CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Toxicon 60 (2012) 934–942 Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus F. Costal-Oliveira a, C.G. Duarte a, R.A. Machado de Avila a, M.M. Melo c, K.C.F. Bordon d, E.C. Arantes d, N.C. Paredes e, B. Tintaya e, C. Bonilla e, R.E. Bonilla a, W.S. Suarez e, A. Yarleque f, J.M. Fernandez f, E. Kalapothakis b, Carlos Chávez-Olórtegui a,* a Departamentos de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil b Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil c Escola de Veterinaria, Universidade Federal de Minas Gerais, Belo Horizonte, 31270-901 Minas Gerais, Brazil d Departamento de Física e Química, Faculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, Ribeirão Preto-SP, Brazil e Instituto Nacional de Salud, Lima, Peru f Universidad Nacional Mayor de San Marcos, Lima, Peru article info abstract Article history: This communication describes the general biochemical properties and some immuno- Received 3 May 2012 logical characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which Received in revised form 1 June 2012 is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic Accepted 20 June 2012 to mice, the LD determined was 0.1 mg/kg and 21.55 mg/kg when the venom was Available online 29 June 2012 50 injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance Keywords: liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two Hadruroides lunatus scorpion venom Phospholipasic activity peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were LD50 Anti-H. lunatus sera produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector. Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. 1. Introduction geographical distribution. This biodiversity has not, however, been properly studied. Peru is a country in western South America. It is Hadruroides (Pocock, 1893) is a scorpion genus included bordered on the north by Ecuador and Colombia, on the in the family Iuridae, subfamily Charaboctoninae. This east by Brazil, on the southeast by Bolivia, on the south by genus comprises sixteen species and there members Chile, and on the west by the Pacific Ocean. This nation has appear brown in color with darker stains and have median a rich and diverse herpetic and arachnid fauna, with wide size of 80 mm (Ochoa and Prendini, 2010; Maury, 1975). Hadruroides scorpions have been reported in Bolivia, Chile, Colombia, Ecuador, Peru, and Venezuela (Mello-Leitão, 1945; Esquivel de Verde, 1968; Kinzelbach, 1973; Maury, * Corresponding author. Departamento de Bioquímica e Imunologia, 1975; Cekalovic, 1983; Sissom and Fet, 2000), but are Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. CP: 486. CEP: 31270-901. Tel.: þ55 31 3409 2625; actually restricted to Ecuador, Peru, northern Chile, and fax: þ55 313 441 5963. several offshore islands, including the Galápagos E-mail address: [email protected] (C. Chávez-Olórtegui). (Cekalovic, 1966; Maury, 1975; Francke and Soleglad, 1981). 0041-0101 Ó 2012 Elsevier Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.toxicon.2012.06.013 F. Costal-Oliveira et al. / Toxicon 60 (2012) 934–942 935 Species of Hadruroides inhabit inter-Andean valleys, Pacific were fed weekly with cockroaches. The venom from desert, and dry forest habitats (Ochoa and Prendini, 2010). mature scorpions was obtained by electrical stimulation Hadruroides lunatus (“escorpion de los pedregales”)is (12 V) of the telsons. The venom collected in micropipettes the most medically relevant species in Peru. According to was diluted in ultrapure water, pooled and stored at À20 C the Health Ministry of Peru (Ministerio de Salud del Perú, until use. The protein concentration was determined by the 2004), the number of human envenomation cases repor- method of Lowry et al. (1951). ted has increased during recent years, with most incidents Tityus serrulatus mature scorpions were collected in the occurring in the Central Coast of the country, which region of Belo Horizonte and maintained at the “Seção de corresponds with the main area of geographical distribu- Animais Peçonhentos” of Ezequiel Dias Foundation tion of H. lunatus scorpions (Zavaleta et al., 1981). Severe (FUNED) of Belo Horizonte, Brazil. The crude venoms were toxic effects by H. lunatus stings have not been noted in obtained by electrical stimulation of the telsons, lyophi- humans; however, intense pain, edema and ulceration are lized and stored at À20 C in the dark until use. The venoms frequently described as symptoms (Zavaleta et al., 1981). from the scorpions Androctonus australis hector and Cen- Different approaches are adopted for the treatment of truroides sculpturatus were obtained from the Laboratoire scorpion envenomations such as local care, analgesics and de Biochimie, Faculté de Médecine, Marseille, France and antihistaminics (Ministério de Salúd, Peru, 2004). Never- from Sigma Chemical Company, St. Louis, USA, respectively. theless, there are no scientific data to support these treat- Male and female Swiss and C57BL/6 mice (weighing 18– ments. The Instituto Nacional de Salud (INS) in Lima, Peru 22 g) and male Wistar rats (weighing 110–150 g) were does not produce specific scorpion anti-venon (Ministério maintained at the Centro de Bioterismo of the Instituto de de Salúd, Peru, 2004). Consequently, the treatment of Ciências Biológicas of the Universidade Federal de Minas scorpion envenomations with specific anti-venom for Gerais (UFMG), Belo Horizonte, MG, Brazil. All animals Peruvian species does not exist. received water and food under controlled environmental Very little is known about the structural and functional conditions. The experimental protocols were approved by characteristics of Peruvian scorpion venoms. The first the “Ethics Committee on the Use of Laboratory Animals of toxicological information was obtained from research on UFMG” (CETEA-UFMG). Eight- to nine-week-old New Zea- the H. lunatus species (Delgado and Pesce, 1967; Aguilar, land rabbits were used to produce anti-H. lunatus and anti- 1968; Aguilar and Meneses, 1970 and Zavaleta et al., T. serrulatus sera. Animals were maintained and handled as 1981). The pharmacological effects described by Zavaleta described previously. et al. (1981) showed that H. lunatus crude venom has a low lethality in mice (LD50, 68 mg/kg i.p.) and, in dogs, 2.2. Toxic activities of H. lunatus venom induces a fall in blood pressure. Neurotoxic activity in insects, crustaceans and mice and antibacterial peptides 2.2.1. Determination of median lethal dose (LD50) from the Hadruroides sp. crude venoms were showed by The lethality was assessed by intraperitoneal (i.p.) and Escobar et al. (2002) and Escobar and Flores, (2008). intracranial (i.c.) routes. For the intraperitoneal route, However, proteolytic and phospholipasic activities of this groups of four mice were injected with different doses of scorpion venoms are not shown in these studies. Despite venom (from 11.53 mg to 32.95 mg per kg of body weight) these published works, the studies with Peruvian scorpions dissolved in 0.5 mL of PBS–BSA 0.1%. For the intra- are still very preliminary. cerebroventricular route, groups of six mice were injected In view of the lack of information on the general with various doses of venom (from 0.075 mg to 0.8 mg per characteristics of Hadruroides scorpion venoms, the main kg) dissolved in 5 mL of PBS–BSA 0.1%. Twenty four hours goal of this work was to report additional biochemical later deaths were recorded and the LD50 was then calcu- and toxic characterization of H. lunatus scorpion venom. lated by Probit analysis (Finney, 1971). In this paper, the hyaluronidase, proteolytic, phospholi- pase, cardiotoxic and lethal activities of H. lunatus crude 2.2.2. Determination of proteolytic activity (PA) venom were investigated. This communication also Proteolytic activity was measured with dimethylcasein describes the separation of the soluble venom compo- (Sigma) as described by Lin et al. (1969) with modifications nents by SDS-PAGE and by high performance liquid described in Sanchez et al. (2000). Dilutions corresponding chromatography (HPLC). Furthermore, the last part of this to 5, 10, 20 and 40 mg of venom were used and absorbance study shows, some immunological characteristics of values were determined at 340 nm. One unit was defined as soluble whole venom using specific polyclonal rabbit anti- DA 340 nm/min. Activity was expressed relative to protein H. lunatus antibodies. concentration (mg). 2. Materials and methods 2.2.3. Determination of phospholipase A2 (PLA2) activity PLA2 activity was measured using an indirect hemolytic 2.1. Animals and venoms assay (Gutierrez et al., 1988). Increasing concentrations of H. lunatus crude venom (0.0625, 0.125, 0.25, 0.5 and 1 mg) were H. lunatus scorpions were collected in the region of prepared in a final volume of 15 mL in PBS and added to 2 mm Atocongo (Lima, Peru) and maintained in the herpetarium wells in agarose gels (0.8% in phosphate buffered saline, pH of the Centro Nacional de Producción de Biologicos of 8.1) containing 1.2% sheep erythrocytes, 1.2% egg yolk as Instituto Nacional de Salud (INS), in Lima, Peru.