Toxicon 74 (2013) 179–192 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Acute Lonomia obliqua caterpillar envenomation-induced physiopathological alterations in rats: Evidence of new toxic venom activities and the efficacy of serum therapy to counteract systemic tissue damage Markus Berger a, Walter Orlando Beys-da-Silva b, Lucélia Santi b, Iuri Marques de Oliveira c, Patrícia Mendes Jorge c, João Antônio Pêgas Henriques c, David Driemeier d, Maria Aparecida Ribeiro Vieira e, Jorge Almeida Guimarães a,* a Laboratório de Bioquímica Farmacológica, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Bento Gonçalves, 9500, Cep 91501-970, Porto Alegre, RS, Brazil b Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, United States c Departamento de Biofísica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil d Departamento de Patologia Clínica Veterinária, Faculdade de Medicina Veterinária, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil e Laboratório de Fisiologia Renal, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil article info abstract Article history: The clinical manifestations of Lonomia obliqua caterpillar envenomation are systemic Received 4 May 2013 hemorrhage and acute kidney injury. In an effort to better understand the physiopatho- Received in revised form 30 July 2013 logical mechanisms of envenomation, a rat model was established to study systemic tissue Accepted 13 August 2013 damage during L. obliqua envenomation. An array of acute venom effects was characterized, Available online 29 August 2013 including biochemical, hematological, histopathological, myotoxic and genotoxic alter- ations. Rapid increases in serum alanine and aspartate transaminases, g-glutamyl trans- Keywords: ferase, lactate dehydrogenase, hemoglobin, bilirubin, creatinine, urea and uric acid were Lonomia Caterpillar observed, indicating that intravascular hemolysis and liver and kidney damage had occurred. fi Venom Treatment with a speci c antivenom (antilonomic serum) for up to 2 h post-venom injection Hemorrhagic syndrome neutralized the biochemical alterations. However, treatment after 6 h post-venom injection Genotoxicity failed to normalize all biochemical parameters, despite its efficacy in reversing coagulation Nephrotoxicity dysfunction. The hematological findings were consistent with hemolytic anemia and neutrophilic leukocytosis. The histopathological alterations were mainly related to hemor- rhage and inflammation in the subcutaneous tissue, lung, heart and kidneys. Signs of congestion and hemosiderosis were evident in the spleen, and hemoglobin and/or myoglobin casts were also detected in the renal tubules. Increased levels of creatine kinase and creatine kinase-MB were correlated with the myocardial necrosis observed in vivo and confirmed the myotoxicity detected in vitro in isolated extensor digitorum longus muscles. Significant DNA damage was observed in the kidneys, heart, lung, liver and lymphocytes. The majority of the DNA lesions in the kidney were due to oxidative damage. The results pre- sented here will aid in understanding the pathology underlying Lonomia’s envenomation. Ó 2013 Elsevier Ltd. All rights reserved. * Corresponding author. Tel.: þ55 51 33086068; fax: þ55 51 33087309. E-mail address: [email protected] (J.A. Guimarães). 0041-0101/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxicon.2013.08.061 180 M. Berger et al. / Toxicon 74 (2013) 179–192 1. Introduction knowledge about the mechanisms involved in kidney damage and its management (Gamborgi et al., 2006). Contact dermatitis and urticarial cutaneous reactions Recently, molecular biology and proteomic studies have are well known signs of accidental contact with the hairs contributed to the increasing number of toxins that have and spines of many lepidopterous larvae (Hossler, 2010). been identified in L. obliqua venomous secretions, The consequences of these reactions are usually limited to providing valuable information regarding how this toxin local skin inflammation without any systemic tissue dam- cocktail acts on biological tissues (Veiga et al., 2005; Ricci- age. However, contact with Lonomia spp. has been associ- Silva et al., 2008). Toxins related to envenomation symp- ated with potentially fatal systemic disorders, such as tomatology, especially those that cause hemostatic distur- hemorrhage and acute kidney injury (AKI) (Arocha- bances, such as serine proteases, phospholipases A2, lectins Piñango et al., 2000; Pinto et al., 2010). One of these spe- and protease inhibitors, were identified. These toxins are cies is the moth Lonomia obliqua (Lepidoptera, Saturniidae), able to directly modulate the victim’s hemostatic system by which is highly venomous in the larval stages. Larval forms proteolytic activation of the coagulation and fibrinolytic occur during spring and summer in the southern regions of cascades, generating high concentrations of intravascular Brazil (mainly in the states of Rio Grande do Sul, Santa thrombin, plasmin, urokinase and kallikrein (Reis et al., Catarina and Paraná) where envenomation by this animal is 2006; Pinto et al., 2008; Berger et al., 2010a). As a conse- an important public health problem due to its high inci- quence, consumption coagulopathy with decreased levels dence (Veiga et al., 2009; Pinto et al., 2010; Guimarães, of fibrinogen, factors V and XIII, pre-kallikrein, plasmin- 2011). In fact, this caterpillar is responsible for severe and ogen, protein C and a2-antiplasmin occurs (Zannin et al., sometimes fatal accidents caused by skin contact with the 2003). Platelet aggregation function is also markedly bristles that cover the animal’s body. Unlike snakes, spiders impaired during envenomation, which contributes signifi- and scorpions, there is no specialized venomous gland in L. cantly to the bleeding disorders (Berger et al., 2010a,b). obliqua. The venom is produced by secretory epithelial cells Moreover, the venom also triggers an acute inflammatory of the tegument and stored in a hollow internal channel in response and disturbances in the vascular system, inducing each bristle. Because the bristles have weak articulations at increases in blood–brain barrier permeability, hypotension their tips, only a slight contact with the skin is enough to and the nociceptive and edematogenic responses (Da Silva break off these chitinous structures, injecting the venom et al., 2004a; De Castro Bastos et al., 2004; Bohrer et al., into the subcutaneous tissue of victims (Veiga et al., 2001). 2007; Nascimento-Silva et al., 2012). Furthermore, accidents frequently involve colonies of Despite understanding the mechanisms involved in the dozens or hundreds of caterpillars that are camouflaged at hemorrhagic syndrome, little is known about the systemic tree trunks, which makes accidental contact more physiopathological effects induced by L. obliqua venom. dangerous due to the venom quantities absorbed by the Although venom components have been detected in several victim. organs (including the kidneys, lungs, liver, spleen, heart and Clinical symptoms include local pain (burning sensa- skeletal muscle) of rats following a single subcutaneous tion) and an inflammatory reaction, which starts immedi- injection of the venom, the systemic tissue damage in these ately after contact, followed by systemic reactions, organs remains poorly characterized (Rocha-Campos et al., including headache, fever, vomiting and hypotension. Signs 2001; Da Silva et al., 2004b). For example, the current level of bleeding diathesis, characterized by hematomas, ecchy- of knowledge regarding the kidney damage is based only on mosis, gross hematuria, hematemesis and melena are a few clinical case reports in which hematuria and high frequently observed between 6 and 72 h after contact. If the levels of serum creatinine are described as the main features victim is not promptly treated, the clinical profile can of L. obliqua-induced AKI (Burdmann et al., 1996). The evolve to intracerebral hemorrhage, AKI and death (Zannin venom-induced pathology in other organs remains et al., 2003; Kowacs et al., 2006; Garcia and Danni-Oliveira, completely unknown. In human patients, the impossibility 2007). Actually, the unique specific treatment available for of conducting early tissue biopsies, due to the coagulation L. obliqua envenomation is the early intravenous adminis- disturbances inherent to the envenomation, has made it tration of anti-lonomic serum (ALS), an animal-derived difficult to analyze the acute anatomopathological alter- antivenom. ALS is a concentrated pool of immunoglobu- ations. For these reasons, we believe that animal models of 0 lins (usually pepsin-refined F(ab )2 fragments of whole IgG) envenomation may be useful not only to characterize the that is purified from the plasma of a horse that has been underlying physiopathology but also to identify previously immunized with the venom (obtained from bristle ho- unknown toxic activities of the venom. Therefore, the aim of mogenates) (Rocha-Campos et al., 2001). In Brazil, ALS is the present work was to develop a rat model to study sys- produced by the Butantan Institute (São Paulo) and has temic tissue damage during L. obliqua envenomation. An been successfully used to re-establish physiological