Tityus serrulatus venom – A lethal cocktail

Accepted Manuscript Tityus serrulatus venom – a lethal cocktail Manuela Berto Pucca, Felipe Augusto Cerni, Ernesto Lopes, Pinheiro Junior, Karla de Castro Figueiredo Bordon, Fernanda Gobbi Amorim, Francielle Almeida Cordeiro, Heloisa Tavoni Longhim, Caroline Marroni Cremonez, Guilherme Honda de Oliveira, Eliane Candiani Arantes PII:

S0041-0101(15)30118-5

DOI:

10.1016/j.toxicon.2015.10.015

Reference:

TOXCON 5230

To appear in:

Toxicon

Received Date: 21 August 2015 Accepted Date: 27 October 2015

Please cite this article as: Pucca, M.B., Cerni, F.A., Lopes, E., Junior, P., Bordon, K.d.C.F., Amorim, F.G., Cordeiro, F.A., Longhim, H.T., Cremonez, C.M., de Oliveira, G.H., Arantes, E.C., Tityus serrulatus venom – a lethal cocktail, Toxicon (2015), doi: 10.1016/j.toxicon.2015.10.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Tityus serrulatus venom – a lethal cocktail

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Manuela Berto Pucca, Felipe Augusto Cerni, Ernesto Lopes Pinheiro Junior, Karla de Castro

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Figueiredo Bordon, Fernanda Gobbi Amorim, Francielle Almeida Cordeiro, Heloisa Tavoni

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Longhim, Caroline Marroni Cremonez, Guilherme Honda de Oliveira, Eliane Candiani

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Arantes*

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Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto,

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University of São Paulo, Av. do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil.

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*Corresponding author: Eliane Candiani Arantes, Department of Physics and Chemistry,

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School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. Do Café,

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s/n, 14040-903, Ribeirão Preto, SP, Brazil.

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Tel.: +55 16 3315-4193; Fax: +55 16 3315-4880

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E-mail address: [email protected] (E.C. Arantes).

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Keywords: Tityus serrulatus; anatomical systems; Brazilian scorpion; envenoming; scorpion

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venom; neurotoxins.

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ACCEPTED MANUSCRIPT Abstract

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Tityus serrulatus (Ts) is the main scorpion species of medical importance in Brazil. Ts venom

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is composed of several compounds such as mucus, inorganic salts, lipids, amines,

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nucleotides, enzymes, kallikrein inhibitor, natriuretic peptide, proteins with high molecular

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mass, peptides, free amino acids and neurotoxins. Neurotoxins are considered the most

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responsible for the envenoming syndrome due to their pharmacological action on ion

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channels such as voltage-gated sodium (Nav) and potassium (Kv) channels. The major goal

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of this review is to present important advances in Ts envenoming research, correlating both

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the crude Ts venom and isolated toxins with alterations observed in all human systems. The

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most remarkable event lies in the Ts induced massive releasing of neurotransmitters

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influencing, directly or indirectly, the entire body. Ts venom proved to extremely affect

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nervous and muscular systems, to modulate the immune system, to induce cardiac disorders,

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to cause pulmonary edema, to decrease urinary flow and to alter endocrine, exocrine,

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reproductive, integumentary, skeletal and digestive functions. Therefore, Ts venom possesses

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toxins affecting all anatomic systems, making it a lethal cocktail. However, its low lethality

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may be due to the low venom mass injected, to the different venom compositions, the body

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characteristics and health conditions of the victim and the local of Ts sting. Furthermore, we

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also described the different treatments employed during envenoming cases. In particular,

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throughout the review, an effort will be made to provide information from an extensive

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documented studies concerning Ts in vitro, in animals and in humans (a total of 151

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references).

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ACCEPTED MANUSCRIPT Over the past three decades (since 1981), our research team has undertaken several

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studies involving the isolation, purification and characterization of toxins from the Brazilian

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yellow scorpion Tityus serrulatus (Ts). The present review deals with Ts venom and toxins

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studies and focuses on their effects on all anatomic systems.

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1. T. serrulatus: biology and epidemiology

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Currently, there are approximately 2,069 scorpions species recorded in the world,

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subdivided in 197 genera and 15 families (Di et al., 2014). The Buthidae family presents

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about 34 species considered potentially dangerous to humans, including species from Tityus

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genus (Chippaux and Goyffon, 2008). In Brazil, the Tityus serrulatus (Ts) species (Fig. 1) is

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the main reported. Originally first described in Brazil (Lutz and Mello, 1922), Ts is nowadays

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considered the most dangerous scorpion species with a wide distribution in the country

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(Pucca et al., 2015b)

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The Ts scorpion prefers tropical weather, although it can be found in cerrado and

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urban areas (Cruz et al., 1995, Lourenço, 2008). The ability to live in cities is explained by

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the high concentration of debris and garbage, which can increase the proliferation of insects

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used for feeding (Pucca et al., 2015b) .

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The Brazilian Ts scorpion measures approximately 6-7 cm (adult) and presents a

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yellowish brown body with yellow pedipalps and legs. This explains why the species is also

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known as yellow scorpion (Lutz and Mello, 1922).

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Anatomically, the body of the Ts scorpion is segmented in prosoma (cephalothorax),

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mesosoma (abdomen) and metasoma (tail). The segments 3 and 4 from metasoma have small

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protrusions on the dorsal face like a serration (this is the reason why the species is named

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“serrulatus”). The last segment of metasoma connects the telson, responsible for injecting the

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venom into the prey or predator. Within this structure, there are a pair of venom production

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glands (Marcussi et al., 2011). The reproduction of Ts is performed using parthenogenesis, in which the embryo is

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developed from an unfertilized ovum (Lourenço, 2008, Lourenço, 2002). Using this type of

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reproduction, it is possible for around 20 new individuals to originate in each offspring

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(Marcussi et al., 2011). Although it is an asexual process, there are reports showing the

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male’s existence (Lourenço and Cloudsley-Thompson, 1999, Dos Santos et al., 2014).

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The parthenogenesis reproduction combined with the easy adaptability to urban areas

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contributed to the increasing number of species around the country and explained the reason

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why Ts is responsible for the highest number of accidents in different regions of Brazil

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(Chippaux and Goyffon, 2008). Data from SINAN (Sistema de Informação de Agravos de

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Notificação), a federal agency

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accidents with venomous animals in Brazil, shows that the number of scorpion accidents

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reported in the last eight years has increased (Fig. 2). In 2013 and 2014, the number of

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scorpion accidents was superior to the total of accidents caused by bees, snakes and spiders

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combined.

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2. T. serrulatus venom and envenoming: target systems

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responsible for collecting the information regarding the

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Ts venom is composed of several compounds such as mucus, inorganic salts, lipids,

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amines, nucleotides, enzymes (such as hyaluronidase, serinoprotease and metalloproteinase),

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kallikrein inhibitor, natriuretic peptide, proteins with high molecular mass, peptides

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(disulfide-bridged and non-disulfide-bridged), free amino acids and neurotoxins (Fig. 3)

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(Verano-Braga et al., 2008, Pessini et al., 2001, Ferreira et al., 1993, Cologna et al., 2009,

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Alvarenga et al., 2012, Carmo et al., 2014, Horta et al., 2014, Bordon et al., 2015, Ortiz et al.,

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2015, Carmo, 2015). However, neurotoxins are considered the main responsible for the

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envenoming syndrome as well as the most studied (Cologna et al., 2009). Based on all these components, it is not surprising that Ts envenoming may present

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several distinct signals and symptoms. In all the reported cases, Ts sting causes local pain

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with variable intensity (mild envenoming / Stage Ia). Additionally, the local pain can be

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accompanied by nausea, sweating, tachycardia, fever, and stirring (mild envenoming / Stage

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Ib). The moderate envenoming (Stage II) is characterized by sweating, epigastric pain,

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pulmonary obstruction, cramps, vomiting, hypotension, diarrhea, bradycardia and dyspnea.

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The severe envenoming (Stage III), which generally affects the children and elderly, can

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present several complications, such as cardio-respiratory failure, which may be lethal

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(Chippaux and Goyffon, 2008, Cupo et al., 1994, Venancio et al., 2013).

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Indeed, different human systems are known to be affected by Ts venom components.

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In this research, we review and discuss the clinical/laboratorial manifestations and

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pathophysiology of Ts envenoming on the different anatomical systems: nervous, muscular,

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immune,

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integumentary, skeletal and reproductive systems.

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3. Nervous and Muscular Systems: massive release of neurotransmitters

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and muscular stimulation

respiratory,

urinary,

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and

exocrine,

digestive,

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cardiovascular,

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Since most of Ts venom components identified are neurotoxins, most of the studies

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found in the literature are focused on neurotoxic effects, which are mainly triggered by direct

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modulation of voltage-gated channels by these toxins. Additionally, any over stimuli and/or

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modulation of the nervous system can indirectly affect the muscles. In this way, the effects of

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Ts venom on nervous and muscular systems will be discussed together.

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The central nervous system comprises the brain and spinal cord; whereas the

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peripheral nervous system encompasses the nerve fibers, which are disseminated throughout

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the body (Guyton and Hall, 2012). Overall, Ts neurotoxins interact with presynaptic and postsynaptic ion channels. As a

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result, the symptomatology of Ts envenoming in the nervous system is a result of primary

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effects of neurotoxins on these ion channels, causing a massive release of neurotransmitters

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(catecholamines, acetylcholine, among others), leading to the stimulation of the autonomic

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nervous system (Vasconcelos et al., 2005). Excitation of the autonomic nervous system is

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characterized by both parasympathetic and sympathetic responses. On the other hand, most of

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the parasympathetic effects tend to occur early. Sympathetic effects persist due to the release

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of catecholamines. Catecholamines play a major role in the peripheral neurotransmission

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and, once released, binds to one of two classes of receptors in the postsynaptic membrane

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including muscarinic receptors (mAChR), the G protein-coupled receptors or nicotinic

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receptors (nAChR) and ligand-gated ion channels (Galanter et al., 2012). In the

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neuromuscular junction, acetylcholine (Ach) is employed by somatic motor neurons to

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depolarize striated muscles. In the autonomic nervous system, Ach is the neurotransmitter

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released and used by all preganglionic neurons and by parasympathetic postganglionic

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neurons (Galanter et al., 2012).

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Sympathetic and parasympathetic nervous systems regulate involuntary responses of

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smooth muscle and glandular tissue. They control the cardiac rhythm (strength and rate), the

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vascular tone, sweating, salivation, piloerection (goosebumps), pupillary constriction, uterine

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contraction, gastrointestinal motility, and bladder function (Galanter et al., 2012).

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Sympathetic/adrenergic effects caused by catecholamines include hypertension, tachycardia,

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hyperglycemia, mydriasis, hyperthermia, agitation, restlessness and severe cardiomyopathic

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effects, while parasympathetic/cholinergic effects caused by Ach can induce lachrymation,

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priapism, and increased respiratory secretions. In severe envenoming, hypertension is often

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followed by hypotension, as well as tachycardia that is followed by bradycardia, depending

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on whether cholinergic or adrenergic effects predominate (Isbister and Bawaskar, 2014). In

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this way, the direct effects of Ts venom on the nervous system indirectly interfere in all

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anatomic systems, which will be discussed later on.

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So far, different neurotoxins from Ts venom active on voltage-gated sodium (Nav)

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and potassium (Kv) channels have been described and characterized. Through their

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discovery, these toxins received different names, which often difficult their identification.

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Here, the nomenclature suggested by Cologna et al. (2009) will be used. Specific toxins to

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Nav channels are considered long-chain peptides and can be categorized into two classes: α-

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and β-scorpion neurotoxins. Classified in these groups, Ts venom presents the toxins: Ts1,

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Ts2, Ts3, Ts4, Ts5, Ts17 and Ts18 (Bordon et al., 2015). Ts1, the major one (16 % of the

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crude soluble venom), contributes significantly to the venom toxicity (Vasconcelos et al.,

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2005). It is also the most-studied toxin from Ts venom: Ts1 influences the activation kinetics

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of Nav1.2, Nav1.4 and Nav1.6, considerably shifting the midpoint of activation of these

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channels towards more negative potentials, causing their opening at resting potentials. It also

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inhibited the sodium peak current through Nav1.5 channel without changes in the properties

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of gating (Peigneur et al., 2015). Ts2 showed to be able to inhibit the inactivation of Nav1.2,

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Nav1.3, Nav1.5, Nav1.6, and Nav1.7 (Cologna et al., 2012). Ts3 induces the release of

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catecholamines, Ach, nitric oxide (NO), gamma-aminobutyric acid (GABA), aspartate (Asp)

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and glutamate (Glu), due to their primary action on the Nav channels (Bordon et al. 2015,

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Cologna et al. 2009). Ts4, referred previous to as a “non-toxic” peptide, induces an allergic

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reaction with lachrymation, spasm in mice hind legs, and a dose dependent neurotransmitter

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release (GABA and Glu) of synaptosomes (Sampaio et al., 1996). Recently, Ts4 showed to

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ACCEPTED MANUSCRIPT affected Nav1.6 channels (Pucca et al., 2015a). Ts5 displays an alpha-effect on mammalian

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sodium channels, inhibiting the rapid inactivation of the Nav1.2, Nav1.3, Nav1.4, Nav1.5,

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Nav1.6 and Nav1.7. Such a broad effect explains its high toxicity in mice, which presents an

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intravenous medium lethal dose (LD50) very similar to the most toxic Ts toxin, Ts1 (Pucca et

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al., 2015c). Ts17 and Ts18 were recently discovered by transcriptomic technique and were

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designated as sodium channel acting toxins due to their high identity with other toxins of this

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group (Alvarenga et al. 2012).

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Although sodium channel toxins play a major role in Ts envenoming, neurotoxins

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affecting potassium channels are extremely important since they work in synergy with other

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neurotoxins, potentiating the venom toxic effect. The voltage-gated potassium channels (Kv)

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are responsible for keeping the membrane potential equilibrium of excitable cells

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(repolarization phase during the action potential) (Mouhat et al., 2008). Ts venom toxins Ts6,

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Ts7, Ts8, Ts9, Ts15, Ts16, Ts19 and Ts19 fragments (Bordon et al., 2015) are classified in

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this group. Ts6 blocks, at low concentration, the Kv1.2, Kv1.3 and Shaker IR channels (Cerni

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et al., 2014). Ts7 showed potent blocking effect on multiple subtypes channels, Kv1.1,

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Kv1.2, Kv1.3, Kv1.6 and ShakerIR (Cerni et al., 2014). Ts8 selectively blocks voltage-gated

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noninactivating K+ channels from rat brain synaptosomes (Rogowski et al., 1994); and Ts15

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blocks Kv1.2 and Kv1.3 channels (Cologna et al., 2011). Ts16 (Saucedo et al., 2012) are

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being studied; however they are classified in this group because of high identity with Kv

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toxins. One of Ts19 fragments, named Ts19 Frag-II, showed to be a specific blocker of Kv1.2

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channels (Alvarenga et al., 2012, Cerni et al., 2015).

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All this modulating and blocking action of Ts neurotoxins on ion channels can be correlated with the effects on neurons and skeletal muscles.

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Different signs and symptoms of Ts venom and pure toxins on nervous system have

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been reported in the literature. The fractions F, H and J of Ts venom (fractions obtained by

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ACCEPTED MANUSCRIPT venom gel filtration using a Sephadex G-50 column) induced convulsions in rat model, and

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when injected directly in the hippocampus, resulted in neuronal damage and seizures

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(Nencioni et al., 2000). In another study, injection of Ts2 (also named TSII) in rat

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hippocampus induced convulsion-related behavioral changes, moderated epileptic discharges

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and neuronal loss from ipsilateral dentate gyrus (Sandoval and Lebrun, 2003). Using

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pharmacological MR imaging (Kodak single-coated Medical x-ray MR Film) of regional

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cerebral blood volume, Ts3 toxin (TsTX or Tityustoxin) recruited the brainstem within the

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first minute after toxin administration, showing a significant reduction of cortical cerebral

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blood flow (CBV) in rats (Guidine et al., 2014). Moreover, microinjections of Ts3 into

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hippocampus showed, after 15 minutes, facial automatisms, rearing, masticatory jaw

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movements, sniffing and wet-dog shakes in rats. One hour after the previous injection, it was

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observed limbic convulsion, characterized by clonus of frontal limbs, rearing, and falling

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after

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electroencephalographic (EEG) recordings showed epileptic discharges in 77.5 % of the

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animals (Sandoval and Lebrun, 2002). Ts5 toxin (TsTX-V), showed a reduction of 3H-

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GABA and 3H-dopamine (3H-DA) uptake in a Ca2+-dependent manner using isolated rat

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brain synaptosomes (Cecchini et al., 2006). Ts6 (TsTX-VI) and Ts7 (TsTX-VII) were able to

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release glutamic acid and gamma-aminobutyric acid from rat brain synaptosomes (Sampaio

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et al., 1997, Sampaio et al., 1996).

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convulsion

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running

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falling).

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Concerning the muscular system, it can be indirectly stimulated by Ts venom, thus

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indicating its activity is mainly mediated through Ach release from nerve terminals. Based on

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that, Ts venom and toxins have extensively demonstrated to affect other muscles such as

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smooth muscles (Savino and Catanzaro, 1985), atria (Couto et al., 1992), uterus (Mendonça

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et al., 1995), penis (Bomfim et al., 2005), diaphragm (Borja-Oliveira et al., 2009) and others

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(detailed effects will be described in the following systems). However, direct effects of Ts

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venom and toxins have also been described in muscles. Ts venom, Ts1 and Ts5 showed to

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interact directly with Nav channels in vascular smooth muscle cells (Neto et al., 2012). In this way, Ts neurotoxins synergically modulate and/or block sodium and potassium

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channels, leading to a massive release of catecholamines and Ach, which will result in an

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autonomic stimulation of peripheric nervous system with, most of the time, an indirect action

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on other anatomic systems, which will be discussed below.

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4. Immune System: an inflammatory reaction

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An immune response is defined as a reaction to components of microorganisms,

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macromolecules (such as proteins), chemicals and even self-antigens that are recognized as

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foreign, regardless of the physiologic or pathologic consequence of such reaction. The

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immune system is composed of lymphoid tissues (bone marrow, thymus and lymph nodes

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including spleen), cellular components (such as macrophages, lymphocytes B and T,

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neutrophils, mast cells, dendritic cells, monocytes, eosinophils and basophils) and soluble

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mediators (such as cytokines, NO and proteins from complement system) (Abbas et al.,

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2012).

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Ts envenoming has shown to trigger a systemic immune response. In humans, severe

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Ts envenoming showed high levels of interleukin (IL)-1α, IL-6, interferon (IFN)-γ and

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granulocyte-monocyte colony-stimulating factor (GM-CSF) (Magalhães et al., 1999). In

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another study, tumor necrosis factor (TNF)-α, IL-1β, IL-6 and IL-8 levels were significantly

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increased in moderate and severe human cases. Levels of these cytokines were positively

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correlated with the severity of the envenoming, as evaluated by clinical profile and plasma

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venom concentration (Fukuhara et al., 2003).

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In animals, Ts venom also showed to be an inflammatory stimulus. In rats, Ts venom

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induces a significant leukocytosis, explained by an increase in the number of neutrophils 10

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(Borges et al., 2000). Recently, rats injected with Ts3 presented a significant increase in

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leukocyte rolling and adhesion and higher levels of TNF-α into cerebral microcirculation

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(Van Fraga et al., 2015). In mice, Ts venom and the major toxin, Ts1, were able to induce an increase of IL-6,

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IL-1α and TNF-α plasma levels (Pessini et al., 2003). Furthermore, also using mice models,

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Ts venom induces systemic alterations characterized by changes in the cell number in

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lymphoid organs and increasing pro- and anti-inflammatory cytokines (IL-10, IL-6 and TNF-

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α) (Fialho et al., 2011). Interestingly, the administration of Ts venom 6 h before the induction

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of sepsis using polymicrobial infection by cecal ligation and puncture (CLP) was able to

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control the harmful effects of sepsis (lethality and lung inflammation), suggesting that both

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systemic IL-10 and oxidative burst are involved in this effect (Maciel et al., 2014).

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Currently, macrophages are considered the main cells responsible for producing the

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inflammatory responses induced by Ts venom. In vitro, Ts venom induced an increase of IL-

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6 and IFN-γ production in the supernatants of peritoneal macrophages (Petricevich, 2002).

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Moreover, isolated Ts toxins were extensively investigated in the macrophage

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immunomodulation: Ts1 was able to increase the production of IL-6 and TNF-α; Ts2

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stimulated the production of TNF-α, IL-10 and inhibited NO release; and Ts6 induced the

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production of

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Zoccal et al., 2013). Recently, Ts5 also showed a pro-inflammatory effect through its ability

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to stimulate the release of macrophage TNF-α and IL-6 and, based on the induced-cytokines

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produced by Ts toxins, a new pathway of macrophage activation was suggested (Pucca et al.,

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2015c). In addition, Ts venom can induce the production of inflammatory mediators by

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interacting with toll like receptor (TLR2) and CD14/TLR4 and PPAR, peroxisome

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proliferator-activated receptor (PPAR)-γ, which can also induce lipid bodies (LB) formation

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IL-6 and NO, and decreased the production of TNF-α (Zoccal et al., 2011,

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and generation of arachidonic-acid-derived lipid mediators prostaglandin (PGE)2 and

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leukotriene (LT)B4 (Zoccal et al., 2014, Zoccal et al., 2015). Other evidences have also demonstrated that Ts toxins can modulate T cell responses.

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Using electrophysiological assays, the toxins Ts6 and Ts15 blocked the voltage-gated

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potassium channel Kv1.3 with nanomolar affinity. The Kv1.3 is a novel target for

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immunomodulation of autoreactive effector memory T cells (TEM) which plays a major role

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in the pathogenesis of autoimmune diseases (Beeton et al., 2005, Bradding and Wulff, 2009,

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Chandy et al., 2004, Wulff et al., 2003, Wulff et al., 2009).

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Besides immune cells activation and cytokine production, Ts venom has also been

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related to modulate the complement system. In vivo, the results showed an increase in serum

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lytic activity of animals injected with venom, reaching values up to 70 % higher than controls

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in classical pathway activity and 120 % in alternative pathway activity. Similar effects were

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obtained for Ts1, but with lower intensity (Bertazzi et al., 2003). Data in vitro from the same

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group showed that Ts venom induced a reduction in hemolytic activity of the classical/lectin

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and alternative complement pathways. Concomitantly, the venom caused chemotactic activity

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for neutrophils suggesting the generation of the complement component C5a (Bertazzi et al.,

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2003).

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Therefore, the production of cytokines and the pro-inflammatory mediators (specially

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IL-6, IL-8, IL-10, TNF-α, IL-1α, IL-1β, NO, LB, PGE2 and LTB4) following the activation

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of complement system causing chemotactic activity (neutrophils recruitment) are the main

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inflammatory mechanisms that are triggered by the immune system during an envenoming by

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Ts venom. Together, these effects could be an important factor in severe envenoming and

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may result in an intense inflammatory reaction - a risk of death.

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5. Cardiovascular System: cardiac arrhythmias and arterial hypertension

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or hypotension The cardiovascular system is composed of the heart and blood vessels, including

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arteries, veins, and capillaries (Guyton and Hall, 2012). Cardiac cells depolarize and

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repolarize themselves, approximately, sixty times per minute in order to promote the cardiac

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action potentials. The activity of protein complexes found in these cells, in which the ion

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channels are present, determine the form and the duration of each action potential. The influx

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of ions through cell membrane creates the ionic stream, which sets the cardiac action

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potentials. The cardiac function, as well as the normal role of ion channels, may be disturbed

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by physiological problems and, in the context of envenoming by poisonous animals, by

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toxins (Brunton et al., 2011).

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The severe morbidity and lethality observed in cases of Ts envenoming are mainly

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due to the ability of certain toxins to activate the cardiovascular system, causing cardiogenic

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shock and pulmonary edema (Cupo et al., 2007, Cupo and Hering, 2002). The releasing of

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cholinergic and adrenergic neurotransmitters induced by neurotoxins is responsible for most

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of these actions, linked to a possible direct toxic effect of the venom on cardiac cells (Karnad,

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1998).

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arrhythmias, arterial hypertension or hypotension and circulatory failure (Cupo et al., 2007,

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Teixeira et al., 2001a). The cardiac involvement is mostly represented by left ventricular

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dysfunction, which can be demonstrated by clinical manifestations, such as increased levels

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of lactate dehydrogenase (LDH), creatinine kinase (CK) and its CK-MB fraction - a dimer

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isoenzymes of creatine kinase with M (muscle) and/or B (brain) subunits, aspartate

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aminotransferase (AST) and by electrocardiogram and echocardiogram alterations (Cupo et

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al., 2007). It has already been established that the main cardiorespiratory events observed

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during the envenoming involve the activation of the autonomic nervous system (Silva et al.,

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2015).

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The studies regarding the effects of Ts toxins in the cardiac system can be bunched in three groups, which comprise case-related observations, in vivo and in vitro assays.

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Amaral et al. (1991) described clinical manifestations in five children, aged between 3

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to 9 years old, with severe envenoming after Ts sting. Sinus tachycardia was detected in all

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patients, while three of them had an acute myocardial infarction-like pattern. One child

312

presented frequent ventricular premature beats with bigeminy. Echocardiographic changes

313

were also identified, including depressed left ventricular systolic function, characterized by

314

reduced motion of the interventricular septum or decreased motion of the left ventricular

315

posterior wall with decreased left ventricular fractional shortening. A combination of these

316

symptoms was also observed. Regarding the laboratory data, two children had a mild increase

317

in CK enzyme activity.

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The well-known class of bradykinin-potentiating peptides (BPPs) is also present in the

319

Ts venom. Ts10 (also known as Peptide T) (Bordon et al., 2015) was the first BPP isolated

320

from this venom, which had its 13 amino acid sequence determined (Ferreira et al., 1993). In

321

vitro and in vivo studies demonstrated that Ts10 potentiates the contractile activity of

322

bradykinin (BK) on isolated guinea-pig ileum. The hydrolysis of BK by angiotensin-

323

converting enzyme (ACE) was inhibited in the presence of this peptide, as well as the

324

conversion of angiotensin I to angiotensin II by kininase II from guinea-pig ileum tissue.

325

Furthermore, the peptide increased the depressor effect of BK on arterial blood pressure in

326

anaesthetized rats (Ferreira et al., 1993). Four other peptides, known as Ts14 (Bordon et al.,

327

2015), with 24-25 amino acids, were discovered later on in Ts venom, being classified as Ts

328

hypotensins (TsHpt) and named from TsHpt-I to TsHpt-IV (Verano-Braga et al., 2008).

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ACCEPTED MANUSCRIPT TsHpt-I was administrated in normotensive rats after BK, potentiating its effects. When

330

administrated alone, it did not significantly alter either the blood pressure nor the heart rate.

331

Furthermore, TsHpt-I did not inhibit ACE in in vivo experiments. On the other hand, this

332

toxin produced a concentration-dependent vasorelaxation effect on rats aortic rings, with a

333

mechanism of action dependent, probably, on NO release. Recently, it was demonstrated that

334

TsHpt-I acts as an agonist of bradykinin receptor 2 (B2) (Verano-Braga et al., 2010).

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Ts1, the major Ts neurotoxin, also presents cardiovascular effects. After Ts1

336

administration on isolated guinea-pig hearts, complex effects were observed, similar to those

337

detected with crude venom administration. An initial phase, characterized by bradycardia,

338

cardiac arrest or tachycardia, was succeeded by a stage of oscillation of the rhythm,

339

possessing a cholinergic nature, with a final term of tachycardia. The effects regarding the

340

electrocardiogram of Ts1 were also similar to those observed with the crude venom,

341

comprising the reduction of heart rate resulted from sinus bradycardia or complete A-V block

342

with junctional or ventricular escapes. Within the period of rhythm oscillation, manifold

343

arrhythmias were also recorded, such as ventricular paroxysmal tachycardia, bigeminy,

344

trigemini and ventricular premature depolarizations. Ts1 also caused a reduction of coronary

345

flow, either during tachycardia and simultaneous increase in contractile force or during

346

cardiac arrest or bradycardia (Silveira et al., 1991).

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Most of the cardiac effects observed during the envenoming by Ts seem to be caused

348

mainly by the capability of the venom to release the neurotransmitters Ach and

349

norepinephrine (NE) from nerve terminals, causing chronotropic and inotropic changes. The

350

former appears to be entirely dependent on the release of the aforementioned

351

neurotransmitters from nerve endings, since the administration of mAchR antagonist is

352

capable of blocking the bradycardia induced by the venom. In addition, the increase of the

353

heart rate after the bradycardia was also inhibited by the pre-treatment with metoprolol, a β1-

15

ACCEPTED MANUSCRIPT 354

adrenoreceptor antagonist. On the other hand, the positive inotropic events induced by the

355

venom were not modified by the pre-treatment with this agent or chemical sympathetic

356

denervation with 6-OH-dopamine. Therefore, these results indicate that the latter changes are

357

not entirely dependent on neurotransmitter releases (Teixeira et al., 2001a). Ts1 (β-neurotoxin) and Ts5 (α-neurotoxin), as well as the crude Ts venom, were

359

capable of arising the cytosolic calcium concentration on isolated rat aorta smooth muscle

360

cells. As a result, a direct effect of these toxins was observed, with a concentration-dependent

361

increasing pattern in the contractile effect on these muscle cells, probably by the interaction

362

of Na+ channels (Neto et al., 2012).

SC

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358

Although such damages do not have a clear mechanism of action, some

364

electrophysiological studies characterized a receptor site for Ts1 at the sodium channels

365

present in the sarcolemma of chick’s heart (Lombet and Lazdunski, 1984). Furthermore, it

366

was also detected that Ts1 acts on sodium channels of rat cardiomyocytes, reducing the

367

transitions among closed and open states of these gates (Yatani et al., 1988), which also

368

contributes to these findings.

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A recent study analyzed the role of dorsomedial hypothalamus (DMH) ionotropic

370

glutamate receptors in the hypertensive and tachycardic responses induced by Ts1, when

371

administrated by the intracerebroventrivular via. The results indicated that the tachycardic

372

and hypertensive responses are also dependent on the activation of glutamate receptors in the

373

DMH, providing new understandings in the central mechanisms regarding the development

374

of symptoms in the severe scorpion envenoming syndrome in infants, whose blood brain

375

barrier is still under development (Silva et al., 2015).

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376

In conclusion, the cardiac changes promoted during the envenoming by Ts sting are

377

one of the most remarkable and dangerous symptoms. Ts1 (β-neurotoxin) seems to be the

378

major toxin involved in damaging this system, but the α-neurotoxins and hypotensins appear

16

ACCEPTED MANUSCRIPT to play an important role as well. Although the precise mechanism of action of Ts toxins on

380

cardiac system is not completely understood, some evidences have already been proposed.

381

However, further studies are necessary in order to achieve a better understanding regarding

382

the cardiac system changes and Ts toxins.

383

6. Respiratory system: pulmonary edema

385

The respiratory system allows the exchange of respiratory gases and, as consequence, the transportation of oxygen to all tissues (NIH, 2012).

SC

384

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379

In Ts severe envenoming, one of the most frequent consequences is the pulmonary

387

edema. The main patients who develop this complication are children, in whom the severe

388

cases are more pronounced. Moreover, respiratory failure, cardiogenic disorders and death

389

can also be observed (Marcussi et al., 2011, Bucaretchi et al., 2014).

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The pulmonary edema in a victim bitten by Ts can be diagnosed through radiological

391

exams associated with symptoms such as tachypnea and pulmonary auscultation with

392

crepitant rales (Bucaretchi et al., 2014). A case report of a 16-year-old boy who died with

393

acute pulmonary edema after Ts sting also shows evidence of cardiac dysfunction. The

394

patient showed an increase in tracheobronchial plasma protein concentration, light

395

microscopic features of the lung compatible with the adult respiratory distress syndrome,

396

electron microscopic findings compatible with acute lung injury and increased alveolar

397

capillary membrane permeability (Amaral et al., 1994).

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Several factors induced by Ts venom are linked with pulmonary edema, but the real

399

mechanism is still obscure. It may be related to cardiogenic or non-cardiogenic disorders,

400

like hemodynamic dysfunctions and the release of substances that increase vascular

401

permeability (Amaral et al., 1993, Bucaretchi et al., 2014, Bahloul et al., 2013, Marcussi et

402

al., 2011).

17

ACCEPTED MANUSCRIPT Bahloul et al. (2013) described that the pulmonary edema induced by scorpion

404

venoms are generally attributed by the increase in capillary permeability, which may be

405

caused by cytokines and consequently extravasation of plasma in the alveolar spaces.

406

Moreover the pulmonary edema is also induced by heart failure, which may occur as a result

407

of the increasing of catecholamines or by the direct effect of scorpion toxins in the

408

myocardium.

RI PT

403

Deshpande and Akella (2012) highlight other non-cardiogenic elements that can also

410

be related to pulmonary edema in scorpion envenomation. The authors report some studies

411

showing the relation between kinins and cardio-respiratory dysfunctions. They also describe

412

that the pre-treatment with aprotinin (a kinin synthase inhibitor) can inhibit the pulmonary

413

edema induced by scorpion venom in rats (Ismail, 1995; Pandey and Deshpande, 2007).

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409

Another study in rats demonstrated that the release of vascular permeability factors,

415

such as platelet-activating factor (PAF), leukotrienes, and prostaglandins may play a role in

416

the initiation of pulmonary edema induced by Ts venom (De Matos et al., 1997).

417

Additionally, after the development of lung edema, the clearance of Ts venom decreased to

418

approximately 60 % in rats (Comellas et al., 2003).

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414

In mice, Ts venom led to an acute lung damage with the increase of perivascular

420

infiltration of mononuclear and polymorphonuclear cells (Paneque Peres et al., 2009). On the

421

other hand, using rats and a culture of mast cells, Ts venom caused a remarkable edema in rat

422

airways which are independent of mast cell activation (Zuliani et al., 2013).

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Regarding isolated toxins, only the major toxin of Ts venom, Ts1 or TsTX-I, was

424

investigated. Ts1 induced an increase of IL-1 and IL-6, which are cytokines observed in high

425

concentrations in patients with pulmonary edema (Pessini et al., 2003). In the severe

426

envenomation by Ts venom, the cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α cytokines are

427

released, indicating that these immunomodulators can be related to severe complications,

18

ACCEPTED MANUSCRIPT 428

such as pulmonary edema (Fukuhara et al. 2003). Furthermore, TNF-α and IL-1β

429

demonstrated to depress the myocardial function, which can lead to pulmonary edema (Cain

430

et al., 1999). Bahloul et al. (2013) reported that most of pulmonary edema arises from the

432

cardiogenic dysfunction. According to these authors, the increase of catecholamines through

433

stimulation of adrenal gland and sympathetic nerve endings are the most responsible for

434

pulmonary edema. Furthermore, the releasing of cytokines increases this complication.

435

Recently, a case report of a 7-year-old boy with severe envenomation by T. serrulatus venom

436

revealed that the release of cathecolamines by sympathetic stimulation is the main

437

responsible for cardiogenic dysfunction and consequently pulmonary edema (Miranda et al.,

438

2015). Cupo et al, (2007) report that the acute ventricular dysfunction and cardiogenic

439

disorders in Ts envenomation are probably the main origin of acute pulmonary edema.

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431

In conclusion, the pulmonary edema presents multifactorial origins. There are

441

evidences that it is mainly linked to a cardiogenic dysfunction, yet other mechanisms can also

442

intensify this complication, such the releasing of cytokines and kinins, as well as the increase

443

of vascular permeability. Together, the cardiogenic disorder and the pulmonary edema are the

444

main causes responsible for death after Ts envenoming. Therefore, understanding the real

445

mechanisms of these disorders may assist in the development of more specific treatments

446

and, consequently, a decrease in complications and deaths due to Ts sting.

447

7. Urinary System: decreased glomerular filtration rate and urine volume

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440

448

The urinary system, also known as the renal system, is a group of organs, which

449

includes two kidneys, two ureters, the bladder, two sphincter muscles, and the urethra. The

450

kidneys are the main organs of homeostasis, being responsible for filtering out excess fluid

19

ACCEPTED MANUSCRIPT 451

and substances such as urea, uric acid and creatinine in the form of urine (Guyton and Hall,

452

2012). Ts venom is capable of reaching the renal system in the first minutes after the

454

envenoming. In rats, maximal levels of venom (injected s.c.) were observed after 15 min in

455

the kidney and liver and, after 30 min, in the serum, lungs, heart and spleen (Revelo et al.,

456

1996). Moreover, another study demonstrated that rats injected with Ts venom have an

457

increased perfusion pressure (PP) and renal vascular resistance (RVR), decreased glomerular

458

filtration rate (GFR) and urinary flow (UF), with no changes on tubular transport. In this way,

459

the hypothesis lays on the fact that Ts venom affects renal hemodynamic by a direct

460

vasoconstrictor action leading to a decreased renal flow (de Sousa Alves et al., 2005). In

461

2013, the first natriuretic peptide from Ts venom (TsNP) was described. In the isolated

462

perfused rat kidney assay, TsNP increased the perfusion pressure (PP), glomerular filtration

463

rate (GFR) and urinary flow (UF). Although, these results were controversial to de Souza

464

Alves et al. (2005) study, it is important to consider that TsNP is an isolated toxin and was

465

tested only in vitro, while the other study used Ts whole venom and rat animal model,

466

resulting in difficulties to compare them (Alves et al., 2013).

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453

In addition, an increase of urea and creatinine levels in mice plasma after Ts

468

envenoming has also been described (Pucca et al., 2012, Pinto et al., 2010). Histological

469

studies in rats challenged with Ts venom or the major toxin (Ts1), showed kidneys

470

congestion and small hemorrhagic areas (Correa et al., 1997). On the other hand, urinalysis of

471

envenomed dogs indicated that the renal function remained normal after envenoming, with no

472

changes regarding the levels of serum urea and creatinine (Ribeiro et al., 2010).

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473

Clinically, proteinuria, hematuria and hemoglobinuria can be manifested in

474

scorpionism (Pipelzadeh et al., 2007). However, in humans, severe renal toxicity caused by

475

Ts venom is rare in comparison to other scorpion stings (Rahmani and Jalali, 2012). For

20

ACCEPTED MANUSCRIPT 476

Hemiscorpius lepturus envenoming, renal toxicity is demonstrated by hemoglobinuria,

477

proteinuria and hematuria, which can progress to severe renal failure, if the antivenom is not

478

administered (Pipelzadeh et al., 2007).

479

Based on that, it is clear that Ts venom can cause changes in the renal function, although the alterations are not severe enough to cause renal failure.

481

8. Endocrine and Exocrine Systems: increased hormones and secretions

RI PT

480

The endocrine system is composed of different tissues and glands such as pituitary,

483

hypothalamus, thyroid, parathyroid, adrenal glands, pancreas, gonads, kidney, stomach, small

484

intestine and adipocytes. They secrete hormones that are carried by the circulatory system

485

and bind to specific receptors in target tissues producing a physiological response in other

486

tissues. Glands that have ducts are called exocrine glands. Some examples of exocrine glands

487

are sweat glands and salivary glands (Guyton and Hall, 2012).

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482

The envenoming after Ts sting extensively affects the endrocrine and exocrine

489

systems, which can be detected by the alterations in the production of many hormones and

490

secretions.

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488

There are reports showing that the injection of Ts venom in dogs increased the insulin

492

levels significantly following an expressively decrease over the time (Ribeiro et al., 2010). In

493

mice, Ts venom increases insulin plasma levels in the early hours, as well as EPI and NE in

494

the first half hour (Vasconcelos et al., 2004). The transitory raise in the insulin levels is

495

probably a consequence of the hyperglycemia, which can be explained by glycogenolysis

496

stimulated by the release of EPI and NE (Correa et al., 1997, Vasconcelos et al., 2004). A

497

study performed with the isolated Ts5 toxin (also named TsTx-V) from Ts venom

498

demonstrated that in rat isolated islets, the toxin (5.6 µg/ ml) increased the insulin secretion

499

4-fold over basal values (Gonçalves et al., 2003).

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21

ACCEPTED MANUSCRIPT 500

The cortisol can also be affected by Ts venom, being increased in dogs envenomed

501

with 250 µg/kg (s.c.) of venom (Ribeiro et al., 2010). However, the concentration of this

502

hormone remained unchanged in rats serum injected (s.c.) with 100 or 450 µg of Ts venom

503

(Pinto et al., 2010). It is important to point out that changes in the cortisol and insulin levels observed in

505

scorpion accidents are also associated with the stress produced by pain. This discomfort may

506

cause vasoconstriction, glucose consumption and an increase of blood flow into the muscles,

507

preparing the body to a state of "fight or flight" (Ranabir and Reetu, 2011).

SC

RI PT

504

No histological alterations in the adrenal gland were noted neither in animals treated

509

with the Ts venom nor with Ts1 toxin (Correa et al., 1997). Indeed, an isolated study

510

indicates that the releasing of EPI and NE is not due to a direct action of the Ts venom in the

511

adrenal gland but a result of an effect on the nervous structure linked to the gland. This result

512

was proven when denervated gland was not affected by Ts venom, with no releasing of

513

catecholamines (Henriques et al., 1968).

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508

Other important signals of Ts envenoming are related to exocrine gland alterations,

515

such as increased volume of saliva (sialorrhea), excessive production of tears and profuse

516

sweating. Ts envenoming causes alterations in the submandibular gland following the

517

increase of saliva volume. Histological studies using the toxin Ts1 showed signs of

518

submandibular gland stimulation: the acini were smaller, the cells appeared empty, and the

519

cytoplasm was scanty and vacuolized. This effect is probably due to the stimulation of

520

cholinergic and adrenergic receptors, leading to an exhaustion of the gland (Correa et al.,

521

1997). Ts venom, Ts3 (Tityustoxin) and Ts1 also produced important morphological changes

522

in the acini and granular convoluted tubules (Clemente et al., 2002).

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514

523

However, there are no studies reporting specific alterations, like histological changes,

524

in the production of tears and sweat so far. Many Ts toxins (e.g. Ts1, Ts2 and Ts5) can

22

ACCEPTED MANUSCRIPT 525

interfere in Navs, resulting in depolarization of the nervous cells and neurotransmitter release.

526

Therefore, these effects may be explained by the interaction of these toxins with the Nav

527

channels (Peigneur et al., 2015, Pucca et al., 2015c, Cologna et al., 2012). In general, the endocrine and, especially, the exocrine systems can be affected during

529

Ts envenoming, which are mainly represented by an increase of the production of insulin,

530

cortisol, sweat, tears and saliva.

531

9. Digestive System: increased volume of pancreas and liver secretions and

532

enzymes

M AN U

SC

RI PT

528

The digestive system is responsible for obtaining nutrients from food, an essential task

534

to maintain all organism functions. The digestive system consists of the gastrointestinal tract

535

(comprised of mouth, esophagus, stomach, small and large intestine) and accessory (or

536

associated) organs (salivary glands, pancreas, liver, and gallbladder). The four basic

537

processes of this system are motility, secretion, digestion and absorption, which are

538

responsible for the metabolism of proteins, carbohydrates and fats (Sherwood, 2015).

TE D

533

Troncon et al. (2000) evaluated the effects of the fraction T1 of Ts venom obtained

540

by molecular exclusion chromatography on gastric emptying and small intestinal transit

541

mechanisms using male Wistar rats. The animals showed a salivary hypersecretion and

542

inhibition of both gastric emptying and intestinal transit. These results corroborate with

543

another study, also performed in rats, which showed that the injection of Ts1 may induce a

544

rapid, intense and sustained inhibition of gastric emptying (0.25 to 48 h) after envenoming

545

(Bucaretchi et al., 1999). The effects of the gastric emptying and intestinal transit were

546

observed using another isolated toxin, Ts3, caused by the releasing of both cholinergic and

547

adrenergic mediators, as well as other neurotransmitters (such as serotonin and substance P)

548

in the smooth muscle (Sninsky et al., 1986).

AC C

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23

ACCEPTED MANUSCRIPT A study with T1 fraction from Ts venom demonstrated effects on gastric volume, acid

550

output and pH reduction in rats (Cunha-Melo et al., 1983, Gonzaga et al., 1979). Moreover,

551

another study showed that T1 fraction was capable of inducing a significant increase in

552

volume of gastric juice, gastrin and pepsin output and an increase of acid and pH reduction

553

15–60 min after injection (Toppa et al., 1998). The acute gastric mucosal lesions induced by

554

this fraction are characterized by the presence of multiple superficial mucosal erosions and

555

ulcerations that play an important role in many cases of upper gastrointestinal bleeding (Melo

556

et al., 2006). Luckily, these acute gastric mucosal lesions can be prevented by different class

557

of acid blockers injected before the intoxication (Melo et al., 2006).

SC

RI PT

549

Diarrhea is an evident consequence of scorpion envenoming on the digestive system,

559

being more common observed in children (≤ 15 years old) than in adults. This condition,

560

observed in severe scorpion envenoming, was associated with a fatal outcome (Bahloul et al.,

561

2005). Diniz et al. (1974) showed the effects of the toxin Ts3 on the longitudinal strip of the

562

guinea-pig ileum, which increases the output of Ach from longitudinal strips of the smooth

563

muscle. Indeed, the release of Ach from excitatory motor neurons can activate the motility

564

and peristaltic reflex (Olsson and Holmgren, 2011). Ach also regulates the intestinal water

565

transport and the cholinergic neurons, when activated, release Ach into the neuroepithelial

566

junction in order to stimulate secretion from the crypts and to inhibit absorption through the

567

surface cells (Keely, 2011). Additionally, a significant change in serum electrolyte

568

concentrations has been shown after Ts envenoming, which may alter cell membrane

569

permeability, causing diarrhea (Sofer et al., 1994, Troncon et al., 2000). Therefore, this

570

intestinal motility disorder associated with the non-absorption of the components can explain

571

the diarrhea.

AC C

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558

572

Ts venom toxins can also cause acute pancreatitis, possibly by activating neural

573

pathways that release Ach from postganglionic nerve fibers on muscarinic receptors, causing

24

ACCEPTED MANUSCRIPT hyperstimulation of the pancreatic secretion (Fletcher et al., 1992). The mechanism by which

575

vagotomy potentiates the pancreatic secretion, evoked by Ts3, is under investigation (Novaes

576

et al., 1982). The Ts toxins increase the concentration of enzymes in pancreatic juice and a

577

synergistic action between the venom components is probably responsible for the pancreatitis

578

often observed in the scorpion envenomed victims (Possani et al., 1991). Alpha (Ts2 and

579

Ts3) and beta (Ts1) toxins from Ts venom stimulate the acinar cells to discharge their

580

zymogen granules, resulting in the appearance of large vacuoles and some loss of

581

morphological integrity (Possani et al., 1991). Additionally, a great increase in the exocrine

582

pancreatic output, associated with an outflow obstruction, can explain the pancreatitis

583

induced by scorpion venom (Bartholomew et al., 1976). Moreover, the proteinases present in

584

the Ts venom may also be responsible for the intrapancreatic activation of trypsinogen, the

585

major digestive protease of pancreas, resulting in pancreatitis (Almeida et al., 2002). The Ts

586

metalloproteinases are capable of penetrating into the intact pancreatic tissue and selectively

587

cleave SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor)

588

proteins within exocrine pancreatic tissue (Fletcher et al., 2010). The action of

589

metalloproteinases on the SNARE proteins leads to the loss of their function, avoiding the

590

pancreas to secrete their products and disturbing homeostasis (Fletcher et al., 1992). The

591

SNAREs are important in the selective transport between cellular compartments in the cell

592

membrane and include the v-SNARE vesicle-associated membrane protein (VAMP; also

593

known as synaptobrevin). Antarease, a metalloproteinase isolated from Ts venom, cleaves

594

VAMP2, a v-SNARE protein. The proteolysis of VAMP2 is associated with zymogen

595

granule membranes in pancreatic acinar cells and, consequently, with the pancreatitis

596

observed after the scorpion envenoming (Fletcher et al., 2010).

AC C

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574

597

Besides acting on the organs aforementioned, there are studies showing the effects of

598

Ts venom and its toxins on the liver and hepatic enzymes. Corrêa et al. (1997) performed an

25

ACCEPTED MANUSCRIPT intravenous sublethal injection of crude Ts venom and Ts1 into rats. These injections

600

increased the serum levels of aspartate aminotransferase, amylase, creatine kinase and lactate

601

dehydrogenase, free fatty acids, as well as reduced the level of hepatic glycogen (30-180 min

602

after injection) (Corrêa et al., 1997). However, the levels of alanine aminotransferase,

603

gamma-glutamyl transferase and alkaline phosphatase were not altered. This study also

604

demonstrated that the crude venom and Ts1 caused hepatic congestion with hemolysis and

605

hydropic degeneration. Corroborating with this study, Pessini et al. (2001) confirmed

606

that animals injected with hyaluronidase and Ts1 had an increase of serum levels of creatine

607

kinase, lactate dehydrogenase and aspartate aminotransferase after 15 min of injection.

SC

RI PT

599

The characterization of the components from Ts venom involved in the effects

609

described above may be helpful to elucidate the mechanisms of action of scorpion toxins on

610

the digestive system. In addition, it could also support the understanding of the effects

611

observed after the scorpion sting, helping in the improvement of antivenom therapies.

612

10. Integumentary system: local pain

TE D

M AN U

608

The integumentary system is composed of skin, skin receptors and glands, nails, hair

614

and nerve endings (Anderson, 2012). The sweat glands are part of this system and are

615

extensively affected by Ts venom (see Endocrine and Exocrine system section). After Ts

616

envenoming, local pain is the primary manifestation (95.5%). Although further studies are

617

still needed in order to understand this event, cutaneous hyperalgesia caused by Ts sting is

618

always documented. In another scorpion sting, Buthus martensi Karch (BmK), it was

619

demonstrated that the spinal extracellular signal-regulated kinase (ERK) signaling possibly

620

lead to BmK venom-induced pain-related behaviors (Panga et al., 2008). However, the

621

swelling observed as a result of the inflammation at the site of the sting and the production of

622

pro-inflammatory mediators can also contribute to the intense pain, see Immune System

AC C

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613

26

ACCEPTED MANUSCRIPT section. Nevertheless, additional studies are needed to understand Ts venom and toxins

624

stimuli into skin nociceptive cells, receptors and nerve endings for eliciting the intense pain

625

sensation. No studies were conducted on hairs and nails after Ts envenoming.

626

11. Skeletal system: osteogenesis

RI PT

623

The skeletal system comprises the bones and connective tissues (Guyton and Hall,

628

2012). Although it is still lacking studies involving the Ts venom in bones, scorpion venom

629

effects on the skeletal system are not novelty. The Indian black scorpion (Heterometrus

630

bengalensis) venom intensified the osteoporosis bone status through increased bone mineral

631

deposits, associated with the coordinated actions of hormones, cytokines and enzyme activity.

632

It seems that the scorpion venom acts at the osteoclasts, which was confirmed by the

633

involvement of IL-1, TNF-α and PTH (parathyroid hormone) (Gomes et al., 2009). Surely,

634

the relationship between the Ts venom and the osteogenesis needs to be further investigated.

635

12. Reproductive System: priapism and uterus contraction

TE D

M AN U

SC

627

The major function of the reproductive system is the production of gametes capable of

637

fertilization and producing a viable offspring that might reproduce with success (Klaassen,

638

2013). Therefore, the reproductive systems of male and female exhibit differences that are

639

appropriate to their different roles in the reproductive process. A pair of testes in the male and

640

a pair of ovaries in the female represents the primary reproductive organs. The mature

641

gonads, in both sexes, present a dual function of producing gametes (spermatozoa in the male

642

and ova in the female) and secreting sex hormones (testosterone in male and estrogen and

643

progesterone in females). Beyond the gonads, the reproductive system includes a

644

reproductive tract encompassing a system of ducts specialized in transporting or storing the

645

gametes after their production, besides the accessory sex glands that empty their supportive

AC C

EP

636

27

ACCEPTED MANUSCRIPT 646

secretions into these passageways. Also, this system presents the external genitalia, which

647

consists of the visible portions of the reproductive system (Sherwood, 2015). There are few studies regarding the effects of Ts venom and their toxins on the

649

reproductive system. Concerning the male reproductive system, Ts venom specifically acts

650

on the erectile function of the penis. This organ is composed of the corpus cavernosum,

651

which is a specialized vascular structure consisting of two bodies of erectile tissue, running

652

parallel within the penis (Andersson and Wagner, 1995). The mechanism of penile erection

653

involves peripheral and central reflexes, associated with nitric oxide (NO) release from

654

endothelial cells, that acts as a vasodilator on smooth muscles of the penile corpus

655

cavernosum (Toda et al., 2005). The main consequence of the Ts envenoming in this system

656

is the priapism, which is defined as a painful and persistent erection unrelated to sexual

657

interest or desire (Anele et al., 2015). In the accidents caused by all scorpions from the

658

Buthidae family, all victims from pediatric age group and 20% of adults patients presented

659

priapism (Bawaskar and Bawaskar, 2012). Teixeira et al. demonstrated, in vitro, that crude Ts

660

venom causes relaxation of both rabbit and human cavernosal smooth muscles through a

661

mechanism dependent on NO release from nitrergic nerves (Teixeira et al., 1998, Teixeira et

662

al., 2001b). Besides, three years later, the same group showed that Ts3 is responsible for the

663

relaxation of human corpus cavernosum by NO release from nitrergic nerves (Teixeira et al.,

664

2004). Studies that elucidate this mechanism can be useful for the discovery of new strategies

665

in tretating priapism after scorpion envenoming or to modulate the penile dysfunction.

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Regarding the female reproductive system, a study using isolated rat uterus

667

preparations stimulated with Ts1 showed that the toxin was capable of inducing contractions

668

in this organ. These effects are due to actions on post-ganglionic autonomic nerve endings,

669

with Ach release and stimulation of mAchR (Mendonça et al., 1995). The primary afferent

670

innervation of the uterus is not completely understood and the participation of neurogenic

28

ACCEPTED MANUSCRIPT control of uterine contractility has not been clearly defined (Herweijer et al., 2014, Garfield,

672

1986, Mendonça et al., 1995). It is generally accepted that neurogenic mechanisms do not

673

participate in the control of myometrial contractility. The uterus of several mammal species is

674

thought to be primarily innervated by post-ganglionic adrenergic fibers from the sympathetic

675

nervous system. Cholinergic innervation would be sparse and mainly related to parametrial

676

blood vessels (Garfield, 1986, Mendonça et al., 1995).

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The effects of Ts venom were also investigated on pregnant rats and their offspring.

678

The study showed that the crude venom (0.3 or 1.0 mg/kg s.c. on either day 5 or day 10 of

679

gestation) did not cause maternal or fetal toxicity, nor any histopathological changes in

680

placentas, lungs, heart, liver or kidneys of the fetuses (Cruttenden et al., 2008). On the other

681

hand, a different study which performed the analysis in two different periods of pregnancy (at

682

day 10 of gestation, a period of intense organogenesis, and at day 16, a period of brain

683

development) demonstrated that pregnant rats envenomed with scorpion venom on specific

684

gestational days, at doses that simulates mild envenoming, presented discreet alterations in

685

some physical and reflexive parameters in their offspring (Barão et al., 2008).

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Moreover, additional studies are still necessary to clarify the effect of a Ts sting in the offspring, including isolated experiments using pure Ts toxins.

688

13. T. serrulatus venom: a lethal cocktail

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Ts venom and purified toxins demonstrate evidences that they affect all of the body

690

anatomic systems (Fig. 4). This makes Ts venom a potent lethal cocktail. However, we

691

sought to understand why Ts presents low lethality (0.12 % in 2014) compared to the number

692

of cases registered, even being so harmful to the body. This can be explained by: (1) The

693

venom volume injected: the low amount of venom injected into a living organism (e.g.

694

human) is, undoubtedly, the main responsible for the low lethality of scorpion envenoming.

29

ACCEPTED MANUSCRIPT In general, during the sting, scorpions injected around 0.1 - 0.9 µL into the victim (van der

696

Meijden et al., 2015), this volume is about 1,000 times lower than the venom volume injected

697

by snakes bite. (2) The content of the venom: the geographic localization of the scorpion

698

(Oliveira et al., 2013), the scorpion prey-specific diet (Pucca et al., 2014a), the age of the

699

species (Herzig et al., 2004) and the venom volume injected may significantly vary the

700

concentration of toxic components injected during the sting. (3) The victim’s body weight

701

and blood brain barrier permeability: infants are more susceptible to severe Ts

702

envenoming due to the low body weight, which allows fast venom distribution (Nunan et al.,

703

2003). In addition, their blood–brain barrier is more permeable to small peptides, such as

704

toxins able to act on voltage-gated Na+ or K+ channels (Nunan et al., 2003), as well as to

705

compromise the areas of neurovegetative control (Guidine et al., 2014). (4) The victim’s

706

health conditions: patients that present previous health problems (e.g. cardiac disorders) and

707

the elderly/children that present an impairment of the immune system response (Amaral et

708

al., 1993) are more susceptible to severe Ts envenoming. (5) The victim’s sex: hormonal

709

effect plays an important role in the envenoming (Pucca et al., 2011), with adult female rats

710

being around 2 times more sensitive to the crude Ts venom than the adult male rats (Nunan et

711

al., 2003). (6) The local of the Ts sting: studies demonstrate that depending on the local of

712

the sting (e.g. foot, hand, penis and head) and if a vein is punched during the accident, the

713

severity of envenoming can be altered (Nishioka et al., 1993, Bahloul et al., 2010).

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714

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Based on that, the Ts lethal cocktail is not always fatal as a result of many variables

715

listed before. Nevertheless, each Ts accident is a unique case and the victim needs to be

716

carefully investigated.

30

ACCEPTED MANUSCRIPT 717

14. General Treatment In Brazil, the treatment used for mild cases of Ts accidents (90 %) as well as for other

719

Tityus species is based mainly on pain relief by infiltration of 2 % lidocaine without

720

vasoconstrictor (3-4 mL for adults and 1-2 mL for children) at the sting site or by

721

administrating dipyrone (metamizole) or other analgesics, orally or parenterally. During

722

severe scorpion envenoming cases (Stage III), the antivenom administration by intravenous

723

route is mandatory. The scorpion antivenoms (SAE or soro antiescorpiônico in Portuguese)

724

or arachnidan antivenoms (SAAr or soro antiaracnídeo in Portuguese) are always used in

725

children under 7 years and in adults with previous health problems (such as hypertension and

726

cardiovascular problems) even if they present mild or moderate clinical manifestations.

727

Nevertheless, independent of the envenoming stage, all patients should be maintained under

728

monitoring in the hospital for 4-6 h after the accident occurred (Marcussi et al. 2011).

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Currently, in Brazil, the scorpion antivenom serum (SAE or SAAr) is routinely

730

produced using horses. Although this heterologous antivenom has proven to be effective in

731

reducing mortality rate in Ts stings, its production may present some problems (Pucca et al.,

732

2015b). The heterologous serum poses a risk of serious complications such as anaphylaxis

733

and serum sickness (Roncolato et al., 2015). Moreover, regarding its production, some of the

734

obstacles encountered are the high cost of maintaining horses, the difficulty in obtaining large

735

amounts of venom and the production of large amounts of non-neutralizing antibodies. In this

736

way, several efforts have been made to produce a better antivenom using recombinant human

737

antibodies, which would mitigate these side effects. A monoclonal human antibody fragment,

738

called Serrumab, has shown to neutralize many toxins present in Ts venom, and could be

739

considered nowadays the best alternative for a neutralizing antibody to formulate a human

740

anti-Ts serum in Brazil (Pucca et al., 2012, Pucca et al., 2014b). However, much more efforts

741

are needed to produce a commercial homologous antivenom specific to Ts venom.

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ACCEPTED MANUSCRIPT 742

Conclusion Many studies have documented that the signs and symptoms displayed by victims of

744

Ts envenoming can be attributed to the induced-alterations of the different anatomic systems

745

caused by the venom. This review has clearly shown that all anatomic systems are, somehow,

746

affected by Ts venom, which leads Ts venom to be considered a lethal cocktail.

747

Ethical statement

748

This submission is a review article. No ethical issues are involved.

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749

Acknowledgments

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We would like to thank the Fundação de Amparo à Pesquisa do Estado de SãoPaulo

752

(FAPESP, São Paulo Research Foundation: post-doctoral scholarship to M.B.P. 2012/12954-

753

6; doctoral scholarship to F.A.Cerni 2012/13590-8 and F.G.A. 2011/12317-3; undergraduate

754

scholarship to H.T.L. 2014/07824-1), Conselho Nacional de Desenvolvimento Científico e

755

Tecnológico (CNPq, The National Council for Scientific and Technological Development;

756

doctoral scholarship to F.A.Cordeiro) and Coordenação de Aperfeiçoamento de Pessoal de

757

Nível Superior (CAPES, Coordination for the Improvement of Higher Education Personnel;

758

master degree scholarship to E.L.P.Jr).

759

Conflict of interest

760

The authors confirm that they have no conflicts of interest in relation to this submission.

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32

ACCEPTED MANUSCRIPT 761

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Figure 1. Tityus serrulatus morphology. The photo shows that the Ts body is divided into

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three major sections: prosoma, mesosoma and metasoma. The venom apparatus or telson and

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the serra (responsible for the species name) are also indicated. Photo obtained from the

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Figure 2. Tityus serrulatus epidemiology. Epidemiological data from Ts and other

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Figure 3. Tityus serrulatus venom composition.

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Figure 4. Ts venom effects in anatomical systems. The figure summarizes the main effects

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reported after Tityus serrulatus sting in different anatomical systems: Nervous,

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Cardiovascular, Immune, Endocrine & Exocrine, Integumentary, Muscular, Skeletal,

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Reproductive, Urinary, Digestive and Respiratory (*lack of supporting studies).

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