocryptopidae (8 genera) (Schileyko,
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Toxicon 52 (2008) 2552631. Introduction
Centipedes are terrestrial arthropods belonging to theclass Chilopoda (Negrea and Minelli, 1995), being charac-terized by the presence of a head, and an externally-segmented body containing a pair of articulate legs ineach segment (Barnes et al., 1995). The ventral region ofthe head contains a pair of forcipules, whose extremities
predators that use venom primarily to arrest or subduepreys. They have noctivagant habits, and are frequentlyfound inside termitaria, under trunks in decompositionand in underground galleries. Moreover, they like to inhabithidden places that allow an easy adaptation, such as thesurroundings and interior of dwellings in urban areas (Kny-sak and Martins, 1999).
About 2800 species are known in the world, and theyVenomScolopendraCryptopsOtostigmus0041-0101/$ see front matter 2008 Elsevier Ltddoi:10.1016/j.toxicon.2008.05.012Centipedes have a venom gland connected to a pair of forceps, which are used to arrestpreys. Human victims bitten by centipedes usually manifest burning pain, paresthesiaand edema, which may develop into supercial necrosis. The aim of this work was tocharacterize and compare toxic activities found in venoms of three species of BraziliancentipedesdOtostigmus pradoi, Cryptops iheringi and Scolopendra viridicornis. By SDSPAGE (420%), important differences were noticed among venoms (between 7 and205 kDa). Few bands showed feeble caseinolytic, brinogenolytic and gelatinolytic activi-ties by zymography, but strong hyaluronidase activity was observed in S. viridicornis andO. pradoi venoms. In addition, such activities could be inhibited by o-phenanthroline, in-dicating that these enzymes are metalloproteinases. All venoms induced nociception,edema and myotoxicity in mice, but only S. viridicornis induced mild hemorrhagic activity.No coagulant activity was detected in centipede venoms. Low phospholipase A2 activitywas observed exclusively in S. viridicornis and O. pradoi venoms, but these venoms had in-tense direct hemolytic activity on human erythrocytes. Cross-reactivity among venomswas observed using species-specic sera raised in rabbits. Differences were noticed amongcentipede venoms, but S. viridicornis is indeed the most toxic venom and thereby it couldinduce a more severe envenomation.
2008 Elsevier Ltd. All rights reserved.a r t i c l e i n f o a b s t r a c tToxic activities of Brazilian centiped
Marlia B. Malta a, Marcela S. Lira a, Sabrina LRosana Martins b, Samuel P.G. Guizze b, Marca Laboratory of Immunopathology, Butantan Institute, Av. Vital Brasil 1500, 0b Laboratory of Arthropods, Butantan Institute, Av. Vital Brasil 1500, 05503-9c Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brasil 1500, 055. All rights reserved.venoms
ares a, Guilherme C. Rocha a, Irene Knysak b,L. Santoro c, Katia C. Barbaro a,*
00, Sao Paulo, SP, Brazilo Paulo, SP, Brazil0, Sao Paulo, SP, Brazil
evier .com/locate/ toxicon
M.B. Malta et al. / Toxicon 52 (2008) 2552632561996; Shelley, 2002). Scolopendromorpha centipedes aregeneralist predators, but they appear chiey to eat insects,spiders and other arthropods. The larger species alsoeat small vertebrates, such as rodents, birds and snakes(Stankiewicz et al., 1999). The Scolopendra genus may reachmore than 200 mm, and is commonly found throughoutBrazil, especially in the states of the northern and north-eastern regions.
Cases of humans bitten by centipedes have beenreported in Brazil (Knysak et al., 1998; Barroso et al.,2001; Medeiros et al., in press), but limited information isavailable about the biological activities found in venomsof Brazilian centipedes. Three genera of centipedes aremedically important in Brazil: Otostigmus, Scolopendraand Cryptops (Buecherl, 1946; Knysak et al., 1998; Barrosoet al., 2001, Medeiros et al., in press). Centipede bites admit-ted to Hospital Vital Brazil (Butantan Institute, Brazil) aremainly caused by genera Cryptops and Otostigmus(Medeiros et al., in press), and patients usually manifestmild symptoms, especially pain, edema and erythema(Knysak et al., 1998; Barroso et al., 2001; Medeiros et al.,in press). However, severe cases of centipede envenoma-tion have also been reported, especially in patients bittenby Scolopendra spp. (Harada et al., 1999; Acosta and Cazorla,2004; Ozsarac et al., 2004; Wang et al., 2004; Hasan andHassan, 2005; Yildiz et al., 2006). Taking into considerationthe clinical picture of patients admitted to Hospital VitalBrazil who were bitten by centipedes, we investigated theenzymatic, immunological and biological properties ofvenoms of Otostigmus pradoi, Cryptops iheringi and Scolo-pendra viridicornis, species that are the major agents of cen-tipede bites in Brazil.
2. Materials and methods
2.1. Animals and venoms
Swiss mice (1820 g) and adult rabbits (34 kg) wereprovided by Butantan Institute Animal House. Animals re-ceived food and water ad libitum. Specimens of Otostigmuspradoi (n 20) and Cryptops iheringi (n 40) centipedeswere collected in Sao Paulo State, Brazil, and of Scolopendraviridicornis (n 10) inTocantins State, Brazil. The specimenswere identied by specialists of Laboratory of Arthropods(Butantan Institute). All specimens were adults, and variedbetween 40 and 80 mm for O. pradoi, 60 and 100 mm forC. iheringi, and 160 and 200 mm for S. viridicornis. These an-imals were maintained in captivity for 34 years (dead ani-mals were replaced during this period) and milked byelectrical stimulation once a month. The average amountof venom obtained per animal in each extraction was25 mg from O. pradoi, 17.5 mg from C. iheringi and 100 mgfrom S. viridicornis. Venom pools of at least six different ex-tractions were stored at20 C, and thawed at the momentof use. The protein content of venom pools was determinedusing bicinchoninic acid according to Smith et al. (1985),using bovine serum albumin as standard. The proceduresinvolving animals were conducted in conformity withnational laws and policies controlled by Butantan InstituteAnimal Investigation Ethical Committee (protocol no. 115/2002).2.2. Production of specic rabbit antivenom
Sera against S. viridicornis, O. pradoi or C. iheringi centi-pede venoms were obtained by immunization of rabbits.Two hundred micrograms of each venom extract wasdiluted in 500 mL of PBS and added to 500 mL of completeFreunds adjuvant, and these mixtures were injected i.m.After 1 month, rabbits received ve consecutive boostersof antigen emulsied in incomplete Freunds adjuvant,with 15-day intervals. Blood was collected and sera wasseparated and stored at 20 C until used.
Rabbit species-specic sera were titrated by ELISA usingS. viridicornis, O. pradoi and C. iheringi centipede venoms(10 mg/mL) to coat plates (Nunc, USA), according to Theak-ston et al. (1977). The reaction was read using an ELISAreader (Multiskan EX) and the titer determined as the re-ciprocal of the highest dilution that causes an absorbancegreater than 0.050 at 492 nm, as non-specic reactionswere observed below this value.
2.4. Sodium dodecyl sulfatepolyacrylamidegel electrophoresis (SDSPAGE)
Proteins of S. viridicornis, O. pradoi and C. iheringi centi-pede venoms (5 mg) were analyzed by SDSPAGE (420%acrylamide resolution gels, Pierce, USA) under non-reducing and reducing conditions (Laemmli, 1970). Afterseparation of proteins by electrophoresis, gels were silverstained (Blum et al., 1987). Myosin, b-galactosidase, bovineserum albumin, carbonic anhydrase, soybean trypsin inhib-itor, lysozyme and aprotinin were used as the molecularmass markers (Kaleidoscope pre-stained standards, Bio-Rad, Hercules, CA, USA).
2.5. Western blotting
Proteins of S. viridicornis, O. pradoi and C. iheringi centi-pede venoms (20 mg) were rst fractionated by SDSPAGE(10%) as described above. Electroblotting was performedas described by Towbin et al. (1979). Nitrocellulose mem-branes were incubated with rabbit species-specic sera,diluted at 1/200. Immunoreactive proteins were detectedusing peroxidase-labeled anti-rabbit IgG and the blotwas developed with 0.05% (w/v) 4-chloro-1-naphthol in15% (v/v) methanol in the presence of 0.03% (v/v) H2O2.Non-immunized rabbit serum was used as control. Pre-stained molecular mass markers (BioRad, Hercules, CA,USA) were used.
2.6. Protease and hyaluronidase assays
Zymographywas employed to evaluate protease and hy-aluronidase activities of centipede venoms, using casein,gelatin and brinogen (Heussen and Dowdle,1980; Barbaroet al., 2005), and hyaluronic acid from rooster comb (Sigma,St. Louis, MO) (Miura et al., 1995; Barbaro et al., 2005), re-spectively, as substrates. Samples of S. viridicornis, O. pradoiand C. iheringi centipede venoms in non-reducing sample
M.B. Malta et al. / Toxicon 52 (2008) 255263 257buffer were loaded, and 10% polyacrylamide gels were runat 20 mA/gel. Clear areas in the gel indicated regions of en-zyme activity. When required, the metal chelating agent1,10-phenanthroline (Sigma Chemicals, St Louis, MO) wasadded in a nal concentration of 3 mM to every gel washingand incubation buffers, and then gels were stained as usual.Pre-stained molecular mass markers (BioRad, Hercules, CA,USA) were used.
2.7. Nociceptive and edematogenic activities
To detect the nociceptive activity, mice (n 6) wereinjected in the right hind paw with 30 mL of PBS containingdifferent doses (0.9, 3.8, 15 and 60 mg) of S. viridicornis, O.pradoi or C. iheringi centipede venoms. Animals wereplaced individually under glass funnels on a mirror. After-wards, the reactivity of animals to lick or bite the injectedfoot was measured, in seconds, during 30 min of experi-mental evaluation (Hunskaar et al., 1985). Animals injectedonly with PBS were used as negative controls.
Edema-forming activity was evaluated at different times(0.25, 1, 4, 24, 48 and 72 h) as the difference of thickness(mm) between the right foot pawdinjected with differentdoses (3.8, 15 and 60 mg) of S. viridicornis, O. pradoi or C.iheringi centipede venoms diluted in PBS or vehicle alone(negative control)dand the left paw of mice (not injected).
2.8. Estimation of myotoxic activity
Mice (n 8) were injected intramuscularly (i.m.) intothe right gastrocnemius muscle with 120 mg of S. viridicor-nis,O. pradoi or C. iheringi centipede venoms in 50 mL of PBS.The control group was injected with PBS alone. After 3 h,blood was collected from the brachial plexus. Sera of micewere separated and immediately assayed for creatine ki-nase (CK) activity (CK-NAC Liquiform, Labtest, Brazil). Oneunit corresponds to the amount of enzyme that hydrolyzes1 mmol of creatine per minute at 25 C. Myotoxic activitywas expressed as U/mg of venom of three independent ex-periments. Bothrops jararacussu snake venom (120 mg) wasused as a positive control.
2.9. Coagulant activity
Clotting time was performed according Santoro andSano-Martins (1993). Scolopendra viridicornis, O. pradoiand C. iheringi centipede venoms (30, 60 and 120 mg) di-luted in 50 mL of PBS were added to 200 mL of humanplasma. Samples (duplicate) were observed for 5 min at37 C to determine the clotting time. After this period,50 mL of thrombin (30 U/mL) (Sigma, St. Louis, MO) wasadded to samples to verify brinogen hydrolysis. As a posi-tive control, 50 mL samples of two-fold serially dilutedBothrops jararaca snake venom (1.56200.0 mg) were usedto determine the minimal coagulant dose (MCD). Experi-ments were carried out in duplicate.
2.10. Direct hemolytic activity
Humanblood (typeO, Rh) was collected in the presenceof 0.15 M sodium citrate (9:1), and centrifuged 1900 g for15 min at 10 C. Red blood cells were obtained after threeconsecutive washes with PBS. Samples (50 mL) of 3% redblood cells were mixed with 100 mL of different doses (3.8,7.5, 15, 30, 60 and 120 mg) of S. viridicornis, O. pradoi and C.iheringi centipede venoms. Each sample (50 mL) was placed(duplicates) in microplates. As controls, distilled water(100% hemolysis) and PBS (0% hemolysis) were used. Micro-plateswerekeptat roomtemperature for3 h.Theabsorbancewas read using an ELISA reader (Multiskan EX) at 595 nm.
2.11. Phospholipase A2 activity
Phospholipase activity was determined as describedelsewhere (Santoro et al., 1999). Scolopendra viridicornis, O.pradoi or C. iheringi (15 mg) venoms, diluted in 15 mL of PBSpH 7.4, were added to 1.5 mL of reaction solution (100 mMNaCl, 10 mM CaCl2, 7 mM Triton X-100, 0.265% soybeanlecithin, 98.8 mMphenol red, pH7.6) in a spectrophotometercuvette. The solution was immediately homogenized andread at 558 nm. Thedenitionof 1 Uof phospholipaseA2 ac-tivitywas taken as the amountof toxin (mgof protein/assay)producing a decrease of 0.001 absorbance units per minuteunder the conditions described. Crotalus durissus terricus(6 mg) was used as a positive control. Phospholipase activitywas expressed as U/mg of two independent experiments.
2.12. Hemorrhagic activity
The hemorrhagic activity was evaluated by the methoddescribed by Kondo et al. (1960) with some modications.Groups of six mice were shaved on the back and then intra-dermically (i.d.) injected with different doses of S. viridicor-nis, O. pradoi or C. iheringi venoms (15, 30, 60 and 120 mg) inphosphate-buffered saline (PBS). Skins were excised 2 hlater and the diameters of hemorrhagic spots were mea-sured on the internal surfaces. Injections of B. jararacasnake venom (1 mg) or PBS were used as positive and neg-ative controls, respectively.
2.13. Statistical analysis
Results are expressed as means S.D. Two-way ANOVAfollowed by Bonferroni test was used to analyze data,employing SigmaStat 3.0 software. Values with p < 0.05were considered statistically signicant.
3.1. Analysis of venoms by SDSPAGE
Electrophoretic proles of O. pradoi, C. iheringi and S. vir-idicornis venoms showed that the composition of thesevenoms is distinct, with many unique bands in each venom(Fig. 1). Major differences were observed between 156 and40 kDa, and more densely stained bands were present in O.pradoi venom. On the other hand, C. iher...