Toxicon 108 (2015) 84e96 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Partial in vitro analysis of toxic and antigenic activities of eleven Peruvian pitviper snake venoms C. Guerra-Duarte a, J. Lopes-Peixoto a, B.R. Fonseca-de-Souza a, S. Stransky a, D. Oliveira a, F.S. Schneider a, L. Lopes-de-Souza a, C. Bonilla b, W. Silva b, B. Tintaya b, A. Yarleque c, * C. Chavez-Ol ortegui a, a Departamento de Bioquímica-Imunologia, Instituto de Ci^encias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Minas Gerais, Brazil b Instituto Nacional de Salud, Lima, Peru c Universidad Nacional Mayor de San Marcos, Lima, Peru article info abstract Article history: This work used eleven Peruvian snake venoms (Bothrops andianus, Bothrops atrox, Bothrops barnetti, Received 2 June 2015 Bothrops castelnaudi, Bothriopsis chloromelas, Bothrocophias microphthalmus, Bothrops neuwiedi, Received in revised form Bothriopsis oligolepis, Bothriopsis peruviana, Bothrops pictus and Bothriopsis taeniata) to perform in vitro 16 August 2015 experimentation and determine its main characteristics. Hyaluronidase (HYAL), phospholipase A2 Accepted 7 September 2015 (PLA2), snake venom metalloproteinase (SVMP), snake venom serine protease (SVSP) and L-amino acid Available online 10 September 2015 oxidase (LAAO) activities; toxicity by cell viability assays using MGSO3, VERO and HeLa cell lineages; and crossed immunoreactivity with Peruvian (PAV) and Brazilian (BAV) antibothropic polyvalent antivenoms, Keywords: Peruvian snakes through ELISA and Western Blotting assays, were determined. Results show that the activities tested in Venom characterization this study were not similar amongst the venoms and each species present their own peculiarities, In vitro assays highlighting the diversity within Bothrops complex. All venoms were capable of reducing cell viability of all tested lineages. It was also demonstrated the crossed recognition of all tested venoms by both antivenoms. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction (Ministerio de Salud de Peru, 2003). In addition, many native venomous snake species remain poorly studied, in spite of the Of all accidents with venomous animals taking place in Peru, acknowledged richness of Peruvian herpetofauna. The National ophidism is the main cause of death (Martinez-Vargas, 2004). Most Institute of Health of Peru (INS) has been collecting venom from of the reported accidents occur within forest regions, since Peru- those native snakes, to encourage further research. vian Amazonia forest takes 58% of the country's territory, but areas The Bothrops complex (following taxonomy classification pro- within coast regions and Andes Mountains also present endemic posed by Carrasco et al., 2012; comprising Bothrops, Bothrocophias, medically relevant species. The great diversity of venomous snakes Bothriopsis, Bothropoides e Rhinocerophis subgroups) is responsible in Peru represents a great potential risk of ophidic accidents. In for the vast majority of the accidents involving venomous snakes in 2013, 2143 accidents with venomous snakes were recorded, ac- Peru (Fig. 1). Classic bothropic envenoming involves local and sys- cording to the Peruvian National Institute of Health. Envenoming temic effects. Locally, edema, hemorrhage and necrosis can lead to cases often occur in secluded areas, posing difficulties in registering tissue loss and permanent disability. Systemic hemorrhage and properly the actual incidence and morbidity/mortality rates. intravascular coagulopathy can lead to acute kidney injury and Therefore, available epidemiological data may be underestimated cardiovascular shock and are the principal systemic complications (White, 2005; Albuquerque et al., 2013). The mentioned effects result from the integrated action of several venom components, * Corresponding author. Departamento de Bioquímica-Imunologia, Instituto de such as metallo proteases (known as Snake Venom Metallo Pro- Ciencias^ Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, CP: 486. teases e SVMP), serine proteases (Snake Venom Serine Proteases e CEP: 30.161-970, Brazil. SVSP), phospholipases A2 (PLA2), L-amino acid oxidase (LAAO), E-mail address: [email protected] (C. Chavez-Ol ortegui). http://dx.doi.org/10.1016/j.toxicon.2015.09.007 0041-0101/© 2015 Elsevier Ltd. All rights reserved. C. Guerra-Duarte et al. / Toxicon 108 (2015) 84e96 85 Bothrocophias hyoprora (12.5%each)] was provided by INS. Brazilian anti-bothropic polyvalent anti-venom (BAV) produced from horse plasma previously immunized with a defined pool of venoms [B. jararaca (50%), Bothrops jararacussu, Bothrops alternatus, Bothrops neuwiedi and Bothrops moojeni (12.5%each)], was provided by Instituto Butantan, Sao~ Paulo, Brazil. Antivenoms were kept at 4 C as indicated on their prescription. 2.2. Enzymatic activities 2.2.1. Hyaluronidase For quantifying hyaluronidase activity, dilution curves from 120 to 0.625 mg of each Peruvian species venom were incubated in ac- etate buffer (0.2M sodium acetateeacetate acid and 0.15 M NaCl, pH Fig. 1. Distribution of ophidic accidents in Peru, according to snake genus. Reports of accidents to the National Institute of Health from Peru (INS) in the year of 2013. 6.0) with 12.5 mg of hyaluronic acid (HA) in 96-well microtiter plates (Horta et al., 2014; with modifications). A control curve was made with 5 points of known HA concentrations, corresponding to 0%, 25%, 50%, 75% and 100% of hyaluronidase activity. The plate was hyaluronidases and others (Calvete, 2010). Although these families incubated at 37 C for 15 min. After the incubation, 200 mLofa of molecules are well characterized as the main bothropic venom solution containing 2.5% (w/v) cetyltrimethylammoniumbromide fi components, their relative abundance and speci city within (CTAB) dissolved in 2% (w/v) NaOH were added to each well and different species venoms (or even individuals) are subjected to produced turbidity was measured at 405 nm in microplate reader variations according to different parameters, such as age and Biorad Model 680. All assays were performed in duplicates. Results geographic localization (Calvete et al., 2011). from four independent experiments were plotted as means in a fi Studying venom properties of less known snakes is scienti cally doseeresponse curve and values for 50% activity were determined. relevant to improve medical management of accidents and to drive antivenom production (Calvete et al., 2014). Moreover, snake 2.2.2. Phospholipase A2 venoms are rich in molecules with interesting pharmacological ® To analyze phospholipase A2 activity, the EnzChek Phospholi- actions, being a valuable source for research in drug discovery pase A2 Assay Kit (Life Technologies) was used. The experiment was (King, 2011). made following EnzCheck's protocol, using 2 mg of each Bothrops In vitro methods for venom characterization are available and spp. venoms. A solution of purified PLA2 from bee venom (10 units/ can provide relevant information. In addition of being more ethical, ml) in 1X PLA2 reaction buffer was used as positive control and the in vitro tests can be cheaper, faster, easily standardized and are not same buffer without PLA2 was considered the negative control. All subjected to the inherent variation present in living animals. assays were performed in duplicates. Means of the results from four Animal-free approaches in venom characterization studies, independent experiments were calculated and plotted as percent- although less frequent than research involving in vivo tests, have age of activity, relating to the positive control. been performed (Lopes-de-Sousa et al., 2015) and must be pursued fi by scienti c community, following the principle of the 3R 2.2.3. Proteolytic activities (SVMP and SVSP) fi (Replacement, Reduction and Re nement) in animal experimen- To measure snake venom metalloprotease activity (SVMP), a tation (Flecknell, 2002). FRET peptide (Abz-LVEALYQ-EDDnp) containing the specific Here, we present the characterization of six venom snakes of the cleavage site for these enzymes, previously produced by our group genera Bothrops (Bothrops andianus, Bothrops barnetti, Bothrops (Schneider et al., 2014) was used. Twenty microliters of the FRET castelnaudi, Bothrops neuwiedi, Bothrops pictus and Bothrops atrox), peptide diluted in 60 mL of TriseHCl 100 nM/NaCl 50 nM buffer was four species of genera Bothriopsis (Bothriopsis chloromelas, incubated with 1 mg of each Peruvian Bothrops venoms. After Bothriopsis oligolepis, Bothriopsis peruviana, and Bothriopsis tae- 30 min of incubation, the plate was read in fluorometer (Synergy, niata) and one species of genera Bothrocophias (Bothrocophias BioTek) at 340 nm for excitation and 440 nm for emission. Results of microphthalmus), using an animal-free approach. means of three independent experiments were plotted as arbitrary units of fluorescence. 2. Materials and methods To measure the snake venom serine protease (SVSP) activity, venoms were pre-incubated with EDTA 0.2 mM for 1 h minutes and 2.1. Venoms and antivenoms then followed the same protocol described above, using a FRET peptide with the specific active site for serine proteases (Abz- Crude venoms from B. andianus, B. atrox, B. barnetti, FLPRSFRQ-EDDnp). All assays were performed in duplicates. Results B. castelnaudi, B. chloromelas, B. microphthalmus, B. neuwiedi, of means three independent experiments were plotted as arbitrary B. oligolepis, B. peruviana, B. pictus