Local and Hematological Alterations Induced by Philodryas Olfersii Snake Venom in Mice

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Toxicon 132 (2017) 9e17

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Toxicon

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Local and hematological alterations induced by Philodryas olfersii snake venom in mice

* Juliana S. Oliveira a, Luciana B. Sant'Anna a, , Manoel C. Oliveira Junior b, Pamella R.M. Souza b, Adilson S. Andrade Souza b, Wellington Ribeiro c, Rodolfo P. Vieira b, e, Stephen Hyslop d, Jose C. Cogo e a Laboratory of Histology and Regenerative Therapy, Institute of Research and Development (IP&D), Vale do Paraíba University (UNIVAP), Avenida Shishima Hifumi, 2911, Urbanova, 12244-000, Sao~ Jose dos Campos, SP, Brazil b Laboratory of Pulmonary and Exercise Immunology (LABPEI), Nove de Julho University (UNINOVE) and Brazilian Institute of Teaching and Research in Pulmonary and Exercise Immunology (IBEPIPE), 01504-000, Sao~ Paulo, SP, Brazil c Laboratory of Pharmacology and Biochemistry, Institute of Research and Development (IP&D), Vale do Paraíba University (UNIVAP), Avenida Shishima Hifumi, 2911, Urbanova, 12244-000, Sao~ Jose dos Campos, SP, Brazil d Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Rua Tessalia Vieira de Camargo, 126, Cidade Universitaria Zeferino Vaz, 13083-887, Campinas, SP, Brazil e Department of Bioengineering and Biomedical Engineering, Brazil University, Rua Carolina Fonseca, 584/235 (Campus I and II), Vila Santana, 08230-030, Itaquera, Sao~ Paulo, SP, Brazil article info abstract

Article history: Envenomation by the South American opisthoglyphous snake Philodryas olfersii causes local pain, edema, Received 25 November 2016 erythema and ecchymosis; systemic envenomation is rare. In this work, we examined the inflammatory Received in revised form activity of P. olfersii venom (10, 30 and 60 mg) in mouse gastrocnemius muscle 6 h after venom injection. 22 March 2017 Intramuscular injection of venom did not affect hematological parameters such as red cell count, he- Accepted 23 March 2017 moglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular Available online 24 March 2017 hemoglobin concentration. The venom caused thrombocytopenia (at all three doses), leukopenia and lymphopenia (both at the two highest doses), as well as neutrophilia (30 mg), monocytosis (30 mg) and Keywords: Acute inflammation basophilia (10 mg). Of the cytokines that were screened [IL-1b, IL-6, IL-10, IL-13, IL-17, TNF-a, IFN-g, MIP-2 fi Cytokines and KC] and IGF-1, only IGF-1 showed a signi cant increase in its circulating concentration, seen with Edema 60 mg of venom; there were no significant changes in the cytokines compared to control mice. Histo- Myonecrosis logical analysis revealed the presence of edema, an inflammatory infiltrate and progressive myonecrosis. Inflammatory infiltrate Edema and myonecrosis were greatest with 60 mg of venom, while the inflammatory infiltrate was Philodryas olfersii venom greatest with 10 mg of venom. All venom doses caused the migration of polymorphonuclear and mononuclear leukocytes into muscle, but with no significant dose-dependence in the response. These findings show that, at the doses tested, P. olfersii venom does not cause hematological alterations and has limited effect on circulating cytokine concentrations. These data also confirm that the principal effects of the venom in mice are local edema, inflammatory cell infiltration and myonecrosis. © 2017 Elsevier Ltd. All rights reserved.

1. Introduction cause of non-front-fanged colubroid envenomations in this conti- nent (Prado-Franceschi and Hyslop, 2002; Weinstein et al., 2011, The back-fanged colubroid snake genus Philodryas (Dipsadidae, 2013), with the main species involved in human envenomations Xenodontinae), commonly referred to as racers, consists of ~20 being P. chamissonis (Otero et al., 2007), P. olfersii (Ribeiro et al., species with a widespread distribution throughout South America 1999; Correia et al., 2010) and P. patagoniensis (Medeiros et al., (Zaher et al., 2008, 2014). Snakes of this genus are the principal 2010); species less commonly involved include P. aestivus (Fowler and Salomao,~ 1994), P. baroni (Küch and Jesberger, 1993) and P. viridissima (Means, 2010). * Corresponding author. The venoms of P. olfersii (Assakura et al., 1992; Acosta de Perez E-mail address: [email protected] (L.B. Sant'Anna). et al., 2003; Rodríguez-Acosta et al., 2006) and P. patagoniensis http://dx.doi.org/10.1016/j.toxicon.2017.03.013 0041-0101/© 2017 Elsevier Ltd. All rights reserved. Author's Personal Copy

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(Acosta et al., 2003; Peichoto et al., 2004, 2005, 2006; Lopes, 2008) groups (n ¼ 10/group): Group 1 e mice injected with phosphate- cause edema, hemorrhage and myonecrosis in experimental ani- buffered 150 mM saline (PBS) solution (control group) in the left mals, while in humans the primary manifestations are local effects gastrocnemius muscle and Groups 2e4 e mice injected with 10 mg, such as pain, edema, erythema and ecchymosis (Ribeiro et al., 1999; 30 mg and 60 mgofP. olfersii venom, respectively, in a volume of 50 Medeiros et al., 2010). A few components have been isolated from ml/gastrocnemius muscle. Six hours after saline or venom injection, these venoms, including a myotoxin (Prado-Franceschi et al., 1998) the mice were anesthetized with a mixture of xylazine hydro- and five fibrinogenolytic proteases (four metalloproteinases and chloride (Xilazin™ 2% injectable solution; 10 mg/kg, i.p.) plus ke- one serine protease, with two of these enzymes also being hem- tamine hydrochloride (Cetamin™ 10% injectable solution; 100 mg/ orrhagic) (Assakura et al., 1992) from P. olfersii, and a metal- kg, i.p.). Once satisfactory anesthesia had been reached, blood was loproteinase (patagonfibrase) (Peichoto et al., 2007, 2010, 2011) and collected via the inferior vena cava in a 1 ml syringe containing cysteine-rich secretory protein (CRISP; patagonin) (Peichoto et al., 0.1 ml of EDTA. Ten microliters of blood were used for a complete 2009) from P. patagoniensis. The identification of these isolated blood count and the remainder was centrifuged (900 g, 10 min, components agrees with proteomic and transcriptomic analyses 4 C) and the plasma then collected and stored at 80 C for sub- indicating the presence of metalloproteinases, serine proteases, sequent quantification of inflammatory mediators. After blood CRISPs and other components in these venoms (Ching et al., 2006; collection, the left gastrocnemius muscle was removed from the Peichoto et al., 2012). exsanguinated mice and three samples of each muscle were placed Philodryas olfersii venom degrades fibrinogen in vitro and in vivo in separate polypropylene microtubes and stored at 80 C. via the action of metalloproteinases and serine proteinases (Assakura et al., 1994), but is devoid of thrombin-like activity; this 2.3. Hematological analysis degradation delays the clotting of fibrinogen by thrombin (Assakura et al., 1992). The venom also has fibrinolytic activity, but Hematological analyses were done in an automated hemato- is devoid of platelet-aggregating or inhibitory effects (Assakura logical analyzer (Sysmex 800i, Roche, Germany) using blood sam- et al., 1992). In contrast to these effects on hemostasis, the effect ples collected from the inferior vena cava. The parameters of P. olfersii venom on general hematological parameters is un- measured included red blood cell count (RBC), hemoglobin, he- known. In addition, compared to P. patagoniensis (Peichoto et al., matocrit, mean corpuscular volume (MCV), mean corpuscular he- 2004; Lopes, 2008), the edematogenic response to P. olfersii moglobin (HCM), mean corpuscular hemoglobin concentration venom (Assakura et al., 1992; Acosta et al., 2003) has not been (CHMC), white blood cell (WBC) count, neutrophils, lymphocytes, investigated in detail, particularly with regard to the profile of in- monocytes and platelets. flammatory cells involved and the possible changes in the concentrations of cytokines in the general circulation. 2.4. Cytokine quantification In this work, we therefore examined the profile of the cellular infiltrate associated with the inflammatory response after the Plasma cytokine levels were quantified by ELISA using com- intramuscular injection of P. olfersii venom in mice. We also mercial kits obtained from Biolegend (San Diego, CA, USA) and R&D quantified a variety of cytokines (IL-1b, IL-6, IL-10, IL-13, IL-17, Systems (Minneapolis, MN, USA). The cytokines investigated were TNFa, IFNg, MIP-2 and KC) and the growth factor IGF-1 known to be IL-1b, IL-6, IL-10, IL-13, IL-17,MIP-2,KC, TNF-a and IFN-g and the involved in the development and modulation of inflammation and growth factor IGF-1. All of the assays were done according to each examined the occurrence of myonecrosis. manufacturer's recommended protocol.

2. Material and methods 2.5. Histological and quantitative analysis of polymorphonuclear and mononuclear cells 2.1. Venom After removal, the gastrocnemius muscle was sectioned into Venom was obtained by manual extraction from an adult female three parts, frozen in liquid nitrogen, fixed in cardboard chips and P. olfersii maintained at the Serpentarium of the Center for Nature embedded in tissue freezing medium mixed with powdered milk to Studies at UNIVAP (Environmental license SMA 15.380/2012). The increase the viscosity and support the tissue during cryotomy. venom was collected using glass capillary tubes, essentially as Sections 10-mm thick were cut with a Leica DM1250 cryostat and described by Ferlan et al. (1983), and was lyophilized and stored at then stained with hematoxylin-eosin (HE) for semi-quantitative 2e6 C until use. For the experiments, venom was dissolved in 0.9% evaluation of edema, inflammatory infiltrate and muscle degener- NaCl immediately before use and injected into the left gastrocne- ation (myonecrosis), as well as quantitative evaluation of poly- mius muscle of mice. Control mice were injected with an equal morphonuclear and mononuclear cells in the muscle tissue. volume (50 ml) of saline. Six hours later, the mice were killed and For HE staining, the slides were immersed in acetone for 7 min, blood and tissue samples were collected for analysis. washed three times with deionized water and then incubated with hematoxylin for 1 min followed by three washes under running 2.2. Animals and experimental groups water. The sections were then submerged in a differentiating solution (9.90 ml of 70% ethanol þ 10 ml of HCl) for 1 s, immersed in Male C57BL/6 mice (18e22 g) were housed (5/cage) in poly- water for 3 min and in 80% ethanol for ~30 s. After this processing, propylene cages with a wood shaving substrate at 22 ± 2 Cona the slides were stained with eosin for 1 min and submerged in 95% 12 h light/dark cycle with lights on at 6 a.m. and free access to food ethanol for ~30 s and then washed in absolute ethanol. The slides ® ® (Purina rodent chow) and water. The experiments were approved were subsequently dried and mounted with Entelan . by the Committee for Ethics in Animal Use of Vale do Paraíba For the semi-quantitative evaluation of edema, inflammatory University (CEUA/UNIVAP, protocol no. A13/CEUA/2015) and were infiltrate and muscle degeneration, the slides were examined with done in accordance with the ethical guidelines for animal experi- a Nikon Eclipse E200 optical microscope at 400 magnification and mentation established by the Brazilian Society for Laboratory Ani- the alterations or extent of damage was scored using the following mal Science (SBCAL). arbitrary scale: 0 e no alteration or damage, 1 e mild alteration or For the experiments, 40 mice were randomly allocated to four damage, 2 e moderate alteration or damage, 3 e intense alteration Author's Personal Copy

J.S. Oliveira et al. / Toxicon 132 (2017) 9e17 11 or damage, 4 e very intense alteration or damage. The individual both cases, tissue area was calculated as described in the preceding responsible for this analysis was blinded or unaware of the treat- paragraph. ments from which the different sections had been obtained. The mean scores of five microscopic fields, one from each of five 2.6. Statistical analysis randomly chosen histological sections from each mouse, were used to create a single score for each mouse in each experimental group. Quantitative (numerical) data were expressed as the mean ± SD The mean of these mouse scores provided the overall mean for the whenever appropriate. Prior to statistical analysis, the Shapiro- corresponding group. Wilk normality test was applied to examine the distribution of For the quantitative analysis of polymorphonuclear and mono- the data. Data with a normal distribution were analyzed by one- nuclear inflammatory cells in gastrocnemius muscle, histo- way analysis of variance (ANOVA) followed by the Newman-Keuls morphometry was used in conjunction with image analysis, in an test for multiple comparisons among groups. For data without a adaptation of similar analyses reported elsewhere (Vieira et al., normal distribution, the non-parametric Kruskal-Wallis test was 2008; Ramos et al., 2010; Gonçalves et al., 2012; Vieira et al., used followed by the Dunn test for multiple comparisons. In all 2012). A total of 20 slides (five from each experimental group) cases, the level of significance was set at 5% (p < 0.05). All data were analyzed. From each slide, five microscopic fields were analyses and statistical comparisons were done using Prism 5.0 randomly selected for analysis. Photomicrographs were captured software (GraphPad Inc., La Jolla, CA, USA). with a digital video camera (Leica DF425) coupled to an optical microscope (Leica DM2500) and scanned at 1024 768 pixels, 24 3. Results bits/pixel resolution at a global magnification of 400. Image-Pro Plus 4.0 software was then used to calculate the total area of the 3.1. Hematological parameters photomicrograph and clear area (area without tissue section). The difference in area obtained by subtracting the clear area from the The intramuscular injection of P. olfersii venom (10, 30 and total area of the photomicrograph yielded the tissue area. 60 mg) produced no significant alterations in the following he- Polymorphonuclear (PMN) and mononuclear (MN) cells were matological parameters: red cell count, hemoglobin, hematocrit, counted manually and the number of polymorphonuclear cells/ mean corpuscular volume, mean corpuscular hemoglobin and 2 mm of tissue was calculated using the formula (number of PMN x mean corpuscular hemoglobin concentration (see Supplemen- 2 1000 cells) ÷ tissue area ¼ PMN/mm . Likewise, the number of tary table). 2 polymorphonuclear cells/mm of tissue was calculated using the In contrast, as shown in Fig. 1, there was a significant decrease in 2 formula (number of MN x 1000 cells) ÷ tissue area ¼ MN/mm .In the number of leukocytes (leukopenia), particularly with 30 mg and

A B C * 0.8 ** ** 5 800 * /ml /ml ** ** 5 5 * 0.6 4 ***

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Fig. 1. Changes in the number of circulating total leukocytes (A), platelets (B), neutrophils (C), monocytes (D), lymphocytes (E) and basophils (F) in BALB/c mice injected with P. olfersii venom. The mice were injected in the left gastrocnemius muscle with venom (10, 30 or 60 mgin50ml of PBS) or the same volume of PBS alone (control mice) and 6 h later they were anesthetized for blood sampling from the inferior vena cava (blood collected into EDTA), followed by exsanguination. The cells in plasma samples were counted in an automated hematological analyzer. The columns represent the mean ± SD (n ¼ 10/group). *p < 0.05, **p < 0.01 and ***p < 0.005 for the comparisons indicated (one-way ANOVA followed by the Newman-Keuls multiple comparisons test). The ends of each horizontal bar indicate the two columns being compared in each case. Author's Personal Copy

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60 mg of venom. There was also a significant reduction (~30%) in 4. Discussion platelet count, but no significant difference in the response to the three doses of venom. There was a significant increase in the Philodryas olfersii and P. patagoniensis are the two species most number of neutrophils and monocytes with 30 mg of venom commonly involved in envenomation by Philodryas snakes in Brazil compared to the control group (PBS solution). For neutrophils, (Ribeiro et al., 1999; Medeiros et al., 2010) and their venoms are the venom doses of 10 mg and 60 mg also produced an increase, but this best studied among South American non-front-fanged colubroid was considerably less marked than with 30 mg of venom. For snakes. However, compared to P. patagoniensis (Acosta et al., 2003; monocytes, the dose of 10 mg caused an increase in cell numbers Peichoto et al., 2004, 2005, 2006, 2007, 2009, 2010, 2011, 2012), whereas with 60 mg the number of monocytes was not significantly relatively few studies have examined the biological activities of different from that of the PBS control. The changes in the number of P. olfersii venom. Assakura et al. (1992, 1994) showed that P. olfersii lymphocytes mirrored those for total leukocytes but were rather venom was hemorrhagic, fibrinogenolytic and edematogenic, and more pronounced, with 30 mg and 60 mg producing the greatest subsequent studies confirmed the edematogenic (Acosta et al., reductions (lymphopenia). There was a significant increase in the 2003) and hemorrhagic (Rocha et al., 2006; Rodríguez-Acosta number of basophils (basophilia) with 10 mg of venom, but not with et al., 2006) activities and the ability of this venom to cause myo- the other doses. necrosis in vitro (Prado-Franceschi et al., 1996, 1998; Collaço et al., 2012) and in vivo (Acosta et al., 2003). In this work, we examined 3.2. Cytokine quantification the edema and myonecrosis caused by P. olfersii venom in mouse gastrocnemius muscle, and also assessed the ability of the venom to Fig. 2 shows the plasma concentrations of IGF-1 and various alter hematologic parameters and increase the circulating cytokine cytokines measured by ELISA in PBS- and venom-treated mice 6 h concentrations. The doses of venom used here were chosen based after venom injection. There was a very large increase (p < 0.001) in on Rocha and Furtado (2007), who reported a minimum hemor- serum IGF-1 levels with 60 mg of venom compared to the control rhagic dose of 24 mg/mouse, a minimum necrotizing dose of 79,1 (PBS) group and the other two doses of venom. There was no sig- mg/mouse and a lethality (LD50) of 62.43 mg/mouse for this venom. nificant increase in KC levels compared to the PBS group. However, An interval of 6 h post-venom was studied because literature re- the concentration with 30 mg of venom was significantly greater ports of envenoming by P. olfersii indicate that this interval shows than that seen with 10 mg and 60 mg, primarily because of a typical changes associated with an acute inflammatory reaction. reduction associated with the latter two doses. There were no The lack of significant changes in the hematological parameters significant changes in the concentrations of various other pro- 6 h after the intramuscular injection of P.olfersii venom indicated inflammatory cytokines (MIP 2, IFN-g TNF-a, IL-1b, IL-6 and IL- that there were no systemic alterations in these parameters asso- 17) or the anti-inflammatory interleukins IL-10 and IL-13 for the ciated with the venom doses, route of administration and time three venom doses. interval examined here. The unaltered hemoglobin and hematocrit also indicated that there was no intravascular hemolysis, perhaps 3.3. Histological analysis reflecting the absence of PLA2 activity in this venom (Assakura et al., 1992; Peichoto et al., 2012). Gastrocnemius muscle inoculated with PBS alone showed the In contrast to the lack of effect on hematological parameters, characteristics of normal skeletal striated muscle, namely, multi- significant thrombocytopenia was seen with all doses of venom, nucleated cylindrical cells, peripheral nuclei and transverse stria- although there was no difference in the extent of the response tions visible in light microscopy, with the presence of mild edema among the three doses. Possible changes in circulating platelet and a discrete inflammatory infiltrate. In muscle inoculated with numbers have not previously been examined after injection of 10 mgofP. olfersii venom, there was edema, an intense inflamma- Philodryas venoms. Metalloproteinases (Assakura et al., 1992, 1994; tory infiltrate characterized by a predominance of poly- Ching et al., 2006; Rocha et al., 2006; Peichoto et al., 2012) and C- morphonuclear cells, and mild degeneration of the muscle fibers type lectins (Ching et al., 2006; Peichoto et al., 2012)inP. olfersii seen as membrane rupture and fibrillar disorganization. In muscle venom could possibly contribute to this decrease in platelet num- inoculated with 30 mg of venom, there was moderate edema and an ber, whereas PLA2, thrombin-like enzymes and procoagulant en- inflammatory infiltrate characterized by a predominance of poly- zymes are unlikely to be involved since the venom is devoid of morphonuclear cells accompanied by foci of muscle degeneration these activities (Assakura et al., 1992; Ching et al., 2006; Peichoto with fiber destruction (myonecrosis). In muscle injected with 60 mg et al., 2012). However, the venoms of P. baroni (Sanchez et al., of venom there was intense edema, inflammatory infiltrate with a 2014) and P. patagoniensis (Peichoto, 2007) do not aggregate hu- predominance of polymorphonuclear cells, and muscle degenera- man washed platelets in vitro, but inhibit collagen- and thrombin- tion, the latter involving membrane rupture, fibrillar disorganiza- induced aggregation. Similarly, the metalloproteinase patagonfi- tion and myonecrosis (Fig. 3). brase from P. patagoniensis venom does not aggregate human Semi-quantitative analysis (Fig. 4) showed significant edema washed platelets but inhibits collagen- and ADP-induced aggrega- and muscle degeneration with 60 mg of venom compared to the tion without affecting that induced by thrombin or ristocetin; the control group (PBS alone) after 6 h. In contrast, the inflammatory inhibitory activity is independent of the protein's enzymatic ac- infiltrate was greatest with 10 mg of venom compared to the control tivity (Peichoto et al., 2007). The thrombocytopenia observed here group. Muscle degeneration, but not edema or inflammatory infil- was similar to that reported by Yamashita (2013) in mice inoculated trate, showed dose-dependence. The intramuscular injection of with Bothropsjararaca venom. In contrast, Graça et al. (2008) found P. olfersii venom caused a significant increase in poly- no significant variations in the platelet count in an experimental morphonuclear leukocyte density/mm2 with 30 mg and 60 mgof study of crotalic envenomation in cattle treated with venom of the venom compared to the control group (PBS alone), but there was no South American rattlesnake Crotalus durissus terrificus. difference in the responses among venom doses. Although all three Philodryas olfersii venom caused leukopenia that was greatest venom doses increased the number of mononuclear leucocytes/ with 30 mg of venom. Leukopenia is generally defined as a global mm2, this increase was not significantly different from the control decrease in the number of white blood cells and is most often (PBS) group, nor was there any difference in the responses among caused by a reduction in the number of neutrophils (the most venom doses (Fig. 5). common type of leukocyte) followed by a reduction in lymphocytes Author's Personal Copy

A B C *** 300 20 *** ** ** 5000 *** 15 200 4000 10 3000 100 2000 KC (pg/mL)

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Fig. 2. Plasma concentrations of (A) IGF-1, (B) KC, (C) MIP 2, (D) IFN-g, (E) TNFa, (F) IL-1b, (G) IL-6, (H) IL-10, (I) IL-13 and (J) IL-17 in BALB/c mice injected with P. olfersii venom. The mice were injected in the left gastrocnemius muscle with venom (10, 30 or 60 mgin50ml of PBS) or the same volume of PBS alone (control mice) and 6 h later they were anesthetized for blood sampling from the inferior vena cava (blood collected into EDTA), followed by exsanguination. Cytokines and other mediators were quantiﬁed by ELISA using commercial kits. The columns represent the mean ± SD (n ¼ 10/group). **p < 0.01 and ***p < 0.005 for the comparisons indicated (one-way ANOVA followed by the Newman-Keuls multiple comparisons test). The ends of each horizontal bar indicate the two columns being compared in each case. Author's Personal Copy

14 J.S. Oliveira et al. / Toxicon 132 (2017) 9e17

Fig. 3. Histological analysis of mouse gastrocnemius muscle injected with P. olfersii venom (10, 30 or 60 mgin50ml of PBS) or PBS alone (control). Male Balb/c mice were injected with venom or PBS in the left gastrocnemius muscle and 6 h later the animals were killed with an overdose of anesthetic and exsanguinated. The muscle was removed and processed for histological analysis as described in section 2.5. Asterisks e edema, triangles e inflammatory infiltrate, and arrows e muscle fiber degeneration. H-E staining. Scale bars: 20 mm in all panels.

(the second most common type of leukocyte) (Stock and Hoffman, values. This increase in basophil number appeared to correlate with 2000). However, as shown here, the significant decrease in leuko- the significant increase in inflammatory infiltrate assessed semi- cytes apparently resulted from a decrease in the number of lym- quantitatively in the gastrocnemius muscle of mice inoculated phocytes rather than neutrophils since the latter showed a with this same dose of venom. Basophils release mediators capable significant increase in number with this dose of venom (30 mg). of enhancing vascular permeability and inducing the migration of Acosta de Perez et al. (2003) also reported a neutrophilic infiltrate inflammatory cells (neutrophils and macrophages) (Cruvinel et al., in mouse gastrocnemius muscle injected with 40 mgofP. olfersii 2010). However, the precise relationship between the increase in venom from Argentina. Our findings contrast with those for venom circulating basophil numbers and the increase in inflammatory of the pitviper B. jararaca that caused an increase in the total infiltrate in envenomed muscle remains to be established. The leukocyte count 3 and 6 h after venom administration i.v. number of circulating neutrophils generally increases 1e6 h after (Yamashita, 2013). injury (Leech, 1997). In agreement with this, 6 h after P. olfersii There was a significant increase in the number of circulating venom administration there was a predominance of neutrophils in basophils in mice injected with 10 mg of venom, but not with the the inflammatory infiltrate present in gastrocnemius muscle other doses, the counts for which were similar to basal (control) injected with different doses of P. olfersii venom, as well as an

A Edema B Inflammatory infiltrate C Muscle degeneration

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Fig. 4. Local tissue responses in mouse gastrocnemius muscle injected with P. olfersii venom in mice: (A) edema, (B) inﬂammatory inﬁltrate and (C) myonecrosis. Mice were injected with venom (10, 30 or 60 mgin50ml of PBS) or PBS alone (control) in the left gastrocnemius muscle and 6 h later the animals were killed with an overdose of anesthetic and exsanguination. The muscle was removed and processed for histological analysis as described in section 2.5. Semi-quantitative analysis was done using an arbitrary scoring system, as follows: 0 e no alteration or damage, 1 e slight alteration or damage, 2 e moderate alteration or damage, 3 e intense alteration or damage, and 4 e very intense alteration or damage. The results are expressed as box-plots showing the median, interquartils and range (n ¼ 5 mice/group). *p < 0.05 and **p < 0.01 for the comparisons indicated (one-way ANOVA followed by the Newman-Keuls multiple comparisons test). The ends of each horizontal bar indicate the two columns being compared in each case. Author's Personal Copy

J.S. Oliveira et al. / Toxicon 132 (2017) 9e17 15

A B 2 2 20 150 * 15 100 * 10

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Fig. 5. Analysis of inﬂammatory cell inﬁltrate (A e polymorphonuclear cells and B e mononuclear cells) in mouse gastrocnemius muscle injected with P. olfersii venom. Mice were injected with venom (10, 30 or 60 mgin50ml of PBS) or PBS alone (control) in the left gastrocnemius muscle and 6 h later the animals were killed with an overdose of anesthetic and exsanguination. The muscle was removed and processed for histological analysis as described in section 2.5. The columns represent the mean ± SD (n ¼ 10/group). *p < 0.05 for the comparisons indicated (one-way ANOVA followed by the Newman-Keuls multiple comparisons test). The ends of each horizontal bar indicate the two columns being compared in each case.

increase in the number of circulating neutrophils, with the greatest of envenomation by P. olfersii in mice. However, local production of increase seen with 30 mg of venom. Graça et al. (2008) reported a at least some of these cytokines could be involved in muscle moderate increase in the total leukocyte count in nine out of ten damage since in mouse phrenic nerve-diaphragm and chick cattle injected with C. d. terrificus venom; this increase was char- biventer cervicis preparations in vitro P. olfersii venom enhances the acterized by neutrophilia, relative lymphopenia, eosinopenia and expression of IFN-g and TNF-a, as assessed immunohistochemically monocytosis, with their findings for lymphopenia and monocytosis (Collaço et al., 2012). being similar to those observed here for P. olfersii. Growth factors such as IGF-1 act as positive regulators in the Cytokines are important mediators in the inflammatory control and activation of satellite cells located between the basal response to snake venoms (Petricevich et al., 2000; Petricevich, lamina and plasma membrane of the muscle fiber and are indis- 2004; Cruz et al., 2008). In this regard, the increase in the num- pensable for muscle regeneration. Takahashi et al. (2003) demon- ber of circulating neutrophils seen with 30 mg of venom was strated that IGF-1 promoted muscle regeneration after transfer by possibly related to the greater circulating concentration of KC seen electroporation in experimentally injured mouse muscle. Lopes with this dose of venom relative to that in mice treated with 10 mg (2008) observed that from the sixth hour after the inoculation of and 60 mg of venom, although the levels of KC were not significantly P. patagoniensis venom onwards there was regression of the myo- different from the control group (injected with PBS). KC belongs to necrotic lesions and the initiation of muscle fiber regeneration, the CXC class of chemokines and is selective for the recruitment of such that 24 h later normal fibers were surrounded by an inflam- polymorphonuclear leukocytes. Wengner et al. (2008) demon- matory infiltrate. As shown here, there was a very marked increase strated that KC participates in neutrophil chemotaxis by activating in the circulating IGF-1 concentration in mice 6 h after the injection the CXCR2 receptor expressed in these cells, leading to an increase of 60 mgofP. olfersii venom, perhaps in response to the extensive in the number of this cell type in the peripheral circulation. This muscle degeneration seen with this dose. mechanism could explain the increase in circulating neutrophils in Philodryas olfersii venom caused edema, an inflammatory infil- mice injected with 30 mg of venom. trate and myonecrosis but no hemorrhage after 6 h. The absence of Petricevich et al. (2000) observed an increase in the serum hemorrhage at 6 h post-venom may reflect a previous finding that levels of TNF-a, IL-1b, IL-6, IL-10 and IFN-g in mice injected intra- the hemorrhagic response to P. olfersii venom peaks at 2e4 h after peritoneally with the mean lethal dose (LD50)ofBothropsasper and injection (Rocha and Furtado, 2007). All venom doses stimulated B. jararaca venoms. Similarly, Cruz et al. (2008) reported an increase the migration of polymorphonuclear and mononuclear cells into in the serum levels of TNF-a, IL-10 and IL-6 15e30 min after the gastrocnemius muscle, but with no clear dose-dependence. An administration of one LD50 of C. d. terrificus venom, with peak inflammatory infiltrate has also been observed with P. olfersii concentrations occurring ~2 h after envenomation, followed by a venom from Argentina (Acosta et al., 2003) and for the venoms of decrease thereafter; there were no significant changes in the serum P. baroni (Sanchez et al., 2014) and P. patagoniensis (Acosta et al., concentration of IFN-g. Overall, pro-inflammatory cytokines pre- 2003; Peichoto et al., 2004), as well as purified proteins such as dominated in the early phases of envenomation, whereas anti- the metalloproteinase patagonfibrase (Peichoto et al., 2007, 2011) inflammatory cytokines predominated in the later stages (Cruz and the CRISP patagonin (Peichoto et al., 2009). et al., 2008). In contrast to these findings, 6 h after the injection The discrepancy between the apparently greater inflammatory of all three doses of P. olfersii venom there were no significant infiltrate observed with 10 mg of venom compared to the other two changes in the circulating concentrations of IL-1b, IL-6, IL-10, IL-13, doses and the lack of significant differences among the doses with IL -17, IFN-g and TNF-a, although there was a trend towards an regard to the number of polymorphonuclear and mononuclear increase in the levels of IL-6, IL-1b, IL-10 and especially TNF-a. This leukocytes in the inflammatory infiltrate probably reflects differ- finding indicates that the release of cytokines from the site of ences in the methodological approaches used for the quantitative venom injection into the general circulation is not a major feature and semi-quantitative assessments, with the semi-quantitative Author's Personal Copy

16 J.S. Oliveira et al. / Toxicon 132 (2017) 9e17 analysis providing a more comprehensive assessment of the muscle Transparency document tissue. It is also possible that the photomicrographs used for the quantitative analysis of mice injected with 10 mg of venom were Transparency document related to this article can be found obtained from areas with a lower inflammatory infiltrate that was online at http://dx.doi.org/10.1016/j.toxicon.2017.03.013. not representative of the true response for this venom dose. Myonecrosis is a common finding in experimental studies with References Philodryas venoms in vitro (Prado-Franceschi et al., 1996, 1998; Carreiro da Costa et al., 2008; Collaço et al., 2012) and in vivo Acosta, O., Leiva, L.C., Peichoto, M.E., Marunak,~ S., Teibler, P., Rey, L., 2003. Hemor- (Acosta et al., 2003; Acosta de Perez et al., 2003; Peichoto et al., rhagic activity of the Duvernoy's gland secretion of the xenodontine colubrid Philodryas patagoniensis from the north-east region of Argentina. Toxicon 41, 2004, 2007; Lopes, 2008; Sanchez et al., 2014). As shown here, 1007e1012. myonecrosis was present 6 h after the inoculation of the three Acosta de Perez, O., Leiva de Vila, L., Peichoto, M.E., Marunak,~ S., Ruíz, R., Teibler, P., doses of P. olfersii venom and similar activity was reported for Gay, C., Rey, L., 2003. Edematogenic and myotoxic activities of the Duvernoy's gland secretion of Philodryas olfersii from the north-east region of Argentina. P. olfersii venom from Argentina (Acosta et al., 2003). Although we Biocell 27, 363e370. did not investigate the time-course of myonecrosis, Lopes (2008) Assakura, M.T., Salomao,~ M.G., Puorto, G., Mandelbaum, F.R., 1992. Hemorrhagic, noted that myonecrosis started within 30 min after the injection fibrinogenolytic and edema-forming activities of the venom of the colubrid snake Philodryas olfersii (green snake). Toxicon 30, 427e438. of 10 mgofP. patagoniensis venom. The venom components Assakura, M.T., Reichl, A.P., Mandelbaum, F.R., 1994. Isolation and characterization responsible for myonecrosis remain poorly studied, although a of five fibrin(ogen)olytic enzymes from the venom of Philodryas olfersii (green myotoxin has been identified in P. olfersii venom (Prado-Franceschi snake). Toxicon 32, 819e831. Carreiro da Costa, R.S., Prudencio,^ L., Ferrari, E.F., Souza, G.H., de Mello, S.M., Prianti et al., 1998). In addition, a CRISP (patagonin) from P. patagoniensis Júnior, A.C., Ribeiro, W., Zamuner, S.R., Hyslop, S., Cogo, J.C., 2008. Neuromus- venom causes myonecrosis but no edema or hemorrhage (Peichoto cular action of venom from the South American colubrid snake Philodryas et al., 2009). Venom metalloproteinases may also contribute to the patagoniensis. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 148, 31e38. Ching, A.T.C., Rocha, M.M.T., Paes Leme, A.F., Pimenta, D.C., Furtado, M.F.D., myonecrosis since Philodryas venoms have consistently been Serrano, S.M.T., Ho, P.L., Junqueira-de-Azevedo, I.L.M., 2006. Some aspects of the shown to be very proteolytic when compared to Bothrops (lance- venom proteome of the Colubridae snake Philodryas olfersii revealed from a head) snake venoms (Assakura et al., 1992; Acosta et al., 2003; Duvernoy's (venom) gland transcriptome. FEBS Lett. 580, 4417e4422. Rocha et al., 2006; Carreiro da Costa et al., 2008; Sanchez et al., Collaço, R.C.O., Cogo, J.C., Rodrigues-Simioni, L., Rocha, T., Oshima-Franco, Y., Ran- dazzo-Moura, P., 2012. Protection by Mikania laevigata (guaco) extract against 2014). PLA2 is not involved since these venoms are generally the toxicity of Philodryas olfersii snake venom. Toxicon 60, 614e622. devoid of this enzyme (Assakura et al., 1992; Peichoto et al., 2004, Correia, J.M., Santana Neto, P.L., Pinho, M.S.S., da Silva, J.A., Amorim, M.L.P., 2012; Ching et al., 2006). Escobar, J.A.C., 2010. Poisoning due to Philodryas olfersii (Lichtenstein, 1823) attended at Restauraçao~ Hospital in Recife, state of Pernambuco, Brazil: case In conclusion, the results described here indicate that P. olfersii report. Rev. Soc. Bras. Med. Trop. 43, 336e338. venom (10e60 mg) does not affect erythrocyte parameters or the Cruvinel, W.M., Mesquita Júnior, D., Araújo, J.A.P., Catelan, T.T.T., de Souza, A.W.S., da serum levels of pro- and anti-inflammatory interleukins, but in- Silva, N.P., Andrade, L.E.C., 2010. Immune system e Part I. Fundamentals of fl innate immunity with emphasis on the molecular and cellular mechanisms of creases the serum levels of IGF-1. The venom-induced in amma- inflammatory response. Rev. Bras. Reumatol. 50, 434e461. tory response was characterized by edema and a cellular response Cruz, A.H., Garcia-Jimenez, S., Mendonça, R.Z., Petricevich, V.L., 2008. Pro- and anti- involving leukopenia and lymphopenia, with neutrophilia, mono- inflammatory cytokines release in mice injected with Crotalus durissus terrificus venom. Med. Inflamm. 2008, 874962. http://dx.doi.org/10.1155/2008/874962. cytosis, basophilia and thrombocytopenia. Myonecrosis without Ferlan, I., Ferlan, A., King, T., Russell, F.E., 1983. Preliminary studies on the venom of hemorrhage was seen 6 h after venom injection. These various al- the colubrid snake Rhabdophis subminatus (red-necked keelback). Toxicon 21, terations are probably mediated by metalloproteinases, serine 570e574. Fowler, I.R., Salomao,~ M.G., 1994. Activity patterns in the colubrid snake genus proteinases, CRISPs and C-type lectins present in the venom. Philodryas and their relationship to reproduction and snakebite. Bull. Chic. Herp. Soc. 29, 229e232. ~ Ethical statement Gonçalves, C.T.R., Gonçalves, C.G.R., Almeida, F.M., Lopes, F.D., Durao, A.C.S., Santos, F.A., Silva, L.F., Marcourakis, T., Castro Faria Neto, H.C., Vieira, R.P., Dolhnikoff, M., 2012. Protective effects of aerobic exercise on acute lung injury 1) This material has not been published in whole or in part induced by LPS in mice. Crit. Care 16, R199. elsewhere. Graça, F.A.S., Peixoto, P.V., Coelho, C.D., Caldas, S.A., Tokarnia, C.H., 2008. Aspectos clínico-patologicos e laboratoriais do envenenamento crotalico experimental 2) The manuscript is not currently being considered for publi- em bovinos. Pesq. Vet. Bras. 28, 261e270. cation in another journal. Küch, U., Jesberger, U., 1993. Human envenomation from the bite of the South 3) All authors have been personally and actively involved in American colubrid snake species Philodryas baroni (Berg, 1895). Snake 25, 63e65. substantive work leading to the manuscript, and will hold them- Leech, S.J., 1997. Review of muscle healing. J. Physiol. X, 15e18. selves jointly and individually responsible for its content. Lopes, P.H., 2008. Local Changes Induced by the Toxic Secretion of Philodryas patagoniensis (Girad, 1857) (Serpentes: Colubridae). PhD thesis (in Portuguese). University of Sao~ Paulo, Sao~ Paulo, Brazil. Available from: http://www.teses.usp. Conflict of interest br/teses/disponiveis/41/41135/tde-06082008-185402/publico/ PriscilaHessLopes.pdf. fl Means, D.B., 2010. Ophidism by the green palmsnake. Wild. Environ. Med. 21, The authors have no con ict of interest with the publication of 46e49. this work. Medeiros, C.R., Hess, P.L., Nicoleti, A.F., Sueiro, L.R., Duarte, M.R., Almeida- Santos, S.M., França, F.O.S., 2010. Bites by the colubrid snake Philodryas patagoniensis: a clinical and epidemiological study of 297 cases. Toxicon 56, Acknowledgments 1018e1024. Neira, P.O., Jofre, L.M., Oschilewski, D.L., Subercaseaux, B.S., Munoz,~ N.S., 2007. JSO was supported by a studentship from Coordenaçao~ de Mordedura por Philodryas chamissonis. Presentacíon de un caso y revision de la literatura. Rev. Chil. Infectol. 24, 236e241. Aperfeiçoamento de Pessoal de Nível Superior (CAPES, grant no. Peichoto, M.E., 2007. Caracterizacion y aislameinto de proteínas del veneno de 33051011009-PO). Philodryas patagoniensis que habita la region nordeste de Argentina. PhD thesis. Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina, p. 173. Appendix A. Supplementary data Peichoto, M.E., Acosta, O., Leiva, L., Teibler, P., Marunak,~ S., Ruíz, R., 2004. Muscle and skin necrotizing and edema-forming activities of Duvernoy's gland secretion of the xenodontine colubrid snake Philodryas patagoniensis from the north-east of Supplementary data related to this article can be found at http:// Argentina. Toxicon 44, 589e596. dx.doi.org/10.1016/j.toxicon.2017.03.013. Peichoto, M.E., Leiva, L.C., Guiamas Moya, L.E., Rey, L., Acosta, O., 2005. Duvernoy's Author's Personal Copy

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