Toxicon 73 (2013) 33–46 Contents lists available at SciVerse ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Another record of significant regional variation in toxicity of Tityus serrulatus venom in Brazil: A step towards understanding the possible role of sodium channel modulatorsq Fagner Neves Oliveira a, Márcia Renata Mortari a, Fabiana Pirani Carneiro b, Jimmy Alexander Guerrero-Vargas a, Daniel M. Santos c, Adriano M.C. Pimenta c, Elisabeth F. Schwartz a,* a Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF 70910-900, Brazil b Departamento de Patologia, Faculdade de Medicina, Universidade de Brasília, Brasília, DF 70910-900, Brazil c Laboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil article info abstract Article history: The scorpion Tityus serrulatus is responsible for the most severe accidents that have been Received 29 April 2013 registered in Brazil, mainly in the state of Minas Gerais (MG), being the lung edema (LE), Received in revised form 24 June 2013 the main cause of death in these accidents. Although an increased in the number of ac- Accepted 27 June 2013 cidents caused to this species in Federal District (Distrito Federal – DF), it seems that this Available online 12 July 2013 particular species is not responsible for severe scorpionism cases in this region. Given this observation, we tested the toxicity in mice and compared the ability of T. serrulatus venom Keywords: from DF (Ts-DF) and Minas Gerais State (Ts-MG) to induce LE in rats. The LD50 of Ts-DF Scorpion venom m Tityus serrulatus venom was 51.6 g/mouse, almost twice (1.98) higher than that obtained for Ts-MG Pulmonary edema venom. The ability of venom (0.5 mg/kg) to induce LE in rats was determined by the DL50 wet weight differences between treated and untreated lungs, by pulmonary morphological þ Na -channel analyses and by pulmonary vascular permeability (PVP) using the Evans blue protocol. Toxin Significant differences in the wet weight of lungs and changes in PVP were found in Ts-MG venom treated rats when compared to rats treated with Ts-DF venom or untreated rats (p < 0.001), but no differences occurred when comparing rats treated with Ts-DF venom and untreated rats (p < 0.05). These results were confirmed by evaluation of pulmonary morphology. Comparison of chromatographic profiles obtained from these venoms (Ts-DF and Ts-MG) using the fractal dimension (D) analysis and the molecular mass fingerprint of the chromatographic fractions showed a higher number of components between 35 and 40% acetonitrile in Ts-MG venom than in Ts-DF venom, indicating a higher diversity of sodium channel modulators in that venom. Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. 1. Introduction q This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works Li- Although all living scorpion species are venomous, less cense, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. than 25 species are considered dangerous to humans * Corresponding author. Tel.: þ55 61 3107 3106; fax: þ55 61 3107 3107. (Lourenço and Eickstedt, 2009). Most medically important E-mail address: [email protected] (E.F. Schwartz). scorpions belong to the Buthidae family, which includes 0041-0101/$ – see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.06.021 34 F.N. Oliveira et al. / Toxicon 73 (2013) 33–46 the South-American Tityus species (Cupo et al., 2009; Probably in the 1970s, this species was introduced in the Lourenço and Eickstedt, 2009). According to Brazilian Federal District in Brazil (Distrito Federal, DF) during Ministry of Health, the morbidity and mortality rates, due the occupancy of this region after the new Brazilian to scorpion stings are reported from various countries, capital was built (Lourenço et al., 1994), the inhabitant especially in children (Funasa-MS, 2001, 2009). The effects population in DF was 141,742, increasing to 537,492 in of the venom on humans are highly variable with severity 1970, and to 2,570,160 in 2010 (http://www.ibge.gov.br/ ranging from localized, self-resolving pain to death home/estatistica/populacao/censo2010/resultados_dou/ (Funasa-MS, 2001, 2009). Overall, the scorpion venom DF2010.pdf). In the last decade (2000–2010), there were consists of a complex mixture of short and long chain 1889 scorpion accidents in DF. Surprisingly, accidents basic peptides associated with small amounts of free caused by T. serrulatus in DF (Brazil) are considered mild amino acids and salts. However, the most important and symptoms such as acute pulmonary edema have not compounds of scorpion venoms are the neurotoxic pep- been reported (Yoshizawa, 2002; Sinan-MS, 2013) while in tides, which act on ion channels resulting in increased Minas Gerais, a vicinal state, envenoming by this same release of acetylcholine, noradrenaline and adrenaline, species might be severe with reported deaths caused by affecting both the sympathetic and parasympathetic sys- acute lung edema (Funasa-MS, 2001, 2009). Given that, in tems. The neurotoxic peptides are responsible for most the present work, we compared the toxicity and the ede- signs and symptoms observed in scorpion poisoning matogenic activity of T. serrulatus venoms obtained from (Dávila et al., 2002; Vasconcelos et al., 2005; Cupo et al., animals captured in the states of DF and MG in Brazil, and 2007; Pinto et al.,. 2010a,b). The scorpionism in Brazil the venom composition of both scorpion populations to has grown extensively in the last decade and has exceeded understand the differences observed. the number of snake bites which used to lead the ranks of accidents caused by venomous animals in the country 2. Material and methods (Funasa-MS, 2001, 2009). In Brazil, 12,704 and 58,608 scorpionism cases were reported in 2000 and 2011 2.1. Animals and venom respectively (http://portal.saude.gov.br/portal/arquivos/ pdf/tabela02_casos_escorpiao2000_2011_01_04_2013. Male Wistar rats (Rattus norvegicus), weighting from pdf). According to the same Brazilian public health agency, 230 to 250 g and male Swiss mice (Mus musculus), from 18 the number of deaths went from 16 in 2000 to 86 in 2011. to 20 g, were supplied by the vivarium facility of the Bio- Tityus serrulatus scorpion, an endemic species from Brazil, logical Sciences Institute, University of Brasilia (Brazil), is considered the most dangerous species in this country where they were kept in cages, maintained under appro- because it produces a potent venom and is responsible for priate conditions and received commercial chow and water the most frequent and serious accidents that have been ad libitum. registered (Barraviera, 1995; Funasa-MS, 2009). Specimens of T. serrulatus scorpions were collected in The scorpionism is classified according to the intensity urban regions of Distrito Federal and the venom was of symptoms such as mild, moderate or severe. The mild extracted by electrical stimulation, resuspended in ultra- accidents are characterized by local symptoms (pain and pure water and lyophilized. The T. serrulatus venom from paresthesia), while in moderate and severe accidents, in urban regions of Minas Gerais was kindly provided by Dr. addition to local symptoms, systemic symptoms are also Consuelo Latorre Fortes-Dias from the Fundação Ezequiel observed (gastrointestinal, respiratory and cardiopulmo- Dias (FUNED, Belo Horizonte/MG), and was obtained by the nary, and neurological symptoms), which are more intense same method. in severe cases (Funasa-MS, 2001, 2009). In fact, death is mainly caused by acute pulmonary 2.2. Lethality assay (LD50) edema (Magalhães et al., 1999; Ghersy de Nieto et al., 2002; Manzoli-Palma et al., 2003; Cupo et al., 2009). The patho- T. serrulatus venoms from Minas Gerais (Ts-MG) and genesis of lung edema induced by scorpion venom is very from Distrito Federal (Ts-DF) were lyophilized, weighed complex, but acute left ventricular failure resulting from dry, and diluted in saline (150 mM NaCl) prior to the assays. massive catecholamine release and myocardial damage The LD50 of the T. serrulatus venom from two populations induced by the venom have been suggested as possible were determined as follows. pathogenic mechanisms (Matos et al., 1997). Lung edema Ts-MG venom was tested in doses of 5.8, 11.6, 17.4, 23.2, may also result from increased pulmonary vascular 34.8, 46.4, 58 and 72 mg/mouse of 20 g by i.p. injection permeability due to vasoactive substances released by the (n ¼ 8). The first tested dose was 23.2 mg/mouse based on venom (Matos et al., 1997). the LD50 obtained by Nishikawa et al. (1994). Ts-DF venom T. serrulatus has originally lived in environments of was tested in doses of 14, 26, 36, 50, 60, 70, 80 e 90 mg/ transition forests, dry forests, savannah and Caatinga mouse of 20 g by i.p. injection (n ¼ 8). The first tested dose (Lourenço et al., 1996). Nowadays, this species has inhabi- was 50 mg/mouse, which corresponded to twice the ted places with minimal vegetation and proliferated widely calculated LD50 from Ts-MG. All doses of venom were in urban areas, and can be easily found in boxes and net- diluted in 100 mL of saline (150 mM NaCl). In both experi- works of sewers, and storm sewers. Is considered parthe- ments, the animals were compared to control animals nogenetic and ecologically opportunistic, with great (n ¼ 8), which received only saline by i.p. injection in the dispersive fitness and high reproductive capacity (Lourenço same volume.