A Step Towards Understanding the Possible Role of Sodium Channel Modulatorsq

Toxicon 73 (2013) 33–46

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Toxicon

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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. The survival rate of animals was observed 24 et al., 1996; Brazil et al., 2009). and 48 h after inoculation of venom or saline. At the end of F.N. Oliveira et al. / Toxicon 73 (2013) 33–46 35 the experiment, the surviving animals were euthanized euthanized. The blood collection was performed using with sodium pentobarbital (225 mg/kg). micropipettes, after the section of the cervical vessels of animals. The samples were immediately centrifuged at 2.3. Effects of T. serrulatus venoms on mice behavior and 20,000 g for 20 min at 10 C. The supernatant was physiology separated and subsequently used for serum CK and CK-MB activities measure, according to CK-NAC Liquiform Ref 117 The Ts-MG and Ts-DF venoms were evaluated for their and CK-MB Liquiform Ref 118 kits (Labtest, Minas Gerais, ability to induce behavioral and physiological changes in Brazil), respectively. mice. All groups of mice that were part of the experiment for the determination of LD50 were observed during the first 2.6. Measurement of vascular permeability changes three hours after venom or saline injection. Those effects observed in animals that received venom and were absent Vascular permeability changes to serum proteins were in control animals (saline) were considered as behavioral analyzed according to the Evans blue protocol (Saria and and physiological changes. A set of behavioral and physio- Lundberg, 1983; Matos et al., 2001). Briefly, Evans blue logical effects was previously defined (see Table 1). (20 mg/kg) was injected (i.v.) just prior to the administration of venom or vehicle (saline). Rats were anesthetized 2.4. Investigation of induction of acute pulmonary edema with a mixture of xylazine hydrochloride (10 mg/kg) and in rats ketamine (75 mg/kg) i.p., and after that, they were injected with Ts-MG venom (0.5 mg/kg, i.v.), or Ts-DF venom The ability of T. serrulatus venom from DF and MG to (0.5 mg/kg, i.v.), or control group (150 nM NaCl). The ani- induce acute pulmonary edema in rats was evaluated as mals were observed for a period of 1 h and after this period done by Matos et al. (1999, 2001), with some modifications, were euthanized with an overdose of sodium pentobarbi- as follows. tone, and cannulas were inserted into the trachea and the Eighteen rats were divided into three experimental bronchoalveolar lavage (BAL) performed in all animals. The groups (n ¼ 6): Ts-MG venom (0.5 mg/kg of T. serrulatus BAL fluid was centrifuged (1000 g for 7 min) and the venom from MG), Ts-DF venom (0.5 mg/kg of T. serrulatus supernatant used for Evans blue determination. The lungs venom from DF) and control group (150 mM NaCl). Saline were excised, chopped, placed in 2 mL formamide and or venom was injected by i.v. route (200uL). The rats were incubated without homogenization at 40 C for 24 h and firstly anesthetized with a mixture of xylazine hydrochlo- used for Evans blue determination. Evans blue was quan- ride (10 mg/kg) and ketamine (75 mg/kg) i.p. The animals tified by spectrophotometry at 620 nm (Shimadzu, Japan). were observed for a period of 1 h after the treatment. After Evans blue levels that were significantly higher in rats that, animals were euthanized by an overdose of sodium injected with scorpion venom than in control animals were pentobarbital (225 mg/kg) and their hearts and lung were assumed to represent increased vascular permeability. rapidly removed; the lungs were weighted and both organs were prepared for histology. 2.7. Total leukocyte count The lungs were immediately weighed and the presence of acute pulmonary edema (APE) was determined ac- The pellet containing cells from the bronchoalveolar cording to Magalhães et al. (1998) using the formula: lavage fluid was resuspended in 1 mL of sodium phosphate APE ¼ Pulmonary Mass 100/Body Mass. The presence of buffered (0.1 M) saline containing 3% bovine serum albu- pulmonary edema activity was assessed by differences min and an aliquot (20 mL) diluted in Turk solution (0.5% of between the APE obtained from animals injected with methylene blue in 30% acetic acid), 1:20 (v:v), and used for venom and the APE obtained from the control group. counting. Total leukocyte counts were then performed in a Hearts and lungs were fixed in 10% buffered formalin Neubauer chamber using an optical microscope (Nikon and embedded in paraffin(Prophet et al., 1992). Histologi- E200, USA). Analysis was carried out under a 100 im- cal sections (4 mm thick) were stained with haematoxylin mersion objective. The leukocytes were quantified in four eosin (HE) and analyzed under an optical microscope. external squares A, B, C and D of the Neubauer chamber. After the morphological analyses, the tissues were clas- The total number of leukocytes/mm3 was determined by A/ sified according to Matos et al. (1997) with some adjust- DV (A ¼ total leukocyte count in the four quadrants, ments, as described: 1) normal tissue: tissue without D ¼ dilution used, and V ¼ volume counts were performed, morphological changes when compared to the control where D and V are constants). group; 2) mild pulmonary edema: usually irregular, sub- pleural, with extravasations of plasma; 3) moderate pulmo- 2.8. Chromatographic analysis by reversed phase high nary edema, multifocal, with large plasma leakage; 4) severe performance liquid chromatography (RP-HPLC) pulmonary edema: diffuse interstitial (intra-alveolar) in all lung lobes, sometimes associated with hemorrhagic foci. The same venom pools used to conduct the toxicity and edematogenic activity were fractioned by RP-HPLC. The 2.5. Blood collection and serum creatine kinase (CK) and its crude venoms (Ts-MG and Ts-DF) were submitted to isoenzyme CK-MB activities chromatography according to Schwartz et al. (2008). Briefly, the crude venom was dissolved in solvent A (0.12% All animals used in the induction of pulmonary edema trifluoroacetic acid, TFA, in water) and centrifuged at were also subjected to blood sampling right after being 10,000 g for 15 min. The soluble supernatant was 36

Table 1 Behavioral and physiological changes seen in mice (n ¼ 8) during the ﬁrst three hours after i.p. injection of T. serrulatus venom from Minas Gerais (Ts-MG) and from Distrito Federal (Ts-DF).

Injected dose ( mg) per mouse of 20 g and percentage of occurrence of the change (%) in group Dose 1 Dose 2 Dose 3 Dose 4 Dose 5 Dose 6 Dose 7 Dose 8 (Ts-MG ¼ 5.8) (Ts-MG ¼ 11.6) (Ts-MG ¼ 17.4) (Ts-MG ¼ 23.2) (Ts-MG ¼ 34.8) (Ts-MG ¼ 46.4) (Ts-MG ¼ 58) (Ts-MG ¼ 72) [Ts-DF ¼ 14] [Ts-DF ¼ 26] [Ts-DF ¼ 36] [Ts-DF ¼ 50] [Ts-DF ¼ 60] [Ts-DF ¼ 70] [Ts-DF ¼ 80] [Ts-DF ¼ 90] Ataxia Staggering gait accompanied (–)[–](–)[–] (12.5) [–] (62.5) [37.5] (87.5) [75] (100) [87.5] (100) [100] (100) [100] by postural imbalance Convulsion Disordered twitches, violent (–)[–] (12.5) [25] (25) [25] (37.5) [37.5] (62.5) [50] (75) [62.5] (100) [75] (100) [100] ..Oier ta./Txcn7 21)33 (2013) 73 Toxicon / al. et Oliveira F.N. and widespread Dyspnea Difﬁculty in breathing (25) [12.5] (37.5) [50] (62.5) [75] (75) [87.5] (87.5) [100] (87.5) [87.5] (100) [100] (100) [100] Throes Involuntary contraction of small (–)[–] (12.5) [–] (37.5) [12.5] (–) [37.5] (62.5) [50] (75) [87.5] (87.5) [100] (87.5) [100] muscle groups ) [50] (62.5) [37.5] (100) [75] (100) [87.5] (100) [100] Hypersensitivity Reaction to sudden stimuli (–)[–](–)[–] (12.5) [12.5] (– Hyperactivity Exacerbation of the normal (25) [12.5] (37.5) [25] (50) [37.5] (75) [50] (62.5) [62.5] (87.5) [75] (100) [87.5] (100) [100] movements Hypoactivity Reduction of normal movements (75) [87.5] (62.5) [75] (50) [62.5] (25) [50] (12.5) [37.5] (12.5) [25] (–) [12.5] (–)[–] Profuse sweating Production and release of (–)[–](–)[–] (25) [25] (37.5) [37.5] (87.5) [75] (75) [62.5] (100) [87.5] (100) [100] excessive sweat Shedding of tears Excessive production and release (–)[–](–)[–] (37.5) [12.5] (75) [37.5] (87.5) [75] (100) [87.5] (100) [62.5] (87,5) [75] of tears Movements of Wave motion in a spiral shape (–)[–](–)[–] (12.5) [–] (37.5) [37.5] (62.5) [62.5] (50) [12.5] (50) [37.5] (62.5) [62.5] –

the tail 46 Piloerection Erection of body hair (–)[–](–)[–] (37.5) [12.5] (100) [37.5] (87.5) [50] (100) [87.5] (87.5) [87.5] (87.5) [100] Priapism Prolonged and painful erection (–)[–] (12.5) [12.5] (62.5) [37.5] (87.5) [75] (100) [100] (100) [75] (87.5) [87.5] (87.5) [87.5] of the penis Prostration Weakening and loss of normal (–)[–](–) [37.5] (25) [50] (37.5) [37.5] (62) [50] (75) [75] (100) [87.5] (100) [100] mobility Defecation Excessive out of control release (25) [–] (50) [37.5] (50) [50] (62.5) [75] (75) [100] (100) [87.5] (100) [87.5] (100) [100] of excreta Drooling Production and release of excess (37.5) [12.5] (50) [37.5] (62.5) [37.5] (62.5) [50] (75) [75] (100) [75] (100) [100] (100) [100] saliva Death Definitive cease of all (–)[–](–)[–] (12.5) [12.5] (12.5) [25] (62.5) [37.5] (75) [62.5] (100) [75] (100) [100] physiological activities F.N. Oliveira et al. / Toxicon 73 (2013) 33–46 37 separated by RP-HPLC in a C18 analytical column (Phe- 2.11. Ethical nomenex, Inc., USA), using a linear gradient from 0% solvent A (0.12% trifluoroacetic acid, TFA, in water) to 60% solvent B The experimental procedure was approved by the Ethical (0.10% TFA in acetonitrile) run for 60 min, at a flow rate of Committee for Animal Experimentation of Brasilia Univer- 1 mL/min. For the purpose of standardizing all procedures, sity (CEUA/UnB) under protocol number 133424/2009. An- solutions A and B were freshly elaborated and the four imals were maintained in accordance with the Brazilian chromatographies (two for each population) were run Society for Neuroscience and Behavior ethical statements, continuously in the same day. The chromatographies were which follow the guidelines for animal care prepared by the done using a Shimadzu 20A HPLC modular equipment Committee on Care and Use of Laboratory Animal Resources, with an SPD-20A detector, and the data were generated National Research Council, U.S.A. Likewise, every effort was and stored using the Shimadzu LC-Solutions Software, made to avoid unnecessary stress and pain to the experi- acquiring the absorbance data at a rate of 1 Hz, resulting in mental animals. The number of animals was kept to a a total of 9005 points which were processed for each minimum necessary to prove the concept. chromatography. 2.12. Statistical analyses 2.9. Venom variability and fractal dimension of chromatograms The LD50 values and their confidence limits were calculated by Probit analysis (Finney, 1971), using the The fractal dimension (D) analysis on the chromato- software BioStat5.0 (Software Informer, Inc.). graphic profiles of the venoms was calculated for the initial Analysis of variance (ANOVA), followed by T test (Tukey) 60 min of venom fractioning. This analyses is an alternative and F test were performed for all variables with normal to study inter and intraspecific venom variability taking distribution (Pulmonary Mass, CK, CK-MB, amount of Evans advantage of the multiple waveforms of these and blue and total leukocyte) and these data are shown as comparing them point to point in all or in partial intervals mean SEM (standard error of the mean). In both cases the of elution time. By this study, it is also possible to calculate significance level was set at 5%. the probability (P) of the difference between two values of D (DD) that is used to indicate the contortedness of wave- 3. Results forms, inter venoms.

The data were transformed to ASCII format (American 3.1. Lethality assay (LD50) Standard Code for Information Interchange) in data pairs, and analyzed under VeFractDim software according to Among the doses of Ts-DF venom tested on mice, the D’Suze and Sevcik (2010). For the phase plot of D analysis a minimal dose capable of causing death was 26 mg/mouse. sliding window sequences (SWS) of the 500 continuous The starting dose of 90 mg/mouse showed 100% lethality of points starting from time 0 of the chromatography was the assayed animals. For Ts-MG venom, the smallest dose used and with a recursive displacing by 1 s for 60 min of causing death of mice (12.5%) was 11.6 mg/mouse, while the elution stored as [(ti,Di) sets]. For this data sets the con- dose 58 mg/mouse was lethal to 100% of the animals tested. tortedness represented by Q ¼ D 1 was calculated, and a The dose/lethality dependence was clearly observed for phase plot with their (ti,Qi) sets was constructed. The both venoms (Fig. 1). In addition, it is noted from the determination coefficient (ds) was calculated squaring the rightward shift of the dose–response curve calculated for Spearman rank correlation coefficient as suggested previ- Ts-DF venom that this venom is less toxic than Ts-MG ously (D’Suze and Sevcik, 2010). The data were plotted venom. It was observed during the course of the experi- using the GraphPad Prism 5 software. ment that most deaths occurred within the first three hours after venom injection and particularly in groups of animals 2.10. Mass fingerprinting MALDI-TOFMS receiving the highest doses (data not shown). After 24 h of venom injection there were no deaths in either group. All fractions obtained from Ts-DF and Ts-MG venom chromatography separation were analyzed by mass spectrometry performed on a MALDI-TOF AutoFlex III (Bruker Ò Daltonics , Germany) in linear and reflector modes and the spectra were processed with MassLynxÔ3.5 (Manchester, Ò UK) and FlexAnalysis 3.3 (Bruker Daltonics , Germany). Briefly, solubilized fractions (0.5 mL of sample, variable concentrations) were spotted onto the target followed by 0.5 mL of CHCA (a-cyano-4-hidroxycinnamic acid) matrix solution (60% acetonitrile/0.3% TFA), and allowed to dry at room temperature (dried-droplet method). Peptide Cali- bration Standard II (700 4000 Da) and Protein Calibration Ò Standard I (3000–25,000 Da) (Bruker Daltonics , Germany) were used as external calibrates. Mass spectra from the Fig. 1. Dose-response curves obtained from injections i.p. of the venom of T. average of 256 laser pulses from m/z 600 to 39,400 were serrulatus from the DF (Ts-DF) and from MG (Ts-MG) in mice. R2 Ts-MG obtained. (0.98) and R2 Ts-DF (0.99). 38 F.N. Oliveira et al. / Toxicon 73 (2013) 33–46

The LD50 (limit of 95%) calculated by Probit analysis for T. serrulatus (MG) venom, the venom of specimens from DF the Ts-DF and Ts-MG venoms were respectively 51.6 (40– was not able to induce acute pulmonary edema in rats. 64.8) mg/mouse and 26.0 (19.8–33) mg/mouse. Thus, the Histological examination of the lungs from rats injected LD50 calculated for the Ts-DF venom was almost twice (1.98) with Ts-DF venom and from control animals showed higher than that calculated for Ts-MG venom, showing that alveolar capillaries slightly congested, accompanied by a the venom of the Ts-DF is less toxic than Ts-MG. few hemorrhagic spots, probably resultant of extended handling of tissue during the lungs weighing. In contrast, 3.2. Determination of venom effects lungs from rats injected with Ts-MG venom showed multifocal intra-alveolar pulmonary edema, characterized The behavioral and physiological changes in mice dur- by dilated alveoli containing liquid inside and precipitated ing the first three hours of injection of the Ts-DF and Ts-MG plasma (Fig. 3). venoms are specified in Table 1. These changes were dose- Additionally, no morphological and histopathological dependent; with increasing doses of venom most of the alterations after T. serrulatus envenomation with either changes listed become more frequent, with only exception venom from DF or MG were observed in heart tissues (data of hypoactivity that was more frequently visualized in an- not shown). imals receiving the lowest doses of venom. The presence of intense salivation usually preceded the onset of spasms, 3.4. Serum creatine kinase and its isoenzyme CK-MB and later convulsions. As shown in Table 2, CK and CK-MB activities in animals 3.3. Evaluation of induction of acute pulmonary edema injected with Ts-DF venom were not significantly different from control group. In relation to Ts-MG venom group As expected Ts-MG venom induced pulmonary edema; values were significantly higher (p < 0.001) than those of the lung mass/body mass ratio of rats receiving Ts-MG the control group (Table 2). venom, when compared with that obtained for the control animals (p < 0.001) and Ts-DF venom (p < 0.001) 3.5. Measurement of pulmonary vascular permeability groups, increased significantly (Fig. 2-A). On the other changes hand, the lung mass/body mass ratio of rats in Ts-DF venom group suffered no significant increase when compared to Pulmonary vascular permeability did not increase the control group (p > 0.05), demonstrating that unlike the significantly in animals treated with Ts-DF venom when

Fig. 2. Evaluation of the Acute Pulmonary Edema. Control groups n ¼ 6 (200 mL of saline i.v.), Ts-MG n ¼ 6 (0.5 mg/kg T. serrulatus venom from Minas Gerais in 200 mL of saline i.v.) and Ts-DF n ¼ 6 (0.5 mg/kg T. serrulatus venom from the Distrito Federal in 200 mL of saline i.v.). Evaluation of lung mass (A), quantiﬁcation of Evans blue in lung (B), and in the bronchoalveolar lavage (C), and leukocytes in the bronchoalveolar lavage (D). One-way ANOVA followed by Tukey test (*p < 0.05 and ***p < 0.001 between control and Ts-MG; ###p < 0.001 between Ts-MG and Ts-DF). F.N. Oliveira et al. / Toxicon 73 (2013) 33–46 39

Fig. 3. Histology of rat lungs 1 h after i.v. injection of the Ts-DF or Ts-MG venom (0.5 mg/kg), or saline (150 mM NaCl). Representative photomicrographs of (A) control group and (B) Ts-DF group, showing in both thickening of alveolar septa and focal hemorrhage. Representative photomicrographs of Ts-MG group showing pulmonary edema multifocal intra-alveolar (C) (arrows) and presenting capillary congestion and severe multifocal hemorrhage (D). HE, 200, scale bar 100 mm. compared to the control group (p > 0.05) (Fig. 2-B).Yet, in more than double the number of the total leukocytes when Ts-MG venom injected group a raise in the pulmonary compared to the control group (p < 0.05). vascular permeability was observed when compared to the control and Ts-DF venom groups (p < 0.001). The same was 3.7. Chromatographic analysis by RP-HPLC and fractal observed with regard to bronchoalveolar lavage of Ts-MG dimension of chromatograms venom group compared to the control and Ts-DF venom groups (Fig. 2-C). Fig. 4 presents the chromatographic proﬁles obtained after fractioning Ts-DF and Ts-MG venoms. These chro- 3.6. Total leukocyte count matograms present visually high similarity, with the same number of collected fractions and only minimal peak in- The amount of total leukocytes present in bron- tensity variations of few fractions. choalveolar lavage of Ts-DF venom group was not statisti- The whole trace values of D calculated for T. serrulatus cally different from the control group (p > 0.05) (Fig. 2-D). venom from DF was 1.15 3.76 10 5 (N ¼ 7200), and The bronchoalveolar lavage of Ts-MG venom animals had 1.16 3.23 10 5(N ¼ 7200) for Ts-MG venom. These values result in DDTs-DF,Ts-MG ¼ 0.01, l ¼ 1.04, and a probability of the difference between Ts-DF and Ts-MG values Table 2 ¼ Values of creatine kinase (CK) and CK-MB isoenzyme in the serum of statistically distinct from zero (P 0.20). This states that animals of control (200 mL of saline i.v.), Ts-MG (0.5 mg/kg of T. serrulatus the chromatogram of Ts-MG is slightly more contorted than venom from MG in 200 mL of saline i.v.), and Ts-DF (0.5 mg/kg of T. ser- the Ts-DF chromatogram. As depicted in Table 3, the fractal m rulatus venom from DF in 200 L of saline i.v.) groups are presented as dimension varies in the time function and, as explained by mean SEM utilizing F test (parametric) and compared with ANOVA. D’Suze and Sevcik (2010), the higher D values correspond to Animals (n) CK (U/L) CK-MB (U/L) intervals with more elution peaks rather than to periods Mean (SEM) Mean (SEM) with peaks with higher amplitudes. Control (6) 1067.5 115 290 82 To further exploit these data, and to identify the elution Ts-MG (6) 2192 365** 810 392** time sections presenting the most divergent D values, the # # Ts-DF (6) 1028 135 295 81 plots of D values calculated for a sliding window of 500 **(p < 0.001);# (p < 0.001); SEM means standard error of the mean. digitized points obtained from Ts-DF and Ts-MG venoms 40 F.N. Oliveira et al. / Toxicon 73 (2013) 33–46

corresponding molecular masses. Moreover, 54 (32.1%) were present only in Ts-DF venom, and 70 (38.0%) were exclusive for Ts-MG venom (Fig. 5-B and Table 4). Ts-DF venom yielded a smaller number of peptides with molecular mass distributed between 6500 and 7500 Da than Ts- MG venom. On the other hand, 5001 to 5500 Da peptides were in higher number in Ts-DF venom than in Ts-MG one (Figs. 5 and 6).

4. Discussion

T. serrulatus is considered the most important scorpion species for Public Health in Brazil (Funasa-MS, 2001, 2009). This is the first study to evaluate the toxicity of the venom Fig. 4. RP-HPLC of Ts-DF and Ts-MG venoms. One mg of soluble venoms was of T. serrulatus from DF, Brazil, and the effects it provokes injected into an analytical C18 RP column (250 mm 10 mm, Phenomenex, in vivo on murine species. USA). Venom components were separated using a linear gradient from so- We demonstrated that the T. serrulatus venom from lution A (0.12% TFA in milli-Q water) to 60% solution B (0.10% TFA in aceto- m Distrito Federal (LD50 of 51.6 g/mouse) is almost twice nitrile) in 60 min at room temperature (19 C), eluted 1 mL/min. (1.98) less toxic than the T. serrulatus (MG) venom (LD50 of 26.0 mg/mouse). Nishikawa et al. (1994) had previously m were overlapped (data not shown). The differences be- shown for T. serrulatus the LD50 of 25.5 g/mouse. The LD50 tween both venoms are observed in the following aceto- of the venom of T. serrulatus from Bahia, a northeastern nitrile elution percentages profile: In the 0–25 and 36–40% Brazilian state also bordering MG, is 96.16 mg/mouse (Silva intervals, Ts-MG venom presents higher D values than et al., 2005a,b), indicating the existence of differences in those of Ts-DF venom; in the 51–60% interval, Ts-DF pre- the venom of this species from different regions of Brazil. sents higher D values than Ts-MG values (Table 3). For this Factors such as milking and storage means of the venom, comparison, those values resulting in a probability other the route of venom administration on mice, and the than zero are considered statistically distinct (D’Suze and observation time course of LD50 experiment could possibly Sevcik, 2010). From the phase plot (ti,Qi), which repre- result in different toxicities. However, as the experiments sents Ts-DF venom (SWS) of Q ¼ D 1 plotted against the conducted here with both venoms followed the same SWS of Q ¼ D 1 from Ts-MG venom (data not shown), and protocols, these factors were controlled, being the origin of based on the non-parametric Spearman rank correlation scorpions the most acceptable hypothesis to reinforce the 2 coefficient (rs, when ds ¼ rs ), the coefficient of determina- assertion for regional venom variation. tion (ds) value obtained was 0.56 and rs (0.75) with P(rs ¼ 0) The neurotoxins were the most important compounds of 3.19 10 20. Considering these values and that plotted of scorpion venom, acting on ion channels and resulting in points do not tend to cluster around a straight line, it is an expressive release of acetylcholine, noradrenaline and strengthened that venoms are different. adrenaline affecting both the sympathetic and parasympathetic systems, inducing physiological and behavioral changes (Henriques et al., 1968; Ismail, 1995; Dávila 3.8. Mass fingerprinting MALDI-TOFMS analyzes et al., 2002; Vasconcelos et al., 2005; Cupo et al., 2007; Pinto et al., 2010a). Such effects were observed by Silva MALDI-TOFMS analyses of Ts-DF and Ts-MG venom et al. (2005a) in mice injected with venom of T. serrulatus chromatographic fractions resulted in the detection of 171 from Bahia, Brazil. Similarly, as shown here, physiological and 174 components whose molecular masses ranged from and behavioral events regulated by the autonomic nervous m/z 1145.6 to 10,988.4 and 1196.8 to 16,457.5, respectively system were exacerbated in mice after injection of T. ser- (Fig. 5-A). Were observed in Ts-DF and Ts-MG venoms 114 rulatus venoms from both MG and DF. However in mice

Table 3 D values obtained for Ts-DF and Ts-MG venoms in three different chromatographic elution time intervals. This table indicates the elution time intervals (in min), the scorpion venom population, the D values for each time interval, the standard error of the mean (SEM), number of data analyzed (n), the delta of D (DD) with its values of l (value of the one tailed case when the probability distribution function is Gaussian) and P (probability different to 0).

Time interval in mina Scorpion venom population D SEM n DD

DDTs_MG Ts_DF l P 0–25 Ts-MG 1.16 6.60 10 5 3001 0.01 1.23 0.14 Ts-DF 1.15 6.22 10 5 3001 36–40 Ts-MG 1.10 2.29 10 4 481 0.02 1.25 0.14 Ts-DF 1.07 2.80 10 4 481 51–60 Ts-MG 1.02 1.49 10 4 1080 0.03b 1.56 0.09 Ts-DF 1.04 1.28 104 1080

a Time interval is equivalent to acetonitrile elution percentages. b This result is as absolute value. F.N. Oliveira et al. / Toxicon 73 (2013) 33–46 41

Fig. 5. Molecular mass distribution of T. serrulatus chromatographic fractions of the venom from Distrito Federal and Minas Gerais, obtained by MALDI-TOF-MS. (A) total molecular mass and (B) distinct molecular mass.

receiving Ts-DF venom such events were more frequent at massive release of catecholamines or myocardial damage higher doses. induced by direct action of the venom induces hyperten- Scorpion stings in humans commonly lead to severe sion, which leads to the left ventricular failure, and acute pulmonary edema that in turn is the main cause of consequently the development of the edema. Moreover, it death provoked by T. serrulatus (Abrough et al., 1991; was reported that stimulation of alpha-adrenergic re- Amaral et al., 1993; Cupo et al., 1994; Bucaretchi et al., ceptors could lead to suppression of insulin secretion and 1995; Yildizdas et al., 2008; Razi and Malekanrad, 2008). damage the heart, inducing the onset of acute pulmonary T. serrulatus venom (0.5 mg/kg i. v.) from DF did not induce edema (Gueron and Yaron, 1970; Freire-Maia et al., 1978, acute pulmonary edema in rats as assessed by index lung 1994; Gueron et al., 1980; Matos et al., 1997, 2001; Joy, mass/body mass, morphological analysis and pulmonary 2009). vascular permeability. As expected, the T. serrulatus venom Several authors have reported evidence of the action of (0.5 mg/kg i.v.) from MG caused severe interstitial and the T. serrulatus venom on the cardiac muscle (Corrêa et al., intra-alveolar edema in rats 1 h after venom injection, as 1997; Teixeira et al., 2001). However in the present study was observed previously by Matos et al. (1997). the hearts of rats that received the venoms of T. serrulatus According to the published data, the pathogenesis of from DF and MG remained without morphological changes acute pulmonary edema induced by scorpion venom is very when observed by optical microscope. On the other hand, intricate. This respiratory affection may result from the only animals subjected to injections with Ts-MG venom activation of both cardiogenic and non-cardiogenic mech- showed enhanced levels of CK and CK-MB. Recently, anisms (Amaral et al., 1993; Freire-Maia et al., 1994). The changes in serum CK and CK-MB of rats subjected to 42 F.N. Oliveira et al. / Toxicon 73 (2013) 33–46

Table 4 Molecular masses that were distinctly detected in T. serrulatus venom from Distrito Federal (Ts-DF) and Minas Gerais (Ts-MG). Data obtained by MALDI-TOF MS.* Retention time (RT). Shaded values: supposedly NaScTx. MM (Da) [M + H]+ MM (Da) [M + H]+ RT* (min) Ts-MG Ts-DF 1’ to 5’ - 2160.0 6’ to 10’ - 3210.5 11’ to 15’ 1535.6 5381.0 1153.6; 1293.0; 1336.6; 1374.4; 16’ to 20’ 3611.8 1564.5; 1963.8; 2850.5; 6197.8 1396.8; 1651.8; 1763.7; 2361.4; 5103.4; 1452.5; 1614.6; 1700.8; 1892.7; 21’ to 25’ 5268.7; 5405.1; 8975.2 2210.1; 2513.2; 3204.2; 7101.0 2058.5; 2262.6; 2380.2; 3265.9; 4219.1; 1262.6; 2190.1; 2642.3; 3190.0; 4236.3; 4514.5; 5011.5; 5177.2; 5188.2; 3760.9; 4195.5; 4250.4; 5038.1; 26’ to 30’ 5248.5; 7647.0; 8349.1; 8486.6; 8640.0; 5233.6; 5478.1; 7508.2; 7968.5; 8783.1 8217.3; 8461.4; 9606.8 4460.0; 5297.3; 5312.6; 5430.2; 5448.0; 1854.1; 1879.7; 2327.3; 5281.4; 31’ to 35’ 5703.6; 5910.5; 6816.2; 7323.4; 7847.8; 7826.9 9082.6 1546.7; 1603.7; 1944.1; 3036.9; 4506.9; 4876.2; 4907.9; 6828.3; 6928.5; 3344.0; 3490.0; 6079.6; 6430.0; 36’ to 40’ 6966.3; 7026.5; 7182.6; 7458.4; 7475.9; 6581.7; 6695.2; 7240.9; 8579.7; 8608.7; 11006.0 10973.2; 10988.4 2227.2; 3356.1; 4986.3; 6330.1; 6715.6; 41’ to 45’ 6738.7; 7694.7; 8241.0; 9958.5; - 11042.0; 11186.6; 11476.5; 16457.5 46’ to 50’ 6362.0; 7217.1; 9972.7 - 51’ to 55’ 1754.9; 2345.4; 6915.5 - 56’ to 60’ 2099.1; 5921.9 6890.2

injections of Tityus fasciolatus and T. serrulatus venom were (prostaglandin E2, leukotriene B4 and thromboxane A2) observed, without any morphological changes on the car- induced by the venom, which would increase pulmonary diac muscle (Pinto et al., 2010a). vascular permeability and hence the appearance of acute The second mechanism suggested to explain the path- pulmonary edema (Freire-Maia et al., 1978, 1994; Matos ogenesis of pulmonary edema in response to the T. serru- et al., 1997). A signiﬁcant increase in pulmonary vascular latus venom is the release of vasoactive substances permeability was observed in mice injected with Ts-MG

Fig. 6. Histogram of the distinct molecular mass distribution of the chromatographic fractions from T. serrulatus venom from Distrito Federal and Minas Gerais obtained by MALDI-TOFMS, distributed in 500 Da classes for 54 (31.57%) and 70 (40.22%) distinctly molecular mass measured, respectively. F.N. Oliveira et al. / Toxicon 73 (2013) 33–46 43 venom (0.5 mg/kg) was observed here and by Matos et al. As it was presented in the earlier fingerprinting studies (2001). However, this increase was not observed in Ts-DF mentioned above and reviewed elsewhere (Rodríguez de la venom injected animals. Vega et al., 2010), in the first 25 min of chromatographic Thus, the inability of the T. serrulatus venom from DF to separation, which corresponds to 0–25% of acetonitrile in a induce pulmonary edema could be related to the absence of 1% acetonitrile/min linear gradient elution, elute mainly both cardiogenic and non-cardiogenic effects, such as low molecular mass peptides (<1500 Da), particularly elevated levels of CK and CK-MB, morphological changes in those without disulfide bridges. Among them, there are cardiac muscle, or increased pulmonary vascular perme- fragments of larger venom toxins and bradykinin potenti- ability. We observed the presence of leukocytes in bron- ating peptides (bpp) that strikingly account for half of the choalveolar lavage of rats injected with Ts-MG venom. molecular masses identified within this molecular mass However, this response was not observed in animals (MM) range in T. serrulatus venom (Rates et al., 2008; injected with Ts-DF venom, just as in previous studies Verano-Braga et al., 2008). It is worth reinforcing that performed by Matos et al. (1999) who suggested that the these studies were done with Ts-MG population. recruitment of leukocytes do not play an important role in Usually, peptides in the range of molecular masses from þ the development of acute pulmonary edema. Otherwise, it 3500 to 4500 Da are short-chain K channel blockers (KTx) was shown that the T. serrulatus venom stimulates the and they start eluting from RP-HPLC usually after 20% release of pro-inflammatory cytokines such as TNF-a acetonitrile. The molecular masses of the six KTxs previ- (tumor necrosis factor alpha) and KC (keratinocyte-derived ously described for T. serrulatus venom were identified in chemokine), and the activity of MPO (myeloperoxidase and the present work in Ts-MG venom (see Table 5). Among nitric oxide) and lung perivascular mononuclear and them, three were not found in Ts-DF venom: alpha-KTX polymorphonuclear cells infiltration (Comellas et al., 2003; 12.1 (P59936), alpha-KTX 22.1 (P86270) and b-TsTXK Andrade et al., 2004, 2007; Coelho et al., 2007; Peres et al., (P69940). The alpha-KTX 12.1 has 4508.3 Da, a LD50 in mice 2009). Andrade et al. (2007) showed that scorpion venom of 826 mg/kg (i.v.) and inhibits high conductance calcium- not only increases the expression of mRNA pulmonary in- activated potassium channels and, to a lesser extent, flammatory cytokines but also non-inflammatory cyto- Shaker B potassium channels, moreover, inhibits Kv 1.3 kines, moreover the expression of IL-1a, IL-1b and IL-6 (Novello et al., 1999; Pimenta et al., 2003b). The alpha-KTX mRNA was shown to be higher among the remaining 22.1 is a 3956.0 Da peptide that preferentially blocks Kv1.2 detectable cytokines. and Kv1.3 channels with IC50 values of 196 25 and Recently, Filho et al. (2011) demonstrated that the T. 508 67 nM, respectively (Cologna et al., 2011). The b- serrulatus venom did not cause local inflammation in mice, TsTXK, the long-chain KTx described for T. serrulatus, has but it induced an increase of blood neutrophils and serum molecular mass of 6716.1 Da and selectively blocks voltage- þ IL-6, TNF-a and IL-10. In addition, after 360 min of enven- gated noninactivating K channels in synaptosomes with omation there was a reduction in the cells number from IC50 values of 30 nM (Legros et al., 1998; Rogowski et al., peritoneum and spleen, but there was an increase in the 1994). cell number from lymph nodes (Filho et al., 2011). Buthidae scorpion venom peptides with 6000 to þ It is widely known that different scorpion species have 7500 Da mostly affect the activity of Na -channels different venom compositions. Interestingly, many studies (NaScTx) and elute from RP-HPLC fractioning at approxi- have reported significant differences in the protein com- mately 33–40% acetonitrile (Batista et al., 2007). In present ponents and venom toxicity within scorpions of the same study, we noticed in Ts-DF and Ts-MG venom the presence species (Kalapothakis and Chávez-Olórtegui, 1997; Pimenta of molecular masses corresponding to the seven NaScTxs et al., 2003a; Newton et al., 2007; Abdel-Rahman et al., previously described in T. serrulatus venom (see Table 5). It 2009, 2010). The present work shows that Ts-MG venom is is known that the most severe cases of scorpionism occur slightly more complex than the Ts-DF and posses a higher with Buthidae scorpions and the most serious symptoms number of compounds eluting between 0–25 and 36–40% result from the action of NaScTxs (see review Rodríguez de acetonitrile than Ts-DF. On the other hand, Ts-DF has a la Vega and Possani, 2005). higher number of compounds elution between 51 and 60% In fact, Kalapothakis and Chávez-Olórtegui (1997) sug- acetonitrile than Ts-MG venom. gested that NaScTx found in T. serrulatus venom was The venom of several scorpions of the Tityus genus has responsible to its toxicity, and verified that the a-type toxin been submitted to proteomic analysis (Pimenta et al., 2001; showed the highest toxicity. It has shown that injection of Diego-García et al., 2005; Nascimento et al., 2006; Batista tityustoxin (TsTX) induced pulmonary edema in rats et al., 2006, 2007; Barona et al., 2006; Rates et al., 2008). (Freire-Maia et al., 1978). TsTX (Gomez and Diniz, 1966)isa According to Pimenta et al. (2001), T. serrulatus venom heterogeneous fraction from T. serrulatus venom (Arantes (obtained from MG species) has a total of 380 different et al., 1992), including the a-type toxin among its compo- molecules identified by MALDI-TOF MS, on line LC/ESI MS nents. The a-toxin Aah II isolated from the venom of an Old and off-line LC/MALDI-TOF MS. In the present study, using World scorpion, Androctonus australis Hector, was also able MALDI-TOF MS, 174 molecular masses were observed in Ts- to induce interstitial lung edema in rats (Sami-Merah et al., MG venom, among them, a total of 142 (around 82%) was 2008). In the interval of 36–40% acetonitrile, Ts-MG venom also detected previously (Pimenta et al., 2001). In a lesser presented a greater number of peptides than Ts-DF venom, extent, from 171 components observed in Ts-DF venom,122 suggesting a greater diversity of NaScTxs in the former, (71%) correspond to components detected by Pimenta et al. which may explain the higher toxicity of Ts-MG venom. (2001). Indeed, Ts-MG venom possesses 9 assumed NaScTxs while 44 F.N. Oliveira et al. / Toxicon 73 (2013) 33–46

Table 5 þ þ Na -Channel Neurotoxins (a-NaTx and b-NaTx); K -Channel Neurotoxins (KTx) and other peptides isolated from Tityus serrulatus venom.

Toxin UniProt Molecular mass (Da) number Experimentala Theoreticalb Ts-MG Ts-DF

Monoisotopic Average NaScTx Ts7 P15226 6882.3 6879.12 6883.98 6883.6 6884.7 Ts2 P68410 6991.1 6986 6991 6996.8 6997.4 Toxin-4 P46115 7193.0 7187 7192 7202.0 7205.4 Toxin-5 P45659 7627.39 7632.64 7635.1 7631.7 Ts6 P45669 6705.66 6709.66 6703.7 6701.6 TsNTxP O77463 6694.4 6691.63 6695.63 6694.7 6695.2 Ts3 P01496 7446.5 7443.28 7448.41 7445.9 7446.6 KTx a-KTx12.1 P59936 4508.3 4502.24 4506.24 4506.9 – a-KTx4.1 P46114 3943.7 3938.92 3941.8 3949.8 3946.6 a-KTx4.2 P56219 3779.4 3775.78 3778.4 3774.4 3774.4 b-TsTXK P69940 6716.1 6716.77 6712.29 6715.6 – Ts16 P86271 3302.95 3300.48 3301.0 3300.6 a-KTx22.1 P86270 3956.0 3955.65 3952.78 3954.9 – Other peptides TsHpt-I P84189 2726.0 2723.44 2725.09 2724.2 2724.3 TsHpt-II P84190 2725.6 2724.42 2726.08 Hpt3 P84191 2652.2 2652.4 2654.01 2652.9 2653.0 Hpt4 P84192 2654.6 2653.38 2655.0 TsPep1 P0C174 2938.9 2936.18 2938.45 2936.6 2936.4 TsPep2 P0C175 2993.7 2992.29 2994.58 2990.7 2990.4 TsPep3 P0C176 3019.6 3017.29 3019.61 3017.0 3017.5

a Experimental molecular mass. b Values were obtained using the Peptide Mass (http://web.expasy.org/peptide_mass/).

Ts-DF has 4 (Table 5). Interestingly, Ts-DF has the all pre- fractionation methodology used in the present study, it was viously described T. serrulatus NaScTxs, including the a- not possible to identify this enzyme in the venoms studied. toxins, whose edematogenic activity has been attributed to. Although, the decisive function of the metalloprotease in The inability to induce acute pulmonary edema of Ts-DF pathogenesis of scorpionism remains to be determined, it is venom can be explained by either smaller concentration plausible that these proteins are related to the scorpion of these toxins, or by the smaller number of supposed venom-mediated changes in the pancreatic morphology NaScTxs that could act synergistically in the induction of and secretory modifications observed in vivo. The ability of the envenoming signs. Ts-DF venom to induce more potent effects on secretory Ts-DF venom presents higher D values than Ts-MG discharge as well as on vesicular transport mechanisms in venom in the last 10 min of elution time (50–60% of aceto- the exocrine pancreas than Ts-MG venom needs to be nitrile). Most high molecular mass components (>9000 Da) verified. elute in acetonitrile percentages greater than 40% and In conclusion, the observation of a smaller diversity of correspond to proteins, such as those from Tityus species supposedly NaScTx in the Ts-DF venom may explain the that have been assigned to lysozyme, proteases or hyal- lower toxicity of this venom when compared to Ts-MG. uronidase enzymes (Batista et al., 2007; Cologna et al., Also, the inability to induce acute pulmonary edema in 2009). The fingerprinting analysis conducted with Ts-MG rats could explain the absence of severe clinical symptoms venom shows there are many peptides greater than and death in patients stung by scorpions in the DF. Given 9000 Da eluting in the last 20 min of fractioning, and their the above and considering the LD50 determined here, it can number are smaller in Ts-DF venom (Fig. 5). In fact, in be inferred that the maximum amount of venom that could T. serrulatus venom from Minas Gerais was previously be inoculated by T. serrulatus from the DF during a sting described a hyaluronidase (P85841) whose full amino acid would not be sufficient to induce the onset of symptoms of sequence is yet to be determined. It is known that these severe poisoning in humans, while the T. serrulatus scor- proteins lack importance and direct action in the poisoning, pion releases about 450 mg of venom after electrical stim- but have fundamental action in the distribution of neuro- ulation (data not shown). However it is noteworthy that toxins in whole organism, because promote random hy- scorpionism in the DF cannot be neglected because of the drolysis of (1–>4)-linkages between N-acetyl-beta-D- increased presence of T. serrulatus in this region. This can glucosamine and D-glucuronate residues in hyaluronate. over time trigger the emergence of a public health problem. Hyaluronidase belongs to the glycosyl hydrolase 56 family (Richardson et al., 2008). Recently, a metalloprotease named Acknowledgments antarease (P86392) was identified in T. serrulatus venom from Minas Gerais, a protein with approximately Financial support: FAPDF, CNPq (306524/2012-0 and 25,500 100 Da, which elutes at 60% acetonitrile, and has 564223/2010-7 to EFS and 308929/2011-0 to AMCP), PPG proteolytic activity (Fletcher et al., 2010). Probably due to BioMol-UnB, CAPES, FAPEMIG and MCT-FINEP. Fagner F.N. Oliveira et al. / Toxicon 73 (2013) 33–46 45

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