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Review

Crotoxin: Novel activities for a classic b-neurotoxin

Sandra C. Sampaio a, Stephen Hyslop b, Marcos R.M. Fontes c, Julia Prado-Franceschi b,Vanessa O. Zambelli a, Angelo J. Magro c, Patrıcia Brigatte a, Vanessa P. Gutierrez a, Yara Cury a,*

a Laboratory of Pathophysiology, Butantan Institute, Av. Vital Brazil, 1500, 05503-900, Sao Paulo, SP, Brazilb Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), CP 6111, 13083-970, Campinas, SP, Brazilc Departament of Physics and Biophysics, Institute of Biosciences, Universidade Estadual Paulista (UNESP), CP 510, 18618-000, Botucatu, SP, Brazil

a r t i c l e i n f o

Article history:Received 27 January 2009Received in revised form 17 December 2009Accepted 9 January 2010Available online 28 January 2010

Keywords:Anti-inflammatoryAnti-microbialAntinociceptionAnti-tumor activityCrotoxinImmunomodulationNeurotoxicity

a b s t r a c t

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom,was the first snake venom protein to be purified and crystallized. Crotoxin is a hetero-dimeric b-neurotoxin that consists of a weakly toxic basic phospholipase A2 and a non-enzymatic, non-toxic acidic component (crotapotin). The classic biological activitiesnormally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity andcardiotoxicity. However, numerous studies in recent years have shown that crotoxin alsohas immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesicactions. In this review, we describe the historical background to the discovery of crotoxinand its main toxic activities and then discuss recent structure–function studies andinvestigations that have led to the identification of novel pharmacological activities for thetoxin.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction and historical background

It has now been 70 years since the first animal venomcomponent, crotoxin, a heterodimeric complex consistingof a basic, weakly toxic PLA2 and an acidic, non-toxic, non-enzymatic component (crotapotin) and the main toxiccomponent of the South American rattlesnake, Crotalusdurissus terrificus, was purified and crystallized. Followingits discovery, numerous studies investigated the majorbiological activities of crotoxin (principally neurotoxicity,but also myotoxicity, nephrotoxicity and cardiotoxicity).However, in recent years, there has been increasingevidence that crotoxin exerts a variety of other importantactions unrelated to these classic activities. These actionsinclude immunomodulatory, anti-inflammatory, anti-

tumor, anti-microbial and analgesic activities. In thisreview, we focus on these novel actions and discuss thepossible mediators and mechanisms involved. We begin,however, with a brief history of the discovery of crotoxinand of the major activities and characteristics attributed tothis toxin in the decades following its crystallization. Ouremphasis in these historical sections is on the seminalstudies that led to the identification of each activity orcharacteristic of crotoxin. We also summarize currentknowledge of the structure of this complex.

By the 1930s, the Instituto Butantan, founded by Dr. VitalBrazil, was an international reference center for toxinology,primarily as a result of Dr. Brazil’s pioneering work on Bra-zilian snake venoms and antivenoms (Brazil, 1911;Hawgood, 1992). During the early years of its existence,the Instituto Butantan received an abundant supply ofvenomous snakes from all over Brazil and this provided anample stock of venom, especially of Bothrops jararaca (jar-araca) and C. durissus terrificus (¼ Crotalus terrificus terrificusin older literature; South American rattlesnake). This stock

* Corresponding author. Laboratorio de Fisiopatologia, InstitutoButantan, Avenida Vital Brazil, 1500, 05503-900, Sao Paulo, SP, Brazil. Tel.:þ55 11 3726 7222; fax: þ55 11 3726 1505.

E-mail address: yarac@attglobal.net (Y. Cury).

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provided the German chemist Karl Heinrich Slotta (1895–1987) with the raw material necessary for his seminalcontribution to toxinology, namely, the isolation and crys-tallization of crotoxin, the main toxin of C. d. terrificus venom(Hawgood, 2001).

From the 1930s onwards, in an effort to boost thequantity and quality of scientific research in the state, thegovernment of the Brazilian state of Sao Paulo, inconjunction with the then director of the Instituto Butan-tan, offered several German scientists positions in state-funded research institutes. In 1937, Dr. Slotta was offeredthe position of director of a newly established ChemicalInstitute located on the premises of the Instituto Butantan,and was allowed to bring with him his laboratory equip-ment. The original aim of the invitation was for Dr. Slotta tocontinue his research on the chemistry of sex hormones, inwhich he was an internationally recognized authority, andto establish a new field of study in the chemistry of coffeesince at that time Sao Paulo had a large surplus of coffeeand was looking for ways to use it industrially (Slotta,1982). However, Dr. Slotta’s interests soon turned toresearch on snake venoms since, like toad venom, snaketoxins were thought to be sterols (Dr. Slotta’s area ofresearch expertise was in sterol hormones) (Hendon andBieber, 1982). In early 1937, he started working on rattle-snake venom and soon concluded that the venom mightcontain proteins. At that time, Dr. Slotta received a grant forthe salary of an assistant for one year, so he invited Dr.Heinz Fraenkel-Conrat, his brother-in-law and an expert inprotein chemistry, to work with him on venom research. In1938, their efforts resulted in the crystallization of a proteinthey called crotoxin, i.e., Crotalus toxin (Slotta, 1938; Slottaand Fraenkel-Conrat, 1938a,b, 1938–1939, 1939). This workwas a landmark in modern toxinology as it definitivelyconfirmed the protein nature of snake venom toxins andlaid the foundation for the subsequent isolation and char-acterization of toxins from animal venoms in general.

Crotoxin behaved as a single, pure protein by the criteriaknown at that time (Slotta and Fraenkel-Conrat, 1938a,1938–1939), including ultracentrifugation (Gralen andSvedberg, 1938) and electrophoresis (Li and Fraenkel-Conrat, 1942). Subsequent work, however, raised doubtsabout the homogeneity of crotoxin and led to a series ofstudies in the 1950s–1970s that showed that crotoxin wasa complex of two proteins, namely, a basic phospholipaseA2 (PLA2) with weak toxicity (basic subunit, component Bor crotoxin B; CB) and an acidic, enzymatically inactive,non-toxic protein known as crotapotin (acidic subunit,component A or crotoxin A; CA) that potentiated thetoxicity of the PLA2 by acting as a chaperone protein(reviewed in Habermann and Breithaupt, 1978; Bon et al.,1979; Hendon and Bieber, 1982; Hawgood and Bon, 1991).Further details delineating how the identity and structureof crotoxin were established can be found in Habermannand Breithaupt (1978) and Hendon and Bieber (1982).Subsequent studies identified isoforms of crotoxin anddemonstrated individual variation in the venom levels ofthese proteins (Faure and Bon, 1987, 1988). In contrast tothe numerous biochemical studies that followed thediscovery of crotoxin, it was not until w20 years after thecrystallization of this protein that investigations of its

pharmacological and physiological actions began(Habermann, 1957a,b; Neumann and Habermann, 1955),with a detailed series of studies being undertaken by Dr.Oswaldo Vital Brazil and co-workers (Vital Brazil, 1966;Vital Brazil et al., 1966a,b).

2. Structural/functional studies

The first major steps towards understanding themolecular structure of crotoxin were reported by Neumannand Habermann (1955) and Fraenkel-Conrat and Singer(1956), who discovered that the crotoxin complex con-sisted of two different, non-covalently bound subunits.Subsequently, Hendon and Fraenkel-Conrat (1971) purifiedthe two components of the complex (named CA and CB)and showed that CB caused hemolysis, whereas CA wasnon-toxic and non-hemolytic. In 1978, Jeng and Fraenkel-Conrat examined the effects of p-bromophenacyl bromide(p-BPB), ethoxyfomic anhydride and acetic anhydride onthe crotoxin complex and its subunits. These authorsobserved that it was not possible to alkylate His48, thecatalytic residue of CB, with p-BPB when this subunit waspart of the crotoxin complex, but that alkylation occurredwhen the complex was dissociated, resulting in catalyti-cally inactive CB. This finding suggested that the active siteof CB was shielded by CA (crotapotin) and providedimportant insights on structural aspects of the crotoxincomplex. These observations were confirmed by Marlasand Bon (1982) based on experiments done with electro-plaque preparations from the electric eel Electrophoruselectricus; the authors concluded that the structuralarrangement of the crotoxin complex was essential forpreventing the non-specific adsorption of CB to membranephospholipids. However, the presence of intrinsic PLA2

activity in the crotoxin complex led Radvanyi and Bon(1982) to suggest that CA did not mask the catalytic siteof CB. Based on a large series of kinetics studies using thecrotoxin complex and its subunits, these authors concludedthat CB was only toxic when combined with anothermolecule of CB in a dimeric arrangement.

Radvanyi et al. (1985) used electron spin resonance (ESR)techniques to provide insights into the structure of crotoxinand its components. By using spin-labeled fatty acids tomeasure the affinity of these molecules for the complex anddissociated subunits, it was observed that two spin-labeledfatty acids were bound per mol of CB and that this subunithad a large hydrophobic cleft that fatty acids were able topenetrate; this cleft was considered to be the binding site forthe acyl chains of specific phospholipid substrates recog-nized by CB. Spin-labeled fatty acids were also found toinhibit the PLA2 activity of CB. In the presence of CA, CB didnot bind spin-labeled fatty acids, indicating that thepotential hydrophobic binding site was not accessible in theintact crotoxin complex. However, in view of the previousfindings regarding the alkylation of crotoxin and its subunitsby p-BPB (Radvanyi and Bon, 1982), Radvanyi et al. (1985)suggested that the inability of spin-labeled fatty acids tobind the crotoxin complex was also related to the apparentoligomeric equilibrium of CB in solution. In this scenario,although CB normally existed in an inert monomeric state orformed a reactive dimer, the binding of spin-labeled fatty

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acids favored the formation of an oligomeric state involvingeight CB monomers. The resulting octameric arrangementproduced conformational changes in CB that preventedaccess to the hydrophobic cleft in non-reactive oligomericstates, i.e., in association with CA.

Although the studies already mentioned have contrib-uted considerably to our understanding of the structuralfeatures of the crotoxin complex and its subunits, there wasstill little structural data for this complex. Aird et al. (1989)used circular dichroism (CD), deconvolution Fourier-transform infrared (FTIR) and fluorescence spectroscopyto examine the structures of four crotoxin homologs andtheir subunits purified from the venoms of C. durissus ter-rificus, Crotalus vegrandis, Crotalus scutulatus scutulatus andCrotalus viridis concolor. The CD spectra showed that theisolated subunits generally had a slightly lower proportionof a-helix and a greater proportion of b-sheet structurerelative to the intact toxins. However, the authors sug-gested that the enhanced fluorescence and alterations insecondary structure seen in the isolated subunits wereartifacts derived from irreversible structural changes thatoccurred during purification by urea ion-exchange chro-matography. In contrast, using CD and fluorescence tech-niques, Iglesias et al. (2005) showed that the flavonoidmorin induced important modifications in the secondarystructure of a CB isoform from C. durissus cascavella venom.Indeed, the partial protein unfolding caused by this flavo-noid resulted in variable effects on the biological activitiesof the isoform, i.e., marked inhibition of enzymatic activity,attenuation of the bactericidal action, but minimal inhibi-tion of edema formation and neuromuscular blockade.These findings suggested that this protein (and presumablyother CB isoforms) contains different molecular regionsresponsible for specific pharmacological activities. SimilarCD and fluorescence spectroscopic analyses of the inter-action between this CB isoform and umbelliferone(7-hydroxycoumarin) showed that this coumarin alsoaltered the secondary structure of the enzyme, reducingthe extent of its a-helical conformation. Moreover, thisstructural change attenuated the edema and myonecrosiscaused by this toxic PLA2 (Toyama et al., 2009).

Parallel to the reports described above, crystallographicstudies have also contributed to our understanding of thestructure of the crotoxin complex and its subunits. Achariet al. (1985) described a crystal X-ray diffraction analysisof crotoxin that provided information on the space group(P4122 or P4322) and unit cell parameters (a ¼ b ¼ 38.5 Å,c ¼ 256.9 Å, 1 molecule/asymmetric unit); no additionalinformation was provided in this report. Subsequently,Abrego et al. (1993) used small technique angle X-raydiffraction (SAXS) experiments to show that the overallshape of CA in solution at pH ¼ 1.5 was that of an oblateellipsoid of revolution with semi-axes a ¼ b ¼ 22 Å andc ¼ 10 Å. Based on X-ray diffraction data of crystallized CBand the analysis of molecular replacement data, Marchi-Salvador et al. (2007, 2008) reported the crystallographicstructure of CB and provided a complete structuraldescription of the crystal structure of this subunit, the firststructural data for a component of the crotoxin complex athigh atomic resolution. This work showed that the asym-metric unit of the crystal consisted of a tetrameric

arrangement of two heterodimers containing the isoformsCB1 and CB2 (Fig. 1). The two CB isoforms that formed thetetrameric crystallographic model possessed seven disul-fide bridges and, like other group IIA PLA2s, had an N-terminal a-helix (h1), a Ca2þ-binding loop, two antiparallela-helices (h2 and h3), two short strands formed by anti-parallel b-sheets (b-wing) and a C-terminal loop. Incontrast, the ‘short helix’ located between helix h1 and theCa2þ-binding loop of other group IIA PLA2s was not wellcharacterized in the CB1 and CB2 models.

The catalytic network for group IIA snake venom PLA2sformed by His48, Tyr52, Tyr73 and Asp99, is also fullyconserved in CB1 and CB2. Interestingly, despite theircommon structural features, only CB1 of the heterodimerscontained a Ca2þ ion in its Ca2þ-binding loop. The absenceof Ca2þ ions in CB2 probably resulted from the CB1/CB2interfacial contacts in the heterodimer that caused struc-tural modification of the CB2 Ca2þ-binding loop. Indeed,the Ca2þ-binding loop, the C-terminal region and the b-wing region with its two adjacent loops (residues 53–91)contain the most important structural differences betweenCB1 and CB2. In their study, Marchi-Salvador et al. (2008)also investigated the possible biological relevance of thetetrameric CB model. SDS-PAGE indicated that non-reduced CB occurred in a dimeric or tetrameric state insolution, although the latter was the predominant quater-nary assembly. Dynamic light scattering (DLS) assaysconfirmed the presence of these two molecular assembliesin solution and the predominance of the tetrameric formduring crystallization (protein concentration ¼ 6.0 mg/mL;T ¼ 291 K). In addition, theoretical data (buriedsurface ¼ 5370 Å2, DGint ¼ �34.3 kcal/mol andDGdiss ¼ 8.8 kcal/mol) obtained with the program PISAindicated that the interface contacts in the crystallographicstructure of CB were not formed by the crystal packing,which suggested that the experimental model was stable in

Fig. 1. Crystallographic model of the quaternary assembly of crotoxin B (CB).The complete structure consists of two heterodimers composed of a pair ofisoforms (CB1 and CB2). The amino acid sequences of the isoforms differ inonly eight residues. Model generated with the program PyMOL (DeLano,2002).

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solution (Krissinel and Henrick, 2007). Ser1 and Glu92,found exclusively in CB2 and CB1, respectively, formed twomutual salt bridges that were involved in the stabilizationof each heterodimer (Marchi-Salvador et al., 2008) (Fig. 2).Since CB1 and CB2 can occur in the venom of the same C. d.terrificus specimen (Faure and Bon, 1987, 1988; Faure et al.,1994) it is possible that after the dissociation of crotoxin atthe neural synapse these two isoforms may combine toform a complex in vivo. Together, these observationssuggest that CB1 and CB2 operate as complementary partsof a functional molecular complex, the quaternary config-uration of which is essential to the biological activities ofCB, especially neurotoxicity. Although the foregoing studieshave focused on only two CB isoforms, the conclusionsprobably also apply to other CB isoforms that have beenidentified in the venoms of C. d. terrificus and other C.durissus ssp. (Faure and Bon, 1988; Faure et al., 1991, 1993;Oliveira et al., 2002; see also Section 4 below).

CB binds to receptors at the presynaptic membrane ofseveral organisms, thereby inhibiting the release of acetyl-choline and, consequently, promoting neuromuscularblockade (reviewed in Kini, 2003). However, the exact loca-tion of the pharmacologically active sites in the structure ofsnake venom neurotoxic PLA2s remains unclear. Based on an

analysis of the amino acid sequences of three ammodytoxins(single chain PLA2 neurotoxins), Kri�zaj et al. (1989) identifiedthe C-terminal of these enzymes as the neurotoxic site. Inanother study, Carredano et al. (1998) showed that somegroup I neurotoxic snake venom PLA2s contain invariantamino acids in the region 70–100, which comprises theb-sheet and its vicinity, when compared with similarnon-neurotoxic toxins. Based on a theoretical model ofammodytoxin A (AtxA), a monomeric neurotoxic PLA2 fromVipera ammodytes ammodytes venom, Chioato and Ward(2003) concluded that presynaptic neurotoxic snake venomPLA2s possess a more extensive biologically active surfacethat also includes the C-terminal segment, the Ca2þ-bindingloop, a short helical turn, and other positions in the N-terminal helix and b-wing. Protein engineering studies ofAtxA have identified structural elements that contribute tothe presynaptic neurotoxicity of this toxin. According toPrijatelj et al. (2002), the C-terminal regions of ammody-toxins are important but not sufficient for neurotoxicity. Inaddition, Petan et al. (2002) indicated that residue Phe24plays an important role in the neurotoxic activity of AtxA. Kini(2003) also tried to identify neurotoxic sites from snakevenom PLA2s based on their primary and tertiary alignments.Rouault et al. (2006) concluded that the N-terminal region

Fig. 2. Salt bridges between the residues Ser1 (found exclusively in the CB2 isoform) and Glu92 (found exclusively in the CB1 isoform). All distances are in Å.Model generated with the program PyMOL (DeLano, 2002).

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was the major contributor to the neurotoxicity of a PLA2 fromOxyuranus scutellatus scutellatus, whereas the C-terminalloop and other regions were considered to have a minimalrole. Recently, Prijatelj et al. (2008) described a structure–function analysis of chimeric mutants of AtxA and VIIIa(a weak neurotoxic PLA2 from Daboia russelli russelli venom)in which they sought to determine the structural featuresresponsible for the different neurotoxic potencies of theserelated toxins. Their results indicated that the C-terminalsegment, interfacial binding surface (IBS) and N-terminalhelix were responsible for the high neurotoxicity of AtxA. Intheir crystallographic analysis, Marchi-Salvador et al. (2008)showed that all of these regions (N- and C-terminal regions,Ca2þ-binding loop and b-wing) are exposed on the surface ofheterodimers that compose the CB model, in an arrangementthat may favor neurotoxicity. Indeed, the association of CB1and CB2 may enhance neurotoxicity by creating new bindingsites not present in the individual isoforms. Hence, theexpression of different CB isoforms in the same venom glandmay serve to maximize the neurotoxicity of crotoxin to thedetriment of PLA2 activity.

More recently, CB isolated from C. durissus collilineatusvenom was crystallized and X-ray diffraction data werecollected to 2.2 Å (Salvador et al., 2009). The structurepresented a dimeric arrangement formed by CB1 and CB2isoforms similar to the dimer structure of CB from C. d.terrificus, thus confirming and reinforcing the findingsdescribed by Marchi-Salvador et al. (2008). Initial analysisof the C. d. collilineatus CB dimer indicated that its oligo-meric arrangement was stable in solution, in a mannersimilar to that reported for C. d. terrificus CB1/CB2. By usingSPOT data derived from the crystallographic structure of C.d. terrificus CB1/CB2, Fortes-Dias et al. (2009) identifiedpotentially important pharmacological sites (segmentsPhe11 to Ala18 and Tyr81 to Gly85) on the surface of the C.d. collilineatus CB dimer. The results of these experimentsidentified amino acids involved in the formation of thecrotoxin complex and the complex between CB and itsinhibitor CNF found in the blood of C. d. terrificus. Since bothCA and CNF inhibit CB, these molecules must interact withsites related to the CB neurotoxicity. Consequently, thesegments identified on the surface of the C. d. terrificus CB1/CB2 crystallographic structure are potentially importantsites involved in the CB neurotoxicity.

3. Neurotoxicity and other classic activitiesof crotoxin

In contrast to the numerous biochemical studies thatfollowed the crystallization of crotoxin (see Habermann andBreithaupt, 1978), detailed pharmacological investigationswere not reported until nearly three decades later when,during the International Symposium on Animal Venomsheld at the Instituto Butantan in Sao Paulo, in 1966, Prof.Oswaldo Vital Brazil and his colleagues presented a series ofpapers in which they described the toxicity (Vital Brazilet al., 1966b), neuromuscular activity (Vital Brazil, 1966),cardiovascular actions (Vital Brazil et al., 1966a) and neph-rotoxicity (Hadler and Vital Brazil, 1966) of crotoxin.

These studies showed that crotoxin was ca. two-fold moretoxic than the venom (which agreed with the observation

that the toxin accounted for w50% of the dry weight ofvenom). The toxicity varied considerably, depending on thetest species used (i.v. LD50 in pigeons and mice was 2.17 mg/kg,and 82 mg/kg, respectively) and the route of administration(i.v. and s.c. LD50 in mice: 82 mg/kg and 178 mg/kg, respec-tively). In various species (rats, guinea-pigs, rabbits, cats, dogsand monkeys), crotoxin produced flaccid paralysis similar tothat observed with curare, whereas in pigeons and mice theparalysis was of an ascendant type (in mice, the hind limbmuscles, but not the forelimb and neck muscles, wereaffected). The onset of paralysis generally occurred aftera long delay, lasted for several hours and was reversible in allspecies except rabbits. Diaphragm muscle was most resistantto blockade. In unanesthetized cats and dogs, intoxicationalso caused defecation, salivation, vomiting and renaldamage. Consciousness was unaffected. These findings con-trasted with the clonic convulsions without paralysisobserved after intracerebroventricular (i.c.v.) injection ofcrotoxin in cats (tachypnea, salivation, vomiting and deathwere also observed with this route of administration) (VitalBrazil et al., 1966b).

In anesthetized cats and dogs, crotoxin produced non-depolarizing neuromuscular blockade that persisted for24–48 h and was unaffected by neostigmine; there was noinhibition of adrenergic or ganglionar transmission. Thesefindings, together with the similarity to the actions ofsubstances such as curare, indicated that the mode ofaction of crotoxin was essentially peripheral. Based on thereduced sensitivity of denervated rat hemi-diaphragmpreparations to acetylcholine, the principal mode ofaction was concluded to be postsynaptic, although at thetime there was insufficient evidence to exclude a presyn-aptic action, i.e., a reduction in the release of neurotrans-mitter at the neuronal end-plate (Vital Brazil, 1966). Inanesthetized dogs, crotoxin (250 mg/kg, i.v.) caused transi-tory hypotension (25–33% decrease in blood pressure) thatdeveloped slowly (2–3 min after injection), in contrast tothe venom (250 mg/kg, i.v.) which produced triphasic bloodpressure changes (immediate hypotension, within 0.5 min,followed by hypertension and then a progressive, sustaineddecrease in blood pressure; 58% decrease); crotoxin had nomarked effects on respiration whereas the venom initiallyincreased the frequency and amplitude of respiratorymovements followed by apnea and then tachypnea (VitalBrazil et al., 1966a).

Together, these studies laid the groundwork for subse-quent investigations on the pharmacology and mechanismof action of crotoxin (for reviews, see Hendon and Bieber,1982; Bon et al., 1986, 1989; Bieber et al., 1990; Hawgoodand Bon, 1991; Choumet et al., 1996; Bon, 1997), the prin-cipal findings of which may be summarized as follows (seealso Fig. 3 and Table 1):

1. Identification of the principal mechanism involved inneuromuscular blockade. Vital Brazil (1966) initiallyconcluded that crotoxin caused blockade principally viaa postsynaptic action. However, although subsequentinvestigations confirmed this postsynaptic activity andshowed that it involves stabilization of the desensitizedform of the nicotinic acetylcholine receptor (Bon et al.,1979; Vital Brazil et al., 2000), several studies in the

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1970s demonstrated that the principal site of action ofcrotoxin at neuromuscular junctions was presynaptic(Vital Brazil and Excell, 1971; Hawgood and Smith,1977; Chang and Lee, 1977). Crotoxin also interfereswith neurotransmission in the myenteric plexus ofguinea-pig ileum via its presynaptic action (Anadonand Martinez-Larranaga, 1985; Muniz and Diniz,1989) and possibly by inhibiting choline uptake(Kattah et al., 2000). Imunohistochemistry hasconfirmed the localization of crotoxin at neuromus-cular junctions in mouse striated muscle (Cardi et al.,1992). Repeated administrations of crotoxin in vivoinduce tolerance to neuromuscular blockade, althoughthe precise mechanism has not been elucidated(Okamoto et al., 1993).

2. Demonstration that the neuromuscular blockadeinvolves a triphasic action on neurotransmitter release.This response includes a rapid initial decrease in themean quantal content of transmitter release followedby a secondary rise and then a third (final) phaseconsisting of a slow, progressive decrease in evokedrelease that eventually results in complete blockade

(Chang and Lee, 1977; Chang et al., 1977; Rodrigues-Simioni et al., 1990). This triphasic response and thecalcium and temperature dependence of the blockadeare characteristics shared with a variety of other snakevenom b-neurotoxins (presynaptic neurotoxins thathave phospholipase A2 activity) (Hawgood and Bon,1991; Bon, 1997; Schiavo et al., 2000; Rossetto andMontecucco, 2008).

3. Demonstration of synergism between the twocomponents of the crotoxin complex. Several studiesdemonstrated synergism between CA and CB, andsuggested that CA acted as a chaperone to direct CB toits site of action (Rubsamen et al., 1971; Hawgood andSmith, 1977; Chang and Su, 1978; Habermann andBreithaupt, 1978; Bon et al., 1979, 1989; Hawgood andSantana de Sa, 1979; Hendon and Tu, 1979; Bon,1982). This chaperone activity potentiates the biolog-ical activity of CB by ensuring interaction of thecomplex with correct binding sites (Bon, 1982;Hawgood and Bon, 1991). Bouchier et al. (1991)showed that CA is generated from a PLA2-likeprecursor (pro-CA) by the removal of three peptides,

Fig. 3. A composite scheme summarizing the principal intracellular actions of crotoxin in nerve and muscle. Green pathway/solid arrows – crotoxin interacts withcells by binding to specific cell surface receptors or is internalized via endocytosis or recycling synaptic vesicles; crotoxin may also interact directly with theplasma membrane to hydrolyze membrane phospholipids. Red pathway/dashed arrows – Crotoxin bound to cell surface receptors hydrolyzes plasma membranelipids, leading to permeabilization of the membrane, loss of ion selectivity and entry of extracellular Ca2þ. PLA2 activity of internalized crotoxin leads to its releasefrom endocytotic vesicles and subsequent interaction with organelle membranes that results in damage to synaptic vesicles and mitochondrial uncoupling (lossof Ca2þ homeostasis and reduced ATP production). Blue pathway/dotted arrows – entry of extracellular Ca2þ following membrane permeabilization markedlyincreases intracellular Ca2þ concentrations, leading to (1) enhanced exocytosis of acetylcholine at nerve terminal, (2) hypercontracture of muscle fibers, (3)activation of endogenous PLA2 that can cause further intracellular phospholipid hydrolysis and (4) activation of calpains involved in the degradation of intra-cellular proteins (myofilaments, cytoskeletal proteins, etc.). The activation of these various pathways leads to neuromuscular blockade and tissue damage(necrosis). Black X indicates inhibition of exocytosis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web versionof this article).

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leaving unchanged the molecule core cross-linked bydisulfide bridges; this structural similarity to CBcontributes to the ability of CA to form complexes withCB from C. durissus ssp. (Faure et al., 1991, 1994; Ponce-Soto et al., 2002) and venom PLA2 from other sources.Thus, Chang and Su (1981) reported that CB inhibitedthe presynaptic actions of notexin and notechis-5 buthad no effect on those of b-bungarotoxin. Later,Choumet et al. (1993) observed that CA formedcomplexes with agkistrodotoxin and ammodytoxin A,which are structurally similar to CB, and modified thebiological activities of these toxins. CB also inhibits theedema-forming activity of Apis mellifera, Naja naja andBothrops PLA2 (Landucci et al., 2000; Cecchini et al.,2004), but inhibits that of Naja mocambique mocambi-que (Landucci et al., 2000).

4. Confirmation of the importance of PLA2 activity forneuromuscular blockade. Phospholipase A2 activity isimportant for neuromuscular blockade (Marlas andBon, 1982; Anadon and Martinez-Larranaga, 1985;Hawgood and Smith, 1989; Hawgood and Bon, 1991)(Fig. 3), although arachidonate metabolites are notnecessarily involved (Edwards et al., 1990). Phospholi-pase A2 activity also contributes to additional effectssuch as the contraction of ileum smooth muscle(Anadon and Martinez-Larranaga, 1985; Muniz andDiniz, 1989), myotoxicity (Soares et al., 2001), andinflammation and pain.

5. Detection of high affinity binding sites. Based on theslow rate of reversal of crotoxin-induced paralysis, VitalBrazil (1966) concluded that there was a strong inter-action (tight binding) between the toxin and its‘‘receptors’’, which suggested the existence of specificbinding sites (proteins) for crotoxin. Various studieshave since confirmed this conclusion by identifyinghigh affinity binding sites and some of the proteinsinvolved (Delot and Bon, 1993; Tzeng et al., 1995; Kri�zajet al., 1997; Faure et al., 2003; reviewed in Kri�zaj andGubensek, 2000; Pungercar and Kri�zaj, 2007;Montecucco et al., 2008) (Fig. 3).

6. Demonstration of the central nervous system actions ofcrotoxin. In addition to its peripheral effects, crotoxinalso has important activity in the central nervoussystem that was noted by Vital Brazil et al. (1966b)following i.c.v. injection of the toxin in cats. Subse-quent studies demonstrated a central action in other

species (Habermann and Cheng Raude, 1975; Dorandeuet al., 1998) and have shown that crotoxin can inducebehavioral changes when administered intraperitone-ally (Moreira et al., 1996).

7. Investigation of the pharmacokinetics of crotoxin.Lomba et al. (1966) described the radiolabeling ofcrotoxin and showed that 131I-crotoxin retained itspharmacological properties. Pharmacokinetic studiesdone with radiolabeled crotoxin (Habermann, 1971;Habermann et al., 1972) and by ELISA (Barral-Nettoand von Sohsten, 1991), together with immunohisto-chemical analysis (Cardi et al., 1998), have investigatedthe clearance of crotoxin from the circulation, its tissuedistribution, and its renal accumulation and excretion.

8. Neutralization studies with antisera, antivenoms andmonoclonal antibodies. Based on neutralizationexperiments with antisera, antivenoms and mono-clonal antibodies, several investigations haveconfirmed that crotoxin is indeed the toxin responsiblefor the lethality (Kaiser et al., 1986; dos Santos et al.,1988; Kaiser and Middlebrook, 1988; Freitas et al.,1990; Choumet et al., 1998; Rodriguez et al., 2006)and neurotoxicity (Oshima-Franco et al., 1999; Beghiniet al., 2004a, 2005) of venoms from C. durissussubspecies. Interestingly, the serum of C. d. terrificuscontains a protein capable of neutralizing crotoxin inthe circulation and at presynaptic sites, and presum-ably protects these snakes against neuromuscularblockade following accidental envenoming (Fortes-Dias et al., 1991, 1994; Perales et al., 1995; Marquesdos Santos et al., 2005). This protein inhibits othersnake venom b-neurotoxins (Faure et al., 2000) andPLA2 from related pitvipers, e.g., Lachesis muta muta(Fortes-Dias et al., 1999).

9. Identification of crotoxin isoforms and homologs.Various studies have reported crotoxin isoforms in C.durissus subspecies (Faure and Bon, 1987; Lennon andKaiser, 1990; Hawgood and Bon, 1991; Faure et al.,1991, 1993, 1994; Beghini et al., 2000, 2004b;Hernandez-Oliveira et al., 2005; Toyama et al., 2003,2005; Ponce-Soto et al., 2002, 2007; Diz Filho et al.,2009; Pereanez et al., 2009) and crotoxin homologs inseveral other rattlesnakes and Asian pitvipers (Hendonand Bieber, 1982; Bieber et al., 1990; Hawgood and Bon,1991; Chen et al., 2004). The pharmacology of thesetoxins is generally similar to that described for crotoxin

Table 1Main classic pharmacological activities of crotoxin and its subunits. See text for discussion of each of these aspects.

Actions Reference

Neuromuscular blockadePresynaptic activity Vital Brazil and Excell, 1971; Hawgood and Smith, 1977; Chang and Lee, 1977; Anadon and

Martinez-Larranaga, 1985;Muniz and Diniz, 1989; Kattah et al., 2000; Cardi et al., 1992

Triphasic action onneurotransmitter release

Chang and Lee, 1977; Chang et al., 1977; Rodrigues-Simioni et al., 1990

Postsynaptic activity Vital Brazil, 1966, Bon et al., 1979; Vital Brazil et al., 2000Myotoxicity Breithaupt, 1976; Gopalakrishnakone and Hawgood, 1984; Gopalakrishnakone et al., 1984;

Kouyoumdjian et al., 1986; Oshima-Franco et al., 1999; Salvini et al., 2001; Soares et al., 2001;Beghini et al., 2004b; Melo et al., 2004; Miyabara et al., 2004a,b; Conte et al., 2008; Gutierrez et al., 2008

Nephrotoxicity Hadler and Vital Brazil, 1966, Monteiro et al., 2001, Martins et al., 2002, Amora et al., 2006Cardiotoxicity Santos et al., 1990; Hernandez et al., 2007

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from C. d. terrificus venom (Hawgood and Bon, 1991;Rangel-Santos et al., 2004).

10. Additional activities. Although historically consideredas a neurotoxin with a predominantly presynapticaction, crotoxin has also been shown to exert additionaleffects, including cardiotoxicity (Santos et al., 1990;Hernandez et al., 2007), nephrotoxicity (Hadler andVital Brazil, 1966; Monteiro et al., 2001; Martins et al.,2002; Amora et al., 2006) and myotoxicity (Breithaupt,1976; Gopalakrishnakone and Hawgood, 1984;Gopalakrishnakone et al., 1984; Kouyoumdjian et al.,1986; Oshima-Franco et al., 1999; Salvini et al., 2001;Soares et al., 2001; Beghini et al., 2004b; Melo et al.,2004; Miyabara et al., 2004a,b; Conte et al., 2008;Gutierrez et al., 2008), as well as having roles ininflammation and pain (see below). Crotoxin is themajor contributor to the systemic myotoxicity seenfollowing bites by C. d. terrificus venom (Azevedo-Marques et al., 1982; Cupo et al., 1988; Gutierrez et al.,2008).

4. Novel pharmacological activities of crotoxin

In addition to the well-known pharmacological activitiessummarized above, numerous studies in recent years haveshown that crotoxin also has a range of actions unrelated toits classic activities such as neurotoxicity and myotoxicity.The discovery of these different activities has considerablyexpanded our understanding of the versatility of thismolecule and is unique in that very few similar studies havebeen reported for other b-neurotoxins. Table 2 and Fig. 4summarize the activities that are discussed below.

4.1. Immunomodulatory and anti-inflammatory activity

Several studies have shown that crotoxin or its isolatedsubunits (CA and CB) modulate immune and inflammatoryresponses by affecting the vascular and cellular compo-nents of these responses (Table 2). Mice pre-treated withcrotoxin and immunized with human serum albumin(HSA) or ovalbumin (OVA) produced lower levels of IgG1antibodies than control mice (Cardoso and Mota, 1997).Similar results were obtained in rats immunized withbovine serum albumin (BSA) (Zambelli, V. and Cury, Y.,unpublished data). Crotoxin also inhibits the concanavalinA (Con-A)-induced proliferation of splenic cells (Cardosoet al., 2001; Rangel-Santos et al., 2004). This inhibitoryaction has been attributed to both subunits of crotoxin.Garcia et al. (2003) showed that CA significantly inhibitedthe Con-A-stimulated proliferation of cultured lympho-cytes. The reduction in the blastogenic response is accom-panied by a significant increase in the production ofprostaglandin E2 (PGE2) by macrophages. Since PGE2

inhibits mitogen-induced lymphocyte responses (Sergeevaet al., 1997), this could explain the inhibitory effect of CA onlymphocyte proliferation. More recently, Castro et al.(2007) reported that CA suppresses the proliferation oflymphocytes obtained from rats with experimental auto-immune neuritis stimulated either with non-specificmitogen or neuritogenic peptides. In contrast to the find-ings for CA, Cardoso et al. (2001) and Rangel-Santos et al.(2004) observed that, unlike the crotoxin complex, CB didnot inhibit the proliferation of spleen cells, a finding thatagrees with the inhibitory action of CA present in the toxincomplex. However, we have observed that lymphocytes

Table 2Novel pharmacological activities of crotoxin, its subunits and their parts. See text for discussion of each of these aspects.

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ImmunomodulatoryInhibition of the humoral immune response to classic protein antigens Cardoso and Mota (1997)Inhibition of Con-A proliferation of splenic/lymphocyte cells Cardoso et al. (2001), Rangel-Santos et al. (2004),

Garcia et al. (2003), Castro et al. (2007)Inhibition of IL-4 and IL-10 production by cultured spleen cells, increase

in adherence to endothelial cells of the microcirculation; decrease inthe number of circulating blood and lymph lymphocytes

Cardoso et al. (2001), Rangel-Santos et al. (2004),Zambelli et al. (2008)

Anti-inflammatoryInhibition of macrophage spreading and phagocytic activity Sampaio et al. (2003, 2005)Enhanced polymerization of F-actin and inhibition of tyrosine

phosphorylation and Rac and RhoA GTPase activitySampaio et al. (2006a)

Inhibition of carrageenin-induced paw edema Landucci et al., (1995), Nunes et al., (2009)

Anti-tumorInhibition of tumor growth Newman et al. (1993), Costa et al. (1998); Cura et al. (2002)Cytotoxicity in tumor cells Baldi et al. (1988), Corin et al. (1993), Rudd et al. (1994),

Donato et al. (1996)Induction of apoptosis Yan et al. (2006)

Analgesic actionCrotoxin

Inhibition of acute phasic and tonic pain and of pain-evoked unit dischargeof neurons in thalamic parafascicular nucleus (activation of centralserotonergic receptors)

Zhang et al. (2006), Zhu et al. (2008)

Inhibition of chronic neuropathic pain (activation of central muscarinicand serotonergic receptors)

Nogueira-Neto et al. (2008), Zhu et al. (2008)

CrotalphineInhibition of acute pain and chronic neuropathic pain (activation of

peripheral opioid receptors)Konno et al. (2008), Gutierrez et al. (2008)

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isolated from mesenteric lymph nodes of rats injected s.c.with CB show a low proliferative response to Con-A(Zambelli, V. and Cury, Y., unpublished data). Culturedspleen cells obtained from mice treated with crotoxinrelease low amounts of interleukin (IL)-4 and IL-10 afterstimulation with Con-A (Cardoso et al., 2001; Rangel-Santos et al., 2004). Alterations in cytokine release couldmodulate the action of crotoxin in the humoral immuneresponse and lymphocyte proliferation.

In addition to the inhibition of lymphocyte activity,crotoxin substantially alters the distribution of theseleukocytes in blood and lymph, and increases the numbersof B and T lymphocytes in lymph nodes (Cardoso et al.,2001; Zambelli et al., 2008). CB is responsible for thedecrease in the number of blood leukocytes (lymphocytes)induced by crotoxin (Zambelli et al., 2008). Crotoxin alsostimulates leukocyte adherence to endothelial cells in themicrocirculation and high endothelial venules in lymphnodes; this adherence could contribute to the decrease inthe number of circulating lymphocytes. Alterations inleukocyte–endothelium interactions caused by crotoxinmay result partly from changes in the expression of adhe-sion molecules since crotoxin increases the expression ofthe adhesion molecule LFA-1 in lymphocytes (Zambelliet al., 2008).

Crotoxin inhibits the spreading and phagocytic activityof macrophages in vivo and in vitro (Sampaio et al., 2003),and this phenomenon is mediated by CB, but not CA(Sampaio et al., 2005). The phagocytic activity of macro-phages involves rearrangement of the actin cytoskeleton

and the activity of small Rho GTPases (Niedergang andChavrier, 2005). Alterations in the levels and activities ofproteins involved in the intracellular signaling pathwaysrelated to phagocytosis may therefore be important in theimpairment of macrophage function by crotoxin. In agree-ment with this, we have observed that the inhibition ofmacrophage function by crotoxin involves an increase incytoplasmic F-actin levels that leads to abnormal actinreorganization; there is also inhibition of tyrosine phos-phorylation and Rac and RhoA GTPase activity (Sampaioet al., 2006a). Interestingly, interference with F-actinorganization has also been implicated in the neurotoxicityof another b-neurotoxin, AtxA, in mouse motoneurone-likecells (Pra�znikar et al., 2008).

The inhibitory effects of crotoxin on macrophages andlymphocytes are mediated by lipoxygenase-derived eicos-anoids (Sampaio et al., 2006b; Zambelli et al., 2008), indi-cating that PLA2 activity is important in this response.Sampaio et al. (2006b) also observed that macrophagesincubated with crotoxin release lipoxin A4, which suggeststhat this lipid mediator could be responsible for theinhibitory action of the toxin on leukocytes. In addition tolipoxin A4, crotoxin also stimulates the formation of PGE2

by macrophages, but this eicosanoid does not mediate theinhibitory effect of the toxin in these cells (Sampaio et al.,2006b; Moreira et al., 2008). The contribution of gluco-corticoids to the action of crotoxin on leukocytes must alsobe considered since Chisari et al. (1998) and Cardoso et al.(2001) observed that crotoxin increases ACTH release andserum corticosterone levels.

Fig. 4. Principal cellular actions of crotoxin in the microcirculation, lymphatic system and interstitial tissue. See Section 4 of the text for details.

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In agreement with studies showing the inhibitoryactivity of crotoxin on immune responses and inflamma-tory cells such as macrophages, various reports haveshown that this toxin also inhibits inflammatory responses(Table 2). Crotoxin induces a long-lasting anti-edemato-genic effect (Nunes at al., 2009) that is mediated by acti-vation of n-formyl peptide receptors. Moreover, CAinhibits carrageenin-induced rat paw edema (Landucciet al., 1995), probably through interaction with secretoryPLA2 released during the inflammatory process. Thissubunit also inhibits rat and mouse paw edema inducedby different PLA2s isolated from bee and snake venoms(Landucci et al., 2000; Cecchini et al., 2004). Despite thisanti-inflammatory activity of crotoxin, Camara et al.(2003) reported that CB increased the microvascularpermeability in rat dorsal skin, probably via mast cellactivation, with the release of histamine and serotonin andthe stimulation of sensory C-fibers, leading to the releaseof substance P.

The demonstration that crotoxin interferes withimmune and inflammatory responses indicates that thistoxin contributes to the well-known anti-inflammatoryand immunomodulatory activities of C. d. terrificus venom(Amorim et al., 1951; Sousa-e-Silva et al., 1996; Cardoso andMota, 1997; Rangel-Santos et al., 2004; Nunes et al., 2009).Furthermore, the immunomodulatory and anti-inflammatory activities of crotoxin could account for thelack of significant local inflammation observed clinicallyafter envenoming by C. d. terrificus (Azevedo-Marques et al.,2009; Warrell, 2004).

4.2. Antibacterial activity

Various reports have shown that CB from C. durissus ssp.has bactericidal activity towards a variety of species (Bur-kholderia pseudomallei, Claribacter michiganensis michi-ganensis, Enterobacter aerogenes, Escherichia coli,Staphylococcus aureus, Pseudomonas aeruginosa and Zan-thomonas axonopodis pv. passiflorae) and that this activity isdependent on PLA2 enzymatic activity (Soares et al., 2001;Oliveira et al., 2003, 2003; Toyama et al., 2003; Samy et al.,2006, 2007; Diz Filho et al., 2009). However, the observa-tion that bactericidal activity can be partially attenuated byacetylation of the Lys residues suggests that regions otherthan the catalytic site of the toxin are also involved in thisphenomenon (Soares et al., 2001). The relevance of thisbactericidal activity to envenoming by C. durissus ssp. isunclear but could perhaps contribute to the very lowfrequency of local bacterial infections following bites bythese snakes.

4.3. Analgesic activity

The venom of C. d. terrificus has long been known tohave analgesic activity in humans (Brazil, 1934), and thishas been confirmed in experimental models of pain (Giorgiet al., 1993; Gutierrez et al., 2008). Various studies haveshown that crotoxin induces antinociception (Table 2). Inexperimental models of acute phasic (hot-plate and tail-flick tests) and tonic (acetic acid writhing test) pain, ananalgesic effect was detected when crotoxin was

administered i.p., i.c.v. or by microinjection into the peri-aqueductal gray area (Zhang et al., 2006). Electrophysio-logical studies in rats demonstrated that crotoxin directlyinhibited the pain-evoked discharge of neurons in thethalamic parafascicular nucleus (Zhu et al., 2008), consid-ered to be the integration center in pain modulation(Weigel and Krauss, 2004). These findings clearly indicatedthat crotoxin-induced analgesia by an action in the centralnervous system. More recently, Nogueira-Neto et al. (2008)demonstrated that crotoxin inhibited neuropathic paininduced by rat sciatic nerve transection when directlyapplied to the nerve stumps and also when administereds.c. In this model of chronic pain, the analgesic effect of thetoxin was long-lasting since it was detected up to 64 daysafter local treatment. Theoretically, the neurotoxicity andmyotoxicity caused by crotoxin (see above) could interferewith the responses of experimental animals to nociceptivestimuli by affecting their locomotor activity. However, thedoses of crotoxin that showed analgesic activity did notalter the locomotor activity of the animals, thus excludingsuch interference in these experiments (Zhang et al., 2006;Nogueira-Neto et al., 2008).

The mechanisms involved in the analgesic action ofcrotoxin vary, depending on the experimental model usedto assess pain. Zhang et al. (2006) and Zhu et al. (2008),who used behavioral and electrophysiological models ofacute pain, showed that muscarinic and opioid receptorsare not involved in the analgesic effect of crotoxin; incontrast, Nogueira-Neto et al. (2008) reported that activa-tion of central muscarinic receptors is involved in crotoxin-induced analgesia in a model of neuropathic pain. Inaddition, central serotonergic and noradrenergic systemshave been implicated in the toxin’s analgesic effect(Nogueira-Neto et al., 2008; Zhu et al., 2008). Centralcholinergic, noradrenergic and serotonergic systemsparticipate in the brain and spinal inhibition of paintransmission, and an interplay between these systems hasbeen proposed (Millan, 2002). The findings of Nogueira-Neto et al. (2008) and Zhu et al. (2008) suggest that suchinterplay may be involved in the analgesic action of cro-toxin. Nogueira-Neto et al. (2008) also observed thateicosanoids derived from the lipoxygenase pathwaymediate the analgesic effect of crotoxin, suggesting thatPLA2 activity is important for the toxin-induced effect. Asdiscussed above, crotoxin can affect the cellular andvascular components of inflammatory and immuneresponses. However, the extent to which these actionscontribute to the analgesic effect of this toxin remains to bedetermined.

Recently, a novel 14-amino acid peptide (crotalphine)with analgesic activity was identified in C. d. terrificusvenom (Konno et al., 2008; Gutierrez et al., 2008). Theprimary sequence of crotalphine is identical to the g-chainof crotapotin, the CA component of crotoxin. As discussedabove, CA has anti-inflammatory and immunomodulatoryactivities (Landucci et al., 1995, 2000; Garcia et al., 2003;Castro et al., 2007), but there are no literature reportsindicating that CA induces antinociception. In contrast toCA, low doses of crotalphine administered orally, s.c. or i.v.to mice and rats produce long-lasting (3–5 days) analgesiathat is mediated by the activation of peripheral k and

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d opioid receptors (Konno et al., 2008; Gutierrez et al.,2008). Compared to crotalphine, crotoxin is inactive whenadministered orally (Giorgi et al., 1993) and its analgesicactivity is not mediated by the activation of opioid recep-tors (Zhang et al., 2006; Nogueira-Neto et al., 2008).

Clinically, crotoxin has been tested in the treatment ofcancer pain (Cura et al., 2002). In a phase I clinical trial inpatients with solid tumors refractory to conventionaltherapy, crotoxin reduced tumor mass and reduced orabolished the accompanying pain. An analgesic effect wasobserved in 78% of the patients, although there was nodirect correlation between analgesia and the reduction intumor mass. We have also observed that crotoxin inhibitstumor growth and induces analgesia in laboratory animals(Brigatte and Cury, unpublished data).

4.4. Anti-tumor activity

Various studies have shown that crotoxin has anti-tumor activity in vivo and in vitro and interferes with tumorgrowth by modifying selective cellular processes associatedwith cell growth (Baldi et al., 1988; Corin et al., 1993;Newman et al., 1993; Rudd et al., 1994; Donato et al.,1996; Cura et al., 2002) (Table 2). Crotoxin alone or incombination with cardiotoxin (VRCTC-310) is cytotoxic toseveral tumor cell lines of human (leukemia as well as lung,colon, renal, ovary and mammary ductal carcinoma,melanoma and brain tumor) and murine (sarcoma, Erhlichtumor and erythroleukemia) origin (Corin et al., 1993;Newman et al., 1993; Rudd et al., 1994). The efficacy ofVRCTC-310 has been demonstrated in humans sufferingfrom advanced cancer (Costa et al., 1997, 1998).

A study of the cytotoxic and anti-tumor activities ofcrotoxin by the National Cancer Institute (DevelopmentalTherapeutics Program, NSC 624244) showed that the toxinwas effective against human melanomas, central nervoussystem tumors and lung carcinomas. Comparative studieswith other anti-tumor compounds indicated that crotoxinhad a unique spectrum of cytotoxicity in vitro, witha distinct anti-tumor mechanism (Paull et al., 1989;Newman et al., 1993).

The cytotoxicity of crotoxin is initially mediated byinteraction with high affinity binding sites on the cellsurface (Kri�zaj and Gubensek, 2000; Montecucco et al.,2008) and involves the PLA2 activity of the toxin (Corinet al., 1993). Donato et al. (1996) suggested that trans-membrane receptors involved in growth signaling, e.g.,epidermal growth factor receptor (EGFr), are the cellulartargets for the anti-proliferative activity of crotoxin andthat this activity is dependent on CB of the toxin. Morerecently, Yan et al. (2006) demonstrated that crotoxincauses collapse of the mitochondrial membrane potential,the release of cytochrome c and activation of caspase-3 inthe chronic myeloid leukemia cell line K562 cells, sug-gesting the involvement of apoptotic mechanisms incrotoxin-induced cell death.

In addition to the activity of crotoxin in cultured tumorcells, anti-tumor activity has also been observed in vivo.Daily i.m. injections of crotoxin inhibited the growth ofLewis lung carcinoma and MX-1 human mammary carci-noma by 83% and 69%, respectively (Newman et al., 1993;

Cura et al., 2002). In contrast, lower activity was observedagainst HL-60 leukemia cells (Cura et al., 2002), perhapsbecause of greater selectivity of the toxin for solid tumors.In an experimental model of cancer pain induced by theintraplantar injection of Walker 256 carcinoma cells(Brigatte et al., 2007), crotoxin reduced tumor growth andcaused analgesia (Brigatte and Cury, unpublished data).Although the mechanisms involved in the anti-tumoraction of the toxin in this model of cancer pain have not yetbeen determined, preliminary histopathological analysissuggests that crotoxin inhibits neovascularization of thetumor mass.

Based on data showing the anti-tumor efficacy ofcrotoxin in vitro and in vivo, a patent (‘‘Crotoxin complexas cytotoxic agent’’) was filed at the US Patent andTrademark Office on November 17, 1992 (http://www.patentstorm.us/patents/5164196.html). In 1995, the Foodand Drug Administration (FDA-USA) approved a phase Istudy in patients with solid tumors refractory toconventional therapy. In this study, the i.m. administra-tion of crotoxin for 30 consecutive days reduced thetumor mass by >50% in some patients, with completeregression of the primary tumor mass in one patient(Cura et al., 2002). Data of the phase II clinical trial havenot yet been published.

5. Future directions

The purification and crystallization of crotoxin by Slottaand Fraenkel-Conrat in 1938 was undoubtedly a landmarkin toxinology. The availability of pure toxin provided theimpetus for detailed pharmacological studies that initiallyfocused on the neurotoxicity and myotoxicity of thismolecule. However, in recent years our knowledge of thistoxin’s biological activities has extended far beyond thesetraditional fields to include immunomodulatory, anti-inflammatory, anti-microbial and anti-tumor and anal-gesic properties (Table 2, Fig. 4). This new information hasgreatly improved our understanding of the pathophysio-logical role of crotoxin in envenoming by C. d. terrificus andrelated subspecies. However, the molecular mechanismsinvolved in these new activities are still not well charac-terized. In this regard, future studies should seek to identifythe possible molecular targets for crotoxin in different celltypes (tumor cells, leukocytes, and neuronal and non-neuronal cells involved in the transmission and regula-tion of pain), in addition to examining how crotoxininteracts with intracellular signaling pathways of thesecells. Future studies should also address the role of PLA2

activity, both of crotoxin itself and of endogenous cytosolicPLA2, in the intracellular effects of this toxin. In addition,the importance of 5-lipoxygenase metabolites in modu-lating intracellular signaling and cellular functions requiresclarification. Knowledge of these mechanisms could help toexplain how a neurotoxic PLA2 can induce a variety ofpharmacological effects that are not directly related toneurotoxicity. Such information could also be useful inassessing potential therapeutic applications of crotoxin orcrotoxin-based drugs in conditions such as inflammation,pain and cancer.

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Conflict of interest

The authors have no conflicts of interest with this work.

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