(Theraphosidae) spider venom - Core

Toxicon 61 (2013) 129–138

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Venomic and pharmacological activity of Acanthoscurria paulensis (Theraphosidae) spider venom Caroline Barbosa F. Mourão a,1, Fagner Neves Oliveira a,1, Andréa C. e Carvalho a, Claudia J. Arenas a, Harry Morales Duque a, Jacqueline C. Gonçalves a, Jéssica K.A. Macêdo a, Priscilla Galante a, Carlos A. Schwartz a, Márcia R. Mortari a, Maria de Fátima M. Almeida Santos b, Elisabeth F. Schwartz a, * a b

Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, Universidade de Brasília ,Instituto de Ciências Biológicas, Brasília 70910-900, DF, Brazil Departamento de Genética e Morfologia, Universidade de Brasília, Brasília 70910-900, DF, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 September 2012 Received in revised form 5 November 2012 Accepted 8 November 2012 Available online 21 November 2012

In the present study we conducted proteomic and pharmacological characterizations of the venom extracted from the Brazilian tarantula Acanthoscurria paulensis, and evaluated the cardiotoxicity of its two main fractions. The molecular masses of the venom components were identified by mass spectrometry (MALDI-TOF-MS) after chromatographic separation (HPLC). The lethal dose (LD50) was determined in mice. Nociceptive behavior was evaluated by intradermal injection in mice and the edematogenic activity by the rat hind-paw assay. Cardiotoxic activity was evaluated on in situ frog heart and on isolated frog ventricle strip. From 60 chromatographic fractions, 97 distinct components were identified, with molecular masses between 601.4 and 21,932.3 Da. A trimodal molecular mass distribution was observed: 30% of the components within 500-1999 Da, 38% within 3500– 5999 Da and 21% within 6500–7999 Da. The LD50 in mice was 25.4 2.4 mg/g and the effects observed were hypoactivity, anuria, constipation, dyspnea and prostration until death, which occurred at higher doses. Despite presenting a dose-dependent edematogenic activity in the rat hind-paw assay, the venom had no nociceptive activity in mice. Additionally, the venom induced a rapid blockage of electrical activity and subsequent diastolic arrest on in situ frog heart preparation, which was inhibited by pretreatment with atropine. In the electrically driven frog ventricle strip, the whole venom and its low molecular mass fraction, but not the proteic one, induced a negative inotropic effect that was also inhibited by atropine. These results suggest that despite low toxicity, A. paulensis venom can induce severe physiological disturbances in mice. © 2012 Elsevier Ltd. Open access under the Elsevier OA license.

Keywords: Acanthoscurria paulensis Tarantula Edematogenic Cardiotoxic Toxicity Toxin

1. Introduction According to the effects of venom in humans, accidents caused by spiders can be categorized in, at least, two distinct groups: those producing necrotic ulceration, and

* Corresponding author. Tel.: þ55 61 31073106; fax: þ55 61 31073107. E-mail address: [email protected] (E.F. Schwartz). 1 These authors contributed equally to the manuscript. 0041-0101 Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.toxicon.2012.11.008

the ones that do not. Arachnidism produced by widow spiders (Theridiidae) will result in systemic symptoms but with minimal tissue damage. Envenomation by Agelenidae family (araneomorph funnel-web spiders, including hobo and grass spiders) results in severe tissue damage (Mattiello-Sverzut et al., 1998; Elston et al., 2000) and, in a minority of accidents, also systemic symptoms. Local necrosis and systemic symptoms are observed in the events incited by Sicariidae (Loxosceles; recluse and fiddlehead spiders) (Madrigal et al., 1972; Barbaro et al., 1992).

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Tarantulas (Theraphosidae, Mygalomorphae) bites are considered to be painful, but do not induce local necrosis or systemic effects (Saucier, 2004). With exception of bites by spiders of the genus Atrax (Hexathelidae), there are no records of serious accidents caused by Mygalomorphae spiders in human probably due to several factors such as venom low toxicity to humans, insufficient amount of venom injected, greater difficulty of Orthognatans’ chelicerae in piercing the skin, and also because some species live in low frequented local by man (Lucas, 2003). Unless infection occurs, a slight inflammation at the puncture site can arise. Spiders of the family Theraphosidae have urticating hairs covering their bodies, which are brushed off by the spider as a mechanism of defense to deter predators. These hairs were found to induce local dermatitis in vertebrates, including humans (Shrum et al., 1999). The puncture wounds from the spider’s fangs require local wound care, follow-up for signs of infection, short-term analgesia and a tetanus booster (Kelley and Wasserman, 1998; Shrum et al., 1999). The spider venom is a diverse mixture of low molecular mass compounds (16% of all compounds), acylpolyamines (11%), linear peptides (6%), cysteine-knotted mini-proteins (60%), neurotoxic proteins (1%) and enzymes (6%) (Jackson and Parks, 1989; Kuhn-Nentwig et al., 2011). It is mainly used to paralyze prey and for defense, and contains toxins that affect the central or peripheral nervous systems. These neurotoxins have been identified mostly as acylpolyamines and peptides or proteins that act on membrane receptors or ion channels (see review Estrada et al., 2007). The acylpolyamine toxins are low molecular mass compounds (20 mg venom/paw) could induce painful response. In fact, 20 mg venom/paw was able to induce hind-paw edema after 10 min of administration. Phoneutria nigriventer (Costa et al., 2001; Zanchet and Cury, 2003) and Loxosceles gaucho (Barbaro et al., 2010) venoms and peptides isolated from Scaptocosa raptoria venom (Ferreira et al., 1998) also produced edema in rodents. These studies showed that pain and swelling caused by these spider venoms are related, as they involve the same molecular cascades. The cardiotoxic activity of A. paulensis venom and its two chromatographic fractions, LMMF and PF, was evaluated by two assays: in situ frog heart and frog heart ventricular slices. In both, the venom induced cardiac arrest inhibited by atropine, suggesting the dependence of acetylcholine receptor activation. Only the LMMF was able to produce similar response, indicating the venom peptides are not responsible for it. The venom of the tarantula spider Lasiodora sp. caused a dose-dependent bradycardia, a transient cardiac arrest and rhythm disturbances on isolated rat heart, effects that were enhanced by anticholinesterase drugs, abolished by atropine, inhibited by an inhibitor of ACh vesicular transport, and not modified by TTX, leading the suggestion that this venom induces the release of ACh from parasympathetic nerve terminals by activating TTXresistant Naþ-channels (Kalapothakis et al., 2003). The dialyzed P. nigriventer venom produced positive inotropic and chronotropic effects in isolated rat heart that were inhibited by b-adrenergic antagonists and potentiated by atropine (Costa et al., 1998). In a previous study, P. nigriventer whole venom induced negative chronotropic and inotropic effects on isolated guinea pig atria, these effects being abolished by atropine (Vital-Brazil et al., 1988). While the sympathetic effect is explained by the presence of venom neurotoxins able to modulate Naþ-channel activity, the parasympathetic response is probably mediated by the presence of biogenic amines in the venom, which were excluded by venom dialysis (Costa et al., 1998). In the present study, it was shown that the parasympathetic-like response produced by A. paulensis venom is also due to low molecular mass compounds, possibly biogenic amines also or polyamines. Alternatively, LMMF component(s),


eluting in more hydrophilic fractions, might induce the release of acetylcholine from parasympathetic nerve terminals, resulting in the negative inotropic effect observed. In conclusion, the A. paulensis venom proteomic and pharmacological profiling was presented for the first time. By means of chromatography and mass spectrometry the venom compounds variability was showed, which featured 60 chromatographic fractions and 97 different components. Noteworthy are the low molecular mass compounds, such as 601.4 and 729.6 Da which are putative acylpolyamines, in addition to many peptide components, among which 60% are between 3500 and 7999 Da. LD50 was defined and is in accordance to the values reported for tarantula spiders, which generally do not provoke severe envenoming. Despite that, A. paulensis venom induced many behavioral and physiological changes in mice, and edematogenic activity in rats. An inotropic effect produced on frog heart is probably due to the low molecular mass compounds present in the more hydrophilic fractions of venom that may act either by inducting the release of acetylcholine from parasympathetic terminals or by directly acting as a cholinergic agonist. Acknowledgments Financial support: CNPq (303003/2009-0, 490068/ 2009-0, 564223/2010-7). CBFM and ACEC receive scholarship from CNPq, and CJA, HMD, JCG, JKAM and PG from CAPES. The authors acknowledge Rafael D. Melani and Karla G. Moreira for their assistance on some bioassays, Dr Paulo César Motta for identifying the spiders, and Dr Carlos Bloch from Mass Spectrometry Laboratory, EMBRAPA, Brazil. Conflict of interest There is no conflict of interest. References Atkinson, R.K., 1993. A comparison of the toxicity of the venoms of twelve common Australian spider species on rodent vital organ systems. Comp. Biochem. Physiol. C. 106, 639–642. Barbaro, K.C., Cardoso, J.L., Eickstedt, V.R., Mota, I., 1992. Dermonecrotic and lethal components of Loxosceles gaucho spider venom. Toxicon 30 (3), 331–338. Barbaro, K.C., Lira, M.S., Araújo, C.A., Pareja-Santos, A., Távora, B.C., Prezotto-Neto, J.P., Kimura, L.F., Lima, C., Lopes-Ferreira, M., Santoro, M.L., 2010. Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom. Toxicon 56, 972–979. Bettini, S., Brignoli, P.M., 1978. Review of the spider families, with notes on the lesser-known poisonous forms. In: Bettini, S. (Ed.), Arthropod Venoms. Springer-Verlag, Berlin, pp. 103–118. Bixel, M.G., Krauss, M., Weise, C., Bolognesi, M.L., Rosini, M., Usherwood, P.N., Melchiorre, C., Hucho, F., 2001. Binding of polyamine-containing toxins in the vestibule of the nicotinic acetylcholine receptor ion channel. Farmaco 56 (1–2), 133–135. Bucherl, W., 1971. Spiders. In: Bucherl, W., Buckley, E.E. (Eds.), Venomous Animals and Their Venoms. Academic Press, London, pp. 197–277. Célérier, M.L., Paris, C., Lange, C., 1993. Venom of an aggressive African Theraphosidae (Scodra griseipes): milking the venom, a study of its toxicity and its characterization. Toxicon 31, 577–590. Chichorro, J.G., Lorenzetti, B.B., Zampronio, A.R., 2004. Involvement of bradykinin, cytokines, sympathetic amines and prostaglandins in formalin-induced orofacial nociception in rats. Br. J. Pharmacol. 141, 1175–1184.

137

Costa, S.K., Hyslop, S., Nathan, L.P., Zanesco, A., Brain, S.D., de Nucci, G., Antunes, E., 1998. Activation by Phoneutria nigriventer spider venom of autonomic nerve fibers in the isolated rat heart. Eur. J. Pharmacol. 18, 139–146. Costa, S.K., Esquisatto, L.C., Camargo, E., Gambero, A., Brain, S.D., De Nucci, G., 2001. Comparative effect of Phoneutria nigriventer spider venom and capsaicin on the rat paw oedema. Life Sci. 69, 1573– 1585. da Silva, S.R., da Silva, R., Lange, A.B., 2011. Effects of crustacean cardioactive peptide on the hearts of two Orthopteran insects, and the demonstration of a Frank-Starling-like effect. Gen. Comp. Endocrinol. 171 (2), 218–224. de Haro, L., Jouglard, J., 1998. The dangers of pet tarantulas: experience of the Marseilles Poison Centre. J. Toxicol. Clin. Toxicol. 36, 51–53. Elston, D.M., Eggers, J.S., Schmidt, W.E., Storrow, A.B., Doe, R.H., McGlasson, D., Fischer, J.R., 2000. Histological findings after brown recluse spider envenomation. Am. J. Dermatopathol. 22 (3), 242–246. Escoubas, P., Rash, L., 2004. Tarantulas: eight-legged pharmacists and combinatorial chemists. Toxicon 43, 555–574. Estrada, G., Villegas, E., Corzo, G., 2007. Spider venoms: a rich source of acylpolyamines and peptides as new leads for CNS drugs. Nat. Prod. Rep. 24, 145–161. Ferreira, L.A., Lucas, S.M., Alves, E.W., Hermann, V.V., Reichl, A.P., Habermehl, G., Zingali, R.B., 1998. Isolation, characterization and biological properties of two kinin-like peptides (peptide-S and peptide-r) from Scaptocosa raptoria venom. Toxicon 36, 31–39. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press. Guette, C., Legros, C., Tournois, G., Goyffon, M., Célérier, M.L., 2006. Peptide profiling by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry of the Lasiodora parahybana tarantula venom gland. Toxicon 47, 640–649. Herold, E.E., Yaksh, T.L., 1992. Anesthesia and muscle relaxation with intrathecal injections of AR636 and AG489, two acylpolyamine spider toxins, in rat. Anesthesiology 77, 507–512. Herzig, V., Wood, D.L., Newell, F., Chaumeil, P.A., Kaas, Q., Binford, G.J., Nicholson, G.M., Gorse, D., King, G.F., 2011. ArachnoServer 2.0, an updated online resource for spider toxin sequences and structures. Nucleic Acids Res. 39, 653–657. Horni, A., Weickmann, D., Hesse, M., 2001. The main products of the low molecular mass fraction in the venom of the spider Latrodectus menavodi. Toxicon 39, 425–428. Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30, 103–114. Isbister, G.K., Seymour, J.E., Gray, M.R., Raven, R.J., 2003. Bites by spiders of the family Theraphosidae in humans and canines. Toxicon 41 (4), 519–524. Jackson, H., Parks, T.N., 1989. Spider toxins: recent applications in neurobiology. Annu. Rev. Neurosci. 12, 405–414. Kalapothakis, E., Kushmerick, C., Gusmão, D.R., Favaron, G.O.C., Ferreira, A.J., Gomez, M.V., Pinto de Almeida, A., 2003. Effects of the venom of a Mygalomorph spider (Lasiodora sp.) on the isolated rat heart. Toxicon 41, 23–28. Kawai, N., Niwa, A., Abe, T., 1982. Spider venom contains specific receptor blocker of glutaminergic synapses. Brain Res. 247, 169–171. Kelley, T., Wasserman, G., 1998. The dangers of pet tarantulas: experience of the Marseilles Poison Centre. J. Toxicol. Clin. Toxicol. 36, 55–56. Kitaguchi, T., Swartz, K.J., 2005. An inhibitor of TRPV1 channels isolated from funnel web spider venom. Biochemistry 44, 15544–15549. Kuhn-Nentwig, L., Stocklin, R., Nentwig, W., 2011. Venom composition and strategies in spiders: is everything possible? Adv. Insect Physiol. 40, 1–86. Le Bars, D., Gozariu, M., Cadden, S.W., 2001. Animal models of nociception. Pharmacol. Rev. 53, 597–652. Lucas, S.M., 2003. Aranhas de interesse médico no Brasil. In: Cardoso, J.L.C., França, F.O., Wen, F.H., Sant’Ana Málaque, C.M., Haddad, V. (Eds.), Animais Peçonhentos no Brasil: biologia, clínica e terapêutica dos acidentes. Sarvier Editora, São Paulo, pp. 141–149. Lucas, S.M., Paula, F.D.S., Gonzalez Filho, H.M.O., Brescovit, A.D., 2010. Redescription and new distribution records of Acanthoscurria paulensis (Araneae: Mygalomorphae:Theraphosidae). Zoologia 27, 563–568. Madrigal, G.C., Ercolani, R.L., Wenzl, J.E., 1972. Toxicity from a bite of the brown spider (Loxosceles reclusus). Skin necrosis, hemolytic anemia, and hemoglobinuria in a nine-year-old child. Clin. Pediatr. (Phila) 11 (11), 641–644. Mattiello-Sverzut, A.C., Fontana, M.D., Diniz, C.R., da Cruz-Höfling, M.A., 1998. Pathological changes induced by PhTx1 from Phoneutria nigriventer spider venom in mouse skeletal muscle in vitro. Toxicon 36 (10), 1349–1361.

138


McNamara, C.R., Mandel-Brehm, J., Bautista, D.M., Siemens, J., Deranian, K.L., Zhao, M., Hayward, N.J., Chong, J.A., Julius, D., Moran, M.M., Fanger, C.M., 2007. TRPA1 mediates formalin-induced pain. Proc. Natl. Acad. Sci. U. S. A. 104, 13525–13530. Mello-Leitão, C., 1923. Theraphosoideas do Brasil. Rev. Mus. Paulista 13, 431–438. Moore, P.K., Oluyomi, A.O., Babbedge, R.C., Wallace, P., Hart, S.L., 1991. LNG-nitro arginine methyl ester exhibits antinociceptive activity in the mouse. Br. J. Pharmacol. 102, 198–202. Mortari, M.R., do Couto, L.L., dos Anjos, L.C., Mourão, C.B.F., Camargos, T.S., Vargas, J.A., Oliveira, F.N., Gati Cdel, C., Schwartz, C.A., Schwartz, E.F., 2012. Pharmacological characterization of Synoeca cyanea venom: an aggressive social wasp widely distributed in the Neotropical region. Toxicon 59, 163–170. Prophet, E.B., Mills, B., Arrington, J.B., Sobin, L.H. (Eds.), 1992. Armed Forces Institute of Pathology: Laboratory Methods in Histotechnology. American Registry of Pathology, Armed Forces Institute of Pathology, Washington DC. Raven, R.J., 2000. Spiders (other arachnids and myriapods). In: Ryan, M., Burwell, C. (Eds.), Wildlife of Tropical North Queensland. Queensland Museum, Brisbane, pp. 21–41. Robinson, G., Griffin, G., 1985. Effects of a bite from a barking spider (Selenocosmia stirlingi Hogg). Vic. Nat. 100, 116–117.

Saucier, J.R., 2004. Arachnid envenomation. Emerg. Med. Clin. N. Am. 22, 405–422. Schwartz, E.N., Schwartz, C.A., Sebben, A., Largura, S.W., Mendes, E.G., 1999. Indirect cardiotoxic activity of the caecilian Siphonops paulensis (Gymnophiona, Amphibia) skin secretion. Toxicon 37, 47–54. Shin, D.J., Jeong, C.W., Lee, S.H., Yoon, M.H., 2011. Receptors involved in the antinociception of intrathecal melatonin in formalin test of rats. Neurosci. Lett. 494, 207–210. Shrum, K., Robertson, D., Baratz, K.H., Casperson, T.J., Rostvold, J.A., 1999. Keratitis and retinitis secondary to tarantula hair. Arch. Ophthalmol. 117, 1096. Skinner, W.S., Dennis, P.A., Lui, A., Carney, R.L., Quistad, G.B., 1990. Chemical characterization of acylpolyamine toxins from venom of a trap-door spider and two tarantulas. Toxicon 28, 541–546. Sutherland, S.K., Tibballs, J., 2001. Australian Animal Toxins: the Creatures, Their Toxins and Care of Poisoned Patient, second ed. Oxford University Press, Melbourne. Vital-Brazil, O., Leite, G.B., Fontana, M.D., 1988. Modo de ação da peçonha da aranha armadeira, Phoneutria nigriventer (Keyserling, 1891), nas aurículas isoladas de cobaia. Ciênc Cult 40, 181–185. Zanchet, E.M., Cury, Y., 2003. Peripheral tackykinin and excitatory amino acid receptors mediate hyperalgesia induced by Phoneutria nigriventer venom. Eur. J. Pharmacol. 467, 111–118.