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Toxicology journal homepage: www.elsevier.com/locate/toxicol

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Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom

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Mariana A.P. Rodrigues, Lourdes Dias, André L. Rennó, Norma C. Sousa, Adriana Smaal, Delano A. da Silva, Stephen Hyslop *

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Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP), Rua Tessália Vieira de Camargo, 126, Cidade Universitária Zeferino Vaz, 13083-887, Campinas, SP, Brazil

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 January 2010 Received in revised form 13 June 2014 Accepted 23 June 2014 Available online xxx

Envenoming by the pitviper Bothrops jararacussu produces cardiovascular alterations, including coagulopathy, systemic hemorrhage, hypotension, circulatory shock and renal failure. In this work, we examined the activity of this venom in rat isolated right atria. Incubation with venom (0.025, 0.05, 0.1 and 0.2 mg/ml) caused concentration-dependent muscle contracture that was not reversed by washing. Muscle damage was seen histologically and confirmed by quantification of creatine kinase-MB (CK-MB) release. Heating and preincubation of venom with p-bromophenacyl bromide (a phospholipase A2 inhibitor) abolished the venom-induced contracture and muscle damage. In contrast, indomethacin, a non-selective inhibitor of cyclooxygenase, and verapamil, a voltage-gated Ca2+ channel blocker, did not affect the responses to venom. Preincubation of venom with Bothrops or Bothrops/Crotalus antivenom or the addition of antivenom soon after venom attenuated the venom-induced changes in atrial function and tissue damage. These results indicate that B. jararacussu venom adversely affected rat atrial contractile activity and muscle organization through the action of venom PLA2; these venom-induced alterations were attenuated by antivenom. ã 2014 Published by Elsevier Ireland Ltd.

Keywords: Antivenom Bothrops jararacussu venom Muscle contracture Myonecrosis Phospholipase A2 Rat atria

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1. Introduction

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Bothrops jararacussu is a large pitviper with a wide distribution in southeastern Brazil (Campbell and Lamar, 2004; Melgarejo, 2009). Bites by this species cause extensive local edema, hemorrhage and necrosis, as well as systemic effects that include coagulopathy, internal hemorrhage, hypotension, circulatory shock and renal failure; death results from circulatory and respiratory failure (Amaral et al., 1985; Milani et al., 1997; Pinho and Burdmann, 2001; Benvenuti et al., 2003; Warrell, 2004). Experimental studies in vivo and in vitro have confirmed these clinical findings by showing that B. jararacussu venom is hemorrhagic (Furtado et al., 1991), myonecrotic (Queiroz et al., 1984; Melo and Suarez-Kurtz, 1987, 1988a,b; Calil-Elias et al., 2002; Santo Neto et al., 2004), cardiotoxic (Sifuentes et al., 2008), neurotoxic (Rodrigues-Simioni et al.,1983; Heluany et al.,1992; Queiroz et al., 2002; Zamunér et al., 2004) and nephrotoxic (Havt et al., 2001; Barbosa et al., 2005). Various studies have characterized venom components that contribute to these different activities, including coagulant enzymes (Zaganelli et al., 1996; Andrião-Escarso et al., 1997; Bortoleto et al., 2002; Pérez et al., 2007; Silva-Junior et al. 2007; Sant'Ana et al., 2008b), (metallo) proteinases (Mazzi et al., 2004; Marcussi et al.,

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* Corresponding author. Tel.: +55 19 3521 9536; fax: +55 19 3289 2968. E-mail addresses: [email protected], [email protected] (S. Hyslop).

2007) and phospholipases A2 (PLA2) (Homsi-Brandeburgo et al., 1988; Bonfim et al., 2001; Andrião-Escarso et al., 2002; Ketelhut et al., 2003; Kashima et al., 2004; Ponce-Soto et al., 2006). Whereas the local actions of B. jararacussu venom, particularly myonecrosis, have been extensively investigated, considerably less is known of the cardiovascular actions of this venom, although venom enzymes (Zaganelli et al., 1996; Andrião-Escarso et al., 2002) and peptides (Ferreira et al., 1992; Wermelinger et al., 2005; Rioli et al., 2008) involved in hypotension have been identified. Sifuentes et al. (2008) showed that B. jararacussu venom is cardiotoxic and that this action can be prevented by the polyanion suramin and antivenom. In this work, we examined the responses of rat isolated right atria to B. jararacussu venom by monitoring changes in contractile force and frequency, and assessed the tissue damage based on the release of the marker enzyme creatine kinase-MB and the presence of histological alterations. We also examined the involvement of venom PLA2 activity in this response and the ability of antivenom to neutralize these effects.

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2. Materials and methods

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2.1. Reagents

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Indomethacin, phospholipase A2 (PLA2, from Naja mossambica mossambica venom), BAY K8644 and verapamil were obtained

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http://dx.doi.org/10.1016/j.tox.2014.06.010 0300-483X/ ã 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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2.2. Venom and antivenoms

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Lyophilized B. jararacussu venom obtained from adult snakes of both sexes was purchased from the Centro de Extração de Toxinas Animais (CETA, Morungaba, SP, Brazil) and stored at 20  C. Fresh stock solutions were prepared daily in Krebs–Henseleit solution

2.3. Animals

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Male Wistar rats (300–400 g) obtained from the Multidisciplinary Center for Biological Investigation (CEMIB/UNICAMP)

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(see Section 2.4 for composition) and stored on ice until used. Antivenoms produced by the Instituto Butantan (São Paulo, SP, Brazil) by immunizing horses with a pool of venoms (B. alternatus, B. jararaca, B. jararacussu, B. moojeni and B. neuwiedi) (Bothrops antivenom) or with the foregoing venoms plus Crotalus durissus terrificus (South American rattlesnake) venom (Bothrops/Crotalus antivenom) (Cardoso et al., 2009) were provided by the Poison Control Center (Centro de Controle de Intoxicações – CCI) of the university teaching hospital at UNICAMP.

0. 05

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from Sigma Chemical Co. (St. Louis, MO, USA). Isoflurane and heparin (sodium salt) were purchased from Cristália (Itapira, SP, Brazil) and commercial kits for the measurement of creatine kinase-MB activity were obtained from LaborLab (São Paulo, SP, Brazil). The salts for Krebs–Henseleit solution were from Baker (Mexico City, DF, Mexico), Mallinckrodt (Phillipsburg, NJ, USA) or Merck (Rio de Janeiro, RJ, Brazil).

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Venom (mg/ml)

Fig. 1. Changes in rat atrial contractility and muscle contracture in response to B. jararacussu venom. (A–E) Recordings showing the responses to Krebs–Henseleit solution alone (control) (A) and increasing concentrations of venom (B – 0.025 mg/ml, C – 0.05 mg/ml, D – 0.1 mg/ml and E – 0.2 mg/ml). The traces are representative of 5–8 experiments for each concentration. Arrows indicate the addition of venom. Note the initial transitory increase in contractile force followed by muscle contracture (the latter especially at the highest concentration). The preparations were allowed to stabilize for 1 h prior to venom addition. (F,G) Peak increase in transient contractility (F) and muscle contracture (G) in response to venom. The columns are the mean  SEM. (n = 5–8). *p < 0.05 compared to basal values (time zero).

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Fig. 2. Effects of B. jararacussu venom on muscle contracture (A), contractile force (B), beating rate (C) and CK-MB release (D) of rat isolated right atria. CK-MB release was measured before (basal) and after (end) a 60 min incubation with venom. Control atria were incubated in Krebs–Henseleit solution without venom. The points and columns are the mean  SEM. (n = 5–8). *,#p < 0.05 compared to basal values (time zero, *) and control (no venom) group (#). Note that the asterisks (*) from 5 min post-venom onwards in panel B apply to the two highest venom concentrations (0.1 and 0.2 mg/ml) although shown only for the highest concentration.

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were housed (12 h light/dark cycle, lights on at 6 a.m.; 22  2  C) in plastic cages (5/cage) with a wood shaving substrate and free access to food and water. All procedures were approved by the institutional Committee for Ethics in Animal Experimentation (CEEA/UNICAMP, protocol no. 1433–1) and the experiments were done according to the general guidelines of the Brazilian Society of Laboratory Animal Science (SBCAL).

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2.4. Rat isolated right atrial preparations

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Rats were anesthetized with isoflurane, exsanguinated and the hearts quickly removed and placed in modified Krebs– Henseleit solution (KHS) of the following composition (in mM): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 0.45, NaHCO3 25, KH2PO4 1.03, D-glucose 11.1 and ascorbic acid 0.14. Spontaneously beating atria were dissected and mounted under a tension of 1 g in an organ bath containing 20 ml of KHS maintained at 37  C and aerated with a mixture of 95% O2–5% CO2. Atrial rate and contractile force were recorded continuously via an isometric transducer connected to a preamplifier (AD Instruments, New Castle, Australia) and a computer loaded with data acquisition software Chart 6 (PowerLab AD Instruments) (Gasparetti et al., 2002; Fatehi-Hassanabad and Fatehi, 2004). After stabilization

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for 40 min, the atria were washed three times with KHS and, 20 min later, venom (in 200 ml of KHS) was added to the organ baths to give final concentrations of 0.025, 0.05, 0.1 or 0.2 mg/ml (only one venom concentration was tested per atrium); in control atria, 200 ml of KHS without venom was added. Changes in atrial rate and contractile force were monitored for 60 min. In all experiments, a 200 ml aliquot of KHS was obtained at time zero (basal, before the addition of KHS or venom to the bath) and at end of the experiment (after 60 min) for quantification of creatine kinase-MB (CK-MB) activity. CK-MB activity was determined by a UV kinetic method in a Beckman DU 800 spectrophotometer with commercial kits, according to the manufacturer's instructions. The results were expressed in U/ml. The time-course of tissue damage and CK-MB release was assessed by incubating atria with venom for 5, 15, 30 and 60 min followed by quantification of CK-MB in the bath solution and histological analysis of the tissue at each interval. To examine the reversibility of the venom-induced responses, in some experiments the preparations were washed three times at 5 min or 15 min after venom addition. In other experiments, the venom was heated (20 min, 100  C) prior to testing in atria. In the latter protocols, samples of the bathing solution were obtained for CK-MB quantification, as already described, and at

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Venom 0.2 mg/ml Control Venom 0.2 mg/ml (wash 5 min) Fig. 3. Influence of washing on rat atrial responses to B. jararacussu venom. (A) Recordings of atrial responses to venom with (I) or without (II) washing. In the upper recording (I), atria were incubated with venom (0.2 mg/ml) for 5 min and then washed three times with Krebs–Henseleit solution. (B–D) Mean responses for venom-induced contracture (B), contractile force (C) and atrial rate (D). The points are the mean  SEM. (n = 5–8). *p < 0.05 compared to preparations without washing.

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the end of the experiments the atria were processed for histological analysis.

2.6. Role of voltage-gated CA2+ channels in venom-induced contracture

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2.5. Venom PLA2 activity, inhibition with p-bromophenacyl bromide (p-BPB) and incubation of atria with indomethacin

To examine whether the influx of extracellular Ca2+ through voltage-gated Ca2+ channels was involved in the atrial responses to venom, atria were pre-incubated with verapamil (10 nM), a selective blocker of these channels, for 20 min prior to testing the response to venom (0.2 mg/ml). This concentration of verapamil effectively inhibited the increase in contractile tension induced by 100 nM BAY K8644 (positive control; basal: 0.12  0.02 g; BAY K8644: 0.21  0.02 g; BAY K8644 + verapamil: 0.13  0.02 g; n = 4; p < 0.05) (Martín et al., 1997; Wolkowicz et al., 2007).

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2.7. Neutralization by antivenom

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Venom (0.2 mg/ml; total of 4 mg for a 20 ml organ bath) was incubated with 0.8 ml of Bothrops or Bothrops / Crotalus antivenom (60 min, 37  C) (1 ml of antivenom neutralizes 5 mg of venom; Instituto Butantan) and the mixture then tested in isolated atria as described above. In some experiments, antivenom (0.8 ml) was added to the organ bath 1, 2 or 5 min after the addition of venom (0.2 mg/ml). Control atria were incubated with antivenom alone. At

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Venom PLA2 activity was assayed using a colorimetric assay (Price, 2007). The involvement of venom PLA2 in the atrial responses was assessed by pre-incubating venom with the histidine (His) aklylating agent p-bromophenacyl bromide (pBPB; 0.6 mM, 24 h, 23  C) (Diaz-Oreiro and Gutiérrez, 1997), with activity being measured before and after incubation with p-BPB. The possible role of arachidonic acid metabolites in the responses to venom was examined by pre-incubating atria with 10 mM indomethacin (a non-selective inhibitor of cyclooxygenase) for 20 min (Santos et al., 1990) prior to testing the response to venom (0.2 mg/ml). The efficacy of treatment with indomethacin was confirmed by its ability to block the atrial contractile responses to PLA2 purified from N. m. mossambica venom (6 mg/ ml) that are mediated by arachidonic acid metabolites generated by cyclooxygenase (contractile tension in the absence and presence of indomethacin: 0.30  0.04 g and 0.17  0.03 g, respectively; 43% inhibition; n = 4; p < 0.05).

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Fig. 4. Tissue damage in rat isolated right atria incubated with B. jararacussu venom for 60 min. Atria were incubated with Krebs–Henseleit solution alone (control; A) or venom (B – 0.025 mg/ml, C – 0.05 mg/ml, D – 0.1 mg/ml and E – 0.2 mg/ml) for 60 min and then processed for histological analysis, as described in Section 2.8. Arrows indicate muscle fiber disorganization and damaged cardiomyocytes. Longitudinal sections stained with hematoxylin-eosin. Scale bars: 20 mm. 158

the end of the experiments, the tissues were processed for histological analysis.

statistical calculations were done using Prism software version 5.0 (GraphPad Software Inc. La Jolla, CA, USA).

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2.8. Histological analysis

3. Results

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Control and venom-treated atria were fixed in buffered (pH 7.2) 10% formalin for 24 h followed by washing three times in distilled water (15 min each). The tissues were dehydrated in an ethanol series of increasing concentrations, cleared in xylol and embedded in paraffin (Histosec, Merck). Sections 5 mm thick were cut on a Leica RM 2245 microtome, stained with hematoxylin-eosin (HE) and analyzed with a Leica DM 5000B light microscope. Images were captured with a Leica DFC 300FX CCD camera and processed and analyzed with LEICA Q Win Plus v.3.2.0 software.

3.1. Effect of B. jararacussu venom on atrial contractility and rate

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The incubation of rat atria with B. jararacussu venom resulted in a transitory increase in contractility followed by a concentration-dependent muscle contracture (increase in baseline tension) that was very marked at the highest concentration (Fig. 1A–G and 2A ); the increase in baseline was reflected in a secondary decrease in contractile activity, particularly at the two highest concentrations (Fig. 2B). The lowest venom concentration (0.025 mg/ml) had no significant effect on atrial contractility (Figs. 1 and 2A and B). The transitory increase in contractility was most noticeable at venom concentrations of 0.05 and 0.1 mg/ml and was attenuated at 0.2 mg/ml because of the marked contracture at this concentration (Fig. 1F). The maximal increase in this contractility occurred 2– 4 min after venom addition. Washing the preparations 5 min after venom addition did not significantly attenuate the marked muscle

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2.9. Statistical analysis

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The results were expressed as the mean  S.E.M. Statistical comparisons were done using Student's t -test (for comparisons involving only two groups) or one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test for multiple comparisons. A value of p < 0.05 indicated significance. All

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Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Time (min) Fig. 5. Time-course of muscle damage (A) and CK-MB release (B) in rat isolated right atria incubated with B. jararacussu venom (0.2 mg/ml). Atria were incubated with venom and after 5, 15, 30 and 60 min samples of organ bath solution were obtained for the measurement of CK-MB release and the tissue then processed for histological analysis. In (A): I – control (no venom), II – 0.025 mg/ml, III – 0.05 mg/ml, IV – 0.1 mg/ml and V – 0.2 mg/ml. Arrows indicate muscle fiber disorganization and damaged cardiomyocytes. Scale bars: 20 mm. In (B), CK-MB release was measured before (Basal) and after (End) incubation with venom for the indicated time. The columns are the mean  SEM. (n = 5– 8). *,#p < 0.05 compared to the corresponding basal value and control (no venom) “End” value, respectively.

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contracture (as judged by peak tension and time to peak) but partially restored muscle contractility (Fig. 3) whereas washing 15 min after venom addition had no effect on the venom-induced responses (data not shown).

There was a trend towards a decrease in atrial rate after 60 min at a venom concentration of 0.1 mg/ml; a decrease was also observed with 0.2 mg/ml, but the marked muscle contracture seen at this concentration attenuated the usual contractile activity so

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Fig. 6. Influence of heating on the atrial responses to B. jararacussu venom (0.2 mg/ml). Venom was heated (100  C for 20 min) and then tested in isolated atria as described in Section 2.4. (A) Records of atrial responses to venom, (B) venom-induced contracture, (C) atrial rate, (D) CK-MB release (basal – before venom addition; end – 60 min after venom addition) and (E) histological analysis (I – control (no venom), II – heated venom; for damage caused by non-heated venom, see Fig. 3E). Note that heating abolished the ability of the venom to cause atrial contracture and tissue damage. The points and columns are the mean  SEM. (n = 5–8). *,#p < 0.05 compared to time zero (0 min, basal) or control (no venom) group (*) and non-heated venom (#).

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that atrial rate could not be reliably measured beyond 15 min after venom addition (Figs. 1 and 2C). The release of creatine kinase-MB (CK-MB), a marker enzyme for cardiac lesions, was generally elevated after a 60 min incubation with venom, particularly at the highest venom

concentration (Fig. 2D). This release suggested the occurrence of muscle damage that was confirmed by histological analysis which showed muscle fiber disorganization and myonecrosis (Fig. 4). Time-course experiments with venom (0.2 mg/ml) showed that the muscle damage and CK-MB release were progressive during the

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Fig. 7. Effect of the PLA2 His-aklyating agent p -BPB on the contracture and tissue damage caused by B. jararacussu venom (0.2 mg/ml). Venom was incubated with p-BPB (0.6 mM, 24 h, 23  C) prior to testing in atria. (A) Records of atrial responses to venom, (B) venom-induced contracture, (C) atrial rate, (D) CK-MB release (basal–before venom addition; end–60 min after venom addition) and (E) histological analysis (I – control (no venom), II – p-BPB alone, III – non-treated venom and IV – p -BPB-treated venom). Note that treatment with p-BPB abolished the venom-induced contracture and tissue damage (histological alterations and CK-MB release). The points and columns are the mean  SEM. (n = 5–8). *,#p < 0.05 compared to time zero (0 min, basal) or the control group (*) and non-heated venom (#).

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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increase in contractility or subsequent contracture (data not shown).

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3.4. Involvement of voltage-gated Ca2+ channels in atrial responses to venom

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To examine the role of the influx of extracellular Ca2+ through voltage-gated Ca2+ channels in the venom-induced contracture, atria were preincubated with verapamil (10 nM, 20 min) prior to the addition of venom. As shown in Fig. 8, verapamil did not significantly affect the atrial responses to venom, suggesting a lack of involvement of Ca2+ entry via voltage-gated Ca2+ channels.

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3.5. Antivenom neutralization of the atrial responses to venom

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Pre-incubation of B. jararacussu venom (0.2 mg/ml) with Bothrops antivenom for 1 h at 37  C had no effect on the venominduced contracture (Figs. 9A and 10 A) or secondary decrease in contractile force during the first 20 min but resulted in partial recovery of contractility at later intervals (Fig. 10B); there was also a recovery in atrial rate at intervals >20 min post-venom (Fig. 10C). When, instead of pre-incubation, antivenom was added to the organ bath 1 min after the addition of venom, there was again no protection against the venom-induced contracture (Figs. 9A and 10D), although the protection against the decrease in contractile force and atrial rate was better than in the pre-incubation experiments (Fig. 10E,F; compare with Fig. 10B,C). Preincubation with Bothrops/Crotalus antivenom resulted in a similar pattern of protection to that seen with Bothrops antivenom in the same protocol (Figs. 9B and 11A–C ), although the reversal seen when the former antivenom was added 1 min post-venom was less marked than with Bothrops antivenom (Figs. 9B and 11D–F). Histological analysis of the atria used in these protocols indicated that Bothrops antivenom protected against the myotoxic action of the venom since there was less muscle damage than with the venom alone (Fig. 12AI–III compared with panel V of Fig. 5A). Antivenom alone did not induce CK-MB release whereas preincubation with venom or the addition of antivenom 1 min after venom attenuated the venom-induced release of this enzyme (Fig. 12B). Bothrops/Crotalus antivenom attenuated the venom-induced muscle damage (Fig. 12AIV–VI) and the release of CK-MB (Fig. 12C) to a similar degree as that seen with Bothrops antivenom. No functional protection was observed when either of the antivenoms was added 2 min after the venom (data not shown).

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The results described here indicate that B. jararacussu venom causes concentration-dependent contracture of rat atrial muscle. The prolonged, accentuated contracture of atrial muscle at the highest concentration was similar to that reported for other snake venoms and toxins (Raina et al., 1978; Sun and Walker, 1986; Alloatti et al., 1991; Huang et al., 1993; Gomes et al., 2001) and reminiscent of the action of cardiotoxins (Harvey, 1985) in isolated heart and atria. Contracture of skeletal muscle has also been reported for chick biventer cervicis neuromuscular preparations incubated with bothropstoxin I, the major myotoxic PLA2 of this venom (Heluany et al., 1992). The concentration- and time-dependent tissue damage seen histologically and confirmed by release of the marker enzyme CKMB agreed with studies showing that B. jararacussu venom is myonecrotic (Melo and Suarez-Kurtz, 1987, 1988a,b; Calil-Elias et al., 2002; Sifuentes et al., 2008), with most of this activity being attributed to the presence of myotoxic PLA2, particularly bothropstoxins (Gutiérrez et al., 1991; Melo et al., 1993; Oliveira et al., 2003), although this venom contains other PLA2 (Bonfim et al.,

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Fig. 8. Lack of effect of verapamil on atrial responses to B. jararacussu venom (0.2 mg/ml). Right atria were preincubated with verapamil (10 nM) for 20 min prior to addition of venom. Verapamil did not alter the atrial responses to venom but completely attenuated the responses to the voltage-gated Ca2+ channel opener BAY K8644 (100 nM) (see Section 2.6). The points are the mean  SEM. (n = 5–8). *p < 0.05 compared to time zero (0 min, basal) or control (no venom) group. 213 214 215

incubation, with minimal histological damage or enzyme release during the first 5 min but marked damage and significant CK-MB release after 60 min (Fig. 5).

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Heating the venom (20 min, 100  C) abolished the increase in muscle contracture and decrease in atrial rate (Fig. 6A–C), as well as the CK-MB release (Fig. 6D) and tissue damage (Fig. 6E). Since these results suggested that enzymatic activity was involved in the venom-induced response, and since Bothrops venom PLA2 are wellknown for their ability to cause myonecrosis, we examined the involvement of PLA2 in the atrial responses.

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3.3. Involvement of venom PLA2 in atrial responses to venom

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Venom PLA2 activity was 28  5 units/mg (n = 3) and this was inhibited 100% by incubation with p -BPB. Treatment with p-BPB abolished the venom-induced transient increase in contractility, the contracture and bradycardia, with the responses of p-BPBtreated venom being similar to those of control preparations incubated with KHS alone (Fig. 7A–C). Treatment with p-BPB also abolished the release of CK-MB (Fig. 7D) and markedly attenuated the muscle damage (Fig. 7E). In contrast to the effectiveness of treatment with p-BPB, pre-treating the atria with indomethacin (10 mM, 20 min) did not affect the venom-induced transient

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Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Fig. 9. Contractile activity of rat isolated right atria incubated with B. jararacussu venom (0.2 mg/ml) in the presence of (A) Bothrops and (B) Bothrops/Crotalus antivenom. The traces (representative of 5–8 experiments each) show the responses to (I) Krebs–Henseleit solution (KHS) containing antivenom alone (control), (II) venom alone, (III) venom pre-incubated with antivenom prior to addition to the organ bath and (V) venom followed 1 min later by the addition of antivenom. Antivenom (0.8 ml) was pre-incubated (p. i.) with venom (4 mg) at 37  C for 60 min or added directly to the organ bath (without pre-incubation) 1 min after the addition of venom (final venom concentration in all cases: 0.2 mg/ml). Control atria were incubated in KHS without venom or with 0.8 ml of antivenom. Although antivenom did not protect against venom-induced contracture, it restored the contractile activity of the muscle. Arrows indicate the addition of venom or venom + antivenom mixture. 295 296 297 298 299 300 301 302 303 304

2001; Andrião-Escarso et al., 2002; Ketelhut et al., 2003; Kashima et al., 2004; Ponce-Soto et al., 2006) that could also contribute to this effect. To examine a possible role for PLA2 in the venom-induced damage, venom was incubated with p-BPB, a classic PLA2 inhibitor (Soares and Giglio, 2003). Pre-treatment with p-BPB significantly attenuated the PLA2 activity and prevented the venom-induced contracture and tissue damage (the latter assessed histologically and by CK-MB release). This finding indicated a prominent role for venom PLA2 in the responses observed and agreed with other

studies showing that p-BPB attenuates the myotoxicity of bothropstoxin I (BthTX-I) (Andrião-Escarso et al., 2000; MarchiSalvador et al., 2006). The initial transitory increase in contractile activity seen before the onset of contracture was also abolished by treatment with p-BPB whereas indomethacin had no effect. The lack of effect of indomethacin, a non-selective inhibitor of cyclooxygenase, on the venom-induced atrial responses (initial transient increase and marked contracture) indicated that, despite the diversity of PLA2 in this venom (Kashima et al., 2004), cyclooxygenase products, e.g., prostaglandins, derived from

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Fig. 10. Atrial responses to B. jararacussu venom in the presence of Bothrops antivenom. (A,D) Contracture, (B,E) contractility and (C,F) atrial rate. Antivenom (0.8 ml) was preincubated (p.i.) with venom (4 mg) at 37  C for 60 min (A–C) or added directly to the organ bath (without pre-incubation) 1 min after the addition of venom (D–F) (final venom concentration in all cases: 0.2 mg/ml). Control atria were incubated in Krebs–Henseleit solution (KHS) without venom or with 0.8 ml of antivenom. Antivenom alone had no significant effect on atrial responses compared to preparations incubated with KHS alone. Antivenom did not significantly affect the venom-induced contracture but led to a recovery in contractile activity and attenuation of the bradycardia. The points are the mean  SEM. (n = 6). *p < 0.05 compared to venom alone.

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arachidonic acid released by these PLA2, were not involved. This conclusion agrees with the general membrane damaging activity of Bothrops myotoxins (Gutiérrez and Ownby, 2003; Montecucco et al., 2008), independent of prostaglandin formation, and with the finding that the atrial actions of other venom PLA2 also do not necessarily correlate with the formation of cyclooxygenase products (Rosenberg, 1986). The inability of washing (within 5 min of venom addition) to protect against the venom-induced contracture indicated that the initiation of contracture was rapid and essentially unaffected by

subsequent venom removal. In addition, the time-course experiments showed that significant contracture occurred when there was still little histologically-detectable tissue damage, e.g., important contracture was observed 5 min post-venom (Fig. 2A) when tissue lesions were apparently minimal and there was no significant CK-MB release (Fig. 5). This lack of correlation between contracture and detectable tissue damage suggested that the latter was not the primary cause of the former, probably because initial membrane damage involved an increase in membrane permeability to ions such as Ca2+ prior to visible tissue damage. On the other

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Fig. 11. Atrial responses to B. jararacussu venom in the presence of Bothrops/Crotalus antivenom. (A,D) Contracture, (B,E) contractility and (C,F) atrial rate. Antivenom (0.8 ml) was pre-incubated (p.i.) with venom (4 mg) at 37  C for 60 min (A–C) or added directly to the organ bath (without pre-incubation) 1 min after the addition of venom (D–F) (final venom concentration in all cases: 0.2 mg/ml). Control atria were incubated in Krebs–Henseleit solution (KHS) without venom or with 0.8 ml of antivenom. Antivenom alone had no significant effect on atrial responses compared to preparations incubated with KHS alone. Antivenom did not significantly affect the venom-induced contracture but led to a recovery in contractile activity and attenuation of the bradycardia. The points are the mean  SEM. (n = 6). *p < 0.05 compared to venom alone. 335 336 337 338 339 340 341 342

hand, the progressive tissue damage probably resulted in a decrease in developed force that contributed to the inability of muscle to maintain the contracture from 15 min onwards. Sustained muscle contracture to snake venoms and toxins generally reflects an excessive (uncontrolled) influx of Ca2+ into myocytes, primarily as a consequence of tissue (membrane) damage (Harvey, 1985; Gutiérrez and Ownby, 2003; Montecucco et al., 2008). Conversely, a decrease in the influx of

extracellular Ca2+ has been suggested in situations where there is reduced contractility with no accompanying contracture (Fletcher et al., 1982; Petkovi c et al., 1983; Santos et al., 1990). The finding here that verapamil, a voltage-gated Ca2+ channel blocker, had no effect on the venom-induced contracture indicated that Ca2+ influx via these channels was not a major contributor to this phenomenon. This finding reinforces the conclusion regarding uncontrolled Ca2+ entry following

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A

Fig. 12. Attenuation by Bothrops and Bothrops/Crotalus antivenoms of the tissue damage and CK-MB release caused by B. jararacussu venom (0.2 mg/ml). Panels AI and AIV show the histological alterations in control atria (60 min incubation with antivenom alone), AII and AV show the alterations when antivenom was preincubated with venom (0.8 ml of antivenom with 4 mg of venom for 60 min at 37  C) prior to testing, and AIII and AVI show the alterations when antivenom was added 1 min after the venom (no preincubation). AI-III – Bothrops antivenom, AIV-VI – Bothrops/Crotalus antivenom. Asterisks indicate muscle fiber disorganization/damage. Longitudinal sections stained with hematoxylin–eosin. Scale bars: 20 mm. (B) and (C) CK-MB release in the presence of Bothrops and Bothrops/Crotalus antivenom, respectively. Note that antivenom attenuated the venom-induced release of CK-MB. The columns are the mean  SEM. (n = 6). *,#p < 0.05 compared to basal values and “End” values of control (no venom) group (*) and venom alone (#).

Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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membrane damage as the primary cause of the venom-induced contracture. The venom concentrations used here were in the same range (10–200 mg/ml) used to investigate the neuromuscular activity of Bothrops venoms in vitro (Rodrigues-Simioni et al., 2004; Zamunér et al., 2004; Abreu et al., 2007; Cavalcante et al., 2011; RodriguesSimioni et al., 2011; Moraes et al., 2012). In contrast, there is very little information on the circulating concentrations of B. jararacussu venom in humans and experimental animals, and it is unclear to what extent the kinetics of Bothrops venoms are similar in humans and rodents. In humans, the determination of circulating venom concentrations is complicated by the interval between the bite and subsequent medical assistance (2–6 h after most Bothrops bites); this time lag makes it difficult to obtain a clear idea of circulating venom levels in the early stages of envenomation when there may be important cardiac/hemodynamic alterations. Thus, in a case involving B. jararacussu , a circulating venom concentration of 0.4 mg/ml was detected 8 h post-bite (Milani et al., 1997), and in Bothrops jararaca, a related species, venom concentrations 20 min, without affecting the onset of damage (in the first 20 min of incubation); histological analysis confirmed this protection against venom-induced damage. In contrast, in the latter experimental protocol, the addition of antivenom 2 min after the onset of incubation with venom offered no protection against the venominduced effects (data not shown). These findings indicate that antivenom can protect against venom-induced atrial damage but that the time of administration is an important factor, with delayed treatment offering little or no protection, possibly because at later stages of incubation the toxins responsible for this damage are either too tightly bound to their receptors or inaccessible to the antibodies. Alternatively, the poor protection by antivenom at later

stages may indicate that the cellular processes involved in muscle damage have already been activated and any toxin neutralization by antivenom will have little effect on the ensuing tissue damage. In agreement with these findings, Bothrops antivenom has also been reported to partially protect mouse phrenic nerve-diaphragm and extensor digitorum longus neuromuscular preparations from damage by B. jararacussu venom and bothropstoxin I in vitro (Oshima-Franco et al., 2000). In the early 1900s, Brazil (1901, 1903, 1911) suggested that envenoming by B. jararacussu shared certain manifestations, e.g., neurotoxicity, with bites by the South American rattlesnake, C. d. terrificus, and that the use of Bothrops/Crotalus antivenom, rather than Bothrops antivenom alone, could be beneficial in the treatment of bites by B. jararacussu. In agreement with this, laboratory studies have shown that Bothrops/Crotalus antivenom offers greater protection than Bothrops antivenom against the myotoxicity, coagulant activity and lethality of B. jararacussu venom (dos Santos et al., 1992). In addition, the neutralization of B. jararacussu venom and bothropstoxin-I by rabbit antiserum against crotoxin, the major neurotoxin of C. d. terrificus venom, has been demonstrated in mouse phrenic nerve-diaphragm preparations (Oshima-Franco et al., 2001). However, clinically, Bothrops antivenom is still widely used for bites by this species (Milani et al., 1997). As shown here, there was no major difference in the profile of protection offered by Bothrops/Crotalus antivenom compared to Bothrops antivenom alone in the two protocols tested; indeed, the reversal of the loss of contractile force by the former antivenom when added 1 min after venom was less than with the latter and the protection against histological damage was not as complete. These findings suggest that, at least in this experimental model, Bothrops/Crotalus antivenom offers no advantage over Bothrops antivenom. In conclusion, the results described here indicate that B. jararacussu venom adversely affects rat isolated rat atria, apparently via the action of venom PLA2. The decrease in muscle contractility and the myonecrosis were attenuated by Bothrops and Bothrops/Crotalus antivenoms.

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Conflict of interest statement

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The authors have no conflicts of interest related to this work.

Uncited references Sant'Ana et al. (2008a) and Wolkowitz et al. (2007). Acknowledgments The authors thank José Ilton dos Santos and Maiara Daguana de Azevedo for technical assistance. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant no. 05/56033-8). LD was supported by FAPESP (grant no. 05/ 50627-3), ALR and DAS were supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and AS, MAPR and NCS by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). SH is supported by a research fellowship from CNPq.

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Please cite this article in press as: Rodrigues, M.A.P., et al., Rat atrial responses to bothrops jararacussu (jararacuçu) snake venom, Toxicology (2014), http://dx.doi.org/10.1016/j.tox.2014.06.010

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Rat atrial responses to Bothrops jararacussu (jararacuçu) snake venom.

Envenoming by the pitviper Bothrops jararacussu produces cardiovascular alterations, including coagulopathy, systemic hemorrhage, hypotension, circula...
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